Page 5 : *, , *, , Pharmacognosy & Phytochemistry-II, Edition 2019, , Copyright © All Rights Reserved, This book is sole subject to t he condition that it shall not, by way of trade or otherwise, be lent, resold,, hired out, or otherwise circulated without the publisher’s prior written consent, in any form of binding or, cover, other than that in which it is published and without including a similar condition. This condition, being imposed on the subsequent purchaser and without limiting the rights under copyright reserved, above, no part of this publication may be reproduced, stored in or transmitted in any form or by any, means (electronic, mechanical, photocopying, recording or otherwise), without the prior written, permission of both the copyright owner and the below mentioned publisher of this book., , Published by :, , Thakur Publication Pvt. Ltd., H.O.-645B/187, Abhishekpuram, Jankipuram Extension,, Lucknow-226021, Mob.: 9415584997/98, 9235318591/94/95/96/97/22/17/24., Website: www.tppl.org.in, , E-mail:
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*, , *, , “Dedicated, to, my Profession”, , Dr. K. Prabhu, , “Dedicated to, Dr.Palani. G. Periasamy,, Chairman, PGP Group of Educational and Research Institutions, Namakkal, &, Mrs.Visalakshi Periasamy,, Vice-Chairman,, PGP Group of Educational and Research Institutions”, , -Dr. G. Arunachalam, , *, , *
Page 7 : *, , *, , Preface, It gives us immense pleasure to place before the, B.Pharm Fifth Semester, pharmacy students the book on “Pharmacognosy and Phytochemistry-II”., This book has been written strictly in accordance with the c, urrent syllabus, prescribed by Pharmacy Council of India, for B.Pharm students. Keeping in view, the requirements of students and teachers, this book has been written to cover all, the topics in an easy, -to-comprehend manner within desired limits of the, prescribed syllabus, and it provides the students fundamentals of secondary, metabolites, phytochemistry, and isolation, industrial production, estimation or, analysis, and utilisation of phytoconstituents which are required by them during, their pharmaceutical career., All efforts have been made to keep the text error -free and to present the subject, in a student friendly and easy to understand. However, any suggestions and, constructive comments would be highly appreciated and incorporated in the, future edition., Please e-mail us at,, Website,, , *, ,
[email protected], www.tppl.org.in, , *
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*, , *, , Acknowledgement, My thanks to Dr. Selvakumar.S, M.Pharm, Ph.D, Professor & HOD in Dept of, Pharmaceutical Chemistry, Cherraan’s College of Pharmacy, Coimb atore who, encouraged me in to this venture and Mr., K. Ponnudurai M.Pharm, (Ph.D),, Professor & HOD in Dept of Pharmacology, Cherraan’s College of Pharmacy,, Coimbatore. I thank the management of Cher, raan foundation trust for their, support., Finally, I thank Thakur Publication Pvt. Ltd., especially, Ms. Tuhina Banerjee, (Copy Editor) and Mr. Sharad Kushwaha (Marketing Coordinator), for providing, me an opportunity to be a part of this book., , -Dr. K. Prabhu, First and foremost, I would like to thank Almighty God. In the process of putting, this book together in front of you, I reali sed how precious gift God has given to, me by giving me the capability and the power to believe in m, y passion and, pursue my dreams, I would also like to thank the Management of PGP College of Pharmaceutical, Science & Research Institute, for their constant motivation and support., I owe a deep sense of appreciation for my academic Colleagues and Students,, for their whole hearted cooperation and support. I would like to extend my, gratitude towards my Family Members, for their cooperation and blessings., , -Dr. G. Arunachalam, , *, , *
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-9-, , Contents, Module 1, , Module 2, , Chapter 1: Metabolic Pathways in, Higher Plants and Their, Determination, , Chapter 3: Secondary Metabolites-I, , 1.1., 1.1.1., 1.1.2., 1.1.2.1., 1.1.2.2., 1.1.2.3., 1.1.3., 1.1.3.1., 1.1.3.2., 1.1.3.3., 1.1.3.4., 1.1.3.5., 1.1.3.6., 1.1.3.7., 1.2., 1.3., , Metabolic Pathways, Introduction, Basic Metabolic Pathways, Shikimic Acid Pathway, (Amino Acid Pathway), Acetate-Mevalonate, Pathway, Acetate-Malonate Pathway, Formation of Different, Secondary Metabolites, Biogenesis of Alkaloids, Biogenesis of Glycosides, Biogenesis of Terpenes, Biogenesis of Steroids, Biogenesis of Isoprenoid, Compounds, Biogenesis of Carotenoids, Biogenesis of Flavonoids, Summary, Exercise, , 13, 13, 13, 15, 17, 18, 19, 20, 23, 26, 27, 28, 29, 30, 31, 32, , Chapter 2: Utilisation of, Radioactive Isotopes, 2.1., 2.1.1., 2.1.2., 2.1.3., 2.1.4., 2.1.5., 2.1.5.1., 2.1.5.2., 2.1.5.3., 2.1.5.4., 2.2., 2.3., *, , Biogenetic Studies, Introduction, Using Isolated Organs/, Tissues, Grafting Methods, Use of Mutant Strains, Utilisation of Radioactive, Isotopes, Significance of Tracer, Technique, Criteria for Tracer/Isotope, Selection, Steps Involved in Tracer, Technique, Applications of Tracer, Technique, Summary, Exercise, , 33, 33, 33, 34, 34, 34, 35, 35, 35, 38, 38, 39, , 3.1., 3.1.1., 3.1.2., 3.2., 3.2.1., 3.2.2., 3.2.2.1., 3.2.2.2., 3.2.2.3., 3.2.2.4., 3.2.2.5., 3.2.2.6., 3.2.3., 3.2.4., 3.2.5., 3.2.6., 3.2.7., 3.2.7.1., 3.2.7.2., 3.2.7.3., 3.2.7.4., 3.3., 3.4., , Secondary Metabolites, Introduction, Classification, Alkaloids, Introduction, Chemistry, Isomerism, Biogenesis, Qualitative Analysis, Quantitative Analysis, Isolation and Extraction of, Alkaloids, Purification of Crude, Extract of Alkaloids, Chemical Classes, Biosources, Therapeutic Uses, Commercial Applications, Individual Drugs, Vinca, Rauwolfia, Belladonna, Opium, Summary, Exercise, , 41, 41, 41, 42, 42, 43, 47, 48, 49, 51, 52, 54, 54, 57, 57, 58, 59, 59, 61, 65, 68, 72, 73, , Chapter 4: Secondary Metabolites - II, 4.1., 4.1.1., 4.1.2., 4.1.3., 4.1.4., 4.1.5., 4.1.5.1., 4.1.5.2., 4.1.5.3., 4.1.5.4., 4.1.5.5., 4.1.5.6., 4.1.6., 4.1.6.1., 4.1.6.2., , Phenylpropanoids, Introduction, Chemical Classes, Biosources, Biosynthesis, Flavonoids, Chemistry, Biosynthesis, Chemical Classes, Biosources, Therapeutic Uses, Commercial Applications, Lignans, Biosources, Preparation, , 74, 74, 74, 74, 75, 77, 77, 78, 78, 80, 80, 81, 81, 82, 82, *
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- 10 -, , 4.1.6.3., 4.1.7., 4.1.8., 4.2., 4.2.1., 4.2.2., 4.2.3., 4.2.4., 4.2.5., 4.3., 4.3.1., 4.3.2., 4.3.2.1., 4.3.2.2., 4.3.2.3., 4.3.2.4., 4.3.3., 4.3.4., 4.3.5., 4.3.6., 4.3.6.1., 4.3.6.2., 4.3.6.3., 4.4., 4.4.1., 4.4.2., 4.4.3., 4.4.4., 4.4.5., 4.5., 4.6., , Characteristic Features, Tea, Ruta, Steroids, Introduction, Chemical Classes, Biosources, Therapeutic Uses, Commercial Applications, Cardiac Glycosides, Introduction, Chemistry, Biosynthesis, Qualitative Analysis, Quantitative Analysis, Isolation and Extraction, Chemical Classes, Biosources, Therapeutic Uses, Individual Drugs, Liquorice, Dioscorea, Digitalis, Triterpenoids, Introduction, Chemistry, Chemical Classes, Biosources, Therapeutic Uses, Summary, Exercise, , 82, 82, 84, 86, 86, 87, 87, 87, 87, 88, 88, 88, 88, 89, 90, 90, 91, 92, 93, 93, 93, 96, 97, 100, 100, 101, 101, 103, 104, 104, 105, , Chapter 5: Secondary Metabolites- III, 5.1., 5.1.1., 5.1.2., 5.1.2.1., 5.1.2.2., 5.1.3., 5.1.4., 5.1.5., 5.1.6., 5.1.6.1., 5.1.6.2., 5.1.6.3., 5.1.6.4., *, , Volatile Oils, Introduction, Chemistry, Qualitative Analysis, Isolation of Volatile Oils, Chemical Classes, Biosources, Therapeutic Uses, Individual Drugs, Mentha, Clove, Cinnamon, Fennel, , 107, 107, 107, 109, 110, 110, 112, 112, 113, 113, 115, 118, 120, , 5.1.6.5., 5.2., 5.2.1., 5.2.2., 5.2.2.1., 5.2.2.2., 5.2.2.3., 5.2.3., 5.2.4., 5.2.5., 5.2.6., 5.2.7., 5.2.7.1., 5.2.7.2., 5.2.7.3., 5.3., 5.3.1., 5.3.2., 5.3.2.1., 5.3.2.2., 5.3.2.3., 5.3.3., 5.3.4., 5.3.5., 5.3.6., 5.3.7., 5.3.7.1., 5.3.7.2., 5.3.7.3., 5.3.7.4., 5.3.7.5., 5.3.7.6., 5.4., 5.5., , Coriander, Tannins, Introduction, Chemistry, Extraction, Qualitative Analysis, Quantitative Analysis, Chemical Classes, Biosources, Therapeutic Uses, Commercial Applications, Individual Drugs, Pale Catechu, Black Catechu, Pterocarpus, Resins, Introduction, Chemistry, Biogenesis, Qualitative Analysis, Quantitative Analysis, Chemical Classes, Biosources, Therapeutic Uses, Commercial Applications, Individual Drugs, Benzoin, Guggul, Ginger, Asafoetida, Myrrh, Colophony, Summary, Exercise, , 123, 125, 125, 125, 125, 126, 126, 127, 128, 129, 129, 130, 130, 133, 135, 136, 136, 137, 138, 138, 139, 142, 143, 143, 144, 144, 144, 147, 149, 151, 153, 154, 156, 158, , Chapter 6: Secondary Metabolites- IV, 6.1., 6.1.1., 6.1.2., 6.1.2.1., 6.1.2.2., 6.1.2.3., 6.1.3., 6.1.3.1., 6.1.3.2., 6.1.3.3., , Glycosides, Introduction, Chemistry, Biogenesis, Qualitative Analysis, Quantitative Analysis, Chemical Classes, On the Basis of Glycone, On the Basis of Aglycone, On the Basis of Glycosidic, , 159, 159, 159, 160, 160, 162, 164, 165, 165, 165, *
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- 11 -, , 6.1.4., 6.1.5., 6.1.6., 6.1.6.1., 6.1.6.2., 6.1.6.3., 6.2., 6.2.1., 6.2.2., 6.2.2.1., 6.2.2.2., 6.2.2.3., 6.2.3., 6.2.4., 6.2.5., 6.2.5.1., 6.2.5.2., 6.2.6., 6.2.7., 6.2.7.1., 6.2.7.2., 6.2.7.3., 6.2.8., 6.2.9., 6.2.9.1., 6.2.9.2., 6.2.9.3., 6.2.9.4., 6.3., 6.4., , Linkage, Biosources, Therapeutic Uses, Individual Drugs, Senna, Aloes, Bitter Almond, Terpenoids, Introduction, Chemistry, Biogenesis, Qualitative Analysis, Quantitative Analysis, Chemical Classes, Biosurces, Iridoids, Biosources, Therapeutic Uses, Other Terpenoids, Individual Drugs, Gentian, Artemisia, Taxus, Naphthoquinones, Carotenoids, Chemistry, Chemical Classes, Biosources, Therapeutic Uses, Summary, Exercise, , 166, 166, 167, 167, 170, 174, 176, 176, 176, 177, 179, 179, 183, 185, 186, 187, 187, 188, 190, 190, 193, 194, 197, 198, 199, 201, 201, 201, 201, 202, , Module 3, Chapter 7: Isolation, Identification, and Analysis of Phytoconstituents, 7.1., , Isolation, Identification, and Analysis of, Phytoconstituents, 7.1.1., Introduction, 7.1.2., Classes of, Phytoconstituents, 7.2., Terpenoids, 7.2.1., Introduction, 7.2.2., Menthol, 7.2.2.1. Isolation, 7.2.2.2. Identification, *, , 204, , 204, 204, 207, 207, 207, 208, 208, , 7.2.2.3., 7.2.3., 7.2.3.1., 7.2.3.2., 7.2.3.3., 7.2.4., 7.2.4.1., 7.2.4.2., 7.2.4.3., 7.3., 7.3.1., 7.3.2., 7.3.2.1., 7.3.2.2., 7.3.2.3., 7.3.3., 7.3.3.1., 7.3.3.2., 7.3.3.3., 7.4., 7.4.1., 7.4.2., 7.4.2.1., 7.4.2.2., 7.4.2.3., 7.4.3., 7.4.3.1., 7.4.3.2., 7.4.3.3., 7.4.4., 7.4.4.1., 7.4.4.2., 7.4.4.3., 7.4.5., 7.4.5.1., 7.4.5.2., 7.4.5.3., 7.5., 7.5.1., 7.5.2., 7.5.2.1., 7.5.2.2., 7.5.2.3., 7.5.3., 7.5.3.1., 7.5.3.2., , Analysis, Citral, Isolation, Identification, Analysis, Artemisin, Isolation, Identification, Analysis, Glycosides, Introduction, Glycyrrhetinic Acid, Isolation, Identification, Analysis, Rutin, Isolation, Identification, Analysis, Alkaloids, Introduction, Atropine, Isolation, Identification, Analysis, Quinine, Isolation, Identification, Analysis, Reserpine, Isolation, Identification, Analysis, Caffeine, Isolation, Identification, Analysis, Resins, Introduction, Podophyllotoxin, Isolation, Identification, Analysis, Curcumin, Isolation, Identification, , 208, 209, 209, 210, 210, 211, 211, 212, 212, 212, 212, 213, 214, 214, 215, 215, 216, 216, 216, 217, 217, 217, 218, 218, 219, 219, 219, 220, 221, 221, 222, 222, 222, 222, 223, 223, 224, 225, 225, 225, 226, 227, 227, 227, 228, 228, *
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- 12 -, , 7.5.3.3. Analysis, 7.6., Summary, 7.7., Exercise, , 229, 229, 231, , Module 4, Chapter 8: Industrial Production,, Estimation and Utilisation of, Phytoconstituents, 8.1., 8.1.1., 8.1.2., 8.1.2.1., 8.1.2.2., 8.1.2.3., 8.1.3., 8.1.3.1., 8.1.3.2., 8.1.3.3., 8.1.4., 8.1.4.1., 8.1.4.2., 8.1.4.3., 8.1.5., 8.1.5.1., 8.1.5.2., 8.1.5.3., 8.1.6., 8.1.6.1., 8.1.6.2., 8.1.6.3., 8.1.7., 8.1.7.1., 8.1.7.2., 8.1.7.3., 8.1.8., 8.1.8.1., 8.1.8.2., 8.1.8.3., 8.1.9., 8.1.9.1., 8.1.9.2., 8.1.9.3., 8.1.10., 8.1.10.1., 8.1.10.2., *, , Industrial Production of, Phytoconstituents, Introduction, Forskolin, Industrial Production, Estimation, Utilisation, Sennoside, Industrial Production, Estimation, Utilisation, Artemisinin, Industrial Production, Estimation, Utilisation, Diosgenin, Industrial Production, Estimation, Utilisation, Digoxin, Industrial Production, Estimation, Utilisation, Atropine, Industrial Production, Estimation, Utilisation, Podophyllotoxin, Industrial Production, Estimation, Utilisation, Caffeine, Industrial Production, Estimation, Utilisation, Taxol, Industrial Production, Estimation, , 8.1.10.3., 8.1.11., 8.1.11.1., 8.1.11.2., 8.1.11.3., 8.2., 8.3., , Utilisation, Vincristine and Vinblastine, Industrial Production, Estimation, Utilisation, Summary, Exercise, , 251, 252, 252, 253, 254, 254, 255, , Module 6, , 232, , Chapter 9: Basics of Phytochemistry, 232, 232, 233, 233, 234, 234, 235, 236, 236, 236, 237, 237, 237, 238, 238, 239, 240, 240, 241, 241, 242, 242, 243, 244, 245, 245, 245, 246, 247, 247, 248, 248, 250, 250, 250, 251, , 9.1., 9.1.1., 9.1.2., 9.1.2.1., 9.1.2.2., 9.1.2.3., 9.1.2.4., 9.1.2.5., 9.1.2.6., 9.1.2.7., 9.1.2.8., 9.1.2.9., 9.1.2.10., 9.1.2.11., 9.1.2.12., 9.1.2.13., 9.1.2.14., 9.1.2.15., 9.1.2.16., 9.1.3., , 9.1.3.1., 9.1.3.2., 9.1.3.3., 9.2., 9.3., , Phytochemistry, Introduction, Extraction, Principles of Extraction, Choice of Solvents, Modern Methods of, Extraction, Maceration, Digestion, Percolation, Continuous Hot Extraction, (Soxhlet Extraction), Supercritical Fluid, Extraction (SFE), Counter Current Extraction, (CCE), Microwave Assisted, Extraction (MAE), Ultrasonic Assisted, Extraction (UAE), Infusion and Decoction, Pressure Cooker Extraction, Extraction by Passage, through a Colloid Mill, Use of Surface Active, Agents in Drug Extraction, Expression and Diacolation, Techniques Used in the, Isolation, Purification, and, Identification of Crude, Drugs, Spectroscopy, Chromatography, Electrophoresis, Summary, Exercise, , 256, 256, 256, 257, 258, 259, 260, 262, 263, 266, 267, 269, 269, 272, 273, 273, 273, 273, 274, 274, , 274, 278, 282, 283, 285, *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , CHAPTER, 1, , 13, , Metabolic Pathways in Higher, Plants and Their Determination, , 1.1. METABOLIC PATHWAYS, 1.1.1., , Introduction, , For facilitating their living, growth, and reproduction, all organisms convert and, interchange a large number of organic compounds. They obtain ATP as energy, and a supply of building block s for making up their tissue s. This requires an, integrated network of enzyme -mediated and cautiously regulated chemical, reactions, which are jointly termed intermediary metabolism, and the involved, pathways are termed metabolic pathways . Carbohydrates, proteins, fats, and, nucleic acids are a few essential molecules of life., Apart from minor variations , the general pathway s involved in the modification, and synthesis of carbohydrates, proteins, fats, and nucleic acids are mainly same, in all organisms. These processes demonstrate the b asic unity of the living, matter, and are collectively termed as primary metabolism , and the involved, compounds are termed primary metabolites. An area of metabolism concerned, with compounds having a limited distribution in nature also exists. These, compounds are secondary metabolites, which are found in certain organisms or, groups of organisms. These compounds are an expression for the distinctiveness, of species. Secondary metabolites are not produced under all condi tions. In large, number of cases , the function of secondary metabolites and their benefit to the, organisms is still to be known., , 1.1.2., , Basic Metabolic Pathways, , Primary metabolism supplies the building blocks for secondary (figure 1.1). This, process shows how the metabolites obtained from photosynthesis, glycolysis, and, Krebs cycle are detached from, the energy -producing p rocesses to deliver, biosynthetic intermediates. The most important building blocks used, for, biosynthesising secondary metabolites are obtained from acetyl co -enzyme A, (acetyl-CoA), shik imic acid, mevalonic acid, and 1 -deoxyxylulose-5-phosphate, intermediates, which are used in the, acetate, shikimate, mevalonate and, deoxyxylulose phosphate pathways, respectively., In natural product synthesis , apart from acetyl -CoA, shikimic acid, mevalonic, acid, and deoxyxylulose phosphate, some other building blocks based on amino, acids are also used. Large number of antibiotics, peptides, proteins, and alkaloids, are derived from amino acids. Figure 1.1 shows the origin of the m ost important, amino acid constituents of these building blocks . For the constructions of these, building blocks, intermediates from glycolytic pathway and Krebs cycle are, employed; however, phenylalanine, tyrosine, and tryptophan are the, aromatic, *, , *
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14, , Pharmacognosy and Phytochemistry-II, , amino acids which are produc ts of shikimate pathway. Ornithine (a non-protein, amino acid) and lysine (its homologue) are alkaloid precursors which have their, origins in Krebs cycle intermediates., , Figure 1.1: Basic Metabolic Pathways, *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 15, , The interrelation of pr imary and secondary metabolism is such that a complete, distinction is meaningles s. To understand their basics, some divisions based on, biosynthetic pathways have to be prepared, . In plants, three main routes,, excluding the primary biosynthesis process of su gar and protein , are found, i.e.,, shikimic acid pathway, acetate -malonate pathway, and acetate -mevalonate, pathway. The interrelation of these pathways is shown in the figure 1.2., Pentose phosphate, pathway, Carbon dioxide, +, Water, Acetate-melonate, pathway, Fatty acids, , Polyketides, , Carbohydrates, Glycolysis, Shikimic acid, pathway, Pyruvic acid, , Aromatic, compounds, Transaminatio, n, Ammonia, , Acetyl CoA Tricarboxylic, acid cycle, , Proteins, Acetate-mevalonate, pathway, , Alkaloids, , Terpenoid, s, , Steroids, Aromatic compounds, Figure 1.2: Biosynthetic Pathways in Plants, , 1.1.2.1., , Amino acids, , Nucleic acids, , Shikimic Acid Pathway (Amino Acid Pathway), , Shikimic acid pathway is an alternate route to aromatic compounds, mainly the Lphenylalanine, L-tyrosine, and L-tryptophan aromatic amino acids. This pathway is, used by the microorganism and plants. Shikimic acid is a compound isolated from, the plants of Illicium species, and is the central intermediate of this pathway. The, following reactions occur in the shikimic acid pathway(figure 1.3):, 1) Conversion of Erythrose -4-Phosphate to 3 -Dehydroquinic Acid: 3Deoxy-D-Arabino-Heptulosonic acid -7-Phosphate (DAHP) is formed when, the p recursors D -erythrose-4-phosphate and phosphoenolpyruvate combine., This reaction is catalysed by phospho-2-oxo-3-deoxyheptonate aldolase. The, 3-dehydroquinate synthase enzyme catalyses the cyclization of DAHP to 3 dehydroquinic acid, and, needs cobalt (II) and Nicotinamide Adenine, Dinucleotide (NAD) as co-factors., 2) Formation of Shikimic Acid: Dehydroquinic acid converts to quinic acid or, 3-dehydroshikimic ac, id, which continues the pathway and producesikimic, sh acid., 3) Formation of Chorismic Acid:, After phosphorylation, catalysed by, shikimate kinase enzyme, shikimic acid attaches to enol pyruvate to form 3 enolpyruvylshikimic acid 5 -phosphate. Enolpyruvylshikimate phosphate, synthase enzyme catalyses this re action. Chorismate synthase enzyme, catalyses its conversion to chorismic acid., 4) Formation of Other Intermediates: Chorismic acid converts to anthranilic, acid in the presence of glutamine, whereas the formation of prephenic acid is, catalysed by chorismate mu tase enzyme. Chorismic acid is also converted to, p-aminobenzoic acid., *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 1.1.3., , 19, , Formation of Different Secondary Metabolites, , Organic compounds like carbohydrates, proteins, fats, membrane lipids, nucleic, acids, chlorophylls and hemes are essential for plant metabolism and found, throughout the plant kingdom. These compounds are termed as, primary, metabolites ., A number o f organic compounds are also synthesised by many plants of certain, genera and families; however, these compounds have no direct function in the, growth and development of plants. These organic compounds are termed, secondary metabolites or secondary plant pr oducts or natural products ., They are extensively numerous and chemically vast in nature, and include, substances like alkaloids, terpenes (including steroids and rubber), tannins,, flavonoids, etc., Figure 1.6 represents the chief biosynthetic pathways of se condary metabolites, and their interrelationship with primary metabolism in plants., CO2, Photosynthesis, Primary Carbon Metabolism, Phosphoenol pyruvate, , Pentose phosphate, pathway, Erythrose-4-phosphate, , Pyruvate, , TCA, Cycle, , Acetyl-CoA, , Aliphatic, amino acids, Shikimic acid, pathway, , Malonic acid, pathway, , Mevalonic acid, pathway, , Aromatic, amino acids, Terpenes, , Nitrogen containing, secondary products, Phenolic, compounds, Secondary Carbon Metabolism, , Figure 1.6: Major Biosynthetic Pathways of Secondary Metabolites and, their Inter-relationships with Primary Metabolism in Plants, , The biogenesis of following secondary metabolites is discussed below:, 1) Biogenesis of alkaloids,, 2) Biogenesis of glycosides,, 3) Biogenesis of terpenes,, 4) Biogenesis of steroids,, 5) Biogenesis of isoprenoid compounds,, 6) Biogenesis of carotenoids, and, 7) Biogenesis of flavonoids., *, , *
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20, , Pharmacognosy and Phytochemistry-II, , 1.1.3.1., , Biogenesis of Alkaloids, , Alkaloids are nitrogen-containing secondary metabolites. Th ey are mainly, synthesised from amino acids in plants. Many compounds which are derived, from ornithine, lysine, phenylalanine , tryptophan, etc. are involved in the, biogenesis of alkaloids., Alkaloids Derived from Ornithine, Studies have shown that ornithine is present in pyrrolidine and tropane alkaloids., In the pyrollidine ring of tropane nucleus, ornithine is present stereospecifically, and unsymmetrically, and the -carbon of ornithine becomes the C -1 of tropane, nucleus., The piperidine moiety is completed by the remaining three carbon atoms , which, are derived from acetate. Methionine, acts as the methyl group donor,, and, phenylalanine serves as the precursor of tropic acid. Different alkaloids derived, from ornithine are grouped together (figure 1.7)., , Figure 1.7: Some Alkaloids Derived from Ornithine, , Alkaloids Derived from Lysine, Lysine and its related compounds are involved in the biogenesis of anabasine,, lupinine, isopelletierine , and associated alkaloids (figure 1.8 ). Lysine is the, precursor of piperidine-type alkaloid . The five amino -pentanal was the, biogenetically functionalised derivative of this precursor., *, , *
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22, , Pharmacognosy and Phytochemistry-II, , Figure 1.10: Biogenesis of Some Indole Alkaloids, , The reactive form of terpenes involves an aldehyde group., During biogenesis,, one carbon atom is comm only lost to give C 9 unit. The combination of acetate, metabolism and tryptophan yields ergot alkaloids. The first step in the pathway is, condensation of dimethy lallyl pyrophosphate at 4 -position of tryptophan. Later,, production of chanoclavine -I and agrocl avine occur in the cycli sation reactions., Agroclavine oxidises in to elymoclavine and finally to lysergic acid. A peptide, linkage along with an amino group of a variety of amino acids is formed by the, carboxyl group of lysergic acid to produce medicinally significant ergot alkaloids, (figure 1.10)., *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 23, , Biosynthesis of Lysergic Acid, Tryptophan and an isoprene unit (figure 1.11) are the building blocks for lysergic, acid. Alkylation of tryptophan with dimethylallyl diphosphate yields, 4dimethylallyl-L-tryptophan, which further undergoes N, -methylation. The, tetracyclic ring system of lysergic acid, proceeds via chanoclavine -I and, agroclavine, however the details of this mechanism are still not clear., Labelling studies have shown that the double bond in dimethylallyl substituent, should become a single bond on two different occasions to allow rotation to form, new rings. This establishment appears as cis-trans isomerisation as 4 dimethylallyl-L-tryptophan converts into chanoclavine-I aldehyde, which, cyclises into agroclavine., In further stages , agroclavine undergoes hydroxylation to produce elymoclavine., Further oxidation of primary alcohol produces paspalic acid, and a spontaneous, allylic isomerisation forms lysergic acid., , Figure 1.11: Biosynthesis of Lysergic Acid, , 1.1.3.2., , Biogenesis of Glycosides, , Two essential parts are involved in the metabolic process of glycoside formation., Formation of several types of aglycones by means of different reactions is the, first part of biosynthesis, and the other part is the metabolic pa thway which, involves the coupling of aglycone and sugar moiety., Transfer of uridylyl group from Uridine Triphosphate (UTP) to sugar, -1phosphate is the principal pathway of glycoside formation. The enzymes which, catalyse this reaction are uridylyl transferases., The enzymatic system glycosyl transferases control the succeeding reaction that, includes transfer of sugar from uridine diphosphate to aglycone moiety, thus, forming glycoside., *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 1.1.3.4., , 27, , Biogenesis of Steroids, , Steroids are o rganic compounds containing a specific arrangement of four, cycloalkane ring joined to each other . Examples of steroids are cholesterol, sex, hormones (estradiol and testosterone), and dexamethasone ( anti-inflammatory)., The steroid core contains 17 carbon atoms bonded together and forming four fused, rings; three cyclohexane rings (A, B, and C rings) and one cyclopentane ring (D, ring). The functional group attached ot this four ring core and theoxidation state of, the rings leads to different types of steroids. Special forms of steroids, i.e., sterol, have a hydroxyl group at position-3 and a cholestane-derived skeleton., Biosynthesis of steroids involves an anabolic pathway that yields steroids from, simple precursors. In animals, this pathway takes place in different ways than in, many other organisms. Due to this reason, the pathway is a common target for, antibiotics and other anti-infective drugs. Metabolism of steroids in humans is the, target of cholesterol -lowering drugs, like statins. Biosynthesis of steroids begins, in the mevalonate pathway in h umans utilising acetyl -CoA as building blocks., DMAPP and IPP are formed, w hich further form the first steroid, i.e., lanosterol, that modifies into the succeeding steroidogenesis., Biogenesis of Cholesterol, , Figure 1.16: Biogenesis of Cholesterol, *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 1.1.3.6., , 29, , Biogenesis of Carotenoids, , Biosynthetic precursor of carotenoids is GGPP (Geranyl, -Geranyl, Pyrophosphate). Carotenoid is a family of C 40 isoprenoids (tetraterpenes) that is, involved in the important functions in the lifecycle of green plants. Carotenoids, are ideal accessory pigments in the light -harvesting steps of photosynthesis due, to the presence of highly, extended, conjugated double bonds., The intense, colouration of carotenoids is also due to the extended sys tems of conjugated, double bonds. The colouration of carotenoids is a property by which plants, harness good effect by using them as pigments to draw ins, ects, birds, and, animals to wards their flowers and fruits., These natural pigments are also, economically beneficial in the ornamental garden and as food additives., Since carotenoids metabolise into vitamin A (retinol), they play an important role, in human diet. Retinol, being the prosthetic group associated with rhodopsin, (light-harvesting pigment ), is significantly involved in the visual proc esses. It, also participates in growth and development process; hence vitamin A deficiency, can give severe results fo r long -term health . Carotenoids have also been used, against heart disease and cancer because of its antioxidant properties., , Figure 1.18: Biogenesis of Carotenoids, *, , *
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30, , Pharmacognosy and Phytochemistry-II, , In carotenoid biosynthesis , the enzymes involved are nuclear encoded but the, biosynthesis sites are chloroplasts or chromoplasts (in case of pigments), which are, plastids without chlorophyll. Condensation of two molecules of GGPP which, appears in head-to-head fashion under the enzymatic action of Phytoene Synthase, (PSY) initiates the biosynthesis, of carote noids ( figure 1.18). Pre-phytoene, pyrophosphate (a cyclopropane ring-containing intermediate) has been involved in, the reaction . Purified phytoene synthase obtained from c apsicum chromoplasts, requires Mn2+ ions to produce action (Mn2+ ions are used by most terpene synthases, to bind pyrophosphate ). In plants , the major product of PSY is the counter, intuitively numbered 15-cis-phytoene. In a sequence of enzymatic steps, phytoene, dehydrogenates into lycopene (pigment responsible for the orange colour of, ripening tomato fruits ). All-trans geometric isomer is the final product. The, pathway gets branched from lycopene; one branch goes through-carotene to form, lutein (the major carotenoid in the light -harvesting complex of chloroplasts), and, the other branch goes through -carotene and the oxygenated xanthophyll series., Branching takes place by the action of two separate pathways for c yclisation of, the terminal unit of carotene chains, catalysed by cyclase enzymes, (although, different but related to each other) . In the -series, the reactions occur in two step processes, one at either end of the carotenoid chain , though the predominant, products are symmetrical. The asymmetrical intermediates are isolable and well, characterised. A series of ferredoxin, -dependent hyd roxylases mediates the, transformation of -carotene via xanthophylls series. In the light-harvesting steps, of photosynthesis , xanthophylls are accessory pigments. Inter-conversion of, zeaxanthin and violaxanthin is important in providing protection to green tissues, from radiation damage through the dissipation of excess energy., , 1.1.3.7., , Biogenesis of Flavonoids, , Acetate metabolism and shikimic acid pathway produce aglycones of flavonol, glycoside. The head-to-tail condensation of two malonyl Co -A units and acetyl, Co-A give rise to ring A. A C6–C3 precursor, which itself can be cinnamic acid,, gives rise to ring B and C3 unit., , Figure 1.19: Biosynthetic Pathways to Flavonoid A glycones, *, , *
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Metabolic Pathways in Higher Plants and Their Determination (Chapter 1), , 31, , 1.2. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Organisms obtain ATP as energy and a supply of building blocks. This, requires an integrated network of enzyme -mediated and cautiously regulated, chemical reactions, which are jointly termed intermediary metabolism, and, the involved pathways are termed metabolic pathways., 2) Synthesis of Carbohydrates, proteins, fats, and nucleic acids demonstrate the, basic unity of the living matter, and are collectively termed as, primary, metabolism, and the involved compounds are termed primary metabolites., 3) An area of metabo lism concerned with compounds having a limited, distribution in nature also exists. These compounds are, secondary, metabolites, which are found in certain organisms or groups of organisms., 4) The most important building blocks used in for biosynthesising second ary, metabolites are obtained from acetyl co -enzyme A (acetyl -CoA), shikimic, acid, mevalonic acid, and 1-deoxyxylulose-5-phosphate intermediates., 5) Shikimic acid pathway is an alternate route to aromatic compounds, mainly, the L -phenylalanine, L -tyrosine, and L-tryptophan aromatic amino acids., This pathway is used by the microorganism and plants., 6) Isopentenyl Pyrophosphate (IPP) and its isomer Dimethylallyl Pyrophosphate, (DMAPP) are produced by mevalonic acid., 7) IPP and DMAPP are the two chief intermediates that set the ‘active isoprene’, unit as the basic building block of isoprenoid compounds., 8) With the involvement of Acyl Carrier Protein (ACP), the acetate pathway, works functionally to produce fatty acyl thioesters of ACP. These acyl, thioesters build the important intermediates in fatty acid synthesis., 9) Alkaloids are nitrogen containing secondary metabolites. They are mainly, synthesised from amino acids in plants., 10) Many compounds which are derived from ornithine, lysine, phenylalanine,, tryptophan, etc. are involved in the biogenesis of alkaloids., 11) Tryptophan and tryptamine (its decarboxylation product) act as precursors, for biosynthesising various indole alkaloids., 12) Formation of several types of aglycones by means of different reactions is the, first part of biosynthesis of glycosides, and the other part is the metabolic, pathway which involves the coupling of aglycone and sugar moiety., 13) The term terpene was previously used for the mixture of isomeric, hydrocarbons (having C 10H16 molecular formula) found in turpentine and, different essential oils., 14) All the terpenes are terpenoids but not vice-versa., 15) Steroids are organic compounds containing a specific arrangement of four, cycloalkane ring joined to each other.Examples of steroids are cholesterol, sex, hormones (estradiol and te, stosterone), and dexamethasone (anti, -inflammatory)., 16) The steroid core contains 17 carbon atoms bonded together and forming four, fused rings; three cyclohexane rings, (A, B, and C rings) and one, cyclopentane ring (D ring)., *, , *
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32, , Pharmacognosy and Phytochemistry-II, , 17) Formation of various isoprenoid co mpounds is based on the biogenetic, isoprene rule stated by Ruzicka in 1953., 18) Carotenoid is a family of C 40 isoprenoids (tetraterpenes) that is involved in, the important functions in the lifecycle of green plants., 19) Biosynthetic precursor of carotenoids is GGP, P (Geranyl -Geranyl, Pyrophosphate)., 20) Acetate metabolism and shikimic acid pathway produce aglycones of, flavonol glycoside., , 1.3. EXERCISE, 1.3.1., 1), 2), 3), 4), 5), 6), , 1.3.2., 7), 8), 9), 10), 11), , True or False, , Organisms obtain ATP as energy and a supply of building blocks., Primary metabolism supplies the building blocks for secondary., Shikimic acid is a compound isolated from the plants of Leguminoseae species., Chorismic acid converts to anthranilic acid in the presence of glutamic acid., Alkaloids are nitrogen containing secondary metabolites., Biosynthesis of steroids begins in the Shikimic acid pathway in humans., , Fill in the Blanks, , The pathways involved in intermediary metabolism are termed __________., Primary metabolism supplies the building blocks for ___________., Chorismic acid converts to anthranilic acid in the presence of __________., Alkaloids are ____________ containing secondary metabolites., All the terpenes are __________ but not vice-versa., , Answers, 1) True, 5) True, 9) Glutamine, , 1.3.3., 1), 2), 3), 4), 5), , 3) False, 7) Metabolic pathways, 11) Terpenoids, , 4) False, 8) Secondary metabolites, , Very Short Answer Type Questions, , Define metabolic pathways., Discuss primary and secondary metabolites., Give the biosynthesis of cyanogenetic glycosides., Discuss biosynthesis of flavonoids., Give the biogenesis of terpenes., , 1.3.4., 1), 2), 3), 4), , 2) True, 6) False, 10) Nitrogen, , Short Answer Type Questions, , Discuss shikimic acid pathway., Give the biogenesis of terpenoids., Write a short note on acetate-malonate pathway., Briefly discuss the basic metabolic pathways., , 1.3.5., , Long Answer Type Questions, , 1) Give a detailed account on shikimic acid pathway., 2) Discuss acetate-mevalonate pathway and acetate-malonate pathway., 3) Give the biogenesis of carotenoids., *, , *
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Utilisation of Radioactive Isotopes (Chapter 2), , CHAPTER, 2, , 33, , Utilisation of Radioactive, Isotopes, , 2.1. BIOGENETIC STUDIES, 2.1.1., , Introduction, , Living plants are the biosynthetic laboratory as many primary and secondary, metabolites are synthesised in them, through various biosynthetic pathways., These pathways involve different intermediate steps that can be identified by, using different scientific techniques. Biogenetic investigation in plants can be, performed by the following methods:, 1) Using isolated organs/ tissues,, 2) Grafting methods,, 3) Use of mutant strains, and, 4) Tracer technique., , 2.1.2., , Using Isolated Organs/Tissues, , In this method, isolated parts of plant (e.g., stem, roots, etc.) are used. Therefore,, this technique is used to investigate the synthesis site of a particular compound., Procedure, From the sample plant, shoots or any desired part are isolated and placed in water, or in a suitable solution. For some days,, the plant part remains turgid and, undergoes normal metabolism; but soon a pathological metabolism starts. This, technique can be modified by connecting the cut end of t he plant part (shoot,, root, petal discs, etc.) to a container of suitable sterile nutrient so that the shoot, remains normal for a longer period. This entire process is carried out under, aseptic conditions. Similar method is used for maintaining the isolated leaves., The factor that may undermine the res ults of this experiment is that generally, roots develop at the cut ends of such shoots, . A rapid growth occurs under, optimum conditions and sub -cultures may be produced, if required. The, biogenetic and growth, studies can be performed by adding the selected, compounds to the culture as per need. By using this method, it has been, investigated that tropane alkaloids are synthesised in the roots of most of the, Solanaceae plants. Also, the inclusion of many precursors into the alkaloids has, been identified., This method is quite useful for investigating various biogenetic pathways, as, isolated tissues and cells can be cultivated in this technique. It involves the use of, pure plant material which can be obtained at al l times and easily managed under, controlled and reproducible conditions . Also, the potential precursors of, metabolites under demonstration can be added to the system and the sample can, be repetitively extracted for analysis. The aseptic culture ensures tha t bacterial, and fungal modifications of the precursors are removed., *, , *
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34, , Pharmacognosy and Phytochemistry-II, , Due to the cultivation of isolated plant organs and tissues, the chances of, interference from the metabolites produced in other plant parts are reduced. This, cultivation method can be us ed to feed experiments utilising labelled compounds, and is also used to determine the synthesis site of a particular compound. For, example, roots and leaves are used to study nicotiana and datura, petal disc s are, used to study rose oil, and tropane alkaloi ds formed in the roots of Solanaceae, plants., , 2.1.3., , Grafting Methods, , The technique of grafting involves the use of grafted plants to study, the, biosynthesis of alkaloids. This method is also preferred for determining the sites, of primary and secondary metabolites of some secondary plant products., For example, Tomato scions grafted on datura shows accumulation of alkaloids,, while Datura scion grafted on tomato contains very small amount of alkaloids., This indicates that the main synthesis site for the formation of datura alkaloids is, root., , 2.1.4., , Use of Mutant Strains, , A number of mutant strains of microbes, that have been developed , lack a n, enzyme that blocks their metabolism at a specific stage. Such microorganisms, may accumulate the intermediate compound just before the blockage, and require, an artificial supply of another intermediate (which occurs after the blockage) for, their survival. Therefore, these microorganisms are useful, for studying the, biosynthetic pathways of various primary and secondary metabolites., For example, mutant strain of Gibberella is used for the synthesis of isoprenoid, compounds; Lactobacillus acidophilus is used in mevalonic acid pathway for the, synthesis of isoprenoid compounds., , 2.1.5., , Utilisation of Radioactive Isotopes, , Radioactive tracers are radioactive isotopes used in biogenetic studies. These are, organic compounds whose one or more atoms, have been replaced with, a radionuclide. Due to their property of radioactive decay, the radioactive tracers, are used to investigate the mechanism of chemical reactions by tracing the path, followed by the radioisotope from reactants to products., Examples of radioisotope tracers used frequently to trace the path of biochemical, reactions are hydrogen, carbon, phosphorus, sulphur, and iodine., Tracer Technique, These radioactive isotopes or tracers are used in tracer techniques for, investigating the biosynthetic pathways. In this technique, a labelled compound is, used to inv estigate the different intermediates and various steps in b iosynthetic, pathways in plants by tracing them at a given rate and time. This technique also, uses a labelled compound which is introduced into the plant system. After which, the compound becomes a part of the general metabolic pool, and gets involved in, *, , *
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Utilisation of Radioactive Isotopes (Chapter 2), , 35, , the related reactions of the particular plant system. For this technique, the, radioactive carbon (14C), hydrogen (3H), and to a lesser extent sulphur (35S) and, phosphorus (32P), are used for analysing the various biosynthetic pathways., , 2.1.5.1., , Significance of Tracer Technique, , The tracer technique has the following significances:, 1) It is highly sensitive., 2) It is used in the living system., 3) A number of isotopes are available that can be used in this technique., 4) It is a more reliable and easy process of administration and isolation., 5) It provides accurate results if used with proper metabolic time and technique., 6) It uses the location and quantity of compound containing 14C labelled glucose, tracer for determining glucose in the biological system., 7) Different studies employ different tracers. For example, for studying, nitrogen and amino acid, labelled nitrogen, is used for providing specific, information., , 2.1.5.2., , Criteria for Tracer/Isotope Selection, , The following two types of isotopes are used for labelling:, 1) Radioactive Isotopes: 1H, 14C, 24Na, 42K, 35S, 35P, and 131I are the examples, of such isotopes., i) Isotope of C and H are used for biological investigation., ii) Isotopes of S, P, and alkali and alkaline earth metals are used for, metabolic studies., iii) Labelled N-atom is used for studying proteins, alkaloids, and amino, acids., 2) Stable Isotopes: 2H, 13C, 15N, and 18O are the examples of such isotopes., i) They are used for labelling compounds as possible intermediates in, biosynthetic pathways., ii) The u sual method s of detection are mass spectroscopy [ 15N, 18O] and, NMR spectroscopy [2H, 13C]., Before selecting an isotope, the following points should be considered:, 1) Its initial concentration should be sufficient enough to hold up dilution, during the metabolic processes., 2) Its physical and chemical properties, should be well -known for proper, labelling., 3) It should have a higher half-life., 4) It should be actively involved in biosynthesis., 5) It should not destroy the reaction system., , 2.1.5.3., , Steps Involved in Tracer Technique, , Following four steps are involved in the tracer technique:, Step 1: Preparation of Labelled Compound, 1) The labelled compound is produced by allowing them to grow in atmosphere, of 14CO2. All compounds containing carbon get 14C labelled., *, , *
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36, , Pharmacognosy and Phytochemistry-II, , 2) The 3H (tritium) labelled compound s are commercially available. Tritium, labelling gets affected by, catalytic exchange in aqueous media by, hydrogenation of u nsaturated compound with tritium gas. Tritium is pure emitter of low intensity having radiation energy lower than 14C., 3) By the use of organic synthesis:, , Step 2: Introduction of Labelled Compound into Biological System, 1) Root Feeding: This method is preferred in plants in which the roots are the, sites of biosynthesis, e.g., tobacco. In this method, the plants are cultivated, hydroponically to prevent them from microbial contamination., 2) Stem Feeding: This method does not require root for biosynthesis. This is, because the substrate can be administered through the cut ends of stem, immersed in a solution. This method of labelling is not suited for plants, containing latex., 3) Direct Injection: This method is preferred for plants having hollow stem,, e.g., plants belonging to Umbelliferae family and capsule bearing plant s, (e.g., opium poppy)., 4) Infiltration or Wick Feeding : This method is preferred when it is required, to feed on plant rooted in soil or other support without disturbing the root., 5) Floating Method: This method is preferred when a small amount of material, is available. In this method, leaf disc or chopped leaves floating on the, substrate solution are used. This technique is used along with vacuum, infiltration so that gases present in the system can be removed., 6) Spray Technique: In this method, the labelled compounds are spraye d on, leaves in aqueous solution, thus the compound s absorb. This method is, preferred for the investigation of steroids., Step 3: Separation and Detection Techniques, Selection of method to be used for t he extraction process depends on the nature, of the drug and its source:, 1) Soft and Fresh Tissue: Infusion and maceration., 2) Hard Tissue: Decoction and hot percolation., 3) Unorganised Drug: Maceration with adjustment., Selection of the solvent to be used for the e xtraction also depends on the nature, of the drug to be extracted:, 1) Fat and Oil: Non-polar solvent., 2) Alkaloid, Glycoside, and Flavonoid: Slightly polar solvent., 3) Plant Phenol: Polar solvent., Techniques like fractional crystallisation and column chromatography are also, used for separation process., *, , *
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Utilisation of Radioactive Isotopes (Chapter 2), , 37, , The f ollowing techniques are used for separation and detection of the plant, metabolites:, 1) GM counters,, 2) Liquid scintillation chamber,, 3) Gas ionisation chamber,, 4) Mass spectrophotometer,, 5) NMR spectrophotometer, and, 6) Auto-radiography., Step 4: Methods for Tracer Technique, The following five methods are involved in the tracer technique:, 1) Precursor Product Sequence: In this method, the presumed precursor of the, plant constituent to be investigated on a labelled fo rm is introduced into the, plant. After some time , the constituent is isolated, purified and, its, radioactivity is measured., Examples, i) In Datura stramonium , the restricted synthesis of hyoscine is different, from hyoscyamine., ii) This method is used to investigate the biogenesis of mo rphine and ergot, alkaloids., 2) Double and Multiple Labelling: This method provides evidences for the, nature of biochemical incorporation of precursor that give rise to double and, triple labelling . This method involves the use of a specifically labelled, precursor and the subsequent degradation of recovered product., Examples, i) This method is used for studying the biogenesis of secondary metabolites, in plants., ii) This method is used for studying morphine alkaloid., 3) Competitive Feeding: This method gives the possible intermediates that are, involved during biogenesis., Examples, i) This method is usedfor elucidation ofthe biogenesis of rtopane alkaloids., ii) This method is used, along with 14C labelled compounds in the, biosynthesis of hemlock alkaloids (coniine, conhydrine, etc.)., 4) Isotope Incorporation: This method is used to investigate the position of, bond cleavage and their formation during reaction., Example: Cleavage of g lucose-1-phosphatase is catalysed by alkaline, phosphatase enzyme; this reaction occurs with the cleavage of either C ─O, bond or P─O bond., 5) Sequential Analysis: In this method, the plant is allowed to grow in, atmosphere of 14CO2 and then the plant growth at given time interval, is analysed, to obtain the sequence in which various correlated compound, s become labelled., Examples, i) In photosynthesis, 14CO2 sequential analysis is used in carbon elucidation., ii) This method is used to determine the sequential formation of opium, hemlock and tobacco alkaloids., *, , *
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38, , 2.1.5.4., , Pharmacognosy and Phytochemistry-II, , Applications of Tracer Technique, , Tracer technique has the following applications:, 1) It is used to trace the biosynthetic pathway of, prunasine ( cyanogenetic, glycoside) by the incorporation of 14C into phenylaniline., 2) It utilises 14C acetate to analyse the relationship between 4-methyl sterols and, 4, 4 dimethyl sterols., 3) It utilises 14C, 3H labelled mevalonic acid for studying squalene cyclisation., 4) It utilises 2 -14C mevalonate to investigate t, erpenoid biosynthesis by, chloroplast isolated in organic solvent., 5) It is used to study cinnamic acid formation from labelled coumarin in, pathway of coumarin., 6) It utilises 14C or 15N labelled precursor to determine the origin of carbon and, nitrogen atoms of purine ring system., 7) It utilises labelled phenylalanine to study scopoletin formation., 8) It utilises 45Ca as a tracer to analyse the uptake of calcium by plants from soil, (CaO and CaCO2)., 9) By adding ammonium phosphate labelled with 32P of known specific activity,, the uptake of phosphorus is followed by measuring the radioactivity as label, reaches first the lower pla nt parts, and then the upper parts, i.e., branches,, leaves, etc., , 2.2. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Living plants are the biosynthetic laboratory as many primary and secondary, metabolites are synthesised in them, through various biosynthetic pathways., 2) The technique of grafting involves the use of grafted plants to study the, biosynthesis of alkaloids., 3) Radioactive tracers are radioactive isotopes used in biogenetic studies., 4) Radioactive tracers are organic compounds whose one or more atoms have, been replaced with a radionuclide., 5) Mutant strain of Gibberella is used for the synthesis of isoprenoid compounds., 6) Lactobacillus acidophilus is used in mevalonic acid pathway for the, synthesis of isoprenoid compounds., 7) Tomato scions grafted on datura shows accumulation of alkaloids, while, Datura scion grafted on tomato contains very small amount of alkaloids., 8) Examples of radioisotope tracers used frequently to trace the path, of biochemical reactions arehydrogen, carbon, phosphorus,sulphur, and iodine., 9) The radioactive isotopes or tracers are used in tracer techniques for, investigating the biosynthetic pathways., 10) In tracer technique, a labelled compound is used to investigate the different, intermediates and various steps in biosynthetic p athways in plants by tracing, them at a given rate and time., *, , *
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Utilisation of Radioactive Isotopes (Chapter 2), , 39, , 11) Tracer technique uses the location and quantity of compound containing, labelled glucose tracer for determining glucose in the biological system., , 14, , C, , 12) Root Feeding method is preferred in plants in which the roots are the sites of, biosynthesis., 13) Direct Injection method is preferred for plants having hollow stem., 14) Infiltration or Wick Feeding method is preferred when it is required to feed, on plant rooted in soil or other support without disturbing the root., 15) Floating Method is preferred when a small amount of material is available., 16) In Spray Technique method, the labelled compounds are sprayed on leaves, in aqueous solution, thus the compounds absorb., 17) Competitive Feeding method gives the possible intermedi, involved during biogenesis., , ates that are, , 18) Isotope Incorporation is used to investigate the position of bond cleavage, and their formation during reaction., , 2.3. EXERCISE, 2.3.1., , True or False, , 1), 2), 3), 4), , Radioactive tracers are radioactive isotopes used in biogenetic studies., Mutant strain of Gibberella is used for the synthesis of terpenes., Floating Method is preferred when a small amount of material is available., Competitive Feeding method gives the possible intermediates that are involved, during biogenesis., 5) Competitive Feeding is used for studying morphine alkaloid., , 2.3.2., , Fill in the Blanks, , 6) Radioactive tracers are ___________ isotopes used in biogenetic studies., 7) Lactobacillus acidophilus is used in __________ pathway for the synthesis of, isoprenoid compounds., 8) ____________ is preferred when a small amount of material is available., 9) ____________ method is preferred in plants in which the roots are the sites of, biosynthesis., 14, 10) ____________ uses the location and quantity of compound containing, C, labelled glucose tracer., Answers, 1) True, 2) False, 3) True, 4) True, 5) False, 6) Radioactive, 7) Mevalonic acid 8) Floating Method, 9) Root Feeding 10) Tracer technique, , 2.3.3., , Very Short Answer Type Questions, , 1) Write a short note on grafting method., 2) Discuss the use of mutant strains for biogenetic studies., *, , *
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40, , Pharmacognosy and Phytochemistry-II, , 3) What are biogenetic studies? Enlist the methods used for biogenetic studies., 4) Write a short note on double and multiple labelling techniques., 5) Give the examples of competitive feeding., , 2.3.4., , Short Answer Type Questions, , 1) Give the criteria for selection of tracer/isotope., 2) Discuss the methods of tracer technique., 3) Give the significance of tracer technique., 4) Discuss the use of isolated organs/tissues for biogenetic studies., , 2.3.5., , Long Answer Type Questions, , 1) What are biogenetic studies? Discuss the methods involved., 2) Give the significance and applications of tracer technique., 3) Discuss the steps involved in tracer technique., , *, , *
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Secondary Metabolites-I (Chapter 3), , CHAPTER, 3, , 41, , Secondary Metabolites-I, , 3.1. SECONDARY METABOLITES, 3.1.1., , Introduction, , The chemical products of plants having no role in the growth, photosynthesis,, reproduction, or other ―primary‖ functions are known as secondary metabolites., The diversity of these chemicals is vast, i.e.,, they ha ve been i dentified in, numerous amounts in many major classes. A characteristic mixture of these, chemicals is produced by plant s of every family, genus, and species . These, chemicals can sometimes be used in plant classification based on their taxonomy., Few of these compounds are used as medicine, flavourings, or recreational drugs, by humans., Several secondary metabolites are toxic or repellent t o herbivores and microbes ,, thus defend the plants in which they are produced. When a plant is attacked by, herbivores or pathogens, the production of these secondary metabolites increases, and few of these compound s are released in air. These com pounds attract the, parasites and predators which kill the herbivores., In recent researches on these chemicals, more of their primary roles in plants like, signals, antioxidants, and other fun ctions are being identified . Consumption of, some specific secondary metabolites can have harmful consequences. Alkaloids, can block ion channels, inhibit enzymes, and, hinder neurotransmission , thus, resulting in hallucination, loss of coordination, convulsions, vomiting, , and, ultimately death. Phenolics can interrupt digestion, slowdown the normal growth,, block enzyme activity and cell division, and also have an unpleasant taste., Secondary chemicals are important in plant use by humans. Thepharmaceuticals are, mostly based on the plant chemical structures, and for recreation and stimulation,, secondary metabolites, e.g., nicotine and cocaine alkaloids and cannabinol terpene, are widely used.Ethnopharmacology is the study of such use ofplant., , 3.1.2., , Classification, , Classification of secondary metabolites is based on the ir chemical structure (e.g.,, having rings, containing a sugar), composition (containing nitrogen or not),, solubility in various solvents,, or the, biosynthetic pathways, (e.g.,, phenylpropanoid, which produces tan nins). Three main groups are included in a, simple classification of secondary metabolites , i.e., terpenes (prepared from, mevalonic acid, and composed of carbon and hydrogen), phenolics (prepared, from simple sugars and composed of benzene rings, hydrogen, and oxygen), and, compounds containing nitrogen (extremely diverse, and also contain sulphur)., *, , *
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42, , Pharmacognosy and Phytochemistry-II, , Table 3.1 shows the examples of secondary metabolites in plants:, Table 3.1: Secondary Metabolites in Plants, Classes, Alkaloids, , Glucosinolates, Monoterpenes, , Sesquiterpenes, Diterpenes, Triterpenes,, Cardiac Glycosides, Terpene Polymers, Sterols, Phenolic Acids, , Coumarins, Lignans, Flavonoids, Tannins, Lignin, , Compounds, Sources, Effects, Nicotine,, cocaine, Tobacco & coca plant Interfere with, and theobromine, neurotransmission; block, enzyme action., Sinigrin, Cabbage, Menthol and linalool Mint and relatives, Interfere with, neurotransmission; block, ion transport; anaesthetic., Parthenolide, Parthenium and, Contact dermatitis., relatives (Asteraceae), Gossypol, Cotton, Block phosphorylation., Digitogenin, Digitalis (foxglove), Stimulate heart muscle ;, alter ion transport., Rubber, Hevea (rubber) trees Gum up insects;, and dandelion, Airplane tires, Spinasterol, Spinach, Interfere with animal, hormone action., Caffeic & chlorogenic All plants, Cause oxidative damage;, browning of fruits and, wine., Umbelliferone, Carrots and parsnip, Cross-link DNA ; block, cell division., Podophyllin, Mayapple poison ivy Cathartic, vomiting, ,, dermatitis., Anthocyanin and, Almost all plants, Provide leaf colour;, catechin, estrogenic., Gallotannin, Oak and legumes, Bind to proteins; block, digestion; antioxidant., Lignin, All land plants, Structure and toughness., , 3.2. ALKALOIDS, 3.2.1., , Introduction, , Alkaloids are chemical compounds that are basic in nature. They are obtained, from plants and are beneficial to the physiological system of our body., Chemically, at least one nitrogen atom is present in its cyclic str, ucture. A, pharmacist, W. Meissner, gave the name alkaloids to nitrogen-containing basic, compounds, obtained from plants., Ladenburg stated alkaloids as, ‘compounds originated from plants with a, basic nature, having one or more nitrogen atom in its ring structure’., Advancement in research of chemistry of alkaloids has added two significant, characteristics to the definition of alkaloids:, 1) Molecular structure of alkaloids is complex, and, 2) Alkaloids produce a significant pharmacological activity., The basic nature of alkaloids is due to the presence of N -atom in the five- or sixmembered ring structure., *, , *
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Secondary Metabolites-I (Chapter 3), , 43, , Hence, based on the above findings, alkaloids can now be defined as,, ‗physiologically active basic compounds of plant origin in which at least one, nitrogen atom forms part of a cyclic system‘., , 3.2.2., , Chemistry, , Chemical characteristics of the alkaloids are taken into account under the, following heads:, 1) N-Atom in the Molecule: Along with carbon, hydrogen, and oxygen, the, most essential compone nt of alkaloids is the N -atom. Alkaloids should have, at least one or more N -atoms in a molecule. For example, cocaine has five, nitrogen atoms in its molecule. In case of quinine, reserpine, strychnine,, vinblastine, and yohimbine molecules, these N, -atoms ar e a part of the, heterocyclic ring, whereas in ephedrine and mescaline, N -atoms are present, in the aliphatic side chain., The N-atom in morphine and reserpine is present as tertiary-amine (R3N), in, ephedrine as secondary-amine (R2NH), and in nor -pseudo-ephedrine as, primary-amine (RNH2). The N -atom in tertiary - or secondary -form is an, integral part of the heterocyclic ring system., In tertiary amine form of N -atom (having only two bonds in the ring), the, methyl moiety makes the third component,, e.g., N-methyl group in, morphine, cocaine, colchicine, dextromethorphan, codeine, physostigmine,, vinblastine, videsine, etc. This concludes that methyl moiety is the only alkyl, group substituted on N-atom., In certain alkaloids, e.g., tubocurarine chloride, N -atom is p resent as, quaternary ammonium (R 4N + X). But as per the logical and technical, perspective, the quaternary ammonium compounds are not alkaloids. This, can be exemplified by the following reasons:, i) N-atom without a H-atom, and, ii) Different chemical properties., 2) O-Atom in the Molecule: Along with C, H and N, some alkaloids carry O atom. Alkaloids of this category are basically solid alkaloids, except certain, oxygenated alkaloids which are non-volatile liquids, e.g., pilocarpine., , Pilocarpine, (Liquid, mp 34°C), , Morphine, (Solid, mp 197°C), , Strychnine, (Solid, mp 268-270°C), , 3) Basicity (Alkalinity): Presence of N-atom in the alkaloid molecule gives it a, characteristic basic or alkaline reaction, thereby resulting in the formation of, their respective salts on reacting with many acids., *, , *
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44, , Pharmacognosy and Phytochemistry-II, , Degree of Basicity, The degree of basicity of alkaloids mainly depen, ds on the following, factors:, i) Electrostatic nature of the N-atom present in the alkaloid molecule, for, example, number and location (either present in the ring or the side, chain) of N-atom, alkyl group (methyl) attached to N-atom, etc., ii) Presence of primary-, secondary-, tertiary-, or quaternary N -atom(s) in a, molecule., Due to these structural variations, the differences arising in the degree of, basicity are understood by the difference in dissociation constant values (i.e.,, pKa values) of alkaloids given below:, Alkaloids, Berberine, Colchicine, Emetine, Morphine, Papaverine, Physostigmine, Quinine, Reserpine, Vinblastine sulphate, Vincristine, , pKa Values, 2.47 (K = 3.35 10–3, 12.35 (at 20C), pK1 = 5.77; pK2 = 6.64, 9.85, 8.07 (at 25C), pKa1 = 6.12; pKa2 = 12.24;, pK1 = 5.07 (at 18C); pK2 = 9.7;, 6.6, pKa1 = 5.4; pKa2 = 7.4;, 5.0;7.4 (in 33% DMF), , Salient Features of Alkaloids, i) The weakly basic alkaloids (having low pKa values) form their salts in a, higher acidic medium with the corresponding acid., ii) The strongly basic alkaloids (having high pKa values) form their salts in, a low acidic medium with the corresponding acid., Note: In a weakly acidic medium, some strongly basic alkaloids produce, their respective salts by reacting quickly with the corresponding acid,, while the weakly basic alkaloids with lower pKa values do not get, transformed and remain as free-bases. This reactivity feature is utilised to, obtain specific alkaloid or a group of alkaloids with identical pKa values,, from alkaloids with very low or high pKa values., iii) Acid neutralises alkaloids to produce salts which gets converted into the, corresponding free-base. This conversion happens by incorporating weak, bases like ammonia, calcium hydroxide or sodium carbonate. NaOH or, KOH solutions are avoided because they decompose the highly sensitive, alkaloids., iv) Amphoteric Alkaloids: These alkaloids have a phenolic ( –OH) gro up, (in morphine) or carboxylic (–COOH) group (in narceine), thus, behave, neither as acid nor as base., *, , *
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Secondary Metabolites-I (Chapter 3), , 45, , v) Unstable Alkaloid Salts: These alk aloids, e.g., piperine, papaverine,, narceine, narcotine, and caffeine show weak basic properties and their, salts are less stable., , vi) Neutral or Slightly Acidic Alkaloids: These alkaloids, e.g., ricinine and, theophylline, are obtained naturally and possess either neutral or slightly, acidic properties., , 4) Stability of Alkaloids: Generally alkaloids are not much stable as they, degrade or decompose not only when exposed to chemical reagents but also, on exposure to air, light, moisture, and heat. Examples of alkaloids along, with their stability are given below:, i) Alkali treatment for long duration decomposes ergotamine but strychnine, withstands such prolonged treatment., *, , *
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46, , Pharmacognosy and Phytochemistry-II, , ii) Fast degradation has been reported for aqueous solutions of alkaloid, s, than their solid forms., iii) For enhancing the stability of alkaloids, they are stored in pure form or, dry extract in desiccators with dehydrating agents like phosphorous, pentoxide (P2O5) or anhydrous calcium chloride (CaCl2)., iv) To minimise the risk of decompos, ition of the desired alkaloid by, preventing its exposure to excessive heat, the solvent used for their, extraction and isolation is eliminated either by distillation carried out, under vacuum or decreased atmospheric pressure, or by evaporation in, rotary thin-film evaporator under vacuum., v) Amber-coloured glass bottles with vacuum desiccators are used for, storing alkaloids., 5) Acid Salts of Alkaloids: Most of the alkaloids showing strong alkalinity, produce well-defined salts. For some cases, it is observed that alkaloids show, weak basic character which reduces its salt formation efficiency with weak, acids (acetic acid, etc.). The salts produced by the reaction of alkaloids with, strong acids (HCl, H 2SO4, etc.) decompose in water , yielding a free base and, an acid. Ve ry few alkaloids have been reported to form alkali carbonates or, alkali hydrogen carbonates which could be employed to yield their, corresponding salts in presence of water., Alkaloids with one or more than one N -atom are monoacidic bases. These, produce a s ingle series of salts with acids designated as ‗BA‘ (where, B =, base; and A = acid)., Exception: Quinine and cinchona alkaloids deviate from the above concept, as they are di-acidic bases. Also many alkaloids having two N -atoms in their, molecule act as mono acidic bases. Therefore, alkaloidal basicity has great, significance for quantitative volumetric estimation., Alkaloidal salts, e.g., morphine hydrochloride, atropine sulphate, quinine, sulphate, ephedrine hydrochloride, etc. are formed with the involvement of, cold and dilute mineral acids. Using concentrated solutions of mineral acids, or heating an alkaloid with dilute acid under pressure results in physical and, chemical changes. The physical changes include colour changes (used for, identifying and characte rising alkaloids) and decomposition with strong, acids. The chemical changes occurring in alkaloids with mineral acids are:, i) Dehydration: Dehydration produce anhydro- or apo-alkaloids, like:, a) Apomorphine (from morphine), and, b) Apoatropine (from atropine)., ii) Demethoxylation: Many alkaloids, e.g., codeine, quinine, narcotine, and, papaverine, carry methoxy moiet ies which are eradicated from the, alkaloid molecule by treating it either with conc. HCl (giving methyl, chloride) or with HI (giving methyl iodide), along wi, th the, corresponding hydroxy base., For example: Narcotine + 3HI, , Nornarcotine + 3CH3I., *, , *
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Secondary Metabolites-I (Chapter 3), , 47, , iii) Hydrolysis: Majority of alkaloids found in nature are isolated in ester, forms which hydrolyse upon heating with either alkali or mineral acids, yielding their conjugate acids and respective alcohols or phenols., A couple of typical examples are given below:, a) Atropine + H2O Tropine + Tropic Acid, b) Cocaine + 2H2O Ecgonine + Benzoic Acid + Methanol, 6) Action of Alkalis:NaOH and KOH alkalis affect alkaloids as illustratedelow:, b, i) Dilute KOH or NaOH solutions degrade alkaloidal salts liberating free, alkaloids., ii) Alkaloids with phenolic hydroxyl groups (morphine) react with alkaline, solutions to produce soluble sodium or potassium salts., iii) The ester alkaloids (atropine and cocaine) get hydrolysed by dilute alkali, solution., iv) Racemic Isomeride: Hyoscyamine in alcohol when treated with alkali, hydroxide solution produces racemic isomeride atropine., v) Alkaloids upon treatment with dry alkali (KOH or NaOH) and heat, undergo decomposition , forming simple heterocyclic bases (pyridine,, quinoline, pyrrolidine, etc.)., vi) Alkaloids fused with alkali hydroxides cause identifiable colour changes., 7) Pharmacological Activity: Presence of a wide -spectrum and complete, diversity of complex structures in alk aloids allow them to show broad range, of pharmacological activities on cardiovascular and central nervous system., Alkaloids have their specific and definite pharmacological action., Observations explore that even 0.1 -1.0mg of alkaloid can result in beneficia l, pharmacological action on organs and tissues of animals and humans., Variation with respect to potency is however reported among alkaloids., 3.2.2.1., Isomerism, The phenomenon dealing with change of one structure into another having th e, same empirical formula but different properties is known as isomerism. Optical, activity of alkaloids is due to the presence of one or more asymmetric carbon, atoms in the molecule. Usually an alkaloid‘s levorotatory form, i.e., ( )-isomer, produces better pharmacological activity than its dextrorotatory form, i.e., (+) isomer. The l -(levo-) and d -(dextro-) rotatory isomer differ from L - and D isomers with respect to optical activity. In this, the latter does not refer to optical, activity but mentions the steric configuration for a traditionally accepted standard, compound. Optical activity relies on alkaloids and their salts. As a matter of fact,, optical activity and the specific rotation show variation with solvents used,, change in temperature, wavelength of light used, etc., Examples, 1) Showing d- and l-Isomers with Distinct Pharmacological Activities, i) Relative Pressor Activities of D ( )-Ephedrine and D (+) -Ephedrine:, The relative pressor activities of D ( )-ephedrine and D (+) -ephedrine, isomer are 36 and 11, respectively. This concludes that D ( )-ephedrine, shows 3½ times more activity than D (+)-ephedrine., *, , *
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50, , Pharmacognosy and Phytochemistry-II, , iii) Wagner’s Reagent (Potassium Tri-Iodide), Iodine = 1.3gm, Potassium = 2.0gm, Distilled water = 100.00ml, Wagner‘s reagent + alkaloids Brown or reddish brown precipitate., iv) Dragendorff’s Reagent (Potassium Bismuth Iodide): Dragendorff‘s, reagent + alkaloids Reddish brown colour precipitate., v) Kraut’s Reagent (Modified Dragendorff’s Re, Bismuth Iodide), Bismuth Nitrate = 8.0gm, Nitric Acid = 20.0ml, Potassium Iodide = 27.2gm, Distilled water = 100.00ml., , agent or Potassium, , vi) Marme’s Reagent (Potassium-Cadmium Iodide Reagent), Cadmium Iodide = 10.0gm, Potassium Iodide = 20.0gm, Distilled water = 100.00ml, vii) Scheibler’s Reagent (Phosphotungstic Acid Reagent), Sodium Tungstate = 20.0gm, Disodium Phosphate = 70.0gm, Distilled water = 1000.00ml, Note: Acidify with nitric acid to litmus paper., viii) Sonnenschein’s Reagent (Phosphomolybdic Acid):, solution of phosphomolybdic acid in ethanol., ix) Bertrand’s Reagent (Silicotungstic Acid):, silicotungstic acid in distilled water., Note: Filter and store in a refrigerator., , A 1%(w/v), , A 1% (w/v) solution of, , x) Reineckate Salt Solution, Ammonium Reineckate (NH4 [Cr. (NH3)2 (SCN)4) = 1.0gm, Hydroxylamine HCl = 0.3gm, Ethanol = 100.00ml, Note: Filter and store in a refrigerator., 2) On the Basis of Colour Reactions: The colour reactions of alkaloids are so, sensitive that alkaloids present in microgram quantities produce a rapid response., Either dehydration or oxidation of alkaloid results in a colour reaction. The, reagents used for producing a colour change consist of conc. sulphuric acid and, some other added compounds , like sulphomolybdic acid, formaldehyde,, sulphovanadic acid, potassium arsenate, hydrogen peroxide, and selenious acid., Following are the examples of reagents used:, i) Froehde’s Reagent: It is a mixture of 5mg of molybdic acid or sodium, molybdate in 5ml of pure conc. H2SO4., Note: The reagent should be freshly prepared before use., *, , *
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Secondary Metabolites-I (Chapter 3), , ii), , 51, , Erdmann’s Reagent: It is a mixture of 10 drops of, 100ml of water in 20ml of pure conc. H2SO4., , iii) Marqui’s Reagent: It is a mixture of 2, solution (40%) in 3ml of conc. H2SO4., , conc. HNO3 and, , -3 drops of formaldehyde, , iv) Mandelin’s Reagent: It is a mixture of 1gm, of finely powdered, ammonium vanadate in 200gm of pure conc. H2SO4., v), , Mecke’s Reagent: It is a mixture of 1gm of selenious acid in 200gm of, pure conc. H2SO4., , vi) Modified Dragendo rff’s Reagent: It is obtained by dissolving 1.6gm, of bismuth sub -nitrate in 60ml of 20% glacial acetic acid. Then adding, to it 5ml of 40% aqueous solution of KI, 5ml of glacial acetic acid and, making up the volume to 100ml with water., vii) Rosenthaler’s Reagent: It is a mixture of 1gm of potassium arsenate in, 100gm of pure conc. H2SO4., viii) Schaer’s Reagent: It is a mixture of 1 volume of pure 30% H, 10 volumes of conc. H2SO4., , 2O2, , with, , Note: The reagent should be freshly prepared before use., , 3.2.2.4., , Quantitative Analysis, , Alkaloids can be quantitatively analysed by the following two techniques:, 1) Gravimetric Method: Ygm of the powdered material is Soxhlet extracted, with hexane for 12 hours in order to defat it. The defatted powder is taken, and the alkaloidal content is extracted with 500ml of ethanol. The extracts, are f iltered and concentrated under reduced pressure using a rotary, evaporator. The residue is mixed with 200ml of 10% aqueous acetic acid and, allowed to stand overnight., The mixture is filtered and the pH of the resulting filtrate is adjusted to 10 by, adding drops of dilute ammonium solution. The alkaloids are extracted with, two equal volumes of 200ml of chloroform., The chloroform extract is dried with anhydrous sodium sulphate, the solvent, is evaporated , and the residue is weighed. The percentage of alkalo, id is, calculated using the following formula:, Total Alkaloid(%) , , W, 100, Y, , Where,, W = Weight of alkaloidal content extracted., Y = Weight of powdered plant material., 2) Bromocresol Green (BCG) Method: A part of extrac t residue is dissolved, in 2N HCl and then filtered. 1ml of this solution is transferred to a separating, funnel and washed 3 times with 10ml chloroform. The pH of this solution is, adjusted to neutral with 0.1N NaOH. Then 5ml of BCG solution and 5ml of, *, , *
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52, , Pharmacognosy and Phytochemistry-II, , pH 4.7 phosphate buffer are added to this solution. The mixture is shaken and, the complex is extracted with 1, 2, 3 and 4ml chloroform by vigorous, shaking. The extract is then collected in a 10ml volumetric flask and diluted, with chloroform., Preparation of Standard Curve, Accurately measured (0.4, 0.6, 0.8, 1 and 1.2ml) atropine standard solution is, transferred to different separating funnels. Then 5ml of pH 4.7 phosphate, buffer and 5ml of BCG solution are taken and the mixture is shaken and the, complex is extracted with 1, 2, 3, and 4ml of chloroform., The extract is collected in a 10ml volumetric flask and diluted to adjust, solution with chloroform. The absorbance of the complex in chloroform is, measured at spectrum of 470nm in UV -spectrophotometer against the blank, prepared as above but without atropine., , 3.2.2.5., , Isolation and Extraction of Alkaloids, , In general, alkaloids may be extracted by any of the following three well -defined, and widely accepted processes:, 1) Stas-Otto Process: Alkaloid extraction is based on its basicity and solubility., Extraction is basically carried out by, Stas-Otto process in which the, moistened drug is treated with alkali to set free the base existing in salt form, and then the free base is separated with an organic solvent., Powdered plant material, Defatted with non-polar solvent, Defatted plant material, , Moist with water and treated with NH 3 dil. lime solution (free alkaloid), Extracted with organic solvent like, chloroform, ether, Extract, concentrate it, Dissolved in dil. acid (alkaloidal salt), , Aqueous phase, , Organic phase impurities, , Basified with ammonia or sodium, bi-carbonate or dil. KOH, , Aqueous phase, , Organic phase (free alkaloid), , Evaporate to dryness, Crude alkaloids, , 2) Manske’s Process: This process is shown below:, *, , *
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Secondary Metabolites-I (Chapter 3), , 53, , Powdered crude material, Defatted with non-polar solvent, Defatted crude material, Extract with methanol, Methanol extract, Concentrate, Dissolve in water and acidified it up to pH 2 (alkaloidal salt), , Steam distillation to remove traces of methanol, Stand for several days in refrigerator, or, Boiled with paraffin, Filter, Filtrate, Shake with organic solvent like, chloroform or ether, , Aqueous phase (alkaloidal salt), , Organic phase, , Basified with ammonia or sodium, bi-carbonate or dil KOH, , Aqueous phase, , Organic phase, , Evaporate to dryness, Crude alkaloids, , 3) Kippenberger’s Process: This process is performed as follows:, i) The powdered and sieved plant substance is first and foremost digested, with solution of tannin in glycerol at a co nstant temperature of 40ºC for, 48 hours., ii) The resulting mixture is heated to 50ºC, to help in the complete, coagulation of proteinous substance., iii) Then the mixture is cooled to ambient temperature and filtered., iv) The resulting filtrate is thoroughly shaken with petroleum ether to get rid, of fa ulty material, and the last traces of petroleum ether are removed, from the extract by heating either on a water bath or, exposing to IR, lamp., v) The fat-free crude plant extract is subsequently acidified and shaken with, chloroform to remove the bulk of alkaloids., vi) The resulting residual extract may still contain narceine and curarine., vii) However, narceine and morphine may be isolated by passing freshly, generated CO2 directly into hydroxide into their corresponding carbonate, which is then ultimately subjected to solvent extraction using alcohol and, chloroform mixture., *, , *
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54, , Pharmacognosy and Phytochemistry-II, , viii) Finally, the third alkaloid, curarine may be extracted by agitation with, mixture of equal volume of ether and chloroform., ix) A combination of Kippenberger‘s process and Stas -otto process by its, application to the final alcoholic extract obtained by the latter process is, found to give a better separation of alkaloids., 4) Steam Distillation: This method is specially employed for volatile liquid, alkaloids like coniine, nicotine, and sparteine. This process is not suitable for, high molecular weight alkaloids., , 3.2.2.6., , Purification of Crude Extract of Alkaloids, , Different alkaloidal crude extracts are purified by different ways:, 1) Direct Crystallisation from Solvent: This simple isolation method is not, used for complex mixtures., 2) Steam Distillation: This method is used for volatile liquid alkaloids like, coniine, sparteine , and nicotine. However, high molecular weight alkaloids, cannot be isolated by this technique., 3) Chromatographic Techniques: This method is ideal for separating majority, of plant alkaloids. The different chromatography techniques such as, TLC,, HPTLC, column, gas, liquid and ion-exchange chromatography, etc. are used, for separating individual alkaloids from complex mixtures., 4) Gradient p H Technique: Though alkaloids are basic in nature, there are, variations in the extent of basicity of various alkaloids of the same plant., Depending on this character, the crude alkaloidal mixture is dissolved in 2%, tartaric acid solution and extracted with benzene so that the first fraction, contains neutral and or very weakly basic alkaloids., The pH of aqueous, solution is increased gradually by 0.5 increments up to pH 9 and extraction is, carried out at each pH level with benzene , ether, or chloroform. By this way,, the alkaloids with different basicity are extracted. Strongly basic alkaloids, are extracted at the end., , 3.2.3., , Chemical Classes, , Based on their properties, alkaloids are chemically classified as follows:, 1) True Alkaloids: These alkaloids have the following characteristic features:, i) Sufficient toxicity., ii) Wide range of physiological activity., iii) Basic nature., iv) Presence of nitrogen atom in the heterocyclic ring., v) Obtained from amino acids., vi) Limited taxonomic distribution., vii) Occur in plants in the form of organic acid salt., Exceptions: Colchicine and aristolochic acid are acidic alkaloids with no, nitrogen atom in their heterocyclic ring., 2) Protoalkaloids: The amino acid nitrogen is not sit uated in the heterocyclic, ring, hence are characterised as simple amines. These alkaloids are, *, , *
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Secondary Metabolites-I (Chapter 3), , 57, , 3) Tyrosine derived alkaloids, e.g., Isoquinoline alkaloid s (morphine, codeine,, emetine, cephaline, berberine,and d- tubocurarine); Amino alkaloid(colchicine)., 4) Tryptophan derived alkaloids,e.g., Indole alkaloids (ergot alkaloid, vincristine,, vinblastine, reserpine, strychnine, physostigmine, and brucine); Quinoline, alkaloids (cinchonine, quinine, quinidine, and camptothecin)., 5) Histidine derived alkaloids, e.g., Imidazole alkaloid (pilocarrpine)., 6) Phenylalanine derived alkaloids, e.g., Amino alkaloid (ephedrine)., , 3.2.4., , Biosources, , Alkaloids are present in varying quantities, such as:, 1) More in dicots than in monocot plants., 2) Lesser common in lower plants., 3) Apocynaceae, Rubiaceae, Solanaceae,, and Papaveraceae families have, abundance of alkaloids, while Rosaceae and Labiatae families are free from, alkaloids., Distribution in Plants, 1) In some plants, alkaloids are found in all the parts, e.g., datura., 2) In some plants, alkaloids are found in barks, e.g., cinchona., 3) In some plants, alkaloids are found in seeds, e.g., nux vomica., 4) In some plants, alkaloids are found in roots, e.g., aconite., 5) In some plants, alkaloids are found in fruits, e.g., black pepper., 6) In some plants, alkaloids are found in leaves, e.g., tobacco., 7) In some plants, alkaloids are found in latex, e.g., opium., Forms of Alkaloids, 1) They may occur in the form of free bases., 2) They may occur in the form of salts with organic acids, e.g., oxalic acid and, acetic acid., 3) They may occur in the form of alts, s with inorganic acids,e.g., HCl and H2SO4., 4) They may occur in the form of salts with specials acids, e.g., meconic acid in, opium and quinic acid in cinchona., 5) They may occur in the form of glycosides, e.g., solanine in solanum., Functions in Plants, 1) Their bitterness and toxic nature provide protection against herbivores., 2) They are the waste products of detoxification., 3) Being the major source of nitrogen, they can cure nitrogen deficiency., 4) They also aid in regulating growth in some metabolic systems., 5) Those containing a sugar moiety may be utilised as a source of energy in, deficiency of carbon dioxide assimilation., , 3.2.5., , Therapeutic Uses, , Presence of a wide -spectrum and complete diversity of complex structures in, alkaloids allow them to show a broad range of pharmacological activities on, cardiovascular and central nervous system. Alkaloids have their specific and, definite pharmacological action. Observations explore that even 0.1 -1.0mg of, *, , *
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58, , Pharmacognosy and Phytochemistry-II, , alkaloid can result in beneficial pharmac ological action on organs and tissues of, animals and humans. Variation with respect to potency is however reported among, alkaloids. Table 3.4 summarises the pharmacological actions of some alkaloids:, Alkaloids, Morphine, Codeine, Brucine, Strychnine, Ergotamine, Atropine, Homatropine, Pilocarpine, Physostigmine, Ephedrine, Reserpine, Quinine, Caffeine, Tubocurarine, Emetine, Hyoscyamine, Cocaine, Colchicine, Lobeline, Arecoline, Protoveratrine A, Conessine, Vasicine, Vinblastine, Vincristine, Piperine, Heroin, Hyoscine, Theophylline, Aconitine, , 3.2.6., , Pharmacological Actions, Narcotic and analgesic, Expectorant and analgesic, CNS stimulant, CNS stimulant, Uterine muscle contractions, Mydriatic, Mydriatic, Miotic, Miotic, Hypertensive, Hypotensive, Antimalarial, CNS-stimulant, Neuromuscular blocking action, Antiprotozoal action, Relief of spasm of urinary tract, CNS stimulant, Anti-gout, Treatment of asthma, Parasympathomimetic action, Management of hypertension in pregnancy, Antiprotozoal and anti-amoebic, Expectorant and bronchodilator, Antineoplastic, Antineoplastic, Carminative and stomachic, Narcotic analgesic, Motion sickness (sedation), Smooth muscle relaxant, Treatment of neuralgia, sciatica, rheumatism ,, and inflammation., , Commercial Applications, , The commercial applications of alkaloids are given below:, 1) As Medicines: In a few serious disorders like hearth -failure, cancer, blood, pressure, etc ., alkaloids serve as lifesaving drugs. Digoxin is used in heart, failure and it also improves heart functioning and maintains life. Vincristine, obtained from vinca is used as an anticancer agent. Ephedrine derived from, ephedra is used in blood pressure. Codeine is used as a cough suppressant., Ergot alkaloids relieve migraine pain., 2) As Insect Repellents and Pesticides: Pyrethrin is an insect repellent and is, added in mosquito coils, mosquito repellents, and pesticides also., *, , *
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Secondary Metabolites-I (Chapter 3), , 59, , 3) For Research and Scientific Study : Alkaloids are used in research and, scientific study because of their specific effects on body., For e xample,, atropine causes pupil dilation. For confirming whether a new substance has, similar or opposite effects, it is compared with atropine. Hence, atropine is, used as a standard for comparison., 4) To Catch Animals: Alkaloids were used earlier for capturing heavy animals, (like deer, elephants, etc. ) by making them immobile. Arrows applied with, arrow poison (D-tubocurarine) were used by the hunters to shoot at animals., D-tubocurarine enter the muscle s and cause s paralysis when the animal is, shot by the poison ed arrow. This is a reversible and short term action. The, animals become immobile, thus they can be easily trapped and transported., , 3.2.7., , Individual Drugs, , The pharmacognostic profile of the following drugs has been discussed below:, 1) Vinca,, 2) Rauwolfia,, 3) Belladonna, and, 4) Opium., , 3.2.7.1., , Vinca, , Vinca (or sadabahar or periwinkle) is commonly, grown in India. It has its origin in Madagascar. Two, varieties of vinca, namely pink and the white, coloured flower varieties are grown for their, medicinal value., Synonyms, Catharanthus, Periwinkle, and Sadabahar., Figure 3.2: Vinca Plant, , Biological Source, Vinca is the dried whole plant of Catharanthus roseus or Vinca rosea., Family, Apocynaceae., Geographical Source, Vinca is native to Madagascar. It is grown as an ornamental plant in India, South, Africa, U.S.A., Europe, Australia, and Caribbean islands., Macroscopic Features, Vinca is an erect, pubescent herb (figure 3.2), and has the following organoleptic, features:, 1) Colour: Leaves are green, roots are pale grey, and flowers are violet pink white or carmine-red coloured., 2) Odour: Characteristic., 3) Taste: Bitter., 4) Roots: Branched tap-root., *, , *
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Secondary Metabolites-I (Chapter 3), , 61, , Ajmalicine, lochner ine, serpentine, and tetrahydroalstonine are the other, alkaloids of vinca. For extracting 1gm of vincristine, around 500kg of crude, drugs is required as vincristine is present in very low content (0.0002%). Hence,, vinca alkaloids are very expensive. The five-ring dihydroindole system present in, their ring is found in other natural drugs also. Thus, synthesis of four -ring indole, system is under process., Chemical Tests, Vincristine sulphatecrystals are obtained from ethanol and are found to be unstable., Uses, 1) Its leaves and stems are a source of alkaloids, which have antitumour and, anticancer properties., 2) Its leaves are used for controlling diabetes and high blood pressure., 3) Its alkaloids are sedative and tranquiliser., 4) It relieves muscle pain and depression., 5) It exhibits the property of detoxification and can counter the effect of poison,, thus is used in wasp stings., 6) It controls nosebleeds, bleeding gums, mouth ulcers, and sore throats., 7) On internal administration , it is helpful in gastritis, cystitis, enteritis,, diarrhoea, diabetes, etc., 8) It ensures brain health as the active ingredients improve blood supply to the, brain and increase the oxygen level that brain can utilise., 9) It also raises the serotonin levels and prevents abnormal blood coagulation., 10) Vincamine alkaloid keeps the blood thin and acts as a memory enhancer, thus, is used in preventing dementia., Adulterants, Commercial samples of Catharanthus roseus are often found adulterated with, certain Solanaceous roots like Solanum melongena (egg plant) and Lycopersicon, esculentum (tomato), which adversely affect the quality, as well as total, alkaloidal yield of the drug., , 3.2.7.2., , Rauwolfia, , Rauwolfia (or sarpagandha or black snakeroot or Indian snakeroot or devil, pepper) is an evergreen plant. In India, roo ts and rhizomes of this plant have, been in use for hundreds of years. The plant is named after its discoverer, a, German doctor and traveller,, Leonhard Rauwolf . Due to its immense, therapeutic properties, it is used in Ayurvedic, Unani, and Homeopathy., Synonyms, Rauwolfia root, Serpentina root, Chhotachand, and Sarpagandha., Biological Source, Rauwolfia is the dried roots of the plant Rauwolfia serpentina Benth., Family, Apocynaceae., *, , *
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62, , Pharmacognosy and Phytochemistry-II, , Geographical Source, Various species of rauwolfia are found in the tropical regions of America, Africa,, and Asia. It is commercially cultivated in America, Thailand, Myanmar, Sri, Lanka, and India. In India, it is cultivated in Gujarat, Maharashtra, Karnataka,, West Bengal, Tamil Nadu, Orissa, Bihar, and Uttar Pradesh., , Longitudinal, wrinkles, , Root scars, Figure 3.4: Pieces of Rauwolfia Roots, , Macroscopic Features, 1) Colour: Root bark is greyish yellow to brown and wood is pale coloured., 2) Odour: Odourless., 3) Taste: Bitter., 4) Size: Diameter is around 1-3cm and length is 10-18cm., 5) Shape: Roots are sub -cylindrical in shape, and tort uous with slight ta pered, end., 6) Fracture: Short and irregular. Transversely cut surface is white, and dense, with finely radiating xylem., Extra Features: Due to longitudinal marking and wrinkled surface, the roots, appear rough. Normally, rootlets are absent but some small c ircular root scars, with tetrastichous arrangements can be observed., Microscopic Features, 1) Cork: It consists of stratified cells below which phelloderm of few rows of, parenchyma is present., 2) Phloem: It is narrow and parenchymatous with small scattered sie, ve, tissues. In parenchyma, starch grains and few latex cells with brown, resinous matter are present. Calcium oxalate c rystals are present in the, secondary phloem., 3) Xylem: About 80% of diameter of root is xylem which includes vessels,, tracheids, wood parenc hyma, and wood fibres. Simple or bordered pits are, present on xylem vessels. These vessels are elongated up to 350 in length, and 50 in width., 4) Stone cells or phloem fibres are absent., *, , *
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64, , Pharmacognosy and Phytochemistry-II, , Ajmalicine, Serpentine, , Ajmaline, Yohimbine, , The other substances present are phytosterols, fatty acids, unsat urated alcohols,, and sugars., Chemical Tests, 1) On adding an acid or on exposing to light, most reserpine solutions give a, distinct yellow colour and also a clear fluorescence on standing., 2) Violet red colour solution appears on treating reserpine with vanillin solution, in acetic acid., 3) On crystallisation of reserpine, crystals of reserpine hydrochloride hydrate, (C33H40N2O9.HCl.H2O) are obtained that decomposes at 224ºC temperature., 4) On treating a freshly fractured surface with concentrated nitric acid, a red, colour is observed along the medullary ray., Uses, 1) It is a hypotensive drug having a strong sedative property., 2) It is a mild tranquiliser used for removing low level of anxiety., 3) It is a rich source of indole alkaloids (such as reserpine, ajmaline, ajmalicine,, and serpentine), which are used in the treatment of circulatory disorders., 4) Its roots are used for managing and lowering, blood pressure due to the, presence of reserpine which dilates the blood vessels, and also depresses the, CNS activity by acting as a hypnotic., 5) Deserpidine and rescinnamine are also used as hypotensive and tranquilliser., 6) It is also used as an antianxiety, antipsychotic (neuroleptic), hypnotic and, sedative drug. It induces sleep and, relieves many mental disorders like, aggression, anxiety, and sleeplessness., *, , *
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Secondary Metabolites-I (Chapter 3), , 65, , 7) Its leaves are used in removing opacities of cornea., 8) Its root decoction is useful for the treatment of snake poison., 9) In Ayurveda, it is best for reducing fever., 10) In Ayurveda, its roots and whole plant are used for treating rheumatism,, insanity, and epilepsy., 11) It also reduces the symptoms of hangover occurring after heavy drinking., 12) It is used to treat uterine pain due to miscarriage as it contracts the uterus and, eliminates the toxins from uterine cavity and further relaxes the uterine, muscles. This stops bleeding and reduces pain., 13) It decreases pain during menstruation by modulating blood flow, which, reduces cramps and throbbing pain., 14) It is also useful for treating dysentery as it reduces the frequency of loose, stools, checks bleeding, and alleviates pain., Substitutes and Adulterants, For substitution purpose, following species of Rauwolfia are used:, 1) Rauwolfia tetraphylla,, 2) R. densiflora, and, 3) R. vomitoria (African rauwolfia)., For adulteration of Rauwolfia serpentine, stem of the same plant is used. But, differentiation between roots and stems is easy as roots are less branched, less, thick, less flexible, and more wrinkled on the surface than the stems. Other plants, used for adulteration are:, 1) Ophiorrhiza mungos L.,, 2) White or red flowered species of Clerodendrum, and, 3) Tabernaemontana divaricata (L.) R.Br. ex Roem. and Schult., The species and varieties of genus Rauwolfia used as adulterants are:, 1) R. tetraphylla L.,, 2) Rauwolfia beddomei Hook.f.,, 3) R. micrantha Hook.f.,, 4) R. verticillata (Lour.) Baill., and, 5) R. densiflora (Wall.) Benth. ex Hook.f., , 3.2.7.3., , Belladonna, , Belladonna (or deadly nightshade) is a perennial herbaceous plant. The foliage, or berries contain tropane alkaloids, and are very poisonous. Its toxicity is due to, the presence of atropine, hyoscyamine, and scopolamine causinghallucinations and, bizarre delirium.Its pharmacological activityis anticholinergic., Synonyms, Belladonna leaf, Belladonna folium, and Deadly night shade leaf., Biological Source, Belladonna is the dried leaves and flowering tops of the plant Atropa belladonna., It should contain 0.30% or more of total alkaloids calculated as hyoscyamine., Family, Solanaceae., *, , *
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66, , Pharmacognosy and Phytochemistry-II, , Geographical Source, Belladonna is native to Europe, North Africa , and Western Asia . In India,, belladonna is found in the western Himalayas from Shimla to Kashmir and also, in some neighbouring areas of Himachal Pradesh. The main habitat of belladonna, includes the forests of Sindh, Chenab valley, and Jammu., Macroscopic Features, 1) Colour, i) Leaves: Green to brownish-green., ii) Flowers: Purple to yellowish-brown., iii) Fruits: Green to brown., 2) Odour: Slight and characteristic., 3) Taste: Bitter and acrid., 4) Size, i) Leaves: 5-25cm long and 2.5-12cm wide., ii) Flowers: Corolla 2.5cm long and 1.5cm, Figure 3.7: Belladonna Herb, wide., iii) Fruits: About 10cm in diameter., 5) Shape, i) Leaves: Ovate, lanceolat e to broadly ovate, with acuminate apex,, decurrent lamina, entire margin, petiolate, brittle, , and transversely broken., ii) Flowers: Campanulate, 5, small reflexed lobes of corolla., iii) Fruits: Berries, sub-globular in shape with numerous flat seeds., Extra Features, The whole plant looks wrinkled and twisted; dropping flowers are present with, many pairs of leaves. Flowers have five stamens, a superior bilocular ovary, having many seeds., Microscopic Features, A bifacial structure appears on the transverse section of A. belladonna leaf. The, epidermal cells are arranged in wavy walls and have a striated cuticle. Stomata, are mainly anisocytic type and some are anomocytic type present on both the, upper and lower surface of leaves but are more commonly present on the low er, side., Epidermis, Palisade layer, Veinlet, , Intra xylary phloem, Xylem, Phloem, Endodermis, Idioblast containing, crystals of calcium oxalate, Collenchyma, Figure 3.8: Atropa Belladonna Leaf, *, , *
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Secondary Metabolites-I (Chapter 3), , 67, , On young leaves, hairs are present abundantly, and are of three types:, 1) First type are uniseriate with two-to four-celled clothing hairs,, 2) Second type resembles the first type but have a unicellular glandular head, and, 3) Third type of hair has a short pedicel and a multicellular glandular head., Microsphenoidal (‗sandy‘) crystals of calcium oxalate are present in few cells of, the spongy mesophyll cells. The midrib has bicollatera l vascular bundle and is, convex-shaped above. In the midri b region, a zone of collenchyma forms an, underlying layer on both epidermises., Chemical Constituents, Around 0.3 -0.60% alkaloids are extracted from, Atropa belladonna in which, hyoscyamine is the main component. Volatile bases like, pyridine and Nmethylpyrroline are present in small quantities. If these are not removed during, the drug assay by heating, on increasing the titration they appear as hyoscyamine., Leaves of belladonna also have a fluorescent substance, i.e., -methylaesculetin, (scopoletin) and cal cium oxalate crystals. The leaves give 4% or less of acid insoluble ash and about 14% of total ash. About 0.28 -0.32% of total alkaloids is, present in finely powdered drug of a prepared belladonna herb., Chemical Test, Belladonna alkaloids give violet colour with fuming HNO 3 and alcoholic KOH, solution employed for their assay., Uses, 1) It has anticholinergic and parasympathetic properties., 2) In case of poisoning of opium and chloral hydrate, it is used as an antidote., 3) It is also used to decrease the secretion of sweat, gastric juice, and saliva., 4) It is used in breathing abnormalities in infants., 5) It is used for reducing sweat and other secretions., 6) It is effective against tonsilitis, meningitis, scarlet fever, whooping cough,, and epilepsy., 7) Belladonna preparations a re used against vesico, -ureteral refluxes (a, condition in which urine flows back towards the kidney from the bladder)., 8) It is used as an analgesic in pain due to kidney stones, sore throat, etc., 9) It is useful in liver and gall bladder disorders., 10) Powdered preparations are used to treat asthma., 11) It can lessen headaches related to migraine., 12) It eases premenstrual syndrome., 13) It can reduce spasms in smooth muscles of digestive tract, but causes tremors, or stiffness in other muscles., 14) Atropine, extracted from belladonna is used to dilate pupils., Adulterants, The most important adulterant among the numerous are:, 1) Phytolacca decandra (family Phytolaccaceae): In this the lamina is denser, and less decurrent than in belladonna. The epidermal cells have straight, walls, stomata are of the anomocytic type, and some of the mesophyll cells, contain bundles of needle-shaped crystals of calcium oxalate., *, , *
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68, , Pharmacognosy and Phytochemistry-II, , 2) Ailanthus glandulosa (family Simarubiaceae): In this the leaves are, triangular-ovate with straight -walled epidermal cells. Cuticl e is strongly, striated, cluster of calcium oxalate crystals are present, and white, lignified, and unicellular clothing hairs are present on both the surfaces., , 3.2.7.4., , Opium, , Opium is the dried latex extracted from the seedpods of the plant opium pop py, (Papaver somniferum). The un-ripened pods are slit open for the sap to seep out, which is then dried on the outer surface of the pod., The resulting latex is scraped off the pod which is yellow -brown in colour and, bitter in taste. Different types of alka loids, e.g., morphine, codeine, thebaine, and, papaverine are present in this latex., Synonyms, Raw Opium, Gum Opium, and Afeem., Biological Source, Opium is the dried latex extracted from the unripe capsules of the plant opium, poppy or Papaver somniferum Linn., Family, Papaveraceae., Geographical Source, Opium is commercially cultivated in, Afghanistan, Yugoslavia, Bulgaria,, Pakistan, Turkey, Persia (Iran) and India. In India, it is mostly grown in Madhya, Pradesh., Macroscopic Features, 1) Colour and Shape: Following are the colours and shapes of different opium:, i) Indian Opium: Dark brown in colour and is found in the form of cubical, pieces enclosed in a tissue paper., ii) Persian Opium: Dark brown in colour and is found in the form of brickshaped masses., iii) Natural Turkish or European Opium: Brown or dark brown in colour, and is found in conical or rounded and somewhat flattened masses., iv) Manipulated Turkish Opium:, Chocolate brown or dark brown, internally and covered with broken poppy leaves externally. The masses, of this type are oval and flattened on upper and lower surface., v) Manipulated European Opium: Dark brown in colour internally and, covered with broken leaves. It is present in the form of elongated masses, with rounded ends., 2) Odour: Strong characteristic., 3) Taste: Bitter., *, , *
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Secondary Metabolites-I (Chapter 3), , 69, , Microscopic Features, Powdered opium is dried latex and brown coloured, amorphous masses of, irregular shape. Small particles of water insoluble vegetable tissues are present in, these masses. The vegetable debris consists of fragments of outer epidermis of, the capsule ( figure 3.9 A). The capsule epidermis consists of un -lignified, polygonal tubular cells (15-40 size in either direction ). These cells have, moderately thick anticlinal walls. Sectional view of vegetable pieces shows, greater thickness of the outer wall, and presence of anomocytic stomata. Some, of these epidermal cells from the stigma are strongly pitted , giving the lumen a, stellate form (figure 3.9 B)., A, C, , B, , F, , E, , D, , Figure 3.9: Fragments of the Capsule Wall of, Papaver, somniferum L. A) Outer Epidermis; B) Outer Epidermis from, the Stigma; C) Inner Epidermis of the Capsule Wall; D), Transverse Section of the Outer Epidermis; E) Transverse, Section of the Inner Epidermis of the Capsule Wall; F) Pollen, Grains., , Pieces of upper and lower epidermis of the foliage leaves of Papaver somniferum, are composed of thin -walled polygonal cells with anomocytic stomata in the, lower epidermis (figure 3.10)., B, C, A, , Figure 3.10: Papaver Somniferum, Foliage Leaf. A) T S. of the, Lamina; B) Upper Epidermis; C) Lower Epidermis., *, , *
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70, , Pharmacognosy and Phytochemistry-II, , Small traces of rounded starch grains (4-8 in diameter) may be present in the, capsule wall. The rarely occurring pollen grai ns are spherical, smooth, with 3, pores and about 20 -32 in diameter ( figure 3.9 F). Fragments of the lignified, inner epidermis of the capsule wall ( figure 3.9 C) are absent; the cells measure, about 70-160 in surface view and are about 40 -50 high; their a nticlinal walls, are pitted and there are a few pits on the inner walls., Chemical Constituents, The alkaloids present in the latex are derived from amino acids, phenylalanine,, and tyrosine. Different alkaloids derived from the opium are grouped under two, categories:, 1) Benzylisoquinoline: Narcotine (noscapine), narceine, and papaverine., 2) Phenanthrene: Morphine, codeine (methyl morphine), and thebaine., Narcotine is a weak, monoacidic base and optically laevorotatory, while its salts, are dextrorotatory. Narcoti ne is insoluble in water and in some polar organic, solvents (alcohol and ether). It is soluble in acetone, benzene, and chloroform., Papaverine is an optically inactive and weak monoacidic base. It is insoluble in, water and slightly soluble in organic solvents., Morphine is a laevorotatory, phenolic and monoacidic alkaloid. At C -6 position,, an alcoholic hydroxyl group is present. Morphine is soluble in alkali hydroxides, (except ammonium hydroxide) due to the presence of phenolic hydroxyl group., Heroin is a diacetyl derivative of morphine., Codeine is a laevorotatory, monoacidic base which is soluble in water and, organic solvents (benzene, ether, etc.)., OCH3, , CH3, │, CH2, N, , CH3, │, CH2, N, , N, OCH3, CH2, , H, , H, , CH2, , CH2, OCH3, │, OCH3, Papaverine, , OH, , O, Morphine, , OH, , OCH3, , O, Codeine, , OH, , The opium alkaloids are present as salts of meconic acid. Few alkaloids are, present in minute concentration, e.g., protopine and hydrocotarnine., , *, , *
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Secondary Metabolites-I (Chapter 3), , 71, , Opium also contains sugar, wax, mucilage , and salts of calcium, potassium and, magnesium. Opium does not contain tannins, starch, and calcium oxalate., Minute, off -white coloured seeds are pre sent in large number within the poppy, fruits. These seeds have 30-35% fixed oil., Chemical Tests, For identification of alkaloids of opium different chemical tests are performed:, 1) In the general test, opium is identified by testing the presence of meconic, acid because opium alkaloids are present as a salt of meconic acid. The test is, performed by dissolving opium in water and adding ferric chloride solution, to the filtrate. This results in a deep reddish purple colour which remains, even on addition of hydrochloric acid., 2) Orange red colour is obtained on sprinkling morphine on nitric acid. It is, noted that codeine does not respond to this test., 3) On treating morphine solution with potassium ferricyanide and ferric, chloride solutions, bluish -green colour is obta ined. Codeine is negative for, this test also., 4) On reacting hydrochloric acid solution of papaverine with ferricyanide, solution, lemon yellow colour is obtained., Uses, 1) Hypnotic, Sedative, and Analgesic Properties: These properties of opium, are due to morph ine. Due to biphasic action on CNS, it shows sedative, effects on cerebrum and medulla. It sedates emetic centre, respiratory centre,, and cough reflex. Morphine has central narcotic effects and causes addiction., Therefore, it is used only in case of severe pain, or when other analgesics are, ineffective., 2) Stimulation of Chemoreceptor Zone: It stimulates the chemoreceptor zone, in medulla which causes nausea and vomiting (a side effect)., 3) Antitussive Properties: Codeine relieves local irritation in the bronchial, tract, thus, used in many cough medicines as an antitussive agent. Narcotine, has a specific depressant action on cough reflex, therefore,, is used in the, formulation of cough syrups., 4) Muscle Relaxant Properties: Papaverine has relaxant effects on intestinal, and bronchial tract smooth muscles, and also on blood vessels., Substitutes and Adulterants, Species used as substitute of poppy are Papaver argemone , P. dubium , P., orientale, P. bracteatum, P. strigosum, P. intermedia, P. paeoniflorum, hybrid of, P. somni ferum and P. oriental e, P. pseu do orientale, and plants from genera, Argemone and Eschscholzia (both belonging to family Papaveraceae)., At the present time no adulterants are found in opium; but previously many, substances like flour, lead shot, powdered po ppy capsule, gum, roasted bread, crumb, pounded dates, etc. were used for adulteration., *, , *
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72, , Pharmacognosy and Phytochemistry-II, , 3.3. SUMMARY, The details given in the chapter can be summarised as follows:, 1) The chemical products of plants having no role in the growth,, photosynthesis, reproduction, or other ―primary‖ functions are known as, secondary metabolites., 2) Ethnopharmacolgy is the study of of plants used for recreation and, stimulation., 3) Alkaloids are chemical compounds that are basic in nature., 4) A pharmacist, W. Meissner, gave the name alkaloids to nitrogen-containing, basic compounds, obtained from plants., 5) Ladenburg stated alkaloids as, ‘compounds originated from plants with a, basic nature, having one or more nitrogen atom in its ring structure’., 6) Along with carbon, hydrogen, and oxygen, the most esse ntial component of, alkaloids is the N-atom., 7) The N-atom in morphine and reserpine is present as tertiary-amine (R3N), in, ephedrine as secondary-amine (R2NH), and in nor -pseudo-ephedrine as, primary-amine (RNH2)., 8) Alkaloids containing O -atom are basically solid alkaloids , except certain, oxygenated alkaloids which are non-volatile liquids, e.g., pilocarpine., 9) Amphoteric Alkaloids have a phenolic ( –OH) group (in morphine) or, carboxylic (–COOH) group (in narceine), thus, behave neither as acid nor as, base., 10) Unstable Alkaloid Salts e.g., piperine, papaverine, narceine, narcotine, and, caffeine show weak basic properties and their salts are less stable., 11) The phenomenon dealing with change of one structure into another having, the same empirical formula but different properties is known as isomerism., 12) Optical activity of alkaloids is due to the presence of one or more, asymmetric carbon atoms in the molecule., 13) In Stas-Otto process the moistened drug is treated with alkali to set free the, base existing in salt form and then the free base is separated with an organic, solvent., 14) Steam Distillation method is specially employed for volatile liquid alkaloids, like conine, nicotine, and spateine., 15) Proto alkaloids are also termed as biological amines., 16) Vinca is the dried whole plant of Catharanthus roseus or Vinca rosea., 17) Rauwolfia is the dried roots of the plant Rauwolfia serpentina Benth., 18) Belladonna is the dried leaves and flowering tops of the plant, Atropa, belladonna. It should contain 0.30% or more of total alk aloids calculated as, hyoscyamine., 19) Opium is the dried latex extracted from the unripe capsules of the plant, opium poppy or Papaver somniferum Linn., *, , *
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Secondary Metabolites-I (Chapter 3), , 73, , 3.4. EXERCISE, 3.4.1., 1), 2), 3), 4), 5), , True or False, , Alkaloids are basic in nature., Vinca is the dried whole plant of Catharanthus roseus., Steam Distillation method is specially employed for solid alkaloids., Proto alkaloids are also termed as biological amines., Papaverine is an optically active and strong monoacidic base., , 3.4.2., , Fill in the Blanks, , 6) In ____________ the moistened drug is treate, d with alkali to set free the base, existing in salt form., 7) ___________ of alkaloids is due to the presence of one or more asymmetric carbon, atoms in the molecule., 8) Rauwolfia serpentina is the source of ____________., 9) Proto alkaloids are also termed as ___________., 10) ___________ have a phenolic (–OH) group or carboxylic (–COOH) group., Answers, 1) True, 4) True, 7) Optical activity, 10) Amphoteric alkaloids, , 3.4.3., , 2) True, 5) False, 8) Rauwolfia, , 3) False, 6) Stas-Otto process, 9) Biological amines, , Very Short Answer Type Questions, , 1), 2), 3), 4), 5), , Define alkaloids., Discuss secondary metabolites., What are amphoteric alkaloids?, Write a short note on pseudoalkaloids., Give the biological source and family of vinca., 6) Give the macroscopic features of opium., , 3.4.4., , Short Answer Type Questions, , 1) Classify secondary metabolites., 2) Give the salient features of alkaloids., 3) Discuss the therapeutic uses of alkaloids., 4) Give the commercial applications of alkaloids., , 3.4.5., , Long Answer Type Questions, , 1) Give the pharmacognosy of rauwolfia., 2) Discuss the qualitative and quantitative analysis of alkaloids., 3) Give a detailed account on the chemistry of alkaloids., , *, , *
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Secondary Metabolites - II (Chapter 4), , 4.1.5., , 77, , Flavonoids, , Flavonoids are a diverse group of phytonutrients (plant chemicals), found in, almost all fruits and vegetables. Along with carotenoids, they are responsible for, the vivid colours in fruits and vegetables., Flavonoids are chemically, polyphenolic compounds. They contain 15 carbon atoms. Two benzene rings, joined by a linear th ree carbon chain. The chemical structure of flavonoids is, based on a C-15 skeleton with a chromane ring bearing a second aromatic ring at, position 2, 3, or 4., Flavonoids are defined as „ a large group of biologically active water-soluble, plant compounds (such as anthocyanins and flavones) that include pigments, ranging in colour from yellow to red to blue, and occurring especially in, fruits, vegetables, and herbs (such as grapes, citrus fruits, peppers, and dill)‟., , 4.1.5.1., , Chemistry, , Flavonoids have a complex chemistry. They can occur in plants with or without, sugar moieties. Their biochemical activities and met abolites are dependent on, their chemical structure and relative orientation of several moieties on the, molecules., Structure of Flavonoids, Flavonoids are low molecular weight polyphenolic substances based on flavan, nucleus. The generic structure of flavon oid is shown in figure 4.2. The structure, is numbered to differentiate the positions of carbon around the molecule. Three, phenolic rings are indicated as A, B, and C (or pyrane) rings . The aglycone of, flavonoid contains a benzene ring (A) condensed with a six membered ring (C), that has a phenyl ring (B) substituent at 2 -position. The s ix-membered ring, condensed with the benzene ring is pyrone or its dihydro derivative., The flavonoid class is divided based on the position of benzenoid substituent into, flavonoids (2 -position) and iso -flavonoids (3 -position). Fl avonols are different, from flavanones because of the presence of hydroxyl group at 3-position and C2C3 double bonds. At position 3, 5, 7, 2′, 3′, 4′, and 5′, the flavonoids get, hydroxylated. The methyl ethers and acetyl esters of alcohol group occur, naturally., After the glycosides of flavonoids are formed, the glycosidic linkage is located in, 3 or 7 position and the carbohydrate can be L, -rhamnose, D -glucose,, glucorhamnose, galactose, or arabinose. The C 6-C3-C6 system of flavonoids is, represented in the skeleton shown in figure 4.2., , Figure 4.2: Basic Skeleton of a Flavonoid, *, , *
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78, , 4.1.5.2., , Pharmacognosy and Phytochemistry-II, , Biosynthesis, , For understanding the diversity of flavonoids , its biosynthesis is very important., The flavonoid molecules are biosynthesised by three molecules of acetic acid and, phenyl propane moiety (precursor of flavonoids)., , Figure 4.3: Biosynthesis of Flavonoid Molecule, , The biosynthesis process of flavonoids involves the interaction of five different, pathways, i.e., glycolytic pathway, pentose phosphate pathway and shikimate, pathway synthesising phenylalanine; phenylpropanoid metabolism that, produces activated cinnamic acid derivati ve (4 -coumaroyl-CoA) and lignin, (structural plant component); and the diverse specific flavonoid pathways., Flavonoids are biosynthesised through condensation of the shikimic acid and, acyipolymalonate pathways. In polyketide synthesis, phenyl propane (a ci nnamic, acid derivative synthesised from shikimic acid) serves as the starting compound., In this synthesis, additional three acetate residues are fused in the structure,, followed by the ring closure., By successive hydroxylation and reductions, plants can, biosynthesise several, classes of flavonoids, like flavones, flavanols (or catechins), anthocyanidins, iso flavones, dihydroflavonols, and chalcones., , 4.1.5.3., , Chemical Classes, , The structures of some flavonoids are discussed as follows (figure 4.4):, 1) Flavones: A benzo-gamma pyrone ring having a phenyl substitution at position, 2 in the ring is present in flavones. A double bond between the 2 nd and 3 rd, positions and a ketone in the 4th position of C ring are present. Vegetables and, fruits having flavones carry a hydroxyl group in 5th position of the A ring. The, taxonomical classification of a vegetable or fruit influences hydroxylation in, other positions, like mostly in position 7 of the A ring or 3′ and 4′ of the B ring., 2) Isoflavone: A benzo -gamma-pyrone ring having a phenyl substitution at, position 3 in the ring is present in isoflavones., *, , *
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82, , 4.1.6.1., , Pharmacognosy and Phytochemistry-II, , Biosources, , Lignans are widely occurring plant compounds and are obtained from roots, heart, wood, foliage, fruits, and resinous exudates of plants. They represent the d imer, stage intermediate between the monomeric propylphenol units and lignin . The, natural occurre nce of tri mers and tetramers has not been reported so far., However, occurrence of lignans in man and animals has been reported. In the, roots and rhizomes of Podophyllum hexandrum Royle (Berberidaceae), -lignan, has been found., , 4.1.6.2., , Preparation, , Lignans are formed by the reduction of ferulic acid to coniferyl alcohol; and then, through oxidative dimerisation of the coniferyl alcohol units and establishment of, linkage via -carbon atom of the C3 side chain., , 4.1.6.3., , Characteristic Features, , Lignans are found as single enantiomeric forms, i.e., as d - or l-isomers. Though,, they als o occur as racemic produc ts, i.e., dl -forms. It has been found that with, respect to their oxidation levels, degree of substitution, and structural complexity,, lignans differ to a large extent., , 4.1.7., , Tea, , Tea is an evergreen shrub having several alternate branches. The leaves are, pointed, lanceolate, or elliptically-oblong having short petiolate. The leaves have, smooth surface on both sides with a shiny green appearance, a prominent midrib,, and pinnately veined on one side., Synonym, Folia thea., Biological Source, Tea is the leaf buds and leaves of the plant, Thea sinensis., Family, Theaceae., , Figure 4.6: Twig of Tea-Herb, , Geographical Source, It is often found in the Sichuan provinces of China and northern part of Burma,, and Yunnan., Macroscopic Features, 1) In the young stage, lea ves are covered with silky hair, which disappear as, they get mature. The surface becomes glabrous (almost) after the silky hair, shed completely., 2) The length of a fully matured or comple tely grown tea leaf is about 5 -10cm., The upper surface of the leaf is glossy and dark green coloured. The outer, lining is elliptical or lanceolate; the apex is acuminate or blunt; and the bases, *, , *
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Secondary Metabolites - II (Chapter 4), , 83, , are tapering having a short stalk. The margins remain indistin ctly short and, serrate with the serrations ending up as characteristically glandular teeth. These, serrations break off readily and are generally absent in the fully grown leaves., The commercial tea leaf is readily distinguished from other tea leaves as the bud, (“flowery” Pekoe) still consists of several hair, whereas the larger and mature, leaves become almost glabrous and smooth., Microscopic Features, The transverse section of the prominent midrib with a broad ridge above shows a, discontinuous palisa de above the meristele with an arc of xylem., Phloem is, present just below the xylem and this entire arrangement is covered by a band of, pericyclic fibres. These fibres are slightly lignified, present in a number of 4, and, are broad at the widest part. Roun ded parenchyma and numerous branching,, lignified sclereids form the ground tissue., Collenchyma, Phloem, Xylem, Astro, sclereids, Dendritic, sclereids, , Figure 4.7: T.S of Midrib of C. Sinensis., , The epidermis is made up of polygonal cells having slightly wavy anticlinal walls., The lower surface of the leaves have numerous stomata. Each of these stomata i s, surrounded by 3 narrow and tangentially elongated cells. The lower surface of young, leaves bears a large number of unicellular, thick -walled, conical-linear trichomes., Palisade is present in 2 rows in the mesophyll. Large lignified sclereids (idioblasts), are also found to be present across the mesophyll from one epidermis to the other., Cluster of calcium oxalate crystals are scattered throughout the parenchyma and, also in the phloem of meristele. Each of the charac teristic marginal teeth ends in, a smal l conical point (glandular in nature) and consist s of an external palisade, layer. This layer covers a small mass of polyhedral parenchyma. These conical, points fall off in older leaves, leaving behind a brown scar., Chemical Constituents, The principal con stituents are caffeine (trimethyl-xanthine), CH3, O, and tannin (10-24%). Adenine, theophylline, theobromine, N, (dimethyl-xanthine) and its isomer, xanthine , and volatile oil HN, are present in trace amounts. Percentage content of caffeine, N, present in tea is 1 -5%, of which 9/10th part appears in O, N, combination with tannin. Later, water decomposes the, CH3, combination complex, resulting in the liberation of caffeine., Theobromine, However, the percentage of free caffeine increases during the, fermentation process., *, , *
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84, , Pharmacognosy and Phytochemistry-II, , Chemical Test, Murexide Test: This test gives a positive result for tea as it is a purine alkaloid., Drug extract is taken in a petridish and potassium chlorate is added along with, hydrochloric acid. The mixture is heated to dryness. The residue is exposed to, dilute ammonia vapours result ing in a purple colour that disappears on addition, of a fixed alkali., Uses, 1) The prime pharmaceutical use of tea is as a caffeine source which produces, stimulatory effect on the heart and nervous system. Caffeine also possesses, diuretic activity but comparatively to a lesser extent than theobromine., 2) It is possibly effective in inhibiting angiogenesis which involves the process, of supporting the growth of blood vessels required for the growth of tumour, as well as metastasis., 3) Caffeine from tea leaves infusi, on when mixed with polyphenols, (epigallocatechin-3-gallate) shows strong antioxidant and free, -radical, scavenging properties., 4) By inhibiting iron absorption by tannates and other ligands, caffeine helps in, treating genetic haemochromatosis., 5) It also helps in treating diabetes -induced blindness, which is an angiogenic, related condition., 6) It also plays a vital role in lowering the risk of, ischemic heart disease in, geriatrics., Adulterants, Foreign leaves and exhausted tea leaves are rolled and dried to be used, adulterants of tea., , 4.1.8., , as, , Ruta, , Originally native to the Mediterranean region , Ruta is an herbaceous perennial, which is now cultivated in many parts of the world., Synonym, Rue., Biological Source, Ruta is obtained from the fresh and dried leaves of the plant Ruta graveolens L., Family, Rutaceae., Geographical Source, It is originally from Mediterranean area and is distributed all around the world., The g enus Ruta has 14 approved species , of which R. graveolens L. and R., chalepensis L. are stated as I ndian floras. Several countries including India ,, cultivate Ruta graveolens as a medicinal and ornamental herb., *, , *
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Secondary Metabolites - II (Chapter 4), , 85, , Macroscopic Features, Ruta graveolens L. is a perennial, scented and glabrous herb or a sub -shrub. The, aerial parts are powdered, aromatic,and pleasant. The colour of the bulk sample is, mild to greyish green. The major part, s of the bulk sampleare stem piecesand leaves:, 1) Stem is slim, smooth, pale green , and goes upto 1m in height. Stem pieces, are 15cm long and 5 mm thick. They are mild green externally and white, internally. They are longitudinally contracted or flattened, hollow or have, white luminous pith. They have a smooth surface, sharp in cut ends, woody, and fibrous fracture., 2) Leaves are of alternate pattern, glaucous, compound, and 2-3 pinnate. They, are a bit fragile and nearly powdery in the bulk sample., They are green to, mild green to greyish green in colour, thin, papery, and have minute dots of, dark green coloured glands everywhere., 3) Leaflets are linear, oval, or oblong. While handling they may get detached or, fragmented., 4) Inflorescence is terminal corymbose with irregularly dichotomous cymes., 5) Flowers are bisexual and dull or dark yellow coloured. The terminal flowers, are pentamerous and others are tetramerous., 6) Petals are wide ly spreading, distinct, undulate, greenish yellow in colour ,, wide and hooded at top, and connected to narrow claw below. They have a, wavy and sometimes toothed margin., 7) Fruits are dry, hard and round. They are 4 or 5 lobed and have blunted tips., Ruta chalepensis L. is somewhat similar in its morphology. It is differentiated, from Ruta graveolens L. by its strong fetid smell, ciliate or fringed petals , and, sharp fruit lobe tops., Microscopic Features, The microscopic description is applied to both the species until unless specified:, 1) The TS of stem appears almost pentagonal in outline with dull corners., 2) It has a single layered outer epidermis followed by hypodermis (single, layered), cortex, vascular zone, and central pith., 3) The cortex is differentiated into few layers of outer chlorenchyma and inner, parenchyma., 4) The chlorenchyma is loosely arranged as aerenchyma with numerous air, spaces., 5) The parenchyma is normal and has intercellular spaces., 6) The pericycle shows patches of lignified fibres., 7) In R. graveolens, the lumen of these fibres is wide and clearly visible; but in, R. chalepensis, the lumen is narrow and represented as dot., 8) Usual elements are shown by the xylem and phloem., 9) The pith is large, parenchymatous, and undifferentiated., 10) The parenchyma cells of cortex and the chlorench yma region have minute, starch grains., *, , *
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86, , Pharmacognosy and Phytochemistry-II, , 11) No starch grains are present in the pith cells., 12) The parenchyma cells of cortex, and pith in R. graveolens have limited, calcium oxalate crystals; while R. chalepensis has abundance of calcium, oxalate crystals., Chemical Constituents, The commonly known phytochemical compounds in R. graveolens are acridone, alkaloids, coumarins, volatile substances , terpenoids, flavonoids, and, fluoroquinolones. Saponin, tannins, and glycosides are also found to be present ., In R. graveolens, the main active flavonoids are rutin and quercetin. Rutin was, first isolated from the leaves of R. graveolens . Aliphatic acids, alcohols, and, ketones are also found in high content in R. graveolens volatile oil., The major components found in the essenti al oil of the flowering aerial parts of, the plants are 2-undecanone (33.9%), 2-heptanol acetate (17.5%), 1-dodecanol, (11.0%), geyrene ( 10.4%), 2-nonanone (8.8%), 2-decanone (1.9%), g eijerene, (1.6%), trans-piperitenone oxide (1.4%), cis-piperitenone oxide (1.2%), 2-methylundecanal (1.1%), 2-dodecanone (1.1%), 2-nonanol (1.1%), and elemol (1.1%)., R. graveolens produces high levels of linear furanocoumarins, mostl y psoralen, and methoxypsoralen. Rutacridone, rutacridone epoxide , and gravacri dondiol, (acridone alkaloids ) are obtained from the roots of, R. graveolens , and, graveoline (an alkaloid) is obtained from its leaves., Uses, 1) It is used as an emmenagogue, haemostat, intestinal antispasmodic, sedative,, uterine stimulant, vermifuge, rheum atism, and in cold and fever in Chinese, medicine., 2) It is used as an aphrodisiac and choleretic in Poland., 3) It is used as a bitter, an aromatic stimulant, ecbolic,and for suppressing menses., , 4.2. STEROIDS, 4.2.1., , Introduction, , Plant steroids are a diver se group of natural products. They are b iosynthetically, derived from S -squalene-2,3-epoxide through acetate mevalonate pathway., Phytosterols are universal steroids in the plant kingdom, and have been reported, to show hypocholesterolemic activity., Withanolides are large steroidal lactones group having many biological, activities. A small group of plant steroids, i.e., brassinosteroids, exhibit plant, growth hormonal activity. Phytoecdysteroids (polyhydroxylated plant steroids ), show anabolic effects with no un wanted side effects. Steroidal alkaloids are, nitrogen-containing plant steroids possessing various biological activities., In, some plants, minute amounts of cholesterol and mammalian steroidal hormones, including progesterone have been detected., *, , *
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Secondary Metabolites - II (Chapter 4), , 87, , Plant ster oid analysis is done by chromatographic methods like thin layer, chromatography (TLC), high performance liquid chromatography (HPLC), ultra, high performance liquid chromatography (UHPLC),and gas liquid chromatography, (GLC); while photodiode array (PDA), ev aporative light scattering (ELS), an, fluorescence (FL) detectors, and mass spectrometry (MS) areused for the detection,, quantification, and identification of plant steroids . Immunoassays and biological, assays are a few less common techniques used in the nalysis, a, of plant steroids., , 4.2.2., , Chemical Classes, , Steroids are chemically classified into the following:, 1) Anabolic Steroids: They interact with androgen recepto r, increase muscle, mass/athlete‟s performance and male sex hormones., 2) Glucocorticoids: They r egulate metabolism and immune function, and, exhibit anti-inflammatory activity., 3) Mineralocorticoids: They maintain blood volume and renal excretion., 4) Progestins: They are involved in the d evelopment of female sex organs and, characteristics., 5) Phytosteroids: They are plant steroids., 6) Ergosteroids: They are the steroids of fungi, and are vitamin D related., , 4.2.3., , Biosources, , Steroidal alkaloids found in the plants of Solanaceae and Melanthiaceae family, (specially the genus Veratrum), cardiac glycosides, phytosterols, , and brassinosteroids, (which include many plant hormones) are the sources of plant steroids., , 4.2.4., , Therapeutic Uses, , Steroids come in several different forms, and the major kinds are:, 1) Tablets, syrups, and liquids, e.g., prednisolone,, 2) Inhalers and nasal sprays, e.g., beclomethasone and fluticasone, and, 3) Creams, lotions, and gels, e.g., hydrocortisone., Steroids are used in treating the following conditions:, 1) Asthma and Chronic Obstructive Pulmonary Disease (COPD),, 2) Hay fever,, 3) Hives and eczema,, 4) Painful joints or muscles, such as arthritis, tennis elbow, and frozen shoulder,, 5) Pain caused by an irritated or trapped nerve, such as sciatica,, 6) Inflammatory bowel disease, such as Crohn‟s disease, and, 7) Multiple Sclerosis (MS)., , 4.2.5., , Commercial Applications, , The converted form of diosgenin, i.e., 16-dehydropregnenolon acetate is an active, ingredient in many steroidal drug, preparations like sex hormones a nd oral, contraceptive pills. For washing silk, wool and hair , saponins of Dioscorea are, used. They are also used as fish poison, and for killing, lice. Solasodine and, hecogenin are used in the production of sex hormones and oral contraceptive pills, ., *, , *
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88, , Pharmacognosy and Phytochemistry-II, , 4.3. CARDIAC GLYCOSIDES, 4.3.1., , Introduction, , The cardiac glycosides are an important class of naturally occurring drugs whose, actions include both beneficial and toxic effects on the heart. Cardiac steroids are, widely used in the modern treatment focongestive heart failure and for the treatment, of atrial fibrillation and flutter. Yet their toxicity remains a serious problem. These, drugs all act by affecting the availability of intracellular Ca +2 ions for myocardial, contraction or increasing the sen, sitivity of myocardial contractile proteins., , 4.3.2., , Chemistry, , Cardiac glycosides are composed of two portions:, 1) Sugars: These are monosaccharide or a polysaccharide with -1,4-glycosidic, linkages. The most common sugars foun d in cardiac glycosides are D glucose, D-digitoxose, L-rhamnose, and D-cymarose., 2), , Non-Sugar (Aglycone) Moiety:, The aglycone portion of the cardiac, glycosides is a steroid nucleus and is usually termed as genin. The aglycone, component is responsib le for phar macological activity, the while sugar, portion is responsible for its absorption. There are two types of aglycones:, i) Cardenolides: These are derived from plant origin and is characterised, by the presence of ,-unsaturated five-membered lactone ring at C -17, position of aglycone like digoxin, digitoxin, etc. It is in -configuration., The presence of this ring is responsible for biological activity., ii) Bufadienolides: These are derived from animal origin and characteris ed, by the presence of six-membered lactone ring with two conjugated double, bonds (generally referredto as -pyrone) at C-17 position like bufotalin., , 4.3.2.1., , Biosynthesis, , The aglycones of cardiac glycosides ar e derived from mevalonic acid, but, condensation of C 21 steroid with a C 2 unit (the source of C -22 and C -23) gives, the final molecules. The condensation products of a C 21 steroid and a C 3 unit are, bufadienolides. Figure 4.8 shows the biosynthesis of cardiac glycosides., *, , *
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Secondary Metabolites - II (Chapter 4), , 89, , Figure 4.8: Biosynthesis of Cardiac Glycosides, , Progesterone, formed with cardiac glycosides, in Digitalis lanata as a res ult of, nourishing pregnenolone acts as a precursor of cardiac glycosides. Work on the, intermediates between progesterone and cardenolides invol ving Strophanthus, kombe, affords reliable indications with the pathway:, Progesterone 5--Pregnenolone 5--Hydroxypregnenolone Cardenolides, In Digitalis purpurea cultures, the same t ransformation has been sho wn but, different pathways exist depending on whether the nucleus hydroxylation takes, place before or after the vital acetate condensation for the, formation of, butenolide ring. A research work involving nine enzymes has shown that, complex interlinking multi -dimensional system of pathways (and not a single, route leading straight to the final product) forms the cardenolide genins., Biogenetic studies involving the side chain shows that the most effective, precursor of digitoxose and sugar side chain of Nerium oleander glycosides is, glucose. In digital is species, around ten enzymes are involved in the sugar side, chain biosynthesis., , 4.3.2.2., , Qualitative Analysis, , The following tests are helpful in the qualitative analysis of cardiac glycosides:, 1) Keller-Kiliani Test: To the alcoholic extract of drug , equal volume of water, and 0.5ml of strong lead acetate solution is added, shaken and filtered. The, filtrate is extracted with equal volume of chloroform. The c hloroform extract, is evaporated to dryness and residue is dissolved in 3ml of glacial acetic acid, *, , *
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90, , Pharmacognosy and Phytochemistry-II, , followed by addition of a few drops of FeCl 3 solution. The resultant solution, is transferred to a test tube contain ing 2ml of conc. H 2SO4. Reddish brown, layer is formed, which turns bluish g reen on standing due to the presence of, digitoxose., 2) Legal Test : To the alcoh olic extract of drug, equal volume of water and, 0.5ml of strong lead acetate solution, is added, shaken, and filtered. The, filtrate is extracted with equal volume of chloroform and the chloroform, extract is evaporated to dryness. The residue is dissolved in 2ml of pyridine, and sodium nitroprus side 2ml is added followed by addition of NaOH, solution to make alkaline. Formation of pink colour results in the presence of, glycosides or aglycone moiety., 3) Baljet Test: Thick section of leaf of digitalis or the part of dru g containing, cardiac glycoside is dipped in sodium picrate solution. It forms yellow to, orange colour due to the presence of aglycones or glycosides., 4) 3,5-Dinitro Benzoic Acid Test: To the alcoholic solution of drug , a few, drops of NaOH followed by 2% solution of 3,5-dinitro benzoic acid is added., A pink colour appears due to the presence of cardiac glycosides., 5) Raymond’s Test: A few ml of 50% ethanol and 0.1ml of 1% m-dinitrobenzene, solution in ethanol are added to the drug sample. To the resultant mixture, 2 3 drops of 20% sodium hydroxide solution is added. A violet colour develops, in the solution indicating the presence of active methylene group., 6) Xanthydrol Test: The crude drug extract is heated with 0.1, -5% of, xanthydrol solution in glacial acetic acid containing 1% hydrochloric acid., Red coloured solution results indicating the presence of 2-deoxysugar., , 4.3.2.3., , Quantitative Analysis, , HPLC technique is used for the qualitative analysis of cardiac glycoside s. In the, HPLC grade methanol, stock solutions of the isolated compounds were preparedat, 100μg/ml concentration and stored in refrigerator till u se. All the samples were, kept at 4 oC, and filtered using a 0.45μm filter before undergoing HPLC analysis., Using a 10μl loop, a 20μl portion of these working standards was added in the, HPLC system at ambient temperature and a 273nm detector wavelengthis selected, for glycosidic compound detection. As stated in the monograph , the extracted, compounds were first identified by direct comparison oftheir retention times., , 4.3.2.4., , Isolation and Extraction, , Cardiac glycosides are isolated and extracted as follows:, 1) Preparation of the Glycoside Crude Extract:, 500gm of lily flowers, contaminated by flower stem impurities are powdered in a mixer and, macerated while stirring with 51 -50% of aqueous methanol for 72 hours at, room temperature. Then, by suction the mixture is filtered and the residue is, washed with 1 lt. of aqueous methanol. The obtained dark brown coloured, drug extract is concentrated to approx. 1 lt. under reduced pressure in the, rotatory evaporator at 35 -40°C ba th temperat ure. Then it is mixed with a, solution of 40gm lead acetate in 220 ml water and centrifuged., *, , *
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Secondary Metabolites - II (Chapter 4), , 91, , From the supernatant solution , lead is removed by adding 10% aqueous, secondary sodium phosphate solution and filtering the mixture. By adding, 20% aqueo us sodium carbonate solution the pH is adjusted to 6.6 , and, then the mixture is concentrated to 800ml by evaporating under reduced, pressure. Thereafter, the concentrate is extracted, with a mixture of, chloroform/methanol in 93:7 ratio at 35 -40°C/140 torr fo r 25 hours. After, every 8 hours, the extracting solvent is replaced., By evaporation in vacuum, the combined organic phases are concentrated to, 5ml and then added with 100 ml of petroleum ether . After standing for 2, hours at 5°C, the supernatant solution containing no Kedde-positive material, is decanted. The remaining dark -brown slurry contains convallatoxin that, cannot form crystals from menthol/water., 2) Preparation of Convallatoxin, -Acetonide from the Glycoside Crude Extract:, The crude glycoside is evaporated to dryness in vacuum and the obtained, dark-brown foam is suspended in 50ml of anhydrous acetone. The mixture, is added with 2,2-dimethoxypropane (25ml) and p-toluene-sulfonic-acidmonohydrate (500mg). After leaving the solu tion undisturbed for 10, minutes at room temperature, it is, neutralised with 5 % aqueous sodium, bicarbonate solution and extracted with chloroform thrice. The combined, extracts are dried with sodium sulphate, filtered, and evaporated to dryness., 348mg of convallatoxin-acetonide m.p. crystallises out(methanol/water) at 0°C., The mother liquor residue is chromatographed on 160 gm of neutral, aluminium oxide Woelm (activity grade II)., For separating the apolar, material, the column is pre-eluted with 600 ml of chl oroform and then with, 300ml of chloroform/methanol in the ration of 99:1 . On elution with, chloroform/methanol (98:2), 770 mg of a TLC -homogeneous fraction is, obtained, from which 246mg of pure convallatoxin-acetonide crystallises out, (methanol/water)., 3) Convallatoxin from Convallatoxin -Acetonide: 250mg of convallatoxin acetonide (obtained above) is dissolved in 20ml of alcohol. The mixture is, then added with 10 ml of 1 % aqueous sulphuric acid, and heated under, nitrogen to 55°C for 2 hours. The mixture is neutralised with 5 % aqueous, sodium bicarbonate solution and thrice extracted with chloroform/ethanol, (8:2). The combined extracts are dried and evaporated in vacuum to yield, 273mg of colourless foam, from which 203mg of pure convallatoxin, crystallises out (methanol/water)., , 4.3.3., , Chemical Classes, , Two classes of cardiac glycosides have been observed in nature , i.e., the, cardenolides and the bufadienolides. The cardenolides have an unsaturated, butyrolactone ring, while the bufadienolides have a pyrone ring. The lactone of, cardenolides has a single double bond and is attached at the C, -17 position of, steroidal nucleus. They are five -membered lactone ring and form C 23 steroids, (Leguminosae, Cruciferae, Eup horbiaceae, etc.), while the lactone of, *, , *
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Secondary Metabolites - II (Chapter 4), , 4.3.5., , 93, , Therapeutic Uses, , Cardiac glycosides have the following therapeutic uses:, 1) It is used in the treatment of congestive heart failure., 2) It is used in the treatment of atrial fibrillation and flutter., 3) It is used in increasing, the contraction force of heart muscles without, increasing the consumption of oxygen., 4) It is used in elevating the cardiac output., 5) It is used as diuretics., , 4.3.6., , Individual Drugs, , The pharmacognostic profile of the following drugs has been discussed below:, 1) Liquorice,, 2) Dioscorea, and, 3) Digitalis., , 4.3.6.1., , Liquorice, , Liquorice is a perennial herb cultivated in the Mediterranean region and Central, and South -West Asia for its sweet taproot (thus, also termed sweet root ). The, plant gives purple with white coloured flowers and its roots grow to a depth of 4, feet (1.2m)., Synonyms, Radix glycyrrhizae and Sweet liquorice., Biological Source, Liquorice is subterranean dried peeled or unpeeled roots and stolon of different, varieties of Glycyrrhiza glabra., Family, Leguminosae., Geographical Source, On a large scale, it is cultivated in Spain, Sicily and Yorkshire (England). Other, species of liquorice like G. glabra var violaceae is found in Iran, while G. glabra, var glandulifera is cultivated in Russia and is named the Russian Liquorice. In, Asian continent, it is cultivated in India (sub-Himalayan tracts) and Baluchistan., Macroscopic Features, 1) Size: Several feet in length, varies in thickness from ¼ to about 1 inch, and, longitudinally wrinkled., 2) Shape: Liquorice root is long, straight, nearly cylindrical,, and occurs as, unpeeled pieces., 3) Colour: Externally greyish brown to dark brown., 4) Taste: Sweet and very slightly acrid., 5) Odour: Weak and somewhat aromatic., 6) Fruit: Legume., *, , *
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94, , Pharmacognosy and Phytochemistry-II, , Liquorice Root, , Microscopic Features, When examined under microscope,, characteristics (figure 4.9):, , Glycyrrhiza Glabra Twig, , stolon of liquorice shows the following, Cork, Phelloderm, Phloem, parenchyma, , Phloem fibres, Vessels, Cambium, , Xylem fibres, , Medullary, rays, Xylem, parenchyma, , Pith, Figure 4.9: Glycyrrhiza Glabra (T.S. of Stolon), , 1) Outer Surface: The outer surface of the unpeeled drug consists of around 10, rows of narrow cork cells., 2) Cork Cells: These are rapidly arranged and thin-walled, polygonal in nature,, beneath which there may be a few parenchyma rows forming the cortex., 3) Parenchymatous Pericycle: It has small groups of fibres at intervals., 4) Phloem Fibres: These are t hick-walled, lignified occur in cylindrical, bundles altering with sieve-tissue, which in the outer part is collapsed to form, ceratenchyma. Sieve tubes are clear near the cambium., *, , *
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Secondary Metabolites - II (Chapter 4), , 95, , 5) Xylem: It consists of strongly lignified xylem fibres and vessels wi th little, xylem parenchyma. The xylem -vessel walls are thickly covered with, bordered pits having slit-like openings., 6) Medullary Rays: These are composed of cellulosic parenchyma, having, rectangular and radially elongated cells. Many parenchymatous cells,, occurring in longitudinal rows adjacent to the fibres of phloem and xylem,, are small in size and contains a solitary prism of, calcium oxalate . All the, parenchyma cells contain either calcium oxalate crystals or starch., 7) Pith: It is parenchymatous., The microscopical characters of roots are similar to st olon, except that at the, centre small four primary xylem bundles are present at right angles to each other,, with protoxylem directed outwards. In root, beneath the cork, phelloderm may be, present., Chemical Constituents, The main constituent of liquorice is glycyrrhizin (6-8%) obtainable in the form, of sweet (50 times sweeter than sucrose ) white crystalline powder consisting of, calcium and potassium salt of glycyrrhizic acid . Glycyrrhizic acid on hydrolysis, yields glycyrrhetic or glycyrrhetinic acid., , It also contains two flavonoids, i.e., liquiritin and isoliquiritin. The fla vonoids, are responsible for its anti-gastric effect and are used in peptic ulcer treatment., Chemical Tests, 1) On addition of 80% sulphuric acid to a section or powder of the drug, orange, yellow colour is produced due to the transformation of, liquiritin ( flavone, glycoside) to isoliquiritin (chalcone glycoside)., 2) Powdered drug on reacting with water in the presence of KOH gives a red, colour., Uses, 1) It is used as a sweetening agent and in bronchial problems (flu and coughs)., 2) It is used as an expectorant and demulcent., *, , *
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96, , 3), 4), 5), 6), , Pharmacognosy and Phytochemistry-II, , It is used in peptic ulcer and as an antispasmodic., It is used as a flavouring agent for chewing tobacco and snuff tobacco., Its prolonged use raises the blood pressure and causes water retention., Its root is used externally in the treatment of herpes and eczema., , Substitutes and Adulterants, 1) Manchurian liquorice is obtained from Glycyrrhiza uralensis . It is pale, chocolate brown in colour with exfoliated cork and wavy medullary rays. It, is free from sugar, but contains glycyrrhizin., 2) Russian liquorice may be peeled and obtained from, Glycyrrhiza glabra, variety Glandulifera. The drug is purplish in colour with numerous long, roots, but no stolons., , 4.3.6.2., , Dioscorea, , In the family Dioscoreaceae, around six hundred species of flowering plants are, classified under the genus Dioscorea. The genus is named after the ancient Greek, physician and botanist, Dioscorides., Synonyms, Yam, Kins, and Singli-mingli., Biological Source, Dioscorea is the dried rhizome of several species of, Dioscorea like Dioscorea deltoidea , D. composita,, and other species of Dioscorea., Family, Dioscoreaceae., , Figure 4.10: Dioscorea, Deltoidea Twig, , Geographical Source, The vast majority of the species are tropical, and only a few species extend into, temperate climates. It is cultivated in North America, Japan, China, Mexico,, India, and Nepal. In India, it grows widel y in Western Himalayas, Karnataka,, Jammu and Kashmir, Maharashtra, Tamil Nadu, West Bengal, etc., Macroscopic Features, 1) Colour: Slightly brown., 2) Odour: Odourless., 3) Taste: Bitter., 4) Size: Varies depending upon age of rhizomes., , Rootlets, Dioscorea Rhizome, , Microscopic Features, Epidermis is normally absent in the transverse section of the drug. The, cork, consists of only a few layers, followed by thin walled cortical parenchymatous, tissue. Stele forms the major part of the drug and consists of several close, collateral fibro-vascular bundles. Endodermis and pericycle are indistinguishable., *, , *
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Secondary Metabolites - II (Chapter 4), , 97, , Chemical Constituents, It mainly contains, diosgenin (a steroidal sapogenin ), epismilagenin (its, glycoside), and yamogenin (a β-isomer). It also contains sapogenase, phenolic, compounds. and starch., , Chemical Tests, When diosgenin (a steroidal sapogenin) is treated with acetic anhydride in the, presence of sulphuric acid, it gives a bluish green colour., Uses, It is a majo r source of diosgenin which is used for the manufacture of, progesterone and other steroid al drugs. These are used as contraceptives and in, diseases like asthma and arthritis., Substitutes and Adulterants, Dioscorea flo ribunda is cultivated in Central Americ a and India (Karnataka, State). It contains 3 -5% of diosgenin. D. villosa Linne mainly from Virginia and, Carolina in U.S.A. is also very rich in diosgenin. D. villosa is a twining perennial, herb with beautiful yellow flowers and triangular capsules., Sikkimensis Prain, occurs in Eastern Himalayas, Nepal, Sikkim, Bhutan, Assam, Bihar and Bengal, up to an altitude of 1600 -2000mt. It contains 2 -2.8% of diosgenin. Costus, speciosus is an alternative potential source for diosgenin (1.5%) and can be used, as a substitute for the genuine drug., , 4.3.6.3., , Digitalis, , The genus Digitalis has around twenty species of herbaceous, perennial or, biennial shrubs. The digitalis plant is also known as foxgloves., Synonyms, Digitalis leaves and Foxglove leaves., Biological Source, Digitalis is the dried leaves of Digitalis purpurea, dried at a temperature below, 60°C, immediately after collection. The leaves should not contain more than 5%, of moisture., Family, Scrophulariaceae., *, , *
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98, , Pharmacognosy and Phytochemistry-II, , Geographical Source, It is cultivated in European countries like England, France, Germany, North, America, and India. In India, it is grown in Kashmir and Nilgiri Hills., , Figure 4.11: Herb and Leaf of Digitalis Purpurea, , Macroscopic Features, 1) Colour: Dorsal surface is deep green and greyish, but ventral surface is pale, green and more greyish., 2) Odour: Slight characteristic., 3) Taste: Bitter., 4) Size: Length 10-40cm and width 4-20cm., 5) Shape: Ovate (egg-shaped), lanceolate (like a lance much longer than broad,, widest in the middle, and tapering. Apex is pointed. Margin of the lea, f is, serrated or crenate (broad rounded teeth). Tips are obtuse (rounded with, narrow spaces). Petiole is small and winged., Microscopic Features, Digitalis is a dorsiventral leaf. It has anomocytic stomata on both surfaces and, water pores at the apex of mo, st of the marginal teeth. The, trichomes are, uniseriate, multicellular (3 to 5 cells) and bluntly pointed., There are also, glandular trichomes with unicellular stalk and unicellular or cellular head. The, glandular trichomes are generally located over the veins., Upper epidermis, Palisade, Spongy mesophyll, layer, Lower epidermis, Trichome, , Collenchyma, Xylem, Phloem, Pericycle, Endodermis, Collenchyma, Figure 4.12: T.S. of digitalis Leaf, *, , *
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Secondary Metabolites - II (Chapter 4), , 99, , Collapsed celled covering trichome is an important characteristic of digitalis., Digitalis is free of calcium oxalate crystals and sclerenchyma. Starch grains are, present in the endodermis. There is collenchyma at 3 different places, i.e. , at the, upper epidermis, lower epidermis, and pericyclic part., Chemical Constituents, Digitalis contains 0.2 -0.45% mixtures of both primary and secondary cardiac, glycosides (cardenolides). Purpurea glycosides A and B and glucogitaloxin are, primary glycos ides possessing at C -3 of the aglycone, a linear chain of 3, digitoxose moieties terminated by glucose. Digitalis also contains several other, glycosides such as odoroside H, gitaloxin, verodoxin, and glucoverodoxin., Purpurea Glycoside A, (Fresh leaves), Enzymatic, hydrolysis, Digitoxin + Glucose, Hydrolysis, Digitoxigenin + 3-Digitoxose, , Purpurea Glycoside B, (Fresh leaves), Enzymatic, hydrolysis, Gitoxin + Glucose, Hydrolysis, Gitoxigenin + 3-Digitoxose, , Additionally, it cont ains 2 saponin glycosides, viz. digitonin and gitonin. The, total number of glycosides reported in the drug is about 30. Apart from the, glycosides, leaves also contain hydrolytic enzymes., , Chemical Tests, 1) Keller-Kiliani Test for Digitoxose: In this test, about 1gm finely powdered, digitalis is boiled with 10 ml 70% alcohol for 2 -3 minutes. The extract is, filtered and the filtrate is added to 5ml water and 0.5ml strong solution of, lead acetate. The mixture is s haken well and the filtrate is separated . T he, clear filtrate is treated with an equal volume of chloroform and evaporated to, yield the extractive. The extractive is dissolved in glacial acetic acid and after, cooling, 2 drops ferric chloride solution is added to it. These contents are, transferred to a test tube containing 2ml of concentrated sulphuric acid. A, reddish brown layer acquiring bluish -green colour after standing is observed, due to the presence of digitoxose., 2) Legal Test: The extract is dissolved in pyridine, sodium nitroprusside, solution is added to it and made alkaline. A pink or red colour is produced., 3) Baljet Test: To a section of digitalis, sodium picrate solution is added. It, shows yellow to orange colour., *, , *
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100, , Pharmacognosy and Phytochemistry-II, , 4) Keddes Reagent: Cardenolides react with this reagent to give blue or violet, colour., 5) Raymond’s Test: Digitalis is dissolved in 1ml of 50% ethanol and 0.1ml of, 1% solution of dinitrobenzene in ethanol or methanol is added. 2 -3 drops of, 20% NaOH are added to the mi xture. A violet colour is produced which, changes to blue., 6) Tollen’s Test: On dissolving the g lycoside in a mixture of pyridine and, ammoniacal silver nitrate solution, a precipitate of silver is obtained., Uses, Digitalis has its major therapeutic effect by increasing the force and velocity of, contraction of the normal and failing h eart. The clinical syndrome of congestive, heart failure results from the inability of heart to pump sufficient blood to meet, the body‟s needs. Digitalis increases the activity of cardiac muscles., Substitutes, 1) Adonidin: The active principle of Adonis vernalis (Ranunculaceae family) is, a glucoside. Adonidin acts more quickly and powerfully than digitalis, and in, some instances, it has more curative power., 2) Foxglove: As digitalis, it has held its place as one of the most valuable, remedies that come from the ve getable kingdom, and the art of using it has, been steadily improved over the course of time., Adulterants, 1) Common Mullein Leaves:, The leaves of, Verbascum thapsus, (Scrophulariaceae) are mixed with the genuine drug and can be distinguished, microscopically bythe presence of large woolly branched candelabra trichomes., 2) Primrose Leaves: The leaves of Primula vulgaris (Primulaceae) are added, to digitalis. They can be detected microscopically by the presence of, uniseriate covering trichomes, which are 8 -9 celled long. The lateral veins of, the leaves of primrose are straight., 3) Comfrey Leaves:, These are the leaves of, Symphytum officinale, (Boraginaceae) and can be detected by the presence of multicellular, trichomes forming hook at the top., , 4.4. TRITERPENOIDS, 4.4.1., , Introduction, , Triterpenoids are terpenoids derived from triterpene molecules . They are useful, in the treatment and prevention of cancer. Triterpenoid saponins are triterpenes ,, which are the members of saponin compounds., Triterpenes consist of 30 c arbon atoms united with sever al oxygen atoms. The, triterpene molecules are assembled from a C, -5 isoprene unit via cytosolic, mevalonate pathway, forming a C-30 compound., *, , *
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104, , 4.4.5., , Pharmacognosy and Phytochemistry-II, , Therapeutic Uses, , Triterpenoids are used in:, 1) It is used in the treatment of dementia., 2) It is used in the treatment of convulsions., 3) It is used in treating depression., 4) It is used in pain and nociception., 5) It is used in treating anxiety., 6) It is used as a sedative and hypnotic., , 4.5. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Phenylpropanoids are a large mixture of naturally occurring phenolic, compounds obtained from phenylalanine and tyrosine (aromatic amino acids), or sometimes from the intermediates of shikimic acid biosynthetic pathway., 2) Flavonoids are a diverse group of phytonutrients (plant chemicals) found in, almost all fruits and vegetables. Along with carotenoids, they are responsible, for the vivid colours in fruits and vegetables., 3) Flavonoids are chemical ly polyphenolic compounds. They contain 15, carbon atoms. Two benzene rings joined by a linear three carbon chain., 4) Flavonoids are biosynthesised through condensation of the shikimic acid, and acyipolymalonate pathways., 5) Flavonoids are essential for the pigmentation of flowers . They also guard, the petals and other parts of flower from UV radiation and oxidative stress., 6) Lignans are low molecular weight polymers formed by the coupling of two, phenylpropene units via their C 3 side chains and between the aromatic ri ng, and the C3 chain., 7) Lignans are widely occurring plant compounds and are obtained from roots,, heart wood, foliage, fruits, and resinous exudates of plants., 8) Lignans are found as single enantiomeric forms, i.e., as d- or l-isomers., 9) Tea is the leaf buds and leaves of the plant Thea sinensis., 10) Ruta is obtained from the fresh and dried leaves of the plant Ruta graveolens, L., 11) Phytosterols are universal steroids in the plant kingdom, and have been, reported to show hypocholesterolemic activity., 12) Withanolides are larg e steroidal lactones group having many biological, activities. A small group of plant steroids, i.e.,, brassinosteroids, exhibit, plant growth hormonal activity., 13) Phytoecdysteroids (polyhydroxylated plant steroids) show anabolic effects, with no unwanted side effects., 14) Steroidal alkaloids found in the plants of Solanaceae and Melanthiaceae, family (specially the genus Veratrum)., *, , *
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Secondary Metabolites - II (Chapter 4), , 105, , 15) The cardiac glycosides are an important class of naturally occurring drugs, whose actions include both beneficial and toxic effects on the heart., 16) Cardiac glycosides act by affecting the availability of intracellular Ca +2 ions, for myocardial contraction or increasing the sensitivity of myocardial, contractile proteins., 17) Two classes of cardiac glycosides have been observed in nature, i.e., the, cardenolides and the bufadienolides., 18) The cardenolides have an unsaturated, butyrolactone ring , while the, bufadienolides have a pyrone ring., 19) Liquorice is subterranean dried peeled or unpeeled root s and stolon of, different varieties of Glycyrrhiza glabra., 20) Dioscorea is the dried rhizome of several species of Dioscorea like, Dioscorea deltoidea, D. composita, and other species of Dioscorea., 21) Digitalis is the dried leaves of Digitalis purpurea , dried at a temperature, below 60°C, immediately after collection. The leaves should not contain, more than 5% of moisture., 22) Triterpenoids are terpenoids derived from triterpene molecules . They are, useful in the treatment and prevention of cancer., 23) Triterpenes consist of 30 carbon atoms united with several oxygen atoms., 24) Cholesterol, phytosterols, phytoecdysteroids, etc. are some, examples of, triterpenes., , 4.6. EXERCISE, 4.6.1., 1), 2), 3), 4), 5), 6), , 4.6.2., 7), 8), 9), 10), 11), , True or False, , The family of liquorice is Leguminoseae., Theaceae is the family of ruta., The cardenolides have an unsaturated butyrolactone ring., Triterpenes consist of 30 carbon atoms., Ruta is the leaf buds and leaves of the plant Thea sinensis., Lignans are found as single enantiomeric forms., , Fill in the Blanks, , Bufadienolides have a ________ ring., ___________ is the family of tea., ____________ consist of 30 carbon atoms united with several oxygen atoms., ____________ are found as single enantiomeric forms, i.e., as d- or l-isomers., ____________ are biosynthesised through condensation of the shikimic acid and, acyipolymalonate pathways., , Answers, , 1) True, 5) False, 9) Triterpenes, *, , 2) False, 6) True, 10) Lignans, , 3) True, 7) Pyrone, 11) Flavonoids, , 4) True, 8) Theaceae, *
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106, , Pharmacognosy and Phytochemistry-II, , 4.6.3., 1), 2), 3), 4), 5), , 4.6.4., 1), 2), 3), 4), , Very Short Answer Type Questions, , Write a short note on phenylpropanoids., Give the chemical classes of phenylpropanoids., Write a short note on flavonoids., Give the family and biological source of liquorice., Give the therapeutic uses of triterpenoids., , Short Answer Type Questions, , Discuss flavonoids. Give their chemistry., Write a note on lignans., Give the biosynthesis of cardiac glycosides., Write a short note on digitalis., , 4.6.5., , Long Answer Type Questions, , 1) Write a detailed note on triterpenoids., 2) Give the qualitative and quantitative analysis of cardiac glycosides., 3) Classify flavonoids and give their biosynthesis., , *, , *
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Secondary Metabolites - III (Chapter 5), , CHAPTER, 5, , 107, , Secondary Metabolites - III, , 5.1. VOLATILE OILS, 5.1.1., , Introduction, , Volatile oils are obtained from various plant parts. They are basically the mixture, of hydrocarbon terpenes, sesquiterpenes, and polyterpenes and their oxygenated, derivatives. Volatile oils carry th e plant‘s active constituents and hence are also, called essential oils . Mostly the volatile oils are already present in special, secretory tissues in the plant, e.g., the oil ducts in umbelliferous fruits, the oil, cells or oil glands present in sub, -epidermal tissue in lemon and orange,, mesophyll in eucalyptus leaves, trichomes in some plants, etc., When volatile oils are not already present in the plant, they are formed by, glycoside decomposition. For example, whole black mustard seeds are although, odourless, but when crushed and added to water a strong odour is produced., Volatile or ethereal oils are defined as, ‗odorous volatile principles of plant and, animal origin which evaporate when exposed to air at ordinary temperature, ‘., , 5.1.2., , Chemistry, , The volatile oils chemically comprise of the following:, 1) Terpene Hydrocarbons, i) Monoterpenes, a) These compounds are present in almost all volatile oils, and have a, 10 carbon atom structure (derived from two isoprene units ) and not, less than one double bond., b) These compounds react to air and heat sources. Due to this , citrus, oils do not last long, since the content of monoterpene hydrocarbons, is high in them and they react quicklywith air and get easily oxidised., c) These compounds have anti -inflammatory, antiseptic, antiviral and, antibacterial therapeutic properties. Some of them possess analgesic, or stimulating effect. They are sometimes also used as decongestants, because few of them have a, stimulating effect on the muc ous, membranes., ii) Sesquiterpenes, a) These compounds contain 15 carbon atoms and exhibit complex, pharmacological actions., b) These compounds show, anti-inflammatory and anti, -allergic, properties. Farnesene is ano ther sesquiterpene that occurs in, chamomile, rose, and other floral oils., *, , *
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108, , Pharmacognosy and Phytochemistry-II, , 2) Oxygenated Compounds, i) Phenols, a) Generally, a carbon side chain is present in phenols found in volatile, oils. Examples of such compounds are thymol, eugenol,and carvacrol., These compounds show antiseptic, anti -bacterial, disinfectant and, stimulating therapeutic properties., b) Volatile oils with high content of phenols should be used for small, period of time in low concentration. This is because their use for, long time period can lead to toxicity as the liver need to work harder, for excreting them from body., c) They show antiseptic properties, but are also considered as irritants, for skin and muc ous membrane, e.g., cinnamon and clove oil can, cause severe skin reactions., ii) Alcohols, a) Monoterpene Alcohols, These alcohols exhibit good antiseptic, anti -viral and anti -fungal, properties, and produce very less side effects like skin irritation, or toxicity. They also show a stimulating effect., Linalool, citronellol, and terpineol found in lavender, rose, and, geranium, juniper and tea tree oil, respectively, are a few, examples of monoterpene alcohols., b) Sesquiterpene Alcohols, These alcohols are rarely present in the volatile, oils, but, bisabolol in German chamomile exhibit properties like liver and, glandular stimulant, anti-allergen, and anti-inflammatory., Sandalwood ( α-santalol), ginger, patchou li, vetiver, carrot seed,, everlasting, and valerian are few other oils contain, ing, sesquiterpene alcohols., 3) Aldehydes, i) These compounds show anti -fungal, anti -inflammatory, disinfectant,, sedative yet stimulating therapeutic properties. These compounds impart, the citrus -like smell in melissa, lemongrass , and citronella. These, properties of aldehydes are utilised in aromatherapy when the volatile oil, is used in low dilution, i.e., around 1%., ii) Volatile oils having high amount of aldehydes, e.g., lemongrass oil can, cause skin irritation and sensitivity. Aldehydes are unstable in nature and, get easily oxidised in the presence of oxygen and low heat., 4) Ketones, i) In thujone (found in thuja ), wormwood oil , and picocamphone , the, ketones present can be toxic; however, these co mpounds also exhibit, therapeutic effects as they facilitate, mucus secretion and tissue, regeneration., ii) Hyssop, eucalyptus and rosemary oils have average amounts of ketones, and their proper use in aromatherapy provide great benefits to the body., *, , *
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Secondary Metabolites - III (Chapter 5), , 109, , iii) Italidone (a ketone found in everlasting ) exhibits mucolytic (mucus easing) properties, and is also used in skin regeneration, wound healing,, and diminishing old scar tissue s like in wound s, stretch marks, and, adhesions., iv) Volatile oils containing high amounts of ketones should be used carefully, in pregnant women., 5) Esters, i) These compounds are obtained from, alcohols and acids. They are, named after both the original molecules, i.e.,, alcohols dropping the, ―ol‖ and acquiring a ―yl‖ and acids dropping the ―lc‖ and acquiring an, ―ate‖., ii) In volatile oils, the esters present have great aroma and are fruity. They, exhibit sedative and antispasmodic therapeutic effects. A f ew esters also, show anti -fungal and anti -microbial properties,, e.g., anti-fungal, properties in geranium oil., iii) The mo st well -known ester must be linalyl acetate, which is found in, lavender, early sage as well as petitgrain., Linalyl acetate is the most well -known ester that is found in lavender, early, sage and petitgrain., 6) Lactones and Coumarins, i) An ester group is attache d to the carbon ring system in lactones ., Coumarins are a kind of lactones. The actions of lactones, coumarins ,, and ketones are slightly similar as they have neurotoxic effects and also, cause skin sensitising and irritation., ii) Helenalin is a sesquiterpene la ctone present in arnica oil and imparts it, the anti-inflammatory action., iii) Lactones and coumarins are found in very low quantity in volatile oils., Lactones have mucus moving and expectorant properties , due to which, elecampane is used in treating bronchitis and chest problems., iv) Furocoumarin and bergaptene are coumarins found in bergamot oil. They, are skin UV sensitive and should be handled with care., 7) Ethers: Phenolic ethers are the most widely occurring ethers in volatile oils., Anethol in aniseed is the only ether of importance together with methyl, chavicol found in basil and tarragon., 8) Oxides: The major therapeutic effect of oxides is expectorant, e.g., 1,8-cineole, (eucalyptol) is the most recognised oxide to be used as an expectorant., , 5.1.2.1., , Qualitative Analysis, , Natural drugs with volatile oils are chemically tested as follows:, 1) A thin section of drug is treated with alcoholic solution of Sudan III. A red, colour appears that confirms the presence of volatile oils., 2) A thin section of drug is treated with a tincture of alkane. A red colour, appears that confirms the presence of volatile oils., *, , *
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110, , Pharmacognosy and Phytochemistry-II, , 5.1.2.2., , Isolation of Volatile Oils, , At particular times, the volatile oil -containing plants exhibit, highest, concentration, e.g., jasmine at sunset; so it is suggested to pluck the plant parts, containing volatile oils at this specific time. Generally, , the following five, methods have been developed for isolating volatile oils:, 1) Expression Method: In this method , the p lant material is crushed and the, juice is screened for removing the large particles. Centrifugation of the, screened juice is done in high speed centrifugal machine. When almost half, of the volatile oils are extracted and the other half is not, the residue is used, for isolating low quality of oils by distillation. Citrus and lemongrass oils, are extracted by expression method., 2) Steam Distillation: In this method, the plant -material is macerated and, steam distilled when the volatile oils travel into the distillate from which, their extraction is done using pure organic volatile solvents (light petroleum)., Some essential oils may get decomposed during the distillation process and, some hydrolyse into fragrant compounds, hence this method should be, performed with great care., 3) Extraction by Means of Volatile Solvents: Some volatile oils decompose, during distillation because of their sensitivity to heat; hence, in such situation, the plant material is treated wi th light petrol at 50°C and the solvent is, removed by distillation under low pressure., 4) Enfleurage (Adsorption in Purified Fats): In this method , the fat is taken, in a glass plate and warmed to 50°C temperature. Then the fat surface is, covered with petals and left undisturbed till the fat gets saturated with the, volatile oils. When this happens the fresh petals are replaced with the old, ones. Thereafter, the p etals are removed and the fat is digested with ethyl, alcohol after the dissolution of volatile oils (present in fat) in ethyl alcohol., The dissolved fat (if an y) is also removed by cooling at 20°C temperature., For removing the solvent, the extract containing ethyl alcohol and volatile, oils is distilled under reduced pressure., 5) Ecuelle: This method is used in citrus oil extraction, where t, he oil cells, present in the rind are mechanically ruptured with pointed projections and by, twisting the raw material in clockwise direction over them, either, mechanically or manually., , 5.1.3., , Chemical Classes, , Volatile oils are classified as given in the table 5.1:, Classes, 1), , *, , Table 5.1: Classification of Volatile Oils, Structures, Constituents, , Drugs, , Hydrocarbon, Volatile Oil, i) Monocyclic, , Limonene, , Cardamom, , ii), , -Pinine, , Coriander oil, , Bicyclic, , *
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Secondary Metabolites - III (Chapter 5), , 2), , Ester Volatile Oil, , Methyl salicylate Wintergreen oil, , 3), , Ether Volatile Oil, , Anethole, , Fennel and, Anise, , 4), , Alcohol Volatile Oils, i) Acyclic, , Citronellol, , Lemongrass, , ii), , Menthol, , Peppermint, , Monocyclic, , 5), , Aldehydes Volatile, Oil, , Citral, , Lemon, , 6), , Ketone Volatile Oil, , Camphor, , Camphor, , Carvone, , Caraway oil, , Cincole, , Eucalyptus oil, , 7), , *, , 111, , Oxides Volatile Oils, , *
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112, , 8), , Pharmacognosy and Phytochemistry-II, , Phenol Volatile Oils, , 5.1.4., , Eugenol, , Clove, , Thymol, , Mint, , Biosources, , Volatile oils are present in the following plant parts:, 1) Glandular Hair: Mint stems and leaves (Labiatae)., 2) Mesophyll: Eucalyptus leaves., 3) Modified: Piperaceae and Parenchyma., 4) Vitale: Anise (Umbelliferae)., 5) Lysigenous: Gossypium species., 6) Schizogenous Gland: Pinaccae and Rutaceae., 7) All Tissues: Conifers., 8) Petals: Rose., 9) Bark: Cinnamon., 10) Rind: Orange., 11) Hear Wood: Sandalwood., , 5.1.5., , Therapeutic Uses, , Volatile oils are employed as flavouring and perfuming agents in pharmaceutical, products, foods, beverages, and in cosmetics., They serve as medicinal agents for therapeutic effects like:, 1) Carminative (e.g., umbelliferous fruits),, 2) Anthelmintic (e.g., chenopodium oil),, 3) Diuretics (e.g., juniper),, 4) Antiseptic (e.g., eucalyptus),, 5) Counter irritant (e.g., oil of wintergreen),, 6) Local anaesthetic (e.g., clove),, 7) Sedative (e.g., jatamansi),, 8) Local irritant (e.g., turpentine),, 9) Insect repellent (e.g., citronella), and, 10) Source of vitamin A (e.g., lemongrass)., *, , *
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Secondary Metabolites - III (Chapter 5), , 5.1.6., , 113, , Individual Drugs, , The pharmacognostic profiles of the following drugs have been discussed below:, 1) Mentha,, 2) Clove,, 3) Cinnamon,, 4) Fennel, and, 5) Coriander., , 5.1.6.1., , Mentha, , Mentha is the oldest known species of medic inal plant in Eastern and Western, traditions. Other than its use as a flavouring agent (from che wing gum to after, dinner mints), it is also used in cosmetics and other pharmaceutical products., Synonyms, Oleum mentha piperita, Colpermin, and Mentha oil., Biological Source, The oil is obtained by the steam distillation of fresh flowering tops of the plant, Mentha piperita Linn. The oil is rectified if required. Mentha oil should contain, not less than 50% of total menthol., Family, Labiatae., Geographical Source, Species of mentha are cultivated in different, parts of the world. It grows wild ly in Europe,, and is cultivated in Japan, England, France,, Italy, U.S.A., Bulgaria , and U.S.S.R. It is, cultivated near Jammu an d in Tarai region of, Uttar Pradesh in India., , Figure 5.1: Mentha Plant (Mentha, piperita), , Preparation of Oil, 1) Mentha plants are dried to 1/4 th of their weight to reduce the bulk. This, saves the distillation time and decreases production cost. Drying in direct, sunlight causes loss of volatile oil., 2) While drying the herb, it should not undergo fermentation, or else the quality, and quantity of oil will be severely affected., 3) The material dried in air is charged into galvanised iron or mild-steel still, having a false perforated bottom (especially designed for this purpose)., 4) Steam under pressure is generated with a boiler, and passed through the drug,, which takes around 3-4 hours for distillation., 5) During the first half of distillation, more than 80% of the oil is distilled off., 6) Distillation should be carefully finished because menthol obtained in the, final stage of distillation is medicinally and commercially important., *, , *
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114, , Pharmacognosy and Phytochemistry-II, , 7) The condenser used should be of aluminium or stainless steel. It should be, coiled to increase the area of condensation. Collection of the distillate, i.e.,, mentha oil is done in separating can., 8) Mentha oil floats in the separating can being lighter in weight and insoluble, in water. This oil is decanted and filtered., The average oil content of the herb is, approximately 0.5 -1% (v/w). For, commercial purposes, around 100kg of the oil per hectare of the crop per year is, satisfactory., Macroscopic Features, 1) Colour: Colourless to yellow., 2) Odour: Characteristic and pleasant., 3) Taste: Pungent followed by cooling sensation., 4) Solubility: Soluble in 70% alcohol, ether and chloroform; insoluble in water., Microscopic Features, Leaves are the most important p art from which oil is extracted . Its upper, epidermis comprises of large, clear epidermal cel ls with sinuous, vertical walls,, few or no stomata, and a few glandular trichomes . Palisade parenchyma, comprises of a layer of columnar cells having abundant chloroplasts. Spongy, parenchyma comprises of 4-6 layers o f irregularly shaped cells containing, chloroplastids and intercellular airspaces. Lower epidermis comprises of small, epidermal cells having sinuous, vertical walls and numerous diacytic stomata., Non-glandular and glandular trichomes are present as outgrowths in the region of, veins and midrib. The non-glandular trichomes are uniseriate, papillose, and 1-8celled; while the glandular trichomes have 1-2-celled stalk and 1-8-celled glandular, head containing the volatile oil. Calcium oxalate crystalsare not present., Chemical Constituents, The chief constituent of mentha oil is L-menthol which exists 70% in free form, and also in form of esters depending on the variety (like American, Japanese, and, Indian). American variety contains 80% of menthol and the Japanese variety, contains 70 -90% of menthol. Menthone, menthofuran, jasmine, menthyl, isovalerate, menthyl acetate , and many other terpene derivatives are other, important constituents of mentha oil., Other te rpenes include L -limonene,, isopelugol, cineole, pinene, c amphene, etc. The pleasant flavour of mentha oil is, because of jasmone and esters; and its dirty smell is due to resinification caused, by menthofuran., CH3, , OH, , *, , CH3, , CH3, , CH3, , O, , O, , CH3 CH3, , CH3 CH3, , CH3 CH3, , Menthol, , Pulegone, , Menthone, , O, CH3, Menthofuran, *
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Secondary Metabolites - III (Chapter 5), , 115, , Chemical Test, A f ew drops of peppermint oil, are mixed wit h 5ml of nitric acid solution, (prepared by mixing 1ml of nitric acid with 300ml of glacial acetic acid). The, resultant mixture is heated on water bath. A, blue colour appears within five, minutes of heating. On further heating, the blue colour darkens to copper colour, fluorescence, which becomes golden yellow after some time to give a clear and, transparent liquid., Uses, 1) It is used as a carminative, stimulant and flavouring agent., 2) It shows mild antiseptic properties., 3) It is used in toothpaste s, tooth powders, shaving creams , and in various, pharmaceutical dosage forms., 4) It is also used for preparing chewing gums, candies, jellies, perfumes , and, essences., 5) It possesses calcium channel blocking activity which produces spasmolytic, and smooth muscle relaxant effects . Therefore, it is used in irritable bow el, syndrome., 6) Due to its muscle relaxant activity, mentha oil is used to relieve spasm during, endoscopy of colon., 7) It stimulates the bile flow, thus exhibits digestant activity., 8) Azulene present in the leaves, shows anti-inflammatory and anti -ulcer, activity., 9) It is considered by U.S.F.D.A. as Generally Regarded as Safe (GRAS) as a, nasal decongestant., 10) It is used for inhalation in steam, and in topical products and lozenges for its, antitussive properties., Substitutes and Adulterants, Many species of mentha contain oil, and sometimes these oils are de-mentholised, and used as drug adulterants., Mentha oil is generally adulterated with cornmint (difficult t o detect even at, 85%) and is the most adulterated oil., , 5.1.6.2., , Clove, , The unopened pink coloured buds of c loves are found in evergreen clove trees., Same as the other spices, they are available throughout the year. Cloves are, known to provide a warm, sweet and aromatic taste., Synonyms, Caryophyllum, Clove flower, and Clove buds., Biological Source, Clove is the dried flower buds of the plant, contain 15% (v/w) or more of clove oil., , Eugenia caryophyllus and should, , Family, Myrtaceae., *, , *
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116, , Pharmacognosy and Phytochemistry-II, , Geographical Source, Cloves were initially grown in the regions of Amboyna and Molucca islands. In, the recent times, they are cultiva ted in Zanzibar, Pemba Penang, Madagascar,, Caribbean islands, Sri Lanka, , and India. Nilgiri, Tenkasi, -hills and in, Kanyakumari district of Tamil Nadu state are the regions in India where cloves, are grown. Kottayam and Quilon districts of Kerala are also fam, ous for their, cultivation., , Figure 5.2: Flowering Twig of, Eugenia caryophyllus Linn, , Figure 5.3: Cloves and Clove Stalks, , Macroscopic Features, 1) Colour: Crimson to dark brown., 2) Odour: Slightly aromatic., 3) Taste: Pungent and aromatic followed by numbness., 4) Size: About 10-17.5mm in length, 4mm in width, and 2mm thick., 5) Shape: Hypanthium is surmounted with 4 thick acute divergent sepals. These, sepals are covered by dome -shaped corolla which consists of unexpanded, membranous petals, several stamens, and a single stiff prominent style., 6) Density: Cloves are heavier than water., 7) Specific Gravity: 1.038-1.06., 8) Refractive Index: 1.527-1.535., 9) The oil is colourless to pale yellow in colour. During storage it becomes, thick and darker in colour., Corolla, , Anther, Cuticle, Epidermis, Oil glands, , Stamen, Style, Calyx, Bilocular ovary, , Fibrovascular, bundle, Collenchyma, , Hypanthium, Oil glands, , Columella, Lacuna, Figure 5.5: Transverse Section of Clove, , Figure 5.4: Longitudinal Section of Clove, *, , *
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Secondary Metabolites - III (Chapter 5), , 117, , Microscopic Features, The epidermis of clove has a thick cuticle c overing and is composed of a large, anomocytic stomata and straight walled cells. Schizolysigenous and ovoid shaped oil glands are present in almost all portions of the drug. The isolated, phloem fibres are rarely found in the spongy tissue. Calcium oxalate crystals, with small number of stone cells are also found in clove. Starch grains are, absent., Chemical Constituents, Clove contains about 15 -20% of volatile oil and 10 -13% of, tannin in the form of gallotannic acid, resin, chromone, and, eugenin. About 70 -90% of eugenol, eugenol acetate ,, caryophyllenes and trace amounts of esters, ketones, and, alcohols are present in the volatile oil of clove., , OH, OCH3, , CH2 CH CH2, Eugenol, , Chemical Test, On treating the transverse section of clove with strong potassium hydroxide, solution, it shows needle-shaped crystals of potassium eugenate., Uses, 1) Clove is used as:, i) Dental analgesic,, ii) Carminative,, iii) Stimulant,, iv) Flavouring agent, and, v) Aromatic and antiseptic., 2) It is also used for preparing cigarettes., 3) Clove oil is used in perfumery and in the production of vanillin., Adulterants, 1) Mother Cloves: These are ovate ripened fruits of clove tree appearing dark, brown in colour. These cloves are slightly aromatic. Starch grains and, minimum quantity of volatile oil are present in them., 2) Blown Cloves: These are lengthened flowers of the clove tree with separated, stamens. These cloves include volatile oil (in content less than that of the, authentic drug) and their colour is similar to that of cloves., 3) Clove Stalks: These adulterate the powdered cloves and get easily mixed, with cloves as they are similar in colour, odour, and taste. The presence of, isodiametric sclereids and calcium oxalate prisms helps in their detection., They possess high content of ash value and crude fibres. To pass the, Pharmacopoeial limit, the authenti c cloves are required to contain less than, 5% of clove stalks. Only 5% of oil is present in clove stalks., 4) Exhausted Cloves: These cloves are darker in appearance and are more, shrunken. When exhausted cloves are pressed with finger nails, oil does not, exude out as it has been removed by distillation. These cloves float on, water., *, , *
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118, , Pharmacognosy and Phytochemistry-II, , 5.1.6.3., , Cinnamon, , Clove obtained from the inner bark of the trees of genus Cinnamomum is majorly, used in sweet and savoury foods., Synonyms, Cinnamon bark, Kalmi-Dalchini, and Ceylon cinnamon., Biological Source, Cinnamon is the dried inner bark of the shoots of coppiced trees of Cinnamomum, zeylanicum Nees., (syn. Cinnamomum verum) and should contain 1.0% or more, of volatile oil., Family, Lauraceae., Geographical Source, Originally, the spice cinnamon was found near Sri Lanka and Malabar Coast of, India. Jamaica and Brazil are also the sites of cinnamon. But mostly the demand, of cinnamon is fulfilled by Sri L anka, therefore, a true cinnamon is referred to as, Sri Lanka cinnamon., , Outer quill, , Middle quill, Inner quill, Figure 5.6: Cinnamon Plant, , Figure 5.7: Compound Quill of Cinnamon, , Macroscopic Features, 1) Colour: The outer surface is dull yellowish-brown, while the inner surface is, dark yellowish-brown., 2) Odour: Fragrant., 3) Shape: Found in the form of compound quills., 4) Size: About 1m in length, 1cm in diameter, and 0.5mm in thickness., 5) Taste: Aromatic and sweet followed by warm sensation., 6) Fracture: Splintery., 7) Surface: On the outer surface of bark , wavy longitudinal striations with, small holes of scars left by th e branches are present, while on the inner, surface longitudinal striations are found., Microscopic Features, Since cinnamon is an inner bark, i t lacks cork and primary cortex (figure 5.8)., However, patches of, primary cortex may be observed in rare cases., Sclerenchymatous pericycle is present. The stelar portion of the cork contains, *, , *
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Secondary Metabolites - III (Chapter 5), , 119, , phloem, phloem fibres, biseriate medullary rays, and secretory cavities with, volatile oil and mucilage. In cortical parenchyma and medullary rays, starch, grains are found. In the cells of parenchyma, calcium oxalate crystals have been, observed., Chemical Constituents, The crude drug contains many essential, constituents such as volatile oil (0.51.0%) and phlobatannins (1.2%). Other, constituents found in cinnamon bark are, mucilage, calcium oxalate, starch grains,, and mannitol. Among these, volatile oil, is considered to be the active constituent, which on distillation appears light, yellow in colour. However, it changes, its colour during storage and becomes, red. Cinnamon bark yields, approximately 14 -16% of 90.0%, alcohol-soluble extractive., , (P.f.), , Pericyclic, fibre (P.f.), Sclereids, Medullary ray, Phloem fibre, Secretory cavity, Figure 5.8: T.S. of Cinnamon Bark, , Cinnamon oil (yellow to red in colour) is composed of a variety of constituents, such as, cinnamaldehyde (60-70%), eugenol (5-10%), benzaldehyde,, cuminaldehyde, and terpenes (phellandrene, pinene, cymene, caryophyllene, etc.)., Chemical Test, 1) When a drop of ferric chloride solution is added to a drop of volatile oil, a, pale green colour appears. This colour is achieved as cinnamic aldehyde on, reacting with ferric chloride gives brown colour and eugenol y, ields blue, colour. The pale green colour is the combination of brown and blue colours., 2) It yields brown colour with oil of cassia due to the presence of cinnamic, aldehyde., Uses, 1) It is highly enriched in carminative, stomachic, and mild astringent properties, ., 2) It has anti-inflammatory properties., 3) It also has anti-diabetic properties., 4) It acts as an anti-ulcer agent, and is active against H. pylori., 5) It is anti-microbial in nature, inhibit the growth of yeast, bacteria, fungi, etc., 6) It helps in reducing LDL levels, and increasing HDL levels, thus lowers, cholesterol levels., 7) It may be employed as a flavouring agent, stimulant, an aromatic, and an, antiseptic., 8) Its anti-oxidant properties protect from the damaging effects of free radicals., 9) On a commercial scale, cinnamon bark is commonly employed as a spice and, condiment., 10) The oil is also used in candies, dentifrices, and perfume preparations., 11) It has been traditionally used in alleviating gastric disorders and dysmenorrhea., *, , *
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120, , Pharmacognosy and Phytochemistry-II, , Adulterants and Substitutes, 1) Jungle Cinnamon: The main source of this cinnamon bark is wild trees. It is, generally dark coloured, less aromatic than the cultivated trees, and slightly, bitter in taste., 2) Cinnamon Chips: These pieces are obtained from untrimmed bark. They are, different from the authentic drug as they show the presence of a large number, of cork cells., 3) Saigon Cinnamon: It is the tree bark of Cinnamomum loureirii, belonging to, the family Lauraceae and is exported from the port of Saigon. China and, Japan are the countries which also favour the g rowth of this cinnamon. Its, bark has a sweet taste and is greyish-brown in colour with light patches on it., Quills are unpeeled (30 × 4 × 0.7cm) and contain 2.5% of volatile oil., 4) Java Cinnamon: It is obtained from the bark of Cinnamomum burma nnii,, belonging to the family Lauraceae. The main characteristic of this cinnamon, is that the bark is peeled, present as double quills, and is less aromatic., Histologically, small tubular crystals of calcium oxalate (not found in the, species of C. zeylanicum) are presen t in medullary rays. The Java cinnamon, oil possesses 75% of cinnamaldehyde., , 5.1.6.4., , Fennel, , All the plant parts of fennel (roots, stalks and leaves, with the spice obtained, from dried seeds) are eatable. Fennel has it origin in the Mediterranean. I t is an, ancient and common plant known to the ancient Greeks and spread throughout, Europe by Imperial Rome., Synonyms, Large fennel, Sweet fennel, Fennel fruit, Saunf (Hindi), Fructus, and Foeniculi., Biological Source, Fennel is the dried, ripe fruits of the plant Foeniculum vulgare Mill., Family, Umbelliferae., Geographical Source, Fennel is native to Mediterranean countries and Asia. It is mainly grown in, France, Saxony, Japan, Galicia, Russia, and Persia. In India, it is cultivated, throughout the country and often grows wild., Macroscopic Features, Fennel herb is stout, about 2m high, glabrous, and aromatic, having pinnately, decompound leaves. The drug consists partly of whole cremocarps and partly of, mericarps (figure 5.9)., 1) Colour: Greenish-brown., 2) Odour: Aromatic., 3) Taste: Distinct, sweet, and aromatic., 4) Size: 0.5-1.0cm long and 2-4mm broad., *, , *
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122, , Pharmacognosy and Phytochemistry-II, 1) A thin section of drug + Alcoholic solution of Sudan III, , Globules become red coloured, Volatile oil is present, , Globules remain unchanged, No volatile oil, , 2) A thin section of drug + Phloroglucinol: Conc. HCl (1: 1), , Pink patches appear, , Section does not show pink patches, , Lignified parenchyma and vascular, bundles are not present, 3) A thin section of drug + Alcoholic picric acid, , The section appears yellow, , Confirms the presence of aleurone grains, Figure 5.11: Chemical Test for Fennel, , Uses, 1) It is used as a stimulant, aromatic, stomachic, carminative, emmenagogue,, and expectorant., 2) It is a common ingredient of cough and stomach mixtures., 3) It is used in diseases related to chest, spleen, and kidney., 4) Anethole is added in mouth and dental preparations., Substitutes, Some varieties of fennel that are used as substituents are given in the table 5.2:, Table 5.2: Characteristics of Different Varieties of Fennel Used as Substitutes, Varieties, Size in mm, Taste, Volatile Oil, Fenchone, Content, Content of, (%), Volatile Oil (%), Saxony, 10 × 4, Aromatic, 4.76, 22.00, Russia or Romanian 4 to 6 × 1 to 2 Camphoraceous, 4.50, 18.00, French sweet or, 7 to 8 × 2 to 3 Sweet aromatic, 2.1, Nil, Roman, Indian, 4 to 7, Camphoraceous, 0.720, 6.70, Japanese, 3 to 4 × 2 to 3 Very sweet, 2.70, 10.20, , Adulterants, 1) Exhausted fennel is generally used to adulterate fennel. The alcohol, exhausted fennel appears as fresh fennel and ha, s 1-2% volatile oil. The, steam-exhausted fennel appears dark, contain small amount of volatile oil ,, and are heavier than water., *, , *
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Secondary Metabolites - III (Chapter 5), , 123, , 2) Its adulteration is also done with undeveloped or mould-attack fruits., 3) It is also adulterated with bitter fennel, synthetic trans-anethole, fenchone,, methyl chavicol, and limonene., , 5.1.6.5., , Coriander, , Coriander herb is grown once in a year. It is also well -known by the names of, cilantro or Chinese parsley. All its parts are edible but fresh leaves and dried, seeds are mostly used for cooking purpose., Synonyms, Coriander fruits, Chinese parsley, and Indian parsley., Biological Source, Coriander is the fully dried ripe fruits of the plant Coriandrum sativum., Family, Umbelliferae., Geographical Source, Coriander is cultivated throughout the European countries, mainly in Russia,, Hungary, and Holland. It is also cultivated in India, Egypt, and Morocco. In, India, it is chiefly grown in the s, tates of Andhra Pradesh (Guntur and, Anantapur), Maharashtra (Jalgaon and Satara), West Bengal (Howrah and 24, Paragana districts), Uttar Pradesh, Rajasthan, and Jammu and Kashmir., Macroscopic Features, 1) Colour: Yellowish-brown to brown., 2) Odour: Aromatic., 3) Taste: Spicy and characteristic., 4) Size: Fruits are 2 -4mm in diameter and 4 30mm in length., 5) Shape: Sub-globular cremocarpous fruit., 6) Extra Features:, 10 wavy and, inconspicuous primary ridges and 8 straight, secondary ridges are present on the outer, surface (figure 5.12). It is further described, as an endospermic and a coelospermic fruit., , -, , Stylopod, Primary, ridge, Secondary, ridge, , Figure 5.12: Coriander Fruit, , Microscopic Features, The epidermis part of the pericarp is formed of polygonal tubular c ells with, stomata. Inner epidermis of pericarp consists ofparquetry cells. Calcium oxalate, prisms are found in epidermal cells. In mesocarp, inner and outer layer of, parenchyma is present along with a layer ofsclerenchyma between them. Presence, of seed i s one of the main characteristic s of umbelliferous fruits. It lacks starch, grains, trichomes, and lignified reticulate parenchyma. In endosperm, fixed oil, globules are found and volatile oil is present in the vittae. The polygonal thick, walled cellulose parenchyma of endosperm containsaleurone grains., *, , *
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124, , Pharmacognosy and Phytochemistry-II, Testa, Secondary, ridge, , Raphe, , Primary ridge, Endosperm, , Carpophore, , Vitta, Cavity, Sclerenchyma, Mesocarp, Vitta, Figure 5.13: T.S. of Coriander Fruit (Cremocarp), , Chemical Constituents, Coriander is chemically constituted with:, 1) Volatile oil (0.3-1%),, 2) Fixed oil (13%), and, 3) Proteins (20%)., The volatile oil contains:, 1) 90% of D-linalool (coriandrol),, 2) Coriandryl acetate, and, 3) L-borneol, geraniol, and pinene (in trace amounts), The coriander leaves contain rich content of vitamin A. Coriander fruits yield 5 7% of ash. The composition of volatile oil varies extensively during the storage, of crude drug., Chemical Test, Prick Test: 2-5mg of powdered coriander is applied on the skin with a drop of, saline solution, and then pricked into the skin. The reactions occurring are, observed after 15 minutes. Reactions with a diameter of at least 3mm larg er than, the negative control (saline solution) are regarded as positive., Uses, 1) The fruits and volatile oil are aromatic, carminative, stimulant and flavouring, agents., 2) The oil is used along with purgatives to prevent gripping., 3) It is an ingredient in orange spirit and cascara elixir., Substitute, Coriander is substituted by ellipsoidal Bombay coriander fruits having a less, content of volatile oil., *, , *
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Secondary Metabolites - III (Chapter 5), , 125, , 5.2. TANNINS, 5.2.1., , Introduction, , The term tannin was first applied by Seguin in 1976 to denote substances, present in plant extracts which are able to combine with proteins of animal hides,, prevent their putrefaction and convert them into leather. On this basis ―tannin is a, substance which is detected qualitatively by a tanning test (the goldbeater‘s skin, test) and is determined quantitatively by its absorption on standard hide powder‖., Tannins are defined as, ‗Complex substances that occur as mixtures of, polyphenols that are very difficult to separate since they do not crystallise‘., Tannins are present in the aerial parts, e.g., leaves, fruits, barks or stem, generally, occurs in immature fruits, but disappears during the ripening process. New leaves, of deciduous plants contain high concentration of tannins. Tannins occur in many, crude drugs. They probably serve as a protection for the plants during growth and, are destroyed or deposited as end-products of metabolism in some dead tissues of, the mature plants., , 5.2.2., , Chemistry, , Tannins are complex organic, non, -nitrogenous derivatives of poly hydroxy, benzoic acid which are widely distributed in the vegetable kingdom. They are the, active constituents of materials like oak bark, which are used in the tanning of, skins. Tannins precipitate and combine with proteins. The protein tannin complex, is resistant to proteolytic enzymes. Following are the structures ( figure 5.14) of, chemical constituents found in tannins:, , 5.2.2.1., , Extraction, , The steps involved in extracting tannins are:, 1) 100gm of the powd ered sample is weighed accurately and refluxed for 15, minutes in a 500ml Erlenmeyer flask. During the reflux process , the entire, sample should be covered with 70% acetone. The flask is removed from the, hot plate and filtered in another flask., 2) The residue o n the filter paper is washed off using a sufficient amount of, 70% acetone., *, , *
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126, , Pharmacognosy and Phytochemistry-II, , 3) The washed solution is combined with the filtrate and the resultant mixture is, subjected to extraction method using diethyl ether. This process is repeated, five times till the diet hyl ether and tannin separate out completely as distinct, upper and lower layer, respectively., 4) The tannin layer is separated, using a separatory funnel. The tannin so, collected is evaporated till a residue, known as the, extracted tannin , is, obtained, which is the final product., 5) The tannin percentage is calculated with the following formula:, %tannin = Wt. of dried tannin / Wt. of the powdered sample × 100, 6) After extracting the tannin, the aqueous and ethereal layers are concentrated,, dried, isolated, and purified using various chromatography techniques., , 5.2.2.2., , Qualitative Analysis, , Tannins undergo the following chemical tests:, 1) Goldbeater’s Skin Test : A small piece of goldbeater‘s skin (a membr ane, prepared from ox intestine) is soaked in 2% hydrochloric acid, rinsed with, distilled water and placed in tannin solution for 5 minutes. The skin piece is, washed with distilled water and kept in a solution of ferrous sulphate. A, brown or black colour is produced on the skin due to the presence of tannins., 2) Gelatin Test: To a solution of tannin (0.5 -1%), aqueous solutions of gelatin, (1%) and sodium chloride (10%) are added. A white buff, -coloured, precipitate is formed., 3) Phenazone Test: A mixture of aqueous extract (5ml) of a drug and sodium, acid phosphate (0.5g m) is heated, cooled and filtered. A solution of, phenazone (2%) is added to the filtrate. A bulky coloured precipitate is, formed., 4) Catechin Test (Matchstick Test): A matchstick is dipped in aqueous plant, extract, dried near burner and moistened with concentrated hydrochloric acid., On warming near a flame , the matchstick wood turns pink or red due to the, formation of phloroglucinol., 5) Chlorogenic Acid Test : An extract of chlorogenic acid containing the drug, is treated with aqueous ammonia. A green colour is formed on exposure to, air., 6) Vanillin-Hydrochloric Acid Test : 1gm vanillin , 10ml alcohol, and 10ml, concentrated hydrochloric acid are mixed . When the drug is treated with, vanillin-hydrochloric acid reagent, pink or red colour is formed due to, the, formation of phloroglucinol., , 5.2.2.3., , Quantitative Analysis, , Assay of tannins can be carried out by either determining some of the specific, functional groups associated with particular structures or by protein, -binding, assays. Among the various types of assays performed, the hide power method is, the standard method adopted in tannin industry. Some of the workers have, carried out oxidative analysis of phenols to measure tannins, or before and after, precipitation of tannins with proteins., *, , *
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Secondary Metabolites - III (Chapter 5), , 127, , The most commonly used method is, Folin-Denis assay which involves, interference from phenolic groups present in proteins and ascorbic acid. Later,, Hagerman and Butler suggested the use of, Prussian blue assay whose, sensitivity to interferences is less, but some other limitations were observed., Hagerman and Butler also suggested the acid-butanol (or proanthocyanidin), assay to be specific and sensitive for proanthocyanidins., Vanillin assay is, another method used for determining flavanols. A frequently used, iodate, method for determining gallotannins is s, ensitive to many interferences., Hagerman and Butler suggested using a newer method of assay with rhodanine., A rapid and highly reproducible method for determining tannins from a large, number of samples is the radial diffusion method which uses agarose -gelcontaining protein., Aspergillus Niger produces tannase enzyme, which forms gallic acid and a polyol, by cleaving the galloyl linkage, but it does not cleave hexahydroxydiphenyl, containing esters. This cleavage reaction is also used for estimating the number of, galloyl units per polyol. This enzyme also cleaves condensed tannins, yielding, flavonoids. Tannins can be purified by various techniques. Hydrolysable tannins, are characterised by hydrolysis and identifying the fragments by using techniques, of paper chromatography, TLC or HPLC and NMR, UVand MS., Condensed tannins undergo cleavage in mild-acidic solution and form a flavan-3ol and a quinone methide. The latter can be captured using a suitable nucleophile, (e.g., —SCH2Ph) to form 4 -thio-benzyl derivat ives, allowing the determination, of constituent flavonoid unit. The 4 -thiobenzyl derivatives may be reduced with, Raney-nickel to yield flavan -3-ols. Condensed tannins are characterised by, structural and conformational analysis by NMR spectroscopy. These co, mplex, 1, 13, structures are assigned with atoms using both H- and C-NMR spectroscopy., The parent ion and fragments of these compounds are observed by Fast Atom, Bombardment (FAB) mass-spectral analysis., , 5.2.3., , Chemical Classes, , Tannins are classified into the following classes:, 1) Hydrolysable Tannins : These may be hydrolysed by acids or, tannase, enzyme. They are formed from several molecules of phenolic acid such, as, gallic acid and hexahydroxy phenic acid whic h are united by ester linkage to, a central glucose molecule. These tannins form blue or black colour with, ferric chloride and pyrogallol on heating. They do not give blue colour with, bromine solution. Two principal types of hydrolysable tannins are:, i) Gallitannins: These are composed of gallic acid units, e.g., rhubarb, red, rose petals, bearberry leaves, Chinese galls, Tu rkish gall, hamamelis,, chestnut, and maple., ii) Ellagitannins: These are composed of hexahydroxydiphe nic acid units., Ellagitannins found in plants are of medicinal interest, e.g., pomegranate, rind, pomegranate bark, myrob alans, eucalyptus leaves, some Australian, kinds and Oak bark., *, , *
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128, , Pharmacognosy and Phytochemistry-II, , 2) Condensed Tannins: These are resistant to hydro lysis and are derived from, flavanol, catechins, and flavan-3,4diols. On treatment with acids or enzymes,, condensed tannins convert into pholaphene (a red insoluble compound )., Pholaphenes give the characteristic red colour to many drugs such as red, cinchona bark which contains these phlobatannins and their decomposition, product. On dry distillation they yield catechol tannins and these tannins, therefore are sometimes called catechol tannins , like catech ol itself, their, solutions turn green with ferric chloride., 3) Complex Tannins : This term has been applied by Okuda to a newly, discovered group of tannin which is biosynthesised from both hydrolysable, tannins mostly a C -glucoside, ellagitannins , and condensed tannins. The, union occurs through a C-C bond between the C -1 of the glucose unit of the, ellagitannin and the C -8 and C -6 of the flavan -3-ol derivative. Complex, tannins have no great relevance to mainstream pharmacognosy; monomers, have been isolated from the Combretaceae., 4) Pseudotannins: These are compounds of lower molecular weight than true, tannins and they do not respond to the goldbeater skin test. They have a wide, occurrence in plants and are usually found in a large quantity in dead or, drying cells. They exert an inhibitory effect on many enzymes due to protein, precipitation and contribute a protective function in bark and heartwoods., , 5.2.4., , Biosources, , Rhubarb, red rose petals, bearberry leaves, Chinese galls, Turkish gall,, hamamelis, che stnut, and maple are rich in, gallotannins. Pomegranate rind,, pomegranate bark, myroba lans, eucalyptus leaves, some Australian kinds , and, oak bark are rich in ellagitannins. Some drugs, e.g., tea, ham amelis bark and, leaves contain both hydrolysable and condensed tannins., Following parts of plants are rich in condensed tannins:, 1) Barks: Cinnamon, wild cherry, cinchona, willow, acacia, oak, , and, hamamelis., 2) Roots and Rhizomes: Krameria and male fern., 3) Flowers: Lime and hawthorn., 4) Seeds: Cocoa, guarana, kola, and areca., 5) Leaves: Hamamelis, hawthorn, and tea., 6) Extracts and Dried Juice: Catechu, acacia, mangrove cutches, East Indian, Kino, butea gum, eucalyptus, and kino., Following are the examples of plants rich in pseudotannins:, 1) Gallic Acid: Rhubarb and most material which contain gallitannins., 2) Catechins: Catechu, acacia cutch, many Australian kinos, cocoa, guarana, and many other drugs containing condensed tannins., , ,, , 3) Chlorogenic Acid: Mate, coffee (particularly unroasted), and nux-vomica (a, small quantity only)., *, , *
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Secondary Metabolites - III (Chapter 5), , 5.2.5., , 129, , Therapeutic Uses, , The medicinal uses of tannins are:, 1) Styptics: Tannin-containing drugs precipitate proteins and have been used, traditionally as styptics and internally for the protection of inflamed surface, of mouth and throat., 2) Anti-diarrhoeal: They act as anti -diarrhoeal and have been employed as, antidotes in poisoning by heavy metals, alkaloids, glycosides. In Western, medicine, their use declined after World War -II when it was found that, absorbed tannic acid can cause severe liver necrosis., 3) Anti-Tumour: They exhibit a strong anti -tumour activity. Ellagitannins, monomer units having galloyl groups at the C -2 and C -3 positions on the, glucose core exhibit anti-tumour activity., 4) Antidote: They are used as antidote s in alkaloid poison ing. They form, insoluble and non -toxic tannates with alkaloids. Only dilute solution is, applied for this purpose., , 5.2.6., , Commercial Applications, , In India, the tannins particularly catechu is used in the kat tha industries . The, industrial uses of these are following:, 1) Kattha: In market, kattha is sold as irregular-shaped pieces or small greyish, coloured square blocks that shows crystalline fracture on breaking. For its, use in preparation of paan and in medicine, kattha has a very large internal, demand. From a long time kat, tha is used in Indian medicine. It has, astringent, cooling, and digestive properties. It is used in relaxed conditions, of the throat, mouth, and gums. It is also used in cough and diarrhoea., Externally, i t is used as an astringent and for providing cooling effect in, ulcers, boils and eruptions of the skin. It is used in various preparations. It is, also an anti-leprotic drug. Kattha is a crucial ingredient i n paans. It imparts, the red colour on chewing of p aan in combination with lime. Regular use of, kattha, however, can cause blackening of teeth, and also oral cancer., Kattha as such or as acacatechin is a highly effective anti-oxidant for its use, in vegetable oils and fats, as it doubles their storage shelf-life. Catechu from, khair wood produces a good stable colour when used as a food additive., 2) Cutch or Dark Catechu: Cutch is sold in market as small cubes or blocks,, rusty is sold in market as rusty brown or dull orange coloured small cubes or, blocks having conchoidal fracture. It is used for printing and dyeing cotton, and silk fabrics., Cutch can be used in admixture with direct dyes , which are selected based, on their capability to bear the action of copper sulphate and dichromate. This, process is inexpensi ve than topping with basic dyes. It can also be used for, dyeing khaki. Variety of bright and attractive shades with enough fastness to, most of the agents and poor fastness, to chlorine can be produced by, combining cutch with diazo salts., *, , *
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130, , Pharmacognosy and Phytochemistry-II, , Cutch is used for dyeing ship sails and mail-bags. Materials dyed with cutch, display greater fastness to weather and sea -water conditions than those dyed, with coal-tar dyes. They are also less vulnerable to attack by d ichromate to, make it rot proof. Cutch is also used for dyeing pulp and paper, and is, widely being used in printing than in dyeing., , 5.2.7., , Individual Drugs, , The pharmacognostic profiles of the following drugs have been discussed below:, 1) Catechu, i) Pale catechu, and, ii) Black catechu., 2) Pterocarpus., , 5.2.7.1., , Pale Catechu, , Pale catechu is an extract occurring as dark or pale-brown coloured cubes having, a dull, powdery fracture. It may also occur in the form of lozenges., Synonyms, Gambier, Pale Catechu, Terra Japonica, and Catechu., Biological Source, Pale catechu is an extract prepared from the leaves and young shoots of, Uncaria gambier Roxburg., , plant, , Family, Rubiaceae., Geographical Source, This plant is a climbing shrub indigenous to the, Malaya Archipelago and largely cultivated on the, small islands between Singapore and Sumatra as well, as in British North Borneo and on other islands of the, Archipelago. The drug was introduced into Europe, towards the end of the 18th century, but was probably, used in India during the primitive era for chewing with, betel leaf (the leaf of Piper betel Linn)., , Figure 5.15: Uncaria, gambier (Herb), , Macroscopic Features, 1) Form: Gambier occurs in the form of regular cubes (measuring 2 -3cm on, each side), in masses of a dherent cubes and sometimes in larger r ectangular, blocks (4cm long), or in irregular broken pieces., 2) Colour: Dark reddish-brown colour., 3) Odour: Odourless., 4) Taste: First bitter and astringent, but afterwards sweetish., 5) Surface: Dull., 6) Fracture: Brittle., *, , *
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Secondary Metabolites - III (Chapter 5), , 131, , Microscopic Features, A little of the powdered drug mounted in lacto phenol and examined under the, microscope exhibits numerous minute acicular crystals of catechin. The residue, left after extraction with alcohol may be examined for starch, which should be, absent., The residue insoluble in water consists mainly of pieces of, epidermis,, trichomes, cork cells , pollen grains (11-18 diameter with three pores ), and, other vegetables debris of the young leafy twigs used in its preparations., , Upper Epidermis of Leaf, , Conical Trichomes Lower Epidermis of Leaf, , Cork Cell, , Calcium, Oxalate, Crystal, , Trichomes, , Epidermis of Stem Acicular Crystal of Catechin, , L.S. of Phloem, , Figure 5.16 Microscopic Features of Catechu, , Chemical Constituents, Pale catechu contains about 7.33% (+) catechin and 22 -50% catechu tannic, acid. These two substances in varying proportions constitute together over 60%, of the drug., Catechu tannic acid gives a green colour with ferric chloride solution, indicating, a phlobatannin. Brown s ubstance, rubea nic and japonic acids, of unknown, chemical nature are also present., OH, H, O, , O, 12, , 7 8, 65, , 4, , OH, H, , 3, , OH, OH, (+) - Catechin, , OH, , O, , H, O, , OH, H, OH, , OH, *, , (+) - Epicatechin, *
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132, , Pharmacognosy and Phytochemistry-II, OH, OH, , O, , H, O, , OR, OH, , O, , Quercetin (R = H), Quercitrin (R = Rhamnose), , Other constituents of the drug are, catechu-red, quercetin, and, fluorescein (a fluorescent substance)., , gambier, , Chemical Tests, Pale catechu gives the chemical tests illustrated in table 5.3:, , 1), , 2), , 3), , 4), , Table 5.3: Chemical Tests of Pale Catechu, Experiments, Observations, Constituents for Test, Petroleum, layer, Due, to the presence of, Gambier Fluorescent Test: A, shows green, fluorescence constituent., little powdered drug is boiled, fluorescence., with alcohol, filtered, and, sodium h ydroxide solution is, added to the filtrate, stirred,, and a few ml of light petroleum, is added., Due to the presence of, Chlorophyll Test:, The A greenish, yellow residue., chlorophyll; chlorophyll is, powdered drug is boiled with, absent in black catechu, thus, 5ml chloroform on, a water, this test is negative., bath, filtered in a white, porcelain dish, and evaporated, in a water bath., By the action of hydrochloric, Matchstick Test: A matchstick Magenta or, purple colour is, acid on catechins or catechol, is dipped in decoction of pale, produced., tannins, phloroglucinol is, catechu, dried in air, dipped in, produced which wit h lignin of, concentrated hydrochloric acid,, the matchstick respond, and warmed near the burner., positively to the test., Pink or red, Due to the formation of, Vanillin Hydrochloric Acid, colour is formed. phloroglucinol., Test: The drug is treated with, vanillin hydrochloric acid, reagent, which is a mixture of, vanillin (1gm), alcohol (10ml),, and concentrated hydrochloric, acid (10ml)., , Uses, 1) Medicinal Uses, i) All parts of the plant have astringent properties., ii) In India, gambier is used as skin lotions since remote times., iii) The Malays (ancient Malaysia) also use gambier as a lotion and apply it, to treat burns., iv) In paste form, it is used to treat scurf., *, , *
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Secondary Metabolites - III (Chapter 5), , 133, , v) It has commonly been used by the Indians and Malays to treat diarrhoea, and dysentery, and as a gargle for sore throat., vi) In Borneo, it has been used in the treatment of sciatica and lumbago., 2) Other Uses, i) Gambier catechu yields a colour known as Cutch Brown, which is used, for dyeing and tanning cotton, wool and silk., ii) It is also used on leather, such as calf and kip skins. The common khaki, colour is obtained from it., Adulterants, The adulteration of gambier has been done with, mineral matter (clay, ferric, hydroxide, etc.), starch and astringent extracts. Few Dutch East Indies gambier, come in last blocks, have about 25% of moisture, and leaves around 30-45% of, water-insoluble remains, calculated on the substance dried at 100°C., , 5.2.7.2., , Black Catechu, , Black catechu occurs in black, shining pieces or cakes, and is sold under the, name of Catechu. It grows in deciduous trees reaching upto a height of 9 -12m., This plant has short hooked spines and bi-pinnately compound green leaves with, 50 pairs of feather-like leaflets., Synonyms, Catechu, Catechu Nigrum, Cutch Khadir (Sanskrit),, Katha (Hindi), Katha (Gujarati), Cashoo, Peru, catechu, and Cachou., Biological Source, Black catech u is dried aqueous extract obtained from, the heartwood of plant Acacia catechu., , Figure 5.17: Catechu Herb, , Family, Leguminosae., Geographical Source, The tree is a native of India and also found in Myanmar., Macroscopic Features, 1) Form: Irregular mass, outer surface is rough and dull and rarely glossy., 2) Colour: Black., 3) Odour: Odourless., 4) Taste: Bitter in the beginning and astringent afterwards., 5) Fracture: Hard and brittle, and the broken surface is dark brown with a dull, gloss and porous., 6) Surface: Rough, dull or slightly glossy and porous., 7) Solubility: Partially soluble in cold water and alcohol; completely soluble in, hot water., *, , *
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134, , Pharmacognosy and Phytochemistry-II, , Microscopic Features, A transverse section of acacia catechu shows numerous uniseriate and bi -seriate, medullary rays with vessels occurring isolated or in small groups of two or four., Xylem fibres with narrow lumen occupy major portion of wood and, xylem, parenchyma is usually predominantly paratracheal, forming a sheath around the, vessels. Wood consists of crystal fibres having prismatic crystals of calcium, oxalate. A few tracheids with scalariform thickening and some cells including, vessels are also present., Chemical Constituents, Black catechu contains tannic acid (7.5-35%), catechin (10-15%), acacatechin, (2-12%), phlobatannin (25-35%), gum (20-30%), and quercitrin. Minor, constituents of black catechu are quercitin, catechu red, and moisture., Acacatechin contains ( –) epicatechin which is the trans-form of acacatechin., During the extraction of heartwood chips with boiled water, epicatechin, undergoes epimerisation and racemisation to dl,-acacatechin., Chemical Tests, Black catechu gives the chemical tests illustrated in table 5.4:, Table 5.4: Chemical Tests of Black Catechu, Experiments, Observations, Results, 1) Gambier Fluorescent Test: Drug Petroleum layer does not Black catechu is present., is boiled with 2ml of ethyl al cohol, show greenish colour., added with 2ml of NaOH solution, followed by 2ml of petroleum ether,, contents are shaken and allowed to, settle., 2) Matchstick Test: Woody side of Magenta/purple, Black catechu may be, matchstick is dipped in solution of coloured is obtained., present, tannin, air dried, dipped in conc. HCl, and warmed over the flame of burner., 3) Vanillin Hydrochloride Test: The Pink or red colour is, Due to the formation of, powdered drug is added with a few observed., phloroglucinol, thus, drops of vanillin hydrochloride, indicating presence of, reagent., tannins., 4) Lime Water Test: Few drops of A brown colour is, Black catechu is present., fresh aqueous extract of the drug are produced and on, added to 10ml lime water., standing for 3 minutes a, precipitate is formed., 5) Ferric Ammonium Sulphate Test: Green colour changes to Black catechu is present., To an aqueous solution of drug (2%), purple colour., solution of ferric ammonium sulphate, is added ; after formation of green, colour sodium hydroxide is added., 6) Chlorophyll Test: The powdered No greenish residue is, Black catechu is present., drug is heated with chloroform in a seen in China dish., water bath for 1 -2 minutes. The, organic layer is filtered in a China, dish and evaporated to dryness in a, water bath., *, , *
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Secondary Metabolites - III (Chapter 5), , 135, , Uses, 1) Internal Uses, i) It possesses cooling and digestive properties, hence used in GIT, disturbance., ii) It is used in relaxed conditions of throat, mouth, and gums., iii) It is also used in cough and diarrhoea., 2) External Uses, i) It is used as an astringent (property to precipitate the body protein)., ii) It is applied to ulcers, boils and skin eruptions., 3) Industrial Uses: It is an ingredient of betel leaf (Paan) and paan masala, so it, is largely used in kattha industry., , 5.2.7.3., , Pterocarpus, , Pterocarpus is a genus of pantropical trees . It is having around 35 species, of, which P. marsupium is one of the most famous members of this genus., Synonyms, Bijasal, Indian kino tree, and Malabar kino., Biological Source, Pterocarpus is the dried juice of Pterocarpus marsupium Linn. The juice is, obtained from the stem and bark by making vertical incisions on them. The, exuding juice is collected and dried., Family, Leguminosae., Geographical Source, Pterocarpus is widely distributed in the hilly regions of, Gujarat, Madhya Pradesh, Uttar Pradesh, Bihar, and, Orissa. It is also cultivated in the forests of Karnal,, Kerala, West Bengal, and Assam., Macroscopic Features, 1) Colour: Ruby-red., 2) Odour: Odourless., 3) Taste: Astringent., 4) Size: 3-10mm granules., , Figure 5.18: Pterocarpus, Marsupium, , 5) Shape: Angular grains., 6) Solubility: Partially soluble in water (about 80 -90%); completely soluble in, alcohol (90%)., 7) Extra Features: Kino pieces are angular, glistening, transparent, break with, a vitreous fracture, sparingly soluble in water (about 80, -90%), and fully, soluble in alcohol (90%)., *, , *
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136, , Pharmacognosy and Phytochemistry-II, , Microscopic Features, Transverse section shows alternating bands of larger and smaller polygonal cells, comprising of tracheids, fiber tracheids, xylem parenchyma, and transversely, arranged xylem rays. Xylem vessels are distributed all over. Tyloses filled with, tannins are present. Tracheids are long,thick walled, have tapering ends and simple, pits. Xylem parenchyma comprises of rectangular cells with simple pits and xylem, rays are uni-to-biseriate. Calcium oxalate crystals are present and starch isabsent., Chemical Constituents, Kino is having around 70 -80% of kino tannic acid, kino-red, k-pyrocatechin, (catechol), resin, and gallic acid. Kino tannic acid is glucosidal tannin, and kinored is a kinoin anhydride. Kinoin i s an insoluble phlobaphene, which is produced, when acted upon by the oxidase enzyme. Its colour is darker than kinotannic acid., Chemical Tests, 1) On treating the solution of pterocarpus with ferrous sulphate, a green, colouration develops., 2) On treating the solution of pterocarpus with alkali (say potassium hydroxide),, a violet colouration develops., 3) On treating the solution of pterocarpus with mineral acid, a precipitate develops., Uses, 1) It acts as a powerful astringent., 2) It is used for treating diarrhoea, dysentery, and passive haemorrhage toothache., 3) The aqueous infusion of the wood is used in diabetes. The alcoholic as well, as the aqueous extract of the drug shows hypoglycaemic action., , 5.3. RESINS, 5.3.1., , Introduction, , The hydrocarbon secretion of various pla nts, especially the coniferous trees is, known as the resin which is considered valuable because of its chemical, constituents and various uses like adhesives, varnishes, perfumes, incense, nail, polish, etc. Apart from this, resins are also used as a raw material for synthesising, organic compounds. The fossilised resins act as a source of amber., Philosophers like Theophrastus of Greece and Pliny the Elder of Rome, mentioned the use of resins in the form of myrrh and frankincense. Since, they, are used in relig ious ceremonies as incense and perfume, they are quite costly., Like natural resins, synthetic resins also possess same properties, i.e., they are, viscous liquids that get hardened on cooling. The manufacturing of synthetic, resins is done by esterifying or soaping the organic compounds., Epoxy resin , a classic variety , is manufactured by using polymerisation, techniques such as polyaddition or polycondensation, which yields thermoset, polymer used as composites and adhesives., *, , *
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Secondary Metabolites - III (Chapter 5), , 137, , Since 1960s, epoxy resin is used for flooring purposes in industries because it is, double in strength when compared to concrete. It forms waterproof coating and, gives seamless finish. However, from 2000 AD epoxy and polyurethane resins, are used for coating interiors, especially in the Western Europe., Unsaturated polyester resin is another category of resins, which makes up, around 75% of the total resins used. Another important resin is the ion exchange, resin applied in purifying water and in catalysis of some specific organic, reactions. Another synthetic polymer is the acetal resin which in comparison to, other synthetics has a simple chain structure with repeated units of ―[CH2O]―., , 5.3.2., , Chemistry, , Resins are chemically composed of the complex mixtures of:, 1) Resin Acids: These are generally mixture of carboxylic acids, oxyacids, and, phenolic acids and are present in either free-state or as esters. Resin acids are, easily dissolved in alkaline solutions and on shaking produces froth (just like, soap). Resin acids include the following examples:, i) Abietic acid in rosin or colophony,, ii) Copavic acid and oxycopavic acid in copaiba,, iii) Guaiaconic acid in guaiac,, iv) Pimaric acid in frankincense,, v) Sandaracolic acid in sandarac,, vi) Aleuritic acid in shellac, and, vii) Commiphoric acid in myrrh., 2) Resin Alcohols: These have complex nature and high molecular weight. In, plants, they remain either in free -state or as esterified derivatives of aromatic, acids, e.g., salicylic acid, cinnamic acid, and benzoic acid. Resin alcohols can, be further divided into the following types:, i) Resinotannols: These are tannins which with ferric chloride gives blue, colouration. The examples of resinotannols are:, a) Aloeresinotannol from aloe,, b) Amoresinotannol and galbaresinotannol from ammoniac,, c) Peruresinotannol from balsam of Peru, and, d) Siaresinotannol and sumaresinotannol from benzoin., ii) Resinols: These do not contain tannins and their examples include:, a) Benzoresinol from benzoin,, b) Storesinol from storax, and, c) Guaiacresinol from guaiac resin., 3) Resenes: These are the neutral compounds wit h complex structures and do, not undergo any type of chemical reaction. They are unable to form salt or, esters as they are insoluble in acids or bases. Resenes can be exemplified as:, i) Alban and fluavil from gutta percha,, ii) Copal resin from copal,, iii) Dammaresene from dammar,, iv) Dracoresene from dragon‘s blood, and, v) Olibanoresene from olibanum., *, , *
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138, , Pharmacognosy and Phytochemistry-II, , 5.3.2.1., , Biogenesis, , Resins can be obtained from carbohydrate produced via photosynthesis. These, carbohydrates breakdown into pyruvate products (simpler compou, nds), from, which terpenoid and phenolic compounds can be synthesised via different, metabolic pathways (figure 5.19)., , 5.3.2.2., , Qualitative Analysis, , The resins can be qualitatively analysed as follows:, 1) Colophony Resin: The tests recommended for colophony resins are:, i) 100mg of resin powder is dissolved in 10ml acetic anhydride. To this, solution, a few drops of sulphuric acid is added which results in purple, violet colouration of the solution., ii) Powdered resin is agitated with petrole um ether and then filtered. To the, filtrate, double volume of copper acetate (dilute) solution is added and, again agitated. The solution obtained is allowed to stand for some time, after which the ethereal layer attains emerald green colour., 2) Guaiacum Resin : A solution of resin is prepared in ethanol and to this, solution ferric chloride is added. This results in deep blue colouration of the, solution., CO2, , Photosynthesis, , Primary Carbon Metabolism, , Erythro-4phosphate, , 3-Phosphoglycerate, , Phosphoenol pyruvate, , Pyruvate, Deoxyxylulose, 5-phosphate, , Acetyl CoA, , Shikimic Acid, Pathway, , Malonic Acid, Pathway, , Mevalonic, Acid Pathway, , Deoxyxylulose 5Phosphate Pathway, , Phenylalanine, Phenylpropanoid, Pathway, Terpenes, Phenolic, compounds, Secondary Carbon Metabolism, Figure 5.19: Generalised Outline of Biosynthesis of Terpenes and Phenolic, Secondary Compounds Constituting Resins, Showing Interconnection with, Relevant Primary Compounds and Processes., *, , *
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Secondary Metabolites - III (Chapter 5), , 5.3.2.3., , 139, , Quantitative Analysis, , Balsam and gum resin s can be estimated on the basis of quantity by the, determination of the following:, 1) Acid value,, 2) Ester value (direct or indirect),, 3) Saponification value, or resin value and gum value,, 4) Percentage of moisture,, 5) Ash,, 6) Percentage soluble in alcohol,, 7) Percentage insoluble in alcohol,, 8) Specific gravity, and, 9) Percentage soluble in other solvents., Some of them are discussed below:, 1) Acid Value: This value can be calculated either by direct titration or by back, titration:, i) By Direct Titration: Acid value by direct titration c an be calculated as, follows:, a) The resinous material to be estimated is dissolved completely in nonpolar solvent like alcohol, chloroform, etc., Performance: 1gm of substance to be titrated is dissolved in an, appropriate solvent, and is titrated against n or n alcoholic caustic, 2, , 10, , potash, using phenolphthalein as an indicator. The end point is, marked by the appearance of light pink colour., For example, such resins that are either soluble, or for which there is, no specific method for preparation, is titrated using this method., b) In case the resin is not completely soluble , its extract is prepared for, titration., Performance: The whole procedure is almost same as that of (a), but it, varies because the calculation is done by using 1gm extract instead of, 1gm crude product.For example, gum resins, benzoin,and storax., c) Such solutions, whose resin content can be extracted partially by, using water and alcohol separately, are used., Performance: Resin is finely grinded and its 1, gm portion is, extracted by boiling with 30ml water for 15 minutes using a reflux, condenser. In the next step, the extract is diluted using 50ml alc ohol, (96%) and then boiled for 15 minutes. On cooling, the extract is not, filtered and its titration is perfo, , rmed directly against n alcoholic, 2, , potassium hydroxide, using phenolphthalein as an indicator. The, endpoint is marked with the appearance of light pink colouration., For example, myrrh, bdellium, opopanax, and sagapenum., *, , *
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140, , Pharmacognosy and Phytochemistry-II, , ii) By Back Titratio n: Acid value by back titration can be calculated as, follows:, a) The resins that are either completely or partially soluble are free, from esters. In this process, the alkali dissolves the whole resin as, well as combines with the acid., Method: 1gm ester -free resin is finely divided and digested for 24, hours with n alcoholic potash (25ml) and benzene (50ml) in a, 2, , stoppered flask. The solution obtained is titrated against n sulphuric, 2, , acid using phenolphthalein as an indic ator. For example, dammar,, sandarach, mastic, guaiacum, copal, etc., b) The partially soluble resins are ast riferous and can be saponified, sparingly. In such resins, the alkali gets fixed with the acid and the, acidic constituents are extracted out., Method: The resin is finely powdered and 1gm of it is kept in, contact with n alcoholic potash (10ml) and n aqueous potash, 2, , 2, , (10ml) for 24 hours in a stoppered flask. The resulting solution is, diluted with 500ml of distilled w ater and its back titration is, performed. For example, asafoetida and olibanum., 2) Ester Value: This value can be determined indirectly by subtracting acid, value from saponification value. However, when a resin value and total, saponification value are present, ester value cannot be calculated., 3) Saponification Value:This value can be calculated either by hot or cold method:, i) By the Hot Method:, Saponification value by hot method can be, determined as follows:, a) Resins that are completely soluble are used., Method: The solution of resin (1gm) is prepared using an, appropriate solvent. This solution is mixed with 25ml of, , n, 2, , potash, , (alcoholic) and is boiled in steam bath for 30 minutes using reflux, condenser. The mixture is diluted using alcohol and, then titrated, against n sulphuric acid using phenolphthalein as an indicator. For, 2, , example, balsam sand resins for which no special methods have, been devised are used by this method., b) The alcoholic extract of resin that is soluble either, sparingly is prepared beforehand., , partially or, , Method: The same procedure as (a) is followed for this, except that, an alcoholic solution of the extract is taken and the results are, calculated for 1gm of crude drug, and not of the extract., For, example, gum resins, benzoin, and storax., *, , *
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Secondary Metabolites - III (Chapter 5), , 141, , ii) Cold Method: Saponification value by cold method can be calculated by, the following two ways:, a) Saponification number for the resins that are completely soluble is, calculated using cold alcoholic solution of alkali along with benzene., Method: 1gm resin is kept in 500ml stoppered glass flask along with, 50ml benzene (specific gravity 0.700 at 15°C.) and 50ml of, , n, 2, , potash (alcoholic). The mixture is allowed to stand for a day at room, temperature and then back t itrated using n sulphuric acid. For, 2, , example, in balsam of Peru, the whole procedure is kept the same, except the addition of 300ml of water to the solution before titration, for dissolving the precipitated salts. Copaiba balsam, benzoin, and, storax can also be used., b) In case of resins that are not perfectly soluble, their fractional, saponification value is calculated using resin value and gum value, with the help of alcoholic and aqueous alkali., Method: Two samples of resins (1gm each) are powdered and kept, in two different 1 litre stoppered flasks containing 50ml of petroleum, ether (specific gravity 0.7 at 15ºC) and 25ml of n potash (alcoholic)., 2, , The flask is stoppered and kept for 24 hours at 25 ºC with frequent, agitation. One of the samples is diluted using 500ml of distilled, water and then back, titrated against n sulphuric acid using, 2, , phenolphthalein as an indicator. This will infer the, Z). The second sample is subjected to treatmen, , resin value (H-, , t with 25ml of, , n, 2, , potash (aqueous) and 75ml of distilled water., The mixture is kept for 24 hours with frequent agitation. Finally this, mixture is diluted with 500ml of distilled water and back titrated, against, , n, sulphuric acid using phenolphthalein as an indicator., 2, , This will infer the total saponification value (G-V-Z). The difference, between total saponification value and resin value gives the, gum, value (G-Z). For example, ammoniacum, galbanum,and gamboge., 4) Melting-Point, Specific Gravity, Ash, Moisture, & Special Determinations:, Other quantitative measures that can be done are:, i) Solubility conditions,, ii) Melting point,, iii) Specific gravity,, iv) Ash value, and, v) Moisture., *, , *
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142, , Pharmacognosy and Phytochemistry-II, , Some special specific tests are also performed like, cinnamein in Peruvian, balsam, the carbonyl, methoxyl, acetyl values, and the investigation of resin, acids and resin alcohols., , 5.3.3., , Chemical Classes, , Resins can be classified, as follows on the basis of their occurrence, in, combination with other compounds:, 1) Balsams: These are resinous substances containing either free form or, combined form or esters of cinnamic and benzoic acids in large proportions., Some typical balsams containing plants are:, i) Tolu balsam,, ii) Benzoin,, iii) Peru balsam, and, iv) Storax., 2) Oleoresins: These resins occur along with the volatile oils. Some of the, plants that contain oleoresins are:, i) Turpentine,, ii) Capsicum,, iii) Ginger,, iv) Male fern,, v) Canada balsam, and, vi) Copaiba., 3) Gum Resins: These resins are present in plants along with the gums. Their, purification is done by dissolving the gum in water. Gum resins are found in, the following plants:, i) Asafoetida,, ii) Gamboge, and, iii) Myrrh., 4) Oleo-Gum Resins: These resins are present along with the gums and volatile, oil. Their purification is done by st, eam distillation which removes the, volatile oil, while removal of gum is done by dissolving in water. The typical, plants containing oleo-gum resins are:, i) Myrrh, and, ii) Frankincense., 5) Glycoresins (Glucoresins): These resins are present naturally, combined, with th e glycosides. Their h ydrolysis yields sugar as a glycine part, while, complex resins as an aglycone part. Glycoresins are present in the following, plants:, i) Ipomoea,, ii) Scammony,, iii) Jalap, and, iv) Podophyllum., *, , *
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Secondary Metabolites - III (Chapter 5), , 5.3.4., , 143, , Biosources, , Below are the examples of different plants whose different structures or, secretion zone contain resins:, 1) Ginger (Zingiber officinale Roscoe) belonging to family Zingiberaceae has, resin cells., 2) Pine Wood (Pinus pa lustris Miller) belonging to family Pinaceae contain, resins in the schizogenous ducts or schizolysigenous ducts or cavities., 3) Cannabis (Cannabis sativa Linn) belonging to family Moraceae contain, resins in their glandular hair., In plants, the formation of resins takes place as a result of their normal, physiological functions. Though, their yield can be increased in certain plants(like, pinus) by inducing a plant injury. Moreover, there are some plants that do not, produce resin on their own until and unless an injury to the plant is deliberately, made in the form of an incision. These incisions lead to oozing of plant secretions, and exudates which are carefully tapped,e.g., Balsam of Tolu and Peru., It can be said that these resins are produced because of pathological conditions., There is a theory which suggests that resins obtained naturally from the secretory, structures are considered as primary flow ; while the one obtained by artificial, means, manmade incisions, or abnormally formed structures are secondary flow., Usually, the production of resins takes place in the ducts and cavities present in, the plant. In some plants, resins are not present in the especially modified, secretory structures but get imbibed in the adjoining tissues,, for example,, Guaiacum resin is obtained from the heartwood of Guaiacum officinale Linn, and, Guaiacum sanctum Linn., ( Zygophyllaceae family), which means they are, present in vessels (xylem and phloem), fibres, parenchyma, medullary rays, etc., In this case, the conduction system of plants or trees is chopped off to obtain, resin in the form of tyloses. This chopping enhances the utilisation of root, pressure and capillary action to force the nutrition and water for reaching the top, end of tall trees. There are rare cases in which the resins are obtained by the, sucking of plant juice by scale, insects. This juice is then converted into a, resinous substance which covers the insect body and plant twigs,, e.g., Shellac, (Laccifer lacca) belonging to family Coccidae., , 5.3.5., , Therapeutic Uses, , Most of the plants, especially th e coniferous trees secrete a gummy hydrocarbon, called the resin, which is quite useful and valuable because of its use in the field, of medicine and for various other purposes, such as:, 1) Drugs comprising resins include:, i) Podophyllum acts as a purgative,, ii) Colocynth, gamboge, ipomoea have cathartic action,, iii) Asafoetida acts as a laxative,, iv) Jalap has hydragogue properties,, v) Cannabis acts as a sedative,, *, , *
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144, , Pharmacognosy and Phytochemistry-II, , vi) Capsicum and turpentine have counter-irritant property,, vii) Asphidium shows anthelmintic action, and, viii) White pine, copaiba, storax, T olu balsam, benzoin, etc. are used as, expectorants., 2) Resins possess mild antiseptic property; hence they are used topically in the, form of plaster, ointments, and cerates., 3) They are also used for preparing emulsions., , 5.3.6., , Commercial Applications, , Resins can also be used for other miscellaneous uses such as:, 1) They act as adhesives., 2) For synthesising organic substances, resins act as valuable raw material., 3) Fossilised resins are considered to be excellent source of amber., 4) Resins are also used in industries related to incense and perfumes., 5) They are also used in cosmetics like nail polish., 6) Resins like copals, mastic, dammars, and sandarac are hard and transparent,, therefore are used in varnishes and cement., 7) For increasin g friction of stringed instruments such as violin, rebec, erhu,, sarangi, etc., rosin (a form of resin) is applied to the bows of such instruments., 8) At the archaeological sites, conservators use resins for joining delicate and, fragile substances like bones. The reason behind using resin is that they bind, the fragile material at molecular level., , 5.3.7., , Individual Drugs, , The pharmacognostic profile of the following drugs has been discussed below:, 1) Benzoin,, 2) Guggul,, 3) Ginger,, 4) Asafoetida,, 5) Myrrh, and, 6) Colophony., , 5.3.7.1., , Benzoin, , Benzoin has four varieties and their names are Sumatra, Palembang, Penang,, and Siam benzoin . They are easily identified by their specific look. Sumatra,, Penang and Siam benzoin are the three derivatives which are derived from three, different plants., Synonyms, Sumatra Benzoin and Loban., Biological Source, Benzoin occurs in two forms, i.e., Sumatra and Siam. The balsamic resin is, derived from Styrax benzoin Dryand or Styrax paralleloneurus Perkins and, another species of Styrax marketed under the n ame of Sumatra Benzoin. It may, *, , *
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Secondary Metabolites - III (Chapter 5), , 145, , also have the balsamic resin obtained from Styrax tonkinensis and other species, known as Siam Benzoin on a commercial scale. It should contain not less than, 25% of total balsamic acids calculated in terms of dry alcohol soluble matter., Family, Styracaceae., Geographical Source, Sumatra benzoin is obtained from the trees grown in south-eastern Asia; while, Siam benzoin is obtained from the trees grown in Thailand and Vietnam., Macroscopic Features, 1) Sumatra Benzoin, i) Colour: Greyish-brown or grey., ii) Odour: Aromatic and characteristic., iii) Taste: Sweetish and slightly acrid., iv) Shape: Lumps of varying sizes or tears, whi ch are yellowish and milky, white in colour., v) Surface: Uneven., vi) Extra Feature: On heating, it produces fumes of benzoic and ci nnamic, acids., 2) Siam Benzoin, i) Colour: Yellowish-brown to rusty-brown., ii) Odour: Agreeable and vanilla-like., iii) Taste: Sweetish and slightly acrid., iv) Shape: Hard and brittle masses., v) Extra Feature: On heating, it softens into plastic., Chemical Constituents, Benzoin contains free balsamic acids and esters of balsamic acids., Sumatra Benzoin: This variety of benzoin has benzoic acid (18% or more) and, cinnamic acid (20%). Cinnamic acid is partially free and partially combined, with benzoresinol and sumarisinotannol. It als o has vanillin (1%), styrol,, styracin, phenyl -propyl cinnamate, and benzaldehyde. With the combination of, these constituents, Sumatra benzoin gets its specific odour., , *, , *
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146, , Pharmacognosy and Phytochemistry-II, , Siam Benzoin: Benzoic acid (38%) is the main component of this variety of, benzoin. It is partially free and partially combined with, benzoresinol and, siaresinotannol. It also consists of vanillin and an oily aromatic liquid. In its, pure form, it should be completely soluble in alcohol and form traces of ash., Chemical Tests, 1) On t reating its alcoholic solution with water, a milky white solution, develops., 2) A small quantity of benzoin is heated in a test tube and the test tube mouth is, covered with a glass plate. The contents of the tube are cooled and on, viewing the glass plate under a microscope, cinnamic acid crystals should be, observed., 3) 2.5gm benzoin is shaken with 10ml ether. 2 -3ml of this extract is poured in, porcelain dish and added with 2 -3 drops of sulphuric acid. A deep brown, colour develops for Sumatra benzoin and a deep purplish red colour develops, for Siam benzoin., 4) 4ml potassium permanganate solution is warmed with 1gm benzoin. Odour, of benzaldehyde is produced for Sumatra benzoin., Uses, 1) It acts as an irritating expectorant, a carminative, and diuretic., 2) It is used externally as an antiseptic., 3) It is used in the form of compound tincture of benzoin, and as an inhalation, for treating upper respiratory tract infection., 4) It is used to delay the rancidity of fats and oil in the preparation of benzoate, lard., Adulterants, L. styraciflua (or sweet gum) is the American variety and is confused because its, product, obtained by exudation, is called liquidambar, liquid storax, or copalm, balsam. It contains cinnamyl cinnamate, with ethyl, benzyl, and other esters of, cinnamic acid., Its other products are obtained by boiling the young branches, and include liquid, storax. It is used in Texas for cough . A syrup of its bark is used in the Western, States for diarrhoea and dysentery., L. storesin is said to be known in Eastern markets., Aromatic resins are obtained from, L. f ormosana in China , and from L., altingea (Altingia excelsa) in Java and Burma, where the s torax-like substances, are white to red in colour., Styrea reticulata and other species in Brazil secrete a fragrant substance (similar, to benzoin), which is used in churches as frankincense., The most common adulterants are sawdust and turpentine., *, , *
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Secondary Metabolites - III (Chapter 5), , 147, , Difference between Sumatra and Siam Benzoin, Table 5.5: Difference between Sumatra and Siam Benzoin, Basis, Sumatra Benzoin, Siam Benzoin, Styrax benzoin Dryand or Styrax tonkinensis, 1) Biological Source, Styrax paralleloneurus, Perkins., South Eastern Asia, chiefly Thailand and Vietnam, 2) Geographical Source, Sumatra, 3) Physical Properties, i) Colour, Greyish brown or grey, Yellowish brown to, rusty brown, ii) Odour, Aromatic and characteristic Aromatic and vanilla Sweetish and slightly acrid like Sweetish and, iii) Taste, slightly acrid, iv) Occurrence, , Occurs as lumps of varying Occurs as hard and, sizes or tears, brittle masses., , 4) Standards, i) Benzoic acid content, Not less than 60%, ii) Alcohol soluble matter, Not less than 76%, 5) Chemical Test, i) 2.5g b enzoin + 10ml, Deep brown colour, ether + sulphuric acid, ii) 4ml solution of potassium Odour of benzaldehyde is, permanganate + 1g, m produced., benzoin + warm the, mixture, , 5.3.7.2., , Not less than 12%, Not less than 90%, Deep purplish red, colour, No reaction, , Guggul, , Guggul is obtained from the sap (gum resin) which exudes from the Indian tree, Commiphora wightii. In Ayurveda, the uses of guggul are mentioned that dates, back to 600 BC. Ancient li terature reveals the use of guggul in treatment of, atherosclerosis., Synonyms, Guggal, Palankasha, and Devadhupa., Biological Source, Guggul is an oleogum-resin (oleoresin) that exudes spontaneously as a result of, injury from the bark of the plant Commiphora wightii., Family, Burseraceae., Geographical Source, The guggul plant may be found from northern Africa to central Asia, but is most, commonly found in northern India., *, , *
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148, , Pharmacognosy and Phytochemistry-II, , Macroscopic Features, 1) Plant: Branched, thorny, dimorphic, and bisexual., 2) Leaves: Small like that of neem and serrate, obovat e, 1-5cm long, and 0.5 2.5cm broad., 3) Flowers: Red or sometimes pink in colour., 4) Fruits: Pulpy and ovate in shape., 5) Juice: Thick, aromatous, inflammable, and melts in sun. When it is, immersed in warm water, it attains milky white colour., 6) Colour: Brown to pale yellow or dull green., 7) Odour: Agreeable, aromatic, and balsamic., 8) Taste: Bitter and characteristic., Chemical Constituents, Guggul comprises of steroids, diterpenoids, aliphatic esters, amino acids, etc. in a, complex mixture. Some phytochemicals like guggulsterone I, II, III, IV, V, Zguggulsterone, E-guggulsterone, and 16--hydroxyprogesterone are also, characterised. Mukulol (a type of alcohol) is also reported to be present in guggul., , Chemical Test, An extract of guggul is prepared us ing ethyl acetate. To this extract, acetic, anhydride is added and boiled. On cooling the resultant solution, 2ml of H 2SO4 is, added which results in the formation of green colour at the junction of liquid due, to the presence of sterols, Uses, 1) As per the Ayurveda, oleogum resin obtained from guggul is used in various, disorders like obesity, arthritis, and rheumatism., *, , *
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Secondary Metabolites - III (Chapter 5), , 149, , 2) It is also used as anti-inflammatory and hypo-cholesteremic drug., 3) Recent scientific researchers have confirmed the anti -hyperlipidemic activity, of guggul in semi -purified form wh ich is patented in the name of Gugulip, and is marketed by Cipla., Adulterants, Guggul can be adulterated with a variety of species of Commiphora such as C., abyssinica, C. roxburghii , C. molmol , and also with Boswellia serra ta that, contains lesser amount and inferior quality of phytochemicals., , 5.3.7.3., , Ginger, , Ginger is an herbal drug which is used in medi cines and also as cookery spice. It, is in use in medical conditions from ancient India and China., Synonyms, Zingiber and Zingiberis., Biological Source, Ginger is rhizomes of Zingiber officinale, Roscoe. Its outer skin is removed by, scraping, after which it is dried in the sun., Its market name is Jamaica ginger., Family, Zingiberaceae., , Node, , Leaf-scar, Scar left by, branch, Bud, Figure 5.20: Ginger Rhizome with, Buds, , Geographical Source, Ginger is nativ e to South East Asia, though it is cultivated in Caribbean islands,, Africa, Australia, Mauritius, Jamaica, Taiwan , and India. More than 35% of, world‘s ginger is produced in India., Macroscopic Features, 1) Colour: Externally buff coloured., 2) Odour: Agreeable and aromatic., 3) Taste: Agreeable and pungent., 4) Size: Rhizomes are 5 to 15×1.5 to 6.5cm., 5) Shape: Rhizomes are laterally compressed, bear short flat, ovate and oblique, branches on the upper side, with bud at the apex., 6) Fracture: Short and fibrous., 7) Surface: Longitudinally striated with occasional projecting fibres., 8) Extra Features: Transverse section shows well-marked endodermis and stele., Microscopic Features, 1) Cork: It comprises of irregularly arranged cells, followed by cortex., 2) Cortex: It comprises of thin walled parenchymatous tissue. It is, distinguished from stele by a w, ell-marked endodermis. Many closed, collateral fibro-vascular bundles are enclosed within the cortical tissue., *, , *
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150, , Pharmacognosy and Phytochemistry-II, , 3) Vascular Bundles: They are present just inside the endodermis and are free, of fibres., 4) Endodermis is free of starch, and oleo -resinous cells and starch grains are, found all over the ground tissue., Chemical Constituents, Ginger contains 1 -4% of volatile oil , 40 60% of starch, 10% of fat, 5% of fibre,, 6% of, inorganic material , 10% of, residual moisture , and 5 -8% of acrid, resinous matter . Ginger oil contains, monoterpene hydrocarbons, ,, sesquiterpene hydrocarbons, oxygenated, mono and sesquiterpenes,, and, phenylpropanoids., All types of ginger oil have, the same, sesquiterpene hydrocarbon content and, contain -zingiberene, -bisabolene, farnesene, -sesquiphellandrene, and curcumene., , Outer cork, Inner cork, Cortex, Starch, , Fibres, Oleo-resin, Cork, Endodermis, Ground tissue, Vascular bundle, Cortex, Figure 5.21: T.S. of Ginger Rhizome, , The main characters of ginger are aroma a nd flavour., CH3, Its a roma is because of the, fragrant principles of, volatile oils, whereas the flavour, pungency and, pharmacological actions are produced by the phenolic, CH3, ketones of oleo-resin. Several volatile oil components, such as isometric terpenic aldehydes like geranial and, citral, produces mild and lemony aroma. Few, CH3, CH3, sesquiterpene oil hydrocarbons produce, the spicy, Zingiberene, flavour., Phenolic ketones of oleo -resin include gingerols such as shogaols, zingerone,, paradols, gingediols hexahydrocurcumin, and their O-methyl ethers., Chemical Test, To 1gm of pulverised ginger, 5ml of dilute acetic acid (prepared by diluting 1, part of glacial ac etic acid with 1 part of water) is added, and shake n for 15, minutes. The resultant mixture is f iltered. A few drops of ammonium oxalate TS, is added to the filtrate, which gives rise to a slight turbidity., Uses, 1) It is used as an anti-emetic, and its aromatic , carminative and absorbent, properties improve the effects of motion sickness directly in the GI tract. It is, also used in controlling vomiting and nausea in hyperemesis gravidarum and, in post-operative nausea and vomiting., 2) Gingerols and shogaols show cardiovascular activity by the inhibition of, prostaglandin synthetase, and hence hindering prostaglandin biosynthesis., *, , *
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Secondary Metabolites - III (Chapter 5), , 151, , 3) It possesses antiplatelet aggregation property because of the inhibition of, thromboxane synthesis., 4) It is also used for lowering cholesterol levels by inhibiting cholesterol, biosynthesis., 5) It also shows antitussive, anti-pyretic and analgesic properties., 6) Its rhizomes show fungicidal, antibacterial and anthelmintic properties., 7) Sesquiterpene hydrocarbons are the reason for its anti-ulcer activity., Adulterants, As ginger grows underground, it gets contaminated with worms and insect, remains. It can also be adulterated with other vegetable adulterants, and this can, be detected by microscopic examination. Adulteration of ginger can also be done, with spent ginger, which is a ginger from which the essence has been extracted,, due to which it produces lower than official standards for extracts. For increasing, pungency, ginger is sometimes adulterated with capsicum or grains of paradise., , 5.3.7.4., , Asafoetida, , The name a safoetida has been derived from the Latin word foetid which means, smelly. Asafoetida is a genus of perennial herbs. It has a very distinct, pungent, odour. It is a spice used as a digestive aid, in food as a condiment, and making, pickles., Synonyms, Gum asafoetida and Devil‘s dung., Biological Source, Asafoetida is the oleo -gum resin obtained by incision from the rhizomes and, roots of Ferula foetida., Family, Umbelliferae., Geographical Source, Asafoetida is found to be distributed from Mediterranean region to Central Asia,, especially in Iran and Afghanistan. Three species of Ferula are found in India., Ferula narthex occurs in Kashmir., Macroscopic Features, 1) Colour: Yellowish-white changing to reddish-brown., 2) Odour: Intense, persistent, penetrating, and alliaceous., 3) Taste: Bitter, alliaceous, and acidic., 4) Shape: Occurs in 2 different forms, i.e., tears and masses. Tears are rounded, or flattened., 5) Size: Tears are 0.5-3cm in diameter., 6) Extra Features: Fresh tears are tough, which on drying becomes hard and, brittle. The inner surface of tears is milky whitish, -yellow, translucent, or, opaque. Mass of asafoetida is agglutinated and mixed with root fragments,, foreign materials, and other impurities., *, , *
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152, , Pharmacognosy and Phytochemistry-II, , Chemical Constituents, Asafoetida contains resin (40-65%), gum (20-25%), and volatile oil (4-20%)., The chief resin of asafoetida is asaresinotannol present either in free form or is, combined with ferulic acid . Asafoetida does not contain free umbelliferone., Ferulic acid when treated with hydrochloric acid, con verts into umbellic acid,, which forms umbelliferone by losing water., HCl, , Ferulic Acid, , Umbellic Acid, , –H2O, , Umbelliferone, , Asafoetida oil is obtained by steam distillation of the oleo -gum resin. The oil, contains secondary butyl propenyl disulphide as its chief constituent., However, di- and tri-sulphides, pinene, and other terpenes are also present in the, oil. The sulphur compounds of the formulae C 7H14S2, C 16H20S2, and C 10H18S2, imparts odour to asafoetida., OCH3, HO, , HO, , CH═CHCOOH, Ferulic Acid, , OH, , CH═CHCOOH, Umbellic Acid, , HO, , O, CH ═ CH CO, Umbelliferone, , Chemical Tests, 1) On treating its fractured surface with sulphuric acid, a red or reddish -brown, colour appears., 2) On treating with 50% nitric acid, it yields a green colour., 3) On triturating with water, it forms a yellowish-orange emulsion., 4) Around 0.5gm of asafoetida is triturated with sand and 5ml of hydrochloric, acid. To the resultant mixture, a small amount of water is added, followed by, filtration. On adding an equal volume of ammonia to the obtained filtrate, a, blue fluorescence appears indicating the presence of umbelliferone., 5) On adding 50% nitric acid to the fracture surface, a green colour appears., 6) Combined Umbelliferone Test:If the drug is in tear form, it is triturated with, sand. About 0.5gm of the drug is boiled with 3ml of hydrochloric acid and 3ml, of water for 5-10 minutes. The resultant mixture is filtered,and an equal volume, of alcohol and excess of strong solution of ammonia are addedto the filtrate. A, blue fluorescenceappears due to the presence of combined umbelliferone., Uses, 1) Sometimes it is used as an antispasmodic, carminative, expectorant, and, laxative., 2) It is also a powerful nerv ing stimulant, used in nervous disorders related to, hysteria., 3) It is also used as intestinal flatulence., 4) Its 2% (w/v) suspension is used as a repellant against dogs, cats, deer,, rabbits, etc., 5) It is used in veterinary to apply over the bandages of dogs so th, ey do not, chew them., *, , *
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Secondary Metabolites - III (Chapter 5), , 153, , Adulterant, Asafoetida is adulterated with gum arabic, rosin, gypsum, red clay, chalk, and, barley or wheat flour., , 5.3.7.5., , Myrrh, , Myrrh is a sap -like resin which is obtained by making incisions in the bark of, trees that belongs to the members of the Commiphora species., Synonyms, Gum, Bol, and Myrrha., Biological Source, Myrrh is an oleo -gum-resin obtained from Commiphora molmol Engler and also, from other Commiphora species., Family, Burseraceae., Geographical Source, Myrrh is widely distributed in North East Africa and Southern Arabia., Macroscopic Features, 1) Colour: Reddish-brown externally and brown, internally., 2) Odour and Taste: Aromatic and agreeable., 3) Size: 3m in height and 1.5-3.0cm in diameter., 4) Shape: Occurs as round or irregular tears., 5) Extra Features: Fractured surface is slightly, granular; fragile and has a translucent surface;, whitish spots are present on the broken pieces., , Figure 5.22: Commiphora, molmol Herb, , Microscopic Features, The bark of Commiphora species consists of:, 1) Outer sclerenchymatous cells., 2) Beneath the sclerenchyma cells, thin walled parenchyma cells are present., 3) Towards the internal surface of the bark , schizolysigenous ducts are present, which consists of resinous secretions., 4) These ducts are intersper sed among the, parenchyma is a part of 2° phloem)., , phloem parenchyma (phloem, , 5) Medullary or vascular rays (made partly of xylem and partly of phloem), are also present in the interior of the bark. These medullary rays are biseriate, and extend towards the pith (parenchymatous cells with intercellular spaces), by passing through fibres made of sclerenchyma., *, , *
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154, , Pharmacognosy and Phytochemistry-II, , Sclerenchyma cells, , Thin walled parenchyma cells, Phloem parenchyma cells, , Resin duct filled with, resinous secretion, Secretory cell, , Schizolysigenous, cavity, , Biseriate medullary rays, Fibres of sclerenchymatous cells, Pith with intercellular spaces, Figure 5.23: Transverse Section of Commiphora (Myrrh) Bark, , Chemical Constituents, Myrrh contains yellowish thick volatile oil (10%), gum (60%), resin (25-40%),, and bitter principle (3-4%). It has many impurities which make up around 5%, of the drug. Ether-soluble resin acids (, , and commiphora acids) are found, in the resin portion of myrrh; the and heerabomyrrholic acids are ether insoluble. Terpenes, cuminic aldehyde, eugenol, etc. are found in the volatile oil, of myrrh. The gum is associated with oxidase enzyme., Chemical Tests, 1) 0.1gm of the drug and 0.5gm of sand are triturated with solvent ether. The, resultant is filtered and evaporated to attain a thin film, which on being, treated with bromine vapours produces violet colour., 2) On triturating the drug with water, a yellowish-brown emulsion is formed., Uses, 1) It is used as a stimulant and an antiseptic., 2) It acts as a protective agent., 3) It is astringent to the mucous membrane; therefore, its tincture is used in, mouthwashes and gargles., Adulterants, It is adulterated with Arabian myrrh, Yemen myrrh, etc., species. Both these, species are less fragrant and less aromatic. Myrrh in India is adulterated with, Balsamodendron mukul, called as Indian bdellium., , 5.3.7.6., , Colophony, , Colophony is a tr anslucent, hard substance and is produced from pi ne oleoresin., It is used particularly in inks and varnishes, and it is also used on the bows of, stringed instruments., *, , *
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Secondary Metabolites - III (Chapter 5), , 155, , Synonym, Rosin, Gandha birojaa (Hindi) and Gandha bihrojaa (Urdu)., Biological Source, Colophony is a yellow resin and abietic anhydride. It, is the residue that remains after distilling the volatile, oil from the oleoresin , which is obtain ed from Pinus, palustris and various other species of Pinus., Generally, it is obtained as wood rosin from s outhern, pine stumps, gum rosin collected as the exudate from, incisions in the living tree, viz., P., palustris and P. caribaea, and finally from tall oil, Figure 5.24: Pinus palustris, rosin., Family, Pinaceae., Geographical Source, The genus Pinus is distributed in United States, France, I taly, Portugal, Spain,, Greece, New Zealand, India (Himalaya n region), China and Pakistan. The c hief, producer of colophony is United States, contributing 80% of world supply of, colophony., Macroscopic Features, Colophony appears as translucent, hard, shiny,, sharp, pale yellow to amber, coloured fragments. Its fracture is brittle at normal temperature. It produces a, smoky flame when burned. It has a turpentine-like odour and taste. It easily melts, on heating. Its density is 1.07-1.09. It is insoluble in water; and readily soluble in, alcohol, benzene, ether, glacial acetic acid oil, carbon disulphide, and alkali, solutions., Microscopic Features, Translucent, pale yellow to brownish -yellow coloured, angular, irregularly shaped, brittle and glassy pieces of different sizes of conchoidal markings appear, on its surface when viewed under a microscope., Chemical Constituents, Colophony contains about 90% of resin acids , fatty oil esters , and resenes., About 90% of the resin acids are isomeric (-, -, and -abietic acids), and the, remaining 10% is a mixture of dehydroabietic acid and dihydroabietic acid., Before distillation, resin comprises of high, quantity of (+) and ( –) pimaric, acids., During distillation, (–) pimaric acid converts into abietic acid and (+) pimeric, acid becomes stable. The other constituents of colophony are sinapinic acid and a, hydrocarbon., *, , *
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156, , Pharmacognosy and Phytochemistry-II, , Abietic acid, , Pimaric acid, , Chemical Tests, 1) A solution of 0.1gm powdered resin in 10ml acetic acid is taken in a dry test, tube and added with a drop of conc. s ulphuric acid. A purple colour appears,, which readily changes to violet., 2) The petroleum-ether solution of powdered colophony is shaken with twice its, volume of dilute solution of copper acetate. The colour of petroleum, -ether layer, changes to emerald-green due tothe formation of copper salt of abietic acid., 3) The alcoholic solution of c olophony is added with sufficient water. The, mixture turns milky white due to the precipitation of chemical compounds., 4) The alcoholic solution of c olophony turn s blue litmus to red due to the, presence of diterpenic acids., Uses, 1) It is used as a hardening agent in ointments, adhesives, plasters and cerates., 2) It is used as a diuretic in veterinary medicines., 3) It is commercially used in manufacturing varnishes, printin g inks, cements,, soap, sealing wax, floor coverings, papers, wood polishes, plastics,, fireworks, tree wax, rosin oil, and for cardboard water proofing., 4) Abietic acids of colophony, exhibit antimicrobial, antiul cer and, cardiovascular activity., 5) Some of them have filmogenic, surfactant, and anti-feedant properties., , 5.4. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Volatile oils, are basically the mixture of hydrocarbon terpenes,, sesquiterpenes, and polyterpenes and their oxygenated derivatives., 2) Volatile oils carry the plant‘s active constituents and hence are also called, essential oils., 3) Volatile or ethereal oils are defined as, ‗odorous volatile principles of, plant and animal origin which evaporate when exposed to air at, ordinary temperature‘., 4) Mentha oil is obtained by the steam distillation of fresh flowering tops of the, plant Mentha piperita Linn. The oil is rectified if required. Mentha oil should, contain not less than 50% of total menthol., *, , *
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Secondary Metabolites - III (Chapter 5), , 157, , 5) Clove is the dried flowe r buds of the plant Eugenia caryophyllus and should, contain 15% (v/w) or more of clove oil., 6) Cinnamon is the dried inner bark of the shoots of coppiced trees of, Cinnamomum zeylanicum Nees., (syn. Cinnamomum verum ) and should, contain 1.0% or more of volatile oil., 7) Fennel is the dried, ripe fruits of the plant Foeniculum vulgare Mill., 8) Coriander is the fully dried ripe fruits of the plant Coriandrum sativum., 9) The term tannin was first applied by Seguin in 1976 to denote substances, present in plan t extracts which are able to combine with proteins of animal, hides, prevent their putrefaction and convert them into leather., 10) Tannins are defined as, ‗Complex substances that occur as mixtures of, polyphenols that are very difficult to separate since they d, o not, crystallise‘., 11) Pale catechu is an extract prepared from the leaves and young shoots of, plant Uncaria gambier Roxburg., 12) Black catechu is dried aqueous extract obtained from the heart wood of, plant Acacia catechu., 13) Pterocarpus is a genus of pantropical tr ees. It is having around 35 species,, of which P. marsupium is one of the most famous members of this genus., 14) The hydrocarbon secretion of various plants, especially the coniferous trees, is known as the resin which is considered valuable because of its che mical, constituents and various uses like adhesives, varnishes, perfumes, incense,, nail polish, etc., 15) Benzoin occurs in two forms, i.e., Sumatra and Siam. The balsamic resin is, derived from Styrax benzoin Dryand or Styrax paralleloneurus Perkins and, another species of Styrax marketed under the name of Sumatra Benzoin., 16) Benzoin should contain not less than 25% of total balsamic acids calculated, in terms of dry alcohol soluble matter., 17) Guggul is obtained from the sap (gum resin) which exudes from the Indian, tree Commiphora wightii., 18) Ginger is rhizomes of Zingiber officinale Roscoe. Its outer skin is removed, by scraping, after which it is dried in the sun. Its market name is, Jamaica, ginger., 19) Asafoetida is the oleo-gum resin obtained by incision from the rhizomes and, roots of Ferula foetida., 20) Myrrh is an oleo-gum-resin obtained from Commiphora molmol Engler and, also from other Commiphora species., 21) Colophony is a yellow resin and abietic anhydride. It is the residue that, remains after distilling the volatile oil from the oleoresin, which is obtained, from Pinus palustris and various other species of Pinus., *, , *
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158, , Pharmacognosy and Phytochemistry-II, , 5.5. EXERCISE, 5.5.1., , True or False, , 1) Black catechu is dried aqueous extract obtained from the heart wood of plant Acacia, catechu., 2) Guggul is obtained from sap of Ferula foetida., 3) Colpermin is the synonym of black catechu., 4) The family of black catechu is leguminosae., 5) Resins can be obtained from carbohydrate produced via photosynthesis., , 5.5.2., 6), 7), 8), 9), 10), , Fill in the Blanks, , The family of benzoin is ___________., Devadhupa is the synonym of __________., __________ occurs in two forms, i.e., Sumatra and Siam., Fennel is obtained from __________., The hydrocarbon secretion of various plants, especially the coniferous trees is known, as ___________., , Answers, 1) True, 2) False, 5) True, 6) Styracaceae, 9) Foeniculum vulgare 10) Resin, , 5.5.3., 1), 2), 3), 4), 5), 6), , 4) True, 8) Benzoin, , Very Short Answer Type Questions, , Discuss volatile oils., Give the qualitative analysis of volatile oils., Write a short note on tannins., Give the biological source of benzoin., What are the macroscopic features of pale catechu?, Give the biogenesis of resins., , 5.5.4., 1), 2), 3), 4), , 3) False, 7) Guggul, , Short Answer Type Questions, , What are resins? Give their chemical classification., Write a note on guggul., Give the qualitative analysis of resins., Discuss the isolation of volatile oils., , 5.5.5., , Long Answer Type Questions, , 1) Write a detailed note on resins., 2) Write a note on cinnamon., 3) Give the qualitative and quantitative analysis of tannins., , *, , *
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Secondary Metabolites - IV (Chapter 6), , CHAPTER, 6, , 159, , Secondary Metabolites - IV, , 6.1. GLYCOSIDES, 6.1.1., , Introduction, , In a glycoside molecule, sugar group is bonded through its anomeric carbon to, another group via glycosidic bond. A glycosidic bond is a covalent bond that, binds the sugar molecule to another mol ecule (th at may or may not be sugar, molecule). Thus, a substance containing a glycosidic bond, is called a, glycoside. On acidic hydrolysis, the glycone and aglycone portions can be, chemically separated. There are also numerous enzymes that can form and break, glycosidic bonds., Thus, in general, glycosides may be defined as „ organic compounds of plants, or animal origin which on enzymatic or acid hydrolysis give one or more, sugar moieties along with non-sugar moiety‟. The sugar group is known as the, glycone and the non-sugar group as aglycone or genin part of the glycoside. The, glycone can consist of a single sugar group (monosaccharide) or several sugar, groups (oligosaccharide). The sugars found in glycosides may be glucose and, rhamnose (monosaccharides) or more rarely , deoxysugars such as the cymarose, found in cardiac glycosides., , 6.1.2., , Chemistry, , Due to the presence of optical activity, glycosides exists in two forms, i.e., α- and, β-glycosides, e.g., methyl-D-glucosides, these α and β structures may be, represented as shown below in figures 6.1 and 6.2:, , Figure 6.1: Methyl-α-D-Glucoside, , Figure 6.2: Methyl-β-D-Glucoside, *, , *
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160, , Pharmacognosy and Phytochemistry-II, , The figures shown above represent the open-chain structure, cyclic structure, and, boat configuration of methyl -α-D-glucoside ( figure 6.1) and methyl -β-Dglucoside ( figure 6.2). T he glycosidic linkage, in the above example, is, established when the hydroxy group of the aglycone part (i.e., methyl alcohol), and the hydroxyl group on the hemiacetal carbon of the carbohydrate undergo, dehydration, thus, forming an acetal type of structur, e. Configuration of, glycosides:, 1) α-Configuration: The glycosidic structure is in α-configuration if the –OCH3, moiety (or –OR') is in opposite steric sense as the–CH2OH moiety on C-5., 2) β-Configuration: The glycosidic structure is of, β-configuration if the, OCH3 moiety is in the same steric sense as the CH2OH group on C-5., , 6.1.2.1., , –, , Biogenesis, , Formation of glycosides involves two steps:, 1) The first step involves the reactions resulting in the formation of aglycones,, and, 2) The second step involves the metabolic pathway inv olving coupling of, aglycones with sugar moiety., To synthesise O-glycosides found in plant cells, the nucleotide glycoside (such, as UDP -glucose) and the alcoholic or phenolic group of the second moiety, (aglycones) interact with each other. The other naturally occurring glycosides are, C-glycosides (involving the linkage is through carbon), N -glycosides (linkage, through nitrogen), or S-glycosides (linkage through sulphur)., The major pathway of glycoside formation involves the following steps:, 1) In the first st ep, uridylyl group is transferred from, Uridine Triphosphate, (UTP) to sugar -1-phosphate in the presence of uridylyl transferases (the, catalysing enzyme)., , 2) In the second step, sugar is transferred from Uridine Diphosphate (UD P) to, aglycone moiety in the presence of glycosyltransferases, thus, forming a, glycoside., , The glycosides may contain either of the types of sugars:, 1) Monosaccharides (rhamnose, glucose, and fructose), or, 2) Deoxysugars (digitoxose or cymarose) as in cardiac glycosides., , 6.1.2.2., , Qualitative Analysis, , Glycosides are the compounds with organic molecules having attached glucose, or any mono -oligo saccharide unit. Usually, these are crystalline or amo rphous, solids; opti cally active, soluble in water and alcohol , but insoluble in organic, solvents like ether, chloroform, and benzene, etc. Generally, aqueous or alcoholic, extracts of crude drugs are tested with specific reagents for the presence of, various types of glycosides., *, , *
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Secondary Metabolites - IV (Chapter 6), , 161, , The tests are as follows:, 1) Chemical Tests for Anthraquinone Glycosides, i) Borntrager’s Test: 1gm of drug is boiled with 5-10ml of dilute HCl on, a water bath for 10 minutes. The mixture is then filtered. The fil trate is, extracted with CCl 4/benzene, and an equal amount of ammonia solution, is added to the filtrate and shake n. A pink or red colour forms in the, ammoniacal layer due to the presence of anthraquinone moiety., ii) Modified Borntrager’s Test: 1gm of drug is boiled with 5ml dilute HCl, and 5ml ferric chloride (5%w/v) for 10 minute s on a water bath. The, mixture is then cooled and filtered. The filtrate is extracted with carbon, CCl4/benzene, and an equal volume of ammonia solution is added to the, filtrate. A pink to red colour forms due to the presence of anthraquinone, moiety. This test is used for C-type of anthraquinone glycosides., 2) Chemical Tests for Saponin Glycosides, i) Haemolysis Test: A drop of blood is taken on a slide and mixed with a, few dro ps of aqueous saponin solution. The, RBCs ruptu re in the, presence of saponins., ii) Foam Test: 1gm of drug is shaken with 10, -20ml of waterfor a few minutes., Frothing occurs thatpersists for1-2 minutes due to the presence of saponins., 3) Chemical Tests for Steroid and Triterpenoid Glycosides, i) Liebermann-Burchard Test: The alcoholic extract of drug is evaporated, to dryness and extracted with CHCl 3. A few drops of acetic anhydr ide, and conc. H 2SO4 are added from the sidewall of the test tube to the, CHCl3 extract. A violet to blue coloured ring forms at the junction of two, liquid, indicating the presence of steroidal moiety., ii) Salkowaski Test: The alcoholic extract of drug is evaporated to dryness, and extracted with CHCl 3 add conc. H 2SO4 from the sidewall of the test, tube to the CHCl 3 extract. A yellow coloured ring forms at the junction, of two liquid, which tur ns red after 2 minutes, indicating the presence of, steroidal moiety., iii) Antimony Trichloride Test: The alcoholic extract of drug is evaporated, to dryness and extracted with CHCl3. Saturated solution of SbCl3 is added, to the CHCl3 extract containing 20% acetic anhydride.Pink colour develops, on heating, which indicates the presence of steroids and triterpenoids., iv) Trichloro Acetic Acid Test : Triterpenes on addition of saturated, solution of trichloroacetic acid forms coloured precipitate., v) Tetranitro Methane Test : A yellow colour appears with unsaturated, steroids and triterpenes., vi) Zimmermann Test : Meta dinitrobenzene solution is added to the, alcoholic solution of dru g containing alkali. On h eating, it develops, violet colour in the presence of ketosteroid., 4) Chemical Tests for Cardiac Glycosides, i) Keller-Kiliani Test : To the alcoholic extract of drug equal volume of, water and 0.5ml of strong lead acetate solution, is added, shaken and, *, , *
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162, , Pharmacognosy and Phytochemistry-II, , filtered. The f iltrate is extracted with equal volume of chloroform. The, chloroform extract is evaporated to dryness and residue is dissolved in, 3ml of glacial acetic acid followed by addition of few drops of FeCl, 3, solution. The resultant so lution is transferred to a test tube contain ing, 2ml of conc. H2SO4. A reddish brown layer is formed, which turns bluish, green after standing due to the presence of digitoxose., ii) Legal Test: To the alcoholic extract of drug , equal volume of water and, 0.5ml of strong lead acetate solution is added, shaken, and filtered. The, filtrate is extracted with equal volume of chloroform, and the chloroform, extract is evaporated to dryness. The residue is dissolved in 2ml of, pyridine and 2ml of sodium nitroprusside is added followed by addition, of NaOH solution to make the residue alkaline. Pink colour develops in, the presence of glycosides or aglycone moiety., iii) Baljet Test : Thick section of leaf of digitalis or the part of dru, g, containing cardiac glycoside when di pped in sodium picrate solution, forms yellow to orange colour in the presence of aglycones or glycosides., iv) 3,5-Dinitro Benzoic Acid Test: To the alcoholic solution of drug , a few, drops of NaOH followed by 2% solution of 3,5 -dinitro benzoic acid are, added. Pink colour develops indicating the presence of cardiac glycosides., 5) Chemical Tests for Coumarin Glycosides, i) FeCl3 Test: To the concentrated alcoholic extract of drug, a few drops of, alcoholic FeCl3 solution are added. Deep green colour forms which turns, yellow on addition of conc. HNO3, indicating the presence of coumarins., ii) Fluorescence Test : The alcoholic extract of drug is mixed with 1N, NaOH solution (one ml each). Development of blu e-green fluorescence, indicates the presence of coumarins., 6) Chemical Tests for Cynophoric Glycoside Sodium Picrate Test:, Powdered drug is moistened with water in a conical flask and few drops of, conc. sulphuric acid are added. Filter paper impregnated with sodium picrate, solution followed by sodium carbonate solution is trapped on the flask neck, using cork. Brick red colour develops due to volatile HCN in the presence of, cynophoric glycosides., 7) Chemical Tests for Flavonoid Glycosides, i) Ammonia Test : Filter paper dipped in alcoholic solution of drug, is, exposed to ammonia vapour. A yellow spot appears on the filter paper., ii) Shinoda Test: To the alcoholic extract of drug,magnesium turnings and dil., HCl are added. Redcolour develops indicatingthe presence of flavonoids., iii) Vanillin HCl Test: Vanillin HCl is added to the alcoholic solution of, drug. Pink colour develops due to the presence of flavonoids., , 6.1.2.3., , Quantitative Analysis, , The therapeutically active glycosides are analysed quantitatively in biological, sample for assay sensitivity as well as for removing interfering substances. This, involves enrichment by extraction and then purific, ation by some specific, *, , *
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Secondary Metabolites - IV (Chapter 6), , 163, , techniques like column or layer chromatography. The, steps of quantitative, analysis of glycosides (e.g., digitalis) are:, 1) Principle: Digitalis (a cardiac glycoside) on reaction with Baljet reagent, (picric acid in alkaline medium) give s an orange -red colour. The intensity, (absorbance) of orange -red colour is proportional to concentration, of, glycosides in the given sample., 2) Materials: The materials required for quantitatively analysing digitalis are:, i) Digitalis tincture, and, ii) Freshly prepared Baljet reagent (95ml of 1% picric acid + 5ml of 10%, NaOH). This reagent is mixed prior to use and filtered through a sintered, glass funnel., 3) Preparation of Tincture: A 10% extract of digitalis is shaken with 70%, alcohol. To this mixture, 1gm of dried powder of digitalis leaves and 20ml of, 70% alcohol are added. The preparation is allowed to stand overnight with, occasional shaking for 2 hours and then filtered., 4) Procedure: The steps involved are:, i) Tincture Purification, a) Around 8ml of digitalis extract or ti ncture is transferred to a 100ml, volumetric flask. In this volumetric flask, 60ml of distilled water and, 10ml of 12.5% lead acetate is also added to precipitate resins, tannins, and pigments. The mixture is shaken well and volume is made with, distilled wate r up to 100ml. Thereafter, the mixture is again mixed, followed by filtration through a filter paper., b) 50ml of the filtrate is pipette d out and transferred to another 100ml, volumetric flask. In this flask, 10ml of 4.77% disodium hydrogen, phosphate (Na 2HPO4) solution is also added to precipitate the Pb ++, ions present in excess amount. The mixture obtained is mixed well, and volume is made with distilled water., c) Thereafter, the mixture is again mixed followed by filtration through, a filter paper. If a non -clear f iltrate is obtained, the solution is re filtered using the same filter paper., ii) Spectrophotometric Determination, a) 10ml of purified and, clear filtrate is transferred, , to a clean dry, stoppered Erlenmeyer flask (conical flask), followed by the addition, of 10ml of Baljet reagent. A blank spectrophotometric determination, is carried out using a suitable spectrophotometer., b) The mixture is left undisturbed for an hour so that it gets sufficient, time for maximum colour development. Thereafter, the solution is, diluted with 20ml of distilled water using automatic burette., c) Spectrophotometric determination of this solution is done and its, colour intensity is observed against the blank at 495nm. The colour, obtained remains unchanged for a few hours., The difference betw een exp eriment and blank (E −B) = original, reading, , *, , *
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164, , Pharmacognosy and Phytochemistry-II, , 5) Calculation: While assaying digitalis tincture, 10ml of 10% tincture, (obtained from 0.8gm powdered leaves) is diluted to 200ml during, purification. From this, 10ml of the solution (around 0.04gm powdered, leaves) is treated with 10ml of Baljet reagent and diluted with 20ml of water, (to a total of 40ml), and then utilised in the assay., Let absorbance of the tincture at 495nm be A. Then, the concentration (%), will be Ag % . This value of concentration is the amount of, glycosides, 170, , present per 100ml of the measured solution., Thus, Percentage of glycosides, , , , A 40 100, g% of total glycosid es, 170 100 0.04, , calculated as digitoxin, and, Percentage of total glycosides A 100 g% of total glycosides calculated as, 17, , digitoxin., Note: In this assay, cardiac glycosides are extracted from digitalis using 70%, alcohol which prevents hydrolysis by enzymatic action and simultaneously, extracts the lowest amount of undesirable materials., , 6) Standard Curve Preparation for Digitoxin, i) „A‟ of digitoxin solution of 0.02% is prepared in chloroform-methanol, (1: 1v/v) mixture (1ml = 0.2mg)., ii) Different volumes of this solution are prepared as 1, 2, 3, 4 and 5ml, (equivalent to 0.2, 0.4, 0.6, 0.8 and 1mg of digitoxin, respectively). Each, volume is transferred to a dry conical flask., iii) The flasks are placed on a water bath to evaporate the solvent present in, each. The residue obtained in each flask is dissolved in 0.35ml of 90%, alcohol. Thereafter, using a graduated pipette volume is made with distilled water up to 10ml., iv) 10ml of freshly prepared Baljet reagent is added to each flask, which is, then left undisturbed at room temperature for an hour. Thereafter, the, solution in each flask is diluted with 20ml of distilled water and a, spectrophotometric determination is carried out at 495nm to record the, absorbance (A) of the orange-red colour developed., v) This determination is carried out against a blank in which 10ml of, distilled water is used instead of digitoxin solution., vi) Three determinations are done for each concentration whose mean value, is calculated to plot a standard curve with the concentration against, absorbance (A)., , 6.1.3., , Chemical Classes, , The glycosides can be classified on the basis of properties of chemical mo, present:, 1) On the basis of glycone,, 2) On the basis of aglycone, and, 3) On the basis of glycosidic linkage., *, , iety, , *
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Secondary Metabolites - IV (Chapter 6), , 6.1.6., , 167, , Individual Drugs, , The pharmacognostic profile of the following drugs has been discussed below:, 1) Senna,, 2) Aloes, and, 3) Bitter almond., , 6.1.6.1., , Senna, , Senna is a herb used as a laxative. It is also known as, wild senna , Cassia, marilandica, or locust plant. In the ancient times, the Arabian physicians used, senna as cathartics., Synonyms, Cassia senna, Folia senna, and Tinnevelles senna., Biological Source, Senna is the dried leaflets of Cassia angustifolia., Family, Leguminosae., Geographical Source, Cassia senna is found to be grown in tropical Africa an d Sudan, while Cassia, angustifolia is native to Arabia, Somalia, Sind, and the Punjab (parts of India)., Macroscopic Features, 1) Colour: Yellowish-green., 2) Odour: Slight., 3) Taste: Mucilaginous, bitter, and characteristic., 4) Size: 7-8mm in width and 25-60mm in length., 5) Shape: Leaves are lanceolate, entire; apex is acute with spine at the top. Base, of the leaflets are asymmetrical with transverse lines, more prominent on, lower surface, while trichomes are present on both the surfaces., , Transverse, line, , Figure 6.3: Indian Senna Twig, , Figure 6.4: Indian Senna Leaflets, , Microscopic Features, Senna represents the typical histological characters of isobilateral leaf in which, the epidermis shows the presence of unicellular, conical, thick -walled warty, trichomes. Trichomes are slightly curved at their bases and are present on both, *, , *
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Secondary Metabolites - IV (Chapter 6), , 169, , Senna leaf also contains other anthraquinone glycosides in small amounts. They, are sennosides C and D, rhein-8-glucoside, rhein-8-diglucoside, aloe-emodin,, 8-glucoside, aloe-emodin, anthrone diglucoside , kaempferol, aloe-emodin,, and isorhamnetin. It also contains phytosterol, mucilage, resin, myricyl alcohol,, salicylic acid, chrysophanic acid, and calcium oxalate., Chemical Tests, 1) Borntrager Test: The leaves are boiled with dilute sulphuric acid and, filtered. The filtrate is added and shaken with organic solvent like benzene,, ether or chloroform. The organic layers are separated and to it ammonia, solution is added. The, ammoniacal layer produces pink to red colour, indicating the presence of anthraquinone glycoside., 2) Little amount of senna leaves is e xtracted with water by boiling and then, filtered. The filtrate is treated with 5N NaOH and sodium hyposulphite. On, heating the mixture, red colour develops., Uses, 1) It is mainly used as a purgative., 2) It is used as a stimulant cathartic., 3) It is also used in manufacturing sennosides., Substitutes, 1) Senna Pods: These are fruits of Cassia acutifolia; occasionally found mixed, with the senna, and imported as a separate article of commerce; very flat, legumes., 2) Cassia Obovata (Dog Senna): It was formerly highly valued as a drug, and, cultivated in Italy and sometimes termed Italian senna ; leaves are broadly, obovate, apex abruptly tapering venation, pinnate, distinct; constituents are, similar to those of senna; total oxymethylanthraquinones 3.8%. The leaves, are sometimes mixed with broken Alexandrian senna., 3) Argel Leaves: These leaves resemble senna in colour and outline, but are, distinguished by their thick, rigid texture and peculiarly curled, curved, or, twisted appearance; finely wrinkled surface; veins not evident; leaf e qual at, the base; three -celled hairs. Taste is distinctly bitter. These were formerly, regularly mixed with senna, but now are of rare occurrence., Following are the other plants which are also imported as senna or have, occurred in commercial senna:, 1) Cassia Holosericea: These are smaller, more obtuse and hairy., 2) Cassia Montana: These have darker, rounded apex,and dark network of veins., 3) Tephrosia Apollinea: It is obovate -oblong, pubescent, emarginate lateral, veins straight and parallel; fruits narrow-cylindrical; recently (1918) found in, Alexandrian senna., 4) Colutea Arborescens: It is green and is very thin., 5) Globularia Alypum: It has spatulate, rounded apex, and mucronate., 6) Coriaria Myrtifolia: It is ovate-lanceolate, greyish-green, has two prominent, lateral veins and conspicuous midrib., *, , *
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170, , Pharmacognosy and Phytochemistry-II, , Adulterants, 1) Cassia brevipes,, 2) Cassia pubescens, and, 3) Solenostemma argel., , 6.1.6.2., , Aloes, , Aloe vera is a plant species of the Aloe genus. It grows wildly in tropical climates, and is also cultivated due to its agricultural and medicinal values., Synonyms, Mussabar, Kumari, and Aloes., Biological Source, Aloe vera is the dried juice obtained from the leaves, of various species of Aloe like:, 1) Aloe barbadensis Miller (or Curacao Aloe),, 2) Aloe ferox Miller (or Cape Aloe),, 3) Aloe perryi Baker (or Socotrine Aloe), and, 4) Aloe africana Miller and Aloe spicata Baker (or Cape Aloe)., Aloe vera is one of the most familiar of all the medicinal herbs. Out of more than, three hundred species of aloe, only four are believed to have medicinal, properties. The most pote nt medicinal effects are believed to come from Aloe, barbadensis, which is frequently produced in gel form., Family, Liliaceae., Geographical Source, 1) Curacao, Barbados, Aruba: Curacao aloes or Barbados aloes and Bonaire, (West Indian Islands)., 2) Cape Town (South Africa): Cape aloes., 3) Socotrine and Zanzibar Islands: Socotrine or Zanzibar aloes., It is also cultivated in Europe and the North West Himalayan region in India., , Figure 6.6: Aloe Plant, *, , *
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Secondary Metabolites - IV (Chapter 6), , 171, , Preparation, 1) Cape aloes are prepared from wild plants of A. ferox and its hybri ds. The, leaves are cut transversely near the base and about 200 of them are arranged, around a shallow hole in the ground, which is lined with plastic sheeting or, more traditionally a piece of canvas or a goatskin., The leaves are arranged such that the cu t ends overlap and drain freely into, the canvas. After about 6 hours when all the juice has been collected, it is, transferred to a drum or paraffin tin and boiled for 4 hours on an open fire., The product is poured while still hot into tins, each holding 25 kg, where it, solidifies. For export the tins are placed in cases holding two, four or eight, tins., 2) Barbados (Curacao) aloes are prepared from cultivated plants of, A., barbadensis. The cut leaves are stacked in V -shaped troughs, arranged on a, slope so that th e juice flows from a hole at one end of the trough into a, collecting vessel. When sufficient juice has been collected, it is evaporated in, a copper vessel., The temperature used is generally lower than in the case of Cape aloes and, the product is, therefo re, usually opaque, although some which is semi, transparent may be produc ed and is known in commerce as, Capey, Barbados., Originally Barbados and Curacao aloes were packed in gourds, now seen, only in museums. The present -day drug is exported in cases each h olding, about 58.5kg., 3) Aloe juice obtained from Socotrine aloes is collected in goat or sheep skin, and allowed to evaporate for about a month till a viscous pasty mass is, obtained., This mass is send by the Arab traders to Zanzibar or Mumbai where they are, packed in barrels, tins or leather packing and then exported to Europe. After, reaching the European countries, it is dried in wooden pans with hot air till, 10% moisture remains behind., Macroscopic Features, , *, , Properties Curacao Aloes, , Cape Aloes, , Socotrine Aloes, , Zanzibar Aloes, , Colour, , Brownish black, opaque mass, , Dark brown or, greenish brown to, olive brown mass, , Brownish yellow, opaque mass, , Liver brown, , Odour, , Strong odour, that resembles, iodoform, , Sour and distinct, odour, , Unpleasant, , Characteristic but, agreeable, , Taste, , Intensely bitter, , Nauseating and, bitter, , Extremely bitter, and nauseous, , Bitter, , Texture, , Waxy and, resinous, , Breaks with a, glassy fraction, , Fractured surface Dull, waxy, smooth,, looks conchoidal and even fracture, *
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172, , Pharmacognosy and Phytochemistry-II, Stomata, Cuticle, , Epidermis, Palisade, Parenchyma, Calcium oxalate, , Aloetic juice, Mucilaginous, parenchyma, , Pericycle, Vascular bundle, Figure 6.7: T. S. of Aloe Leaf, , Microscopic Features, T.S. of th e leaf exhibits ( figure 6.7) outermost cuticle followed by epidermis,, palisade tissue, and mucilaginous parenchymatous mesophyll . Mesophyll, encloses vascular bundles , which are covered with, pericycle. Inside the, pericycle, few large elongated thin walled aloetic cells are located. These cells, contain highly viscous, yellow juice (aloe gel). The leaves are sessile and when, they are cut from the base, the juice flows down which is to be collected. A few, calcium oxalate crystals are present in the parenchyma., The microscopy of different varieties of aloe is more significant for identification, of their powdered forms. The microscopic characters are studied in lactophenol,, because the particles in this reagent are gradually solubilised so that crystals are, clearly observed:, 1) Curacao Aloes: It shows fragments consisting of a large number of very, small needles or slender prisms., 2) Cape Aloes: This variety appears as transparent, brown, angular or irregular, fragments., 3) Socotrine Aloes: It is characterised by fragments consisting of quite large, prisms either present in group or in dispersed form., , Zanzibar, , Cape, , Curacao, , Socotrine, , Figure 6.8: Microscopic Characters of Aloe Powder, , Commercially, aloes are available as both crystalline and amorphous substance., It generally appears that amorphous form is cape aloe and crystalline one is, curacao al oe, depending on their response to the tests. If both the varieties are, mounted in cresol and viewed under polarised light, crystalline aloe, insoluble in, cresol shows a shining bright colour. Amorphous aloe, as it gets quickly, dissolved, remains invisible., *, , *
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Secondary Metabolites - IV (Chapter 6), , 173, , Chemical Constituents, Aloe is made up of 30% of aloin which is a mixture of barbaloin, β-barbaloin,, and isobarbaloin isomers. Barbaloin is a crystalline glycoside which is present in, all the four varieties of aloe . It is slightly yellow -coloured, bitter in taste, and, water-soluble. Barbaloin is a C-glycoside compared to common O-glycosides., , β-barbaloin is amorphous in nature and is present in Cape aloe. It can be obtained, by heating barbaloin. Isobarbaloin exists in crystalline form and is prese, nt in, Curacao and, Cape aloe (in trace amounts), but absent in Socotrine and Zanzibar, aloe. It is a mixture of barbaloin and polyphenols (responsible for its colour tests)., Aloinsides A and B, O -glycosides of aloin in whi ch L -rhamnose is combined, with −OH of hydroxymethyl group at 11 -C atom are also present in aloe. Aloeemodin anthrone and glucose are the hydrolysis products of barbaloin. Aloeemodin anthranol and aloe-emodin are also present in aloes. It also contains a, resin which is an ester of p-coumaric acid or p-hydroxy cinnamic acid esterified, with aloeresinotannol., Chemical Tests, 1) Schonteten’s or Borax Test: To 5ml of aqueous solution of aloe , borax, (0.2gm) is added and heated to dissolve completely., A f ew drops of this, liquid are poured in a test, tube filled with water. A green fluorescence, develops., 2) Bromine Test : A pale yellow precipitate of tetrabromaloin is formed on, adding bromine to aqueous solution of aloe., 3) Nitric Acid Test: On addition of nitric acid (2ml) to a solution of aloe (5ml),, the following changes occur:, i) Cape aloes form a brown colour which changes rapidly to green., ii) Curacao aloes give a deep brownish-red colour., iii) Socotrine aloes give a pale brownish-yellow colour., iv) Zanzibar aloes give a yellow-brown colour., 4) Nitrous Acid Test : On adding sodium nitrite crystals and small amount of, acetic acid to 5ml of aloe solution, the following changes occur:, i) Curacao Aloes: A sharp pink to car mine colour develops due to the, presence of isobarbaloin., ii) Cape Aloes: A faint pink colourdevelops due to the presence ofisobarbaloin., iii) Socotrine and Zanzibar aloes: A comparatively lesser change in colour, is observed., *, , *
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174, , Pharmacognosy and Phytochemistry-II, , 5) Klunge’s Isobarbaloin Test: On adding a drop of saturated solution of, copper sulphate, immediately followed by 1gm of NaCl and 20 drops of, ethanol (90% v/v) to 10ml of a 0.4% (w/v) aqueous solution of aloe, different, shades of colours depending on the variety of aloes used develops:, i) Curacao Aloes: A wine red colour lasting for a few hours., ii) Cape Aloes: A faint colouration that quickly changes to yellow., iii) Socotrine Aloes: No colouration., iv) Zanzibar Aloes: No colouration., 6) Modified Anthraquinone or Borntrager’s or Cupraloin Test: 0.1gm of, aloe is boiled with dilute HCl and 5ml of 5% solution of FeCl 3 for 5 minutes., The solution is cooled, filtered, and the filtrate is shaken with benzene. The, benzene layer is separated; ammonia solution is added to the resultant, mixture producing a pink colouration., Uses, 1) It is used as a purgative because of its intensely irritating effects on the, delicate mucosal lining., 2) It is one of the ingredients of compound benzoin tincture in which it is a, pharmaceutical adjunct., 3) It causes gripping, and therefore is administered with carminatives., 4) Ointment of aloe gel is used in the treatment of skin irritations, burns caused, by sun, heat or radiation., Substitutes and Adulterants, A. candelabrum (Natal aloes) is a dull greenish black to dull brown in col our,, and opaque. When scraped , it gives a pale greyish green or a yellow powder. It, can be distinguished as it responds negatively to borax test and produces a deep, blue colour. Jafferabad aloes and Mocha aloes are the other two type of aloe,, which are used as adulterants., , 6.1.6.3., , Bitter Almond, , Bitter almonds are used as food flavouring agent, almond are found in middle East and Asia., , s or in oils. Trees of bitter, , Synonym, Amygdala amara., Biological Source, Bitter almond is the dried ripe kerne ls of Prunus amygdalus Batch var. amara, (DC.) Focke; Prunus communis Arcang; P. amyg dalus Bail ; and Amygdalus, communis Linn., Family, Rosaceae., Geographical Source, Bitter almond tree s are native to Persia and Asia Minor. They are cultivated in, cooler clima te such as in Italy, Sicily, Portugal , Spain, Southern France , and, Morocco. In India, they are produced in cooler areas of Punjab and Kashmir., *, , *
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Secondary Metabolites - IV (Chapter 6), , 175, , Macroscopic Features, 1) Colour: Brown., 2) Odour: Odourless., 3) Taste: Bitter., 4) Size: About 20mm in length, 125mm in width and 10mm in thickness., 5) Shape: Flattened, oblong, ovoid in shape with markings on testa., , Entire seed Kernel, , Kernel cut, Bitter almond herb, transversely, Figure 6.9: Bitter Almond, , Chemical Constituents, Bitter almond oil contains 80% of benzaldehyde and about 2 to 6% of, hydrocyanic acid. Bitter almonds have 40 -50% of bland fixed oil and 20%, protein. It also co ntains emulsion enzyme and 1-3% amygdalin (a colourless, crystalline bitter glycoside ). About 0.5 % of volatile oil is also present in bitter, almond. Amygdalin hydrolyses in presence of water and, decomposes int o, benzaldehyde (80%) and hydrocyanic acid (2-6%). Bitter almond is not, suitable for internal consumption because, of being highly poisonous due to, hydrocyanic acid. Amygdalin on the contrary is not present in sweet almonds;, therefore, they do not have bitter taste, and do not yield volatile oil on hydrolysis., , Chemical Tests, 1) Ferric Ferrocyanide Test: 1gm of powdered drug is macerated with 5ml of, alcoholic KOH (5% w/v) for 5 minutes. The resultant mixture is transferred, to an aqueous solution containing FeSO 4 (2.5% w/v) and FeCl3 (1% w/v),, and is maintained at 60 -70°C temperature for 10 minutes. Thereafter, the, contents are transferred to HCl (20%) and a distinct Prussian blue colour, appears indicating the presence of HCN., 2) Precipitation of Hg from HgNO 3: The aqueous mercurou s nitrate solution, (3% w/v) reduces to metallic Hg in the presence of HCN, forming a black, metallic Hg in the cells., 3) Grignard Reaction Test:A strip of white filter paper is dipped into picric acid, solution (1% w/v in water), drained, again dipped in sodium carbonate solution, (10% w/v in water), and drained. The crushed and moistened drug material is, taken in a small Erlenmeyer flask. Thestrip of prepared sodium picrate paperis, suspended above the material,and the flask is stoppered with an air tight cock., The flask is kept in a warm place for an hour when the liberated HCN changes, the colour of sodium picrate paper from yellow to brick red becaus e of the, formation of sodium isopurpurate (this is theGrignard‟s reaction)., *, , *
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176, , Pharmacognosy and Phytochemistry-II, , 4) Cuprocyanate Test: Filter paper pieces are saturated with freshly prepared, solution of guai ac resi n dissolved in absolute ethanol, and allowed to dry, completely in air. Then a piece of this filter paper is moistened with a dilute, solution of CuSO 4 and placed in contact with the freshly exposed surface of, the drug. If HCN is produced, a distinct stain appears on the paper., Uses, 1) Due to the presence of hydrocyanic acid, it is used as a sedative., 2) Its oil is used in demulcent skin lotion., 3) It is also used in preparation of amygdalin, bitter almond water, perfumes ,, and liquors., , 6.2. TERPENOIDS, 6.2.1., , Introduction, , The volatile and aromatic substances present in plants and giving them their, characteristic fragrance or odour are terpenoids. Generally, most of the terpenoids, are a group o f compounds present naturally in plants with some exceptions (i.e.,, obtained from other sources) including civet musk, etc. In plants, terpenoids are, present in leaves and fruits of higher plants, conifers, citrus plants, and other plants., Volatile liquid, turpentine obtained from pine trees yields terpene on isolation., The sap and tissues of some specific plants and trees yield simpler, mono and, sesquiterpenes which are the major constituents of essential oils. The other, derivatives of terpenoids like di- and triterpenoids are obtained from gums and, resins of trees and plants, and do not get volatilised by steam., Carotenoids are another group of compounds formed by the tetraterpenoids., The term terpene was coined to be used initially for the mixture of isom, eric, hydrocarbons (molecular formula C 10H16) present in the essential oils yielded, from tissues and sap of trees and plants. However, the general term now, employed is terpenoids containing hydrocarbons, as well as its oxygenated, derivatives. Most recently terpenoids have been defined as, „ hydrocarbons of, plant origin of the general formula (C 5H8)n as well as their oxygenated,, hydrogenated and dehydrogenated derivatives‟., , 6.2.2., , Chemistry, , In 1887, Wallach proposed Isoprene Rule that helps in illustrating the chemistry, of terpenoids. According to this rule, terpenoids are chemical compounds whose, carbon skeleton is made up of isoprene units joined head -to-head or tail -to-tail, (rarest) or head -to-tail (most common) . Hence, isoprene (2 -methylbuta-1, 3 diene) are considered as the building blocks or structural unit of terpenoids., , *, , *
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Secondary Metabolites - IV (Chapter 6), , 177, , Isoprene rule can be applied only to the skeletal unit of carbon. The carbon, skeletons of open chain monoterpenoids and sesquiterpenoids are shown below:, , Below are the examples of terpenoids:, , Ingold, in 1921 came out with a conclusion that terpenoids, ‟ stability gets, affected by a gem alkyl group and is summarised as gem dialkyl rule. According, to this rule, the pres ence of gem dialkyl group destabilises the cyclohexane ring,, but provides stability to 3 -, 4- and 5-membered rings. This rule has limitation as, it limits the number of possible structures in closing the open chain to ring, structure, therefore, the open cha in mono-terpenoids results in only one structure, of monocyclic monoterpenoid (structure of p-cymene)., , P-cymene structure, , The bicyclic monoterpenoids comprise of 2 rings made up of 6- and 3-membered, carbons. Therefore, a 10 -membered open carbon chain of monoterpenoid, on, closure results into 3 bicyclic structures., , Camphor (6 + 5) Pinane (6 + 4) Carane (6 + 3), system, system, system, , 6.2.2.1., , Biogenesis, , Terpenoids are the secondary metabolites synthesised by plants, fungi, and, marine organisms by conjoining the head and tail of different isoprene units., They are also found in animal kingdom and in fossils and rocks. The following, steps are involved in the biosynthesis of terpenoids (figure 6.10):, *, , *
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178, , Pharmacognosy and Phytochemistry-II, , (2), , (1), (2), , (3), , (4, ), , (5), , (6, ), (7), , (8), , (9), , (8), (7), , (11), , (10), , Figure 6.10: Biosynthesis of Terpenoids, , 1) In this step, glucose is formed via photosynthesis using 6 molecules of, carbon dioxide and oxygen. The glucose so formed undergoes glycolysis to, form pyruvate (1)., 2) The pyruvate changes into acetyl coenzyme -A (2). Further formation of, acetoacetyl coenzyme -A (3) takes place by conjunction of two acetyl, coenzyme molecules., 3) Reaction b etween acetoacetyl -coenzyme-A and another molecule of acetyl, coenzyme-A results in hydroxyl methyl glutaric acid (4). This is the key intermediate for 3R-Mevalonic Acid (MVA) (5)., 4) MVA obtained undergoes phosphorylation by ATP to give mevalonic acid, MVA-5-diphosphate (6)., *, , *
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180, , Pharmacognosy and Phytochemistry-II, Air Dried/Pulverised Plant Material, Extraction with Organic Solvents*, (By Percolation or Soxhlet Extraction Methods), , Petroleum Ether, Extract, , Benzene, Ether and, Chloroform Extract, , Ethyl acetate and, Acetone Extract, , Mono and sesquiterpenoids,, less polar flavonoids,, Carotenes, di- and triterpene, hydrocarbons, fats and, waxes, , Sesquiterpene lactose,, diterpenes, sterols and, less polar tri-terpenoids, xanthophylls and, oxygenated flavonoids, , Oxygenated, diterpenoids,, sterols and triterpenoids, , Ethanol, Methanol, and Water Extract, , Highly oxygenated and, hence polar triterpenoids,, steroidal glycosides, free, sugars and amino acids, , *Extraction is perfo rmed with organic solvents with increasing polarity , e.g., petroleum ether, benzene,, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, methanol and water., , Isolation, As the presence of terpenoids is quite diversified in nature, there is no general, method that can be employed for isolating and separating terpenoids. Although,, mono- and sesquiterpenoids share common source (essential oils), and so their, isolation involves the following steps:, 1) Isolation of Essential Oils: The concentration of essential oils in plants, changes with time, so it is beneficial to extract essential oils at a specific time, (in case of jasmine it is done at sunset ). Generally, various methods have, been developed for extracting essential oils based on different principles., These methods include:, i) Expression Method: This method is now out -dated, and has only, historical importance. This method involves fi, ne chopping of plant, material, and then crushing to yield plant juice. This juice is subjected to, screening for getting rid of larger particles. Post -screening, the juice is, centrifuged in a high speed centrifuge. On obtaining half amount of the, oil, the residue along with the rest half of oil is subjected to distillation to, obtain an oil of low quality, e.g., lemon grass oil, oils of citrus plants., ii) Steam Distillation Method: This is the most widely used method in, which the plant material is macerated and t, hen subjected to steam, distillation to obtain essential oil containing distillate. Pure non, -polar, solvents (benzene, etc. ), which are later removed by distillation under, reduced pressure are used for extracting essential oils from the distillate., Because o f the following disadvantages, this method should be used, with proper precautions:, a) During steam distillation some essential oils may decompose., b) Some constituents of essential oils like esters (responsible for odour and, fragrance of the oil) may also decomp, ose giving a low quality perfume., iii) Extraction by Means of Volatile Solvents: In perfumeries, this method, is widely used. The plants giving low yield of essential oils, on steam, distillation due to their decomposition are subjected to this method. In, this method, the plant material at 50ºC temperature is treated with light, *, , *
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Secondary Metabolites - IV (Chapter 6), , 181, , petrol, which makes conditions favourable for the solvent to dissolve the, colouring material and oil for the vegetable matter. The solvent is, removed afterwards via distillation under reduce d pressure yielding the, essential oil in pure state., iv) Adsorption in Purified Fats: This is the French method and is called, enfleurage. It results in a high yield of essential oil. This method can be, employed for various plants for extracting essential oils like jasmine and, rose. This method involves warming the fat to 50ºC and then covering its, surface with petals of the flower. This is kept for several days till the fat, gets saturated with essential oils. The older petals are replaced with fresh, ones and th e process is continued several times. The petals are removed, and the fat is dissolved in ethanol along with the essential oil. The, amount of fat which remains with the alcohol is removed by cooling the, mixture at 20ºC. At this temperature, fat solidifies and separates out. The, alcohol is removed from the distillate via fractional distillation under, reduced pressure., Nowadays instead of fat, coconut charcoal is used as it has, a greater, stability and high adsorption capacity., Coconut charcoal is kept in, contact with the petals for a few days and then its steam distillation is, performed to yield essential oils., 2) Separation of Terpenoids from Essential Oils: Generally, there are various, terpenoids present in the essential oils separated in step (1), for which various, chemical and physical methods are employed:, i) Chemical Methods: The following chemical methods are now, considered obsolete for separating terpenoids from the essential oils:, a) Treatment of essential oils (containing terpenoid hydrocarbons) with, nitrosyl chloride in chloroform results in the formation of crystalline, adducts of hydrocarbons (having sharp melting points) which, decompose into their corresponding hydrocarbons., b) Treatment of essential oils (containing alcohols) with phthalic, anhydride to form diesters involves a rapid reaction of primary, alcohols with phthalic anhydride, whereas a slower reaction with, secondary and tertiary alcohols. After extracting with sodium, bicarbonate, diesters are decomposed by alkali to parent terpenoid ‟s, alcohols., c) Terpenoid aldehydes and ketones are separated from essential oils by, forming their adducts with the common carbonyl reagents (NaHSO 3,, 2,4-dinitrophenylhydrazine, phenyl hydrazine, semi -carbazide, etc.)., After separation, these are decomposed into terpenoid ald ehydes and, ketones., ii) Physical Method - Fractional Distillation: This method is employed, for separating terpenoids from the esse ntial oils. At first, terpenoids, hydrocarbons and then their oxygenated derivatives are distilled. The, residue obtained is distil, led under reduced pressure to yiel, d, sesquiterpenoids, which are separated by fractional distillation. At, *, , *
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182, , Pharmacognosy and Phytochemistry-II, , industrial l evel, specially designed stills are employed and an efficient, condensing system is required for reducing the loss of more volatile, hydrocarbons. There are cases when thermol abile and oxygen -sensitive, terpenoids are subjected to fractional distillation under reduced pressure, using inert gas., 3) Chromatographic Methods: Terpenoids can also be separated by the, following chromatography methods:, i) Gas-Liquid Chromatography (GLC): This chromatography technique, is suited well for both mono - and sesquiterpenoids because of their, property of volatility. In GLC method, nanogram quantities of terpenoid, can also be used for analysis by using capillary columns, and electron, capturing techniques. Mono - and sesquiterpenoids in pure form can be, isolated using preparative scale GLC. The fusion of GC -MS along with, computer analysis has made revolutionary changes in analytical methods, and increased its application at both laboratory as well as industrial level., Di- and triterpene hydrocarbons, diterpene resin acids, acetates and, trimethylsilyl ether derivatives of di -terpene and tri -terpene alcohols are, analysed by GLC., Flame Ionisation Detector (FID) and Thermal Cond, uctivity Detector, (TCD) are the commonly used detectors in GLC. Stationary phase, comprises of both polar and non-polar columns. Polar column consists of, polyester, while carbowax (polyethylene glycol), Apiezon (a paraffin, grease), and SE -30 (silicon oil) a re used as non -polar column s. Tenax GC and g raphitised carbon black are the recently introduced new, columns., ii) Thin-Layer Chromatography (TLC): This method provides quick, screening and can be applied in the analysis of different class of, terpenoids. Commonly, stationary phase comprises of silica gel of mesh, size 200 -300, but for attaining better resolution silica gel of size 400, mesh can also be used. Silica coated plates impregnated with 10, -15%, silver nitrate are used for the resolution of unsaturated terpen oids having, olefinic bonds., In case of monoterpenes and some sesquiterpenes , single solvent is used, as the mobile phase. However, in most of the cases, mobile phase, comprises of a combination of polar and non -polar solvent in different, ratios, e.g., hexane : acetone; hexane : ethyl acetate; hexane : chloroform;, hexane : diethyl ether; chloroform : methanol; toluene : ethyl acetate;, chloroform : acetone; toluene : ethyl acetate; acetic acid : n -butanol; and, n-butanol : acetic acid : water., This method can be used for isolating terpenoids ranging in milligrams, that can be further characterised. This method has an advantage of being, the fastest method for isolating constituents that can be subjected to, characterisation and bioassay. Nowadays, new version of t his method is, available in the form of High Performance Thin Layer Chromatography, (HPTLC)., *, , *
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Secondary Metabolites - IV (Chapter 6), , 183, , iii) Column Chromatography (CC): This method is common but important, and is used extensively for separating different terpenoids like sesqui -,, di-, tri-, and tetraterpenoids., In column chromatography, stationary phase involves the use of silica, gel passed from 60 -120 mesh, alumina, s ephadex, cellulose, etc. The, commonly used solid support is SiO 2. In some cases, when there is a, requirement of high quality separation, SiO 2 of mesh size 300 -400 is, used but this increases the elution time. Silica coated plates impregnated, with 10 -15% silver nitrate are used for the resolution of unsaturated, terpenoids having olefinic bonds., , 6.2.3., , Chemical Classes, , (C5H8)n is the general formula for most of the natural occurring terpenoid, hydrocarbon. Based on the number of structural carbon atoms or the value of „n‟,, terpenoids are classified as:, Table 6.1: Classification of Terpenoids, Number of, Value of n, Class, Carbon Atoms, 10, 2, Monoterpenoids (C10H16), 15, 3, Sesquiterpenoids (C15H24), 20, 4, Diterpenoids (C20H32), 25, 5, Sesterpenoids (C25H40), 30, 6, Triterpenoids(C30H48), 40, 8, Tetraterpenoids(C40H64), >40, >8, Polyterpenoids(C5H8)n, Based on the number of rings present in the structure, each class of terpenoids are, further divided into the following sub-classes:, 1) Acyclic Terpenoids: They contain open structure., 2) Monocyclic Terpenoids: They contain one ring in the structure., 3) Bicyclic Terpenoids: They contain two rings in the structure., 4) Tricyclic Terpenoids: They contain three rings in the structure., 5) Tetracyclic Terpenoids: They contain four rings in the structure., Below are the examples of mono-, sesqui-, and diterpenoids:, 1) Monoterpenoids, i) Acyclic Monoterpenoids, , *, , *
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186, , Pharmacognosy and Phytochemistry-II, , In day -to-day practice, it has been observed that fruits, flowers, leaves, stems,, barks, and roots of most of t he plants have some type of pleasant smell. This, smell is because of the volatile oils that can be distilled using steam and are, known as essential oils . These oils are complex mixtures of hydrocarbons and, their derivatives in oxygenated form. However, the, re are constituents like, benzaldehyde and methyl salicylate which are present in oil of bitter almond and, oil of wintergreen, respectively. Terpenoids are the main constituent of essential, oils and contain carbon atoms up to C, - and, 15 in the form of mono, sesquiterpenoids. Alcohols, aldehydes and ketones which are the oxygenated, derivatives of terpenoids and some open chain alcohols, aldehydes and ketones, that are closely related are also the chief constituents of essential oils., Some of the important essential oils with their terpenoid constituents are given in, the table 6.2:, Table 6.2: Important Essential Oils with their Main Terpenoid Constituents, Essential, Terpenoids, Essential Oils, Terpenoids, Oils, Turpentine Pinene, Lemon, d-Limonene and citral, Bergamot Linalool and its acetate, Sweet orange, d-Limonene, Caraway, Peppermint, Menthol and its esters, Carvone and -limonene, Citronella, , Geraniol, citronellal, and Rose, farnesol, Coriander Linalool and pinene, Sandalwood, Eucalyptus Cineole, Cardamom and, cajeput, Geranium Geraniol esters and, Camphor, citronellol, Jasmine, Linalool, Neroli, Lavender, Linalool, Ginger, , Geraniol, citronellol, and, farnesol, Santalol, Terpineol, Camphor, Nerolidol, Zingiberene, , Since the mono - and sesquiterpenoids have a pleasant smell, they are, commercially valuable, especially in perfumery and fragrance industry. Apart, from this, terpenoids also exhibit pharmacological properties such as insecticidal,, anthelmintic, or antiseptic property which is considered beneficial in, pharmaceutical industry., , 6.2.5., , Iridoids, , Iridoids are a large group of monoterpenoids. They are characterised by skeletons, in which a 6-membered ring, having an oxygen atom is attached to cyclopentane, ring (iridane skeleton). They occur in plants combined with sugar and hence are, classified as glycosides, further divided into four main groups:, 1) Iridoid glycosides (aucubin and harpagoside),, 2) Non-glycosylated or simple iridoids (loganin),, 3) Secoiridoids (gentio-picroside), and, 4) Bisiridoids, formed by dimerisation of iridoids and secoiridoids (table 6.3)., *, , *
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Secondary Metabolites - IV (Chapter 6), , 187, , Table 6.3: Chemical Structures, Resources, and Habitats of Iridoids, Name of, Structures, Resources, Habitat, Compounds, Aucubin`, Euphrasia officinalis Europe, L., Euphrasia, rostkoviana, Hayne, (Scrophulariaceae), Plantago lance olate Europe,, L.(Plantaginaceae), Asia, Harpagoside, , Harpagophytum, Native to, procumbens (Burch.) Southern, DC. Ex Meisn., Africa, (Pedaliaceae), , Gentiopicroside, , Gentiana lutea, (Gentianaceae), , 6.2.5.1., , L. Native to the, mountains of, central and, Southern, Europe, , Biosources, , Iridoids are monoterpenes obtained from iridane skeleton that is derived from, geranyl pyrophosphate. These are natural esterified products and are common i n, plants of, Lamiaceae, Gentianaceae and Valerianaceae, families. The se, compounds are rich in oxygen and the esters are derived from hemiterpenes; for, example, valeric acid gets esterified, into valrate and didroval trate; t hese, compounds are derived from vale rian ( Valeriana officinalis of Valerianaceae, family)., , 6.2.5.2., , Therapeutic Uses, , There is a wide range of health benefits of iridoids. They deliver support against, variety of physical, chemical, or biological stressors. On human body , iridoids, exert a normalising e ffect, which targets imbalances without affecting the, systems already functioning well. They produce these effects without any side, effects. Few benefits of iridoids are as follows:, 1) Neuroprotective: Many iridoids defend, the nervous system against, environmental and chemical stressors like pesticides or heavy metals; for, example, in amalgam tooth fillings. Some studies have shown that iridoids, can also enhance memory., 2) Anticancer and Anti-Tumour: Asperulosidic acid is obtained from the, dried morinda or noni fruit, and inhibits formation of melanoma (skin cancer), cells. Aucubin is a newly discovered iridoid, and produces anti -tumour, effects on cell culture s of breast colon, blood, skin, lung, throat, and nose, cancers., 3) Anti-Ageing: In a recent study, noni seeds w ere believed of no value and, were discarded while producing noni fruit juice and oil. However, in a more, recent study in Japan , it was reported that the iridoid americanin in noni, *, , *
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188, , Pharmacognosy and Phytochemistry-II, , seed can be a useful ingredient in cosmetics for whitening skin and, preventing wrinkles due to its ability to increase elastin production (elastin is, a vital skin protein and is the reason for soft, glowing, and youthful skin)., 4) Antibacterial: Some iridoids have strong natural antibacterial activity., 5) Antiviral: This property of iridoids has been reported in a number of studies., These compounds are also active against viruses that cause encephalitis and, herpes simplex type 1., 6) Anti-Inflammatory: Many iridoids show strong anti, properties when used topically or orally., , -inflammatory, , 7) Antioxidant: This property of iridoids is supposed to be more powerful than, that of more traditional plant antioxidants like flavonoids or polyphenols, obtained from citrus fruits and green tea., The highest concentration of, iridoids is in gentian plant family, and one of the best sources of simple and, complex iridoids is noni fruit., 8) Liver Protection: Iridoids are used in recovery from drug and alcohol liver, damage., 9) Cardiovascular Protection: Two iridoids present in olive oil, i.e., oleacein, and oleuropein, have great cardioprotective properties. People who consume, olive oil have low fat deposition in the abdomen, liver,, and heart, thus, causing less cardiac stiffness. They tend to have better resilience against, stress, and suffer less from cholesterol problems and hypertension. Iridoids in, olive oil protect the heart and arteries from harmful effects of stress., 10) Blood Sugar Control: are also useful in diabetic p atients because it can, protect pancreas. They are isolated from different plants , and tend to have, blood sugar control properties., 11) Protection of the Arteries:In a study it has been reported that iridoids prevent, the furring up of arteries and have effective cholesterol lowering properties., 12) Anti-Allergic: This property of iridoids originate s from their ability to, inhibit histamine release, thus making them natural anti -histaminics. Th ey, can also reduce inflammation, which accompanies allergic reactions, and this, makes them an ideal natural remedy for all kinds of allergies., , 6.2.6., , Other Terpenoids, , Following are some other types of terpenoids:, 1) Hemiterpenoids: These compounds are the simplest terpenoids . The mos t, important hemiterpene is isoprene that is produced from the leaves of many, trees ( e.g., conifers, poplars, oaks , and willow) and herbs ( e.g., Hamamelis, japonica). Tiglic, angelic, isovaleric, senecio acids, and isoamyl alcohol are, few other known hemiterpenoids., 2) Monoterpenoids: These compounds contain a 10 carbon backbone (2 isoprene, units) structure, and are divided into acyclic, monocyclic and bicyclic sub groups. In each group , monoterpenoids are either simple unsaturated, hydrocarbons or have alcohols, aldehydes, and ketones as functional groups., *, , *
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190, , Pharmacognosy and Phytochemistry-II, , Structurally, carotenoids have the following characteristics:, i) The carotenoids that are most widely known are either simple unsaturated, hydrocarbon with the basic lycopene str ucture or t heir corresponding, oxygenated analogues, i.e., xanthophylls (lutein and zeaxanthin), ., ii) In lycopene , eight isoprene units are joined head to tail to provide it a, conjugated system responsible for the chromophoric, property (i.e.,, colour producing characteristic) of the molecule., iii) Cyclisation at both terminals of the lycopene molecule gives a bicyclic, hydrocarbon, known as -carotene that occurs abundantly in higher, plants., In flowers and fruits of higher plants , combined forms of carotenoids occur, which are xanthophylls esterified with fatty acid residues, e.g., palmitic, oleic,, or linoleic acids. In higher plants, the rarely occurring glycosides are crocin (a, water-solube gentiobiose derivative of an uncommon C 20-carotenoid) and, crocetin (yellow pigment of meadow saffron, Crocus sativus L.)., 8) Polyterpenoids: These compounds are polymeric isoprenoid hydrocarbons, containing more than eight isoprene units., These compounds have been, confirmed to include rubbers. The natural rubber molecule is a high, molecular weight polymer containing isoprene units in the cis-configuration., A polyisoprene with trans double bond is also produced by some plants, e.g.,, balata from Mimusops balata (Sapotaceae) and gutta-percha from Palaquium, gutta (Sapotaceae)., 9) Irregular Terpenoids, i) Irregular Monoterpenoids: These compounds are of two major types:, a) Tropanes (the substituted cycloheptane monoterpenes, e.g., nezukone), arise by an unknown ring expansion of cyclohexane skeleton., b) Artemisia ketone, chrysanthemic acid, and lavandulol are compounds, produced by head -to-head condensation of isoprene units. They are, generally found in Asteraceae and Lamiaceae families., ii) Ionones and Damascones:, These compounds belong to C13 norisoprenoids (norterpenoids), which are aroma compounds derived, from carotenoids. The commonly known compounds of this class are and -ionone, and -and -damascone that occur in several essential oil., , 6.2.7., , Individual Drugs, , The pharmacognostic profile of the following drugs has been discussed below:, 1) Gentian,, 2) Artemisia,, 3) Taxus, and, 4) Carotenoids., , 6.2.7.1., , Gentian, , The name of the genus Gentian is derived from the name of an ancient King of, Illyria, Gentius who discovered the medicinal value of this plant. During the, Middle Ages, this plant was majorly used as an antidote to poison., *, , *
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Secondary Metabolites - IV (Chapter 6), , 191, , Synonyms, Gentian root, Gentiana, and Radix Gentianae., Biological Source, Gentian is the dried and partially fermented rhizome and root of yellow gentian,, i.e., Gentiana lutea., Family, Gentianaceae., Geographical Source, Gentian is a perennial herbaceous tree which is native of the hilly zones in, Central and Southern Europe. It is grown on Vosges Mountains, Yugoslavia, (now known as Serbia and Croatia), and Jura., Macroscopic Features, 1) Colour: Whitish roots., 2) Odour: Odourless., 3) Taste: Sweet which later becomes bitter., 4) Shape: Simple or branched, rhizome is cylindrical., 5) Size: Rhizome is 20cm long and 1 -4cm in diameter;, roots are 1m long., 6) Surface: Rhizomes are transversely wrinkled and roots, are longitudinally wrinkled when dried., 7) Fracture: Brittle when in dried, form but becomes, tough by absorbing atmospheric moisture., Microscopic Features, The transverse section of gentian root (, microscopic features:, , Figure 6.11: Gentian, Lutea Twig, , figure 6.12) shows the following, , Cork, , Cortex, Oil globule, Starch, Aci. Raphides, Medullary ray, Sec. phloem, , Periderm, Sec. phloem, Cambium, Xylem, , Cambium, Vessel, Wood parenchyma, , Figure 6.12: T.S. of Gentian Root, *, , *
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192, , Pharmacognosy and Phytochemistry-II, , 1) Periderm: It consists of:, i) Cork: It consists of 4 -5 layers, t hin walled, rectangular and orange, brown coloured cells., ii) Phellogen and Phelloderm: It cannot be distinguished from cork;, present as a few layers of thick walled tangentially elongated, parenchyma immediately below the cork., 2) Cortex: It is a thin zone of tang entially elongated thick walled parenchyma, cells. These cells contain oil globules, few starch grains, and minute acicular, raphides., 3) Secondary Phloem: This comprises of a wider zone containing tangentially, elongated thick walled parenchyma cells. These cel ls contain oil globules,, few starch grains, and minute acicular raphides. Sieve elements are seen, clearly but phloem fibres are absent., 4) Cambium: It is clearly visible as a yellow ring comprised of 3, thin walled, and small rectangular cells., , -5 layers of, , 5) Secondary Xylem: This forms the bulk of the root and consists of, parenchymatous cells, vessels, and medullary rays. The vessels are present, individually but mostly found in groups towards the cambium., Chemical Constituents, Gentian contains bitter glycosides , i.e., gentiopicrin (or gentiopicroside). It is a, water-soluble, crystalline compound whose bitter value is 12,000. When, fermented and dried, it gives gentiogenin and glucose., The drug also has amarogentin, amaroswerin, gentioside, and gentinin (a mixtu re, of gentiopicrin and gentisin)., O, ║, O, , HO, , O, , O, ║, , OH, │, , O, CH2 ═ CH, , H ( D Glucosyl), , Gentiopicroside, , O, , OCH3, , Gentianin (Gentisin), , Chemical Test, An extract of gentian gives out light-blue fluorescence under UV radiation., Uses, 1) It is used as a bitter tonic for stimulating gastric secretion, and thus,, improving appetite., 2) It is also used as an anthelmintic, anti-inflammatory, antiseptic, cholago gue,, febrifuge, refrigerant and stomachic., *, , *
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Secondary Metabolites - IV (Chapter 6), , 193, , Substitutes and Adulterants, 1) Gentiana purpurea,, 2) Gentian kurroa,, 3) Gentiana punctata,, 4) Getiana pannonica, and, 5) Rumex alpinus., , 6.2.7.2., , Artemisia, , Artemisia, also known as mugwort, wormwood, and sagebrush is a plant of the, daisy family of Asteraceae. It belongs to a diverse variety of plants having, around 200-400 species., Synonyms, Santonica and Worm seeds., Biological Source, Artemisia is an unexpanded flower -head obtained from the plant Artemisia cina, Berg, Artemisia brevifolia Wall, Artemisia maritima Linn., and other species of, Artemisia., Family, Asteraceae., Geographical Source, Artemisia plant is found in the Kurran valley regions of Pakistan, Turkey, and, widespread from Kashmir to Kumaon in Himalayas, and in West Tibet. The plant, is also cultivated in the states of Punjab, Uttar Pradesh, and Haryana., Macroscopic Features, 1) Colour: Flowers are yellow in colour, while other parts are whitish-grey., 2) Odour: Aromatic and sweet., 3) Taste: Bitter and camphoraceous., T, C, F, , Figure 6.13: Artemisia China Herb, , Figure 6.14: TEM of Pollen, , It contains yellowish or brownish, oval -shaped flower -heads. The flowers are, fertile in presence of tubular corolla and short cylindrical tube and narrow limb., It lacks calyx., *, , *
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194, , Pharmacognosy and Phytochemistry-II, , Microscopic Features, The furrow membrane part of the pollen is smooth with a circular pore. The, spines are small, and widely and evenly arranged. By using TEM, the walls of, the pollen can be clearly observed containing three main layers:, 1) The outermost layer tectum (T) of thin branching collumella,, 2) An inner layer of thick columella (C), and, 3) A thick foot layer (F)., Chemical Constituents, Santonica possesses oil and two crystalline substances, santonin and artemisin., The essential oil content ranges from 1.0 -2% and that of santonin is 2.0%. The, oil contains many essential constituents like cineole, pinene, and resin, while the, main constituent of the plant is santonin (a sesquiterpene lactone which, is, anhydride of santonic acid). The yield of santonin is not same throughout the, year. In some conditions when the flower-heads are unexpanded and can be dried, quickly, it yields 3.0% of santonin., , Chemical Test, 1gm of finely powdered drug is mix ed with 10ml alcohol and filtered. Sodium, hydroxide is added to the filtrate and the mixture is heated. The colour of liquid, appears red., Uses, Santonica is a strong anthelmintic acting against roundworm infections. It shows, no effect on hook worms and ta pe worms. Today, the crude drug is rarely used, for therapeutic purpose, and has been totally replaced by santonin., Substitutes, The plant can be substituted by a tall aromatic shrub of Artemisia vulgaris Linn., (belonging to family Compositae) distributed in all mountainous regions of India., , 6.2.7.3., , Taxus, , The genus Taxus consists of small coniferous trees or shrubs in the yew family,, Taxaceae. The plant grows very slowly and is very long-lived. The plant length is, 1-40m (3.3-131.2 ft.) and the trunk thickness is 5m (16 ft.)., Synonyms, Yew, Talispatra, and Himalayan yew., Biological Source, Taxus is the dried leaf, stem, bark and roots of different species:, 1) Leaves of Taxus baccata (English or European yew)., *, , *
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Secondary Metabolites - IV (Chapter 6), , 195, , 2) Stem bark of Taxus brevifolia (Pacific yew)., 3) Leaves and roots of Taxus canadensis (Canadian or American yew)., 4) Leaves of Taxus cuspidata (Japanese yew)., Family, Taxaceae., Geographical Source, Taxus is relatively slow -growing evergreen gymnospermous tree. They are the, natives of India, Canada, and A, merica. In India, it is found in temperate, Himalayan regions at an altitude of 2000-3500 metres., Macroscopic Features, Taxus baccata (Yew), indicated in figure 6.15 is a short evergreen tree of height, 9-20m with a huge trunk. The parts of the plant are described below:, 1) Stem: It is highly branched and is surrounded by a thin brown-coloured bark., 2) Branches: The branches grow throughout their life, forming a very dense, canopy (covering). Due to this reason this plant is renowned as a shade providing, evergreen tree. Only the green leaves are available on the vegetative branches., 3) Leaves: They are linear, small, 2-3 cm long, and spirally arranged on the, stem. Each leaf contains a single strong vein with re -curved margins. The, upper surface appears dark green, while the lower surface is pale or rusty red, in colour. Due to deposition of silica , the apex part is sharply pointed and, may cause death of cattle eating them. Each leaf has a short stalk which, extends into a flat persistent base showing a slight twist. On the fertile shoot,, opposite and decussate scaly leaves are present., 4) Root: Long, well- developed, deep-feeders, and highly branched., 5) Seed: Covered with a red fleshy aril and resembles a berry., 6) Odour: Agreeable., 7) Taste: Bitter and acrid., , Male cones, Pollens, , Leaves, Twig, Figure 6.15: Twig of Taxus Baccata Bearing Male, Cones at Different Stages of Development, *, , *
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196, , Pharmacognosy and Phytochemistry-II, , Microscopic Features, Transversally, the outer sketch of the stem shows an irregular structure ( figure, 6.16) and seems to appear as Pinus. The plant is covered with a dense, cuticularised single -layered epidermis. Below the epidermis, the, parenchymatous cortex is present consisting of tannin -filled cells. Subsequently,, the endodermis and sclerenchymatous pericycle are also found., The young stem of Taxus baccata contains a ring of conjoint, collateral, open,, and endarch vascular bundles with centrally enclosed pith. In protoxylem,, spiral tracheid is found, while the phloem part (lacking companion cells), contains sieve cells, sieve plates, and phloem parenchyma., Pericycle, , Cuticle, , Epidermis, Cortex, Phloem, , Xylem, , Pith, Figure 6.16: Taxus Baccata T.S Stem (Young), , Chemical Constituents, One of the chief constituent of the plant is taxol found in all part s of the plant,, but majorly available in leaves, roots, and bark. Taxanes are typical cyclic, diterpenoids, and 40 different varieties of t axanes have been reported till now., Among these, the three important members are taxol, cephalomannine, and 10deacetyl baccatin., The concentration of taxol in all species is 0.007-0.01%, however, some variation, may occur. It is mainly derived from the stem bark of, Taxus brevifolia . Its, isolation technique is tedious and yield is poor (as found in vinca). Extraction of, taxol from the trees is not easy, and requires at least, 3-4 trees of 60 years to, obtain 1gm of taxol., , *, , *
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Secondary Metabolites - IV (Chapter 6), , 197, , The leaves contain 10 -deacetyl baccatin-III which may get easily transformed to, taxol. Regeneration of leaves occurs after harvesting. Various species of the plant, are used in the treatment of cancer. The most potent constituents of the plant are, as follows:, 1) Taxol (containing a rare oxetane ring and amide side chain),, 2) Cephalomannine (about 0.031%),, 3) Baccatin-III (about 0.084%), and, 4) 10-Deacetyl baccatin-III., Taxotere is a potent compound of taxol possessing better bioavailability and, pharmacological properties and is considered as an anticancer agent., Uses, 1) The plant grows as an ornamental plant., 2) The timber part of Taxus baccata and some other species is oily and heaviest, of the soft woods. Due to its strength and toughness, the plant is used for, making decorative veneers, turnery, flowers, and posts., 3) The wood of the plant is used for making various decorative pieces., 4) All parts of the plant (leaves, shoots, and seeds) are believed to be poisonous, due to the presence of taxine (a toxic alkaloid)., 5) In some countries, the plant is used as a fish poison., 6) Trace amount of ephedrine is found in the leaves of plant, thus, used to trea t, bronchitis, asthma, and epilepsy disorders., 7) The seeds are used to produce sedative effect., , 6.2.8., , Naphthoquinones, , The class of organic compounds derived from naphthalene, is, naphthoquinones. Volatile and yellow coloured triclinic crystals having a sharp, odour of benzoquinone are formed by 1,4 -naphthoquinine. It is insoluble in cold, water, slightly soluble in petroleum ether, and more soluble in polar organic, solvents. It produces a reddish-brown colour in alkaline solutions. A derivative, *, , *
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198, , Pharmacognosy and Phytochemistry-II, , of 1,4 -naphthoquinone is vitamin K . Higher plants, fungi and actinomycetes, bacteria produce naphthoquinones, and they have important biological actions, such as fungicidal, antibacterial, anti-carcinogenic, cytostatic and insecticidal., Biosynthesis, Naphthoquinones are biosynthesised through several pathways that includeacetate, and malonate (e.g., plumbagin), shikimate/succinyl CoA combined pathway, (e.g., lawsone), and shikimate/mevalonate combined pathway (e.g., alkannin), (figure 6.17). They occur in reduced or glycosidic form in plants. One of the most, appropriate constituents is juglone (5-hydroxy-1,4-naphthalenedione), which, naturally occurs in the leaves, roots, husks, and bark of the plants of Juglandaceae, family, especially in black walnut (Juglans nigra). It is toxic or growth stunting for, many types of plants. It is used as a herbicide, cloth dye and inks, and a colouring, agent in foods and cosmetics. Juglone is a type of allelopathic compounds that is, synthesised by one type of plant and affects the growth of another plant., , Figure 6.17: Biosynthesis of Naphthoquinone, , Qualitative Analysis, 1) Juglone Test: 2ml of chloroform extract and 2ml of ethyl ether are treated, with dilute ammonia solution. A pink colour develops indicating the presence, of naphthoquinones., 2) Dam-Karrer Test: 10% potassium hydroxide solution is added to the, chloroformic plant extract. A blue colour develops., , 6.2.9., , Carotenoids, , Carotenoids are polyenes and mostly hydrocarbon s. They can also be called as, tetraterpenes because of the presence of polyisoprene structure in the ir carbon, skeleton. Extended conjugation of centra l chain imparts colour to carotenoids., Most of the naturally occurring carotenoids are trans-isomers but some are cistrans-isomers, and this has been prov ed by X -ray analysis . If the ends of the, chain are attached with R (may be an open -chain structure or a ring system), all trans-carotenes can be presented as:, *, , *
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Secondary Metabolites - IV (Chapter 6), , 199, R, , R, , 6.2.9.1., , Chemistry, , Carotenoids are lipid-soluble C-40 tetraterpenoids. Majority of them are derived, from a 40 -carbon polyene chain, which is the backbone of the molecule. This, chain is terminated by cyclic end-groups (rings) and complemented with oxygencontaining functional groups. Carotenoids are mostly present in nature in all trans configuration. Though, food and vegetab le processing produces a 10 -39%, of increase in cis-isomer. The isomerisation degree is directly connected to the, duration and intensity of heating process. But, when Rock et al. fed processed, vegetables to subjects, no increase in 9-cis-β-carotene plasma concentration was, observed. Rather, the plasma response was characterised by an increase of all, trans--carotene, due to isomerisation of cis-isomers to all trans-- carotene or a, rapid tissue uptake., , *, , *
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Secondary Metabolites - IV (Chapter 6), , 6.2.9.2., , 201, , Chemical Classes, , According to the structure, carotenoids are classified as follows:, 1) Hydrocarbon Carotenoids or Carotenes: An example is β-carotene., 2) Oxygenated Carotenoids: These are, derivatives of, xanthophyll, hydrocarbons. Examples are zeaxanthin and lutein (hydroxy), spirilloxanthin, (methoxy), echinenone (oxo), and antheraxanthin (epoxy)., , 6.2.9.3., , Biosources, , Carotenoid pigment is found in large amount in all the parts of green plants., Their presence is confirmed by the colour s like red, orange, green, and yellow ., These colours are imparted to foods (tomatoes, carrots, and apricots), by the, carotenoids. Thus, they are present in any fruit or vegetable having red, orange,, green, and yellow colour. They are also found in high concentration in dark leafy, green vegetables like spinach, broccoli, and kale., , 6.2.9.4., , Therapeutic Uses, , Carotenoids have the following uses:, 1) They are helpful in vision protection and fighting cellular damage., 2) They also provide support to the cardiovascular system., 3) They have antioxidant property, which protects the health of sperm and male, reproductive system., 4) Lycopene is the red pigme nt in tomatoes. It helps in the prevention of, prostate and breast cancer and arterial ag eing. It also keeps the liver, colon,, and lungs healthy., 5) β-Carotene and β-cryptoxanthin protect the skin, tissue s, and cells from, environmental toxins and disease., 6) Non-provitamin A carotenoids (e.g., lutein, zeaxanthin, and astaxanthin), are good for skin., , 6.3. SUMMARY, The details given in the chapter can be summarised as follows:, 1) A glycosidic bond is a covalent bond that binds the sugar molecule to, another mol ecule (that may or may not be sugar molecule). Thus,, a, substance containing a glycosidic bond is called a glycoside., 2) In general, glycosides may be defined as „ organic compounds of plants or, animal origin which on enzymatic or acid hydrolysis give one or more, sugar moieties along with non-sugar moiety‟., *, , *
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Secondary Metabolites - IV (Chapter 6), , 6.4.2., 7), 8), 9), 10), 11), , Fill in the Blanks, , The family of senna is ____________., A derivative of 1, 4-naphthoquinone is ___________., Iridoids are a large group of ____________., Talispatra is the synonym of ____________., The sugar group of glycoside is known as the _________ ___., , Answers, 1) True, 2) False, 5) False, 6) True, 9) Monoterpenoids 10) Taxus, , 6.4.3., 1), 2), 3), 4), 5), , 3) True, 7) Leguminoseae, 11) Glycone, , 4) False, 8) Vitamin K, , Very Short Answer Type Questions, , Write a short note on glycosides., Give the chemical tests for coumarin glycosides., Give the macroscopic features of bitter almond., Write the biological sources of taxus., Write a short note on terpenoids., , 6.4.4., 1), 2), 3), 4), , 203, , Short Answer Type Questions, , Write a short note on aloe., What are glycosides? Classify them., Give the pharmacognosy of gentian., Give the therapeutic uses of iridoids., , 6.4.5., , Long Answer Type Questions, , 1) Give a detailed account on glycosides., 2) Give the classification and chemistry of terpenoids., 3) Discuss the pharmacognosy of senna., , *, , *
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204, , Pharmacognosy and Phytochemistry-II, , CHAPTER, 7, , Isolation, Identification and, Analysis of Phytoconstituents, , 7.1. ISOLATION,, IDENTIFICATION, ANALYSIS OF PHYTOCONSTITUENTS, 7.1.1., , AND, , Introduction, , Natural products are secondary metabolites of plant or animal origin. They are, chemical compounds found in nature, and exhibit pharmacological and biological, actions. Natural products are used in drug discovery and designing. Separating a, single molecular entity from complex mixtures of fats, oils, alka loids, tannins,, and glycosides is a difficult task., According to the WHO, a medici, nal plant contains thera peutically active, substances or precursors for chemopharmaceutical semi-synthesis in one or more, of its parts (like leaves, roots, rhizomes, stems, barks, flowers, fruits, grains, or, seeds). These parts are thus employed in the control or treatment of a disease, and, therefore contain medically active chemical components., These non -nutrient plant chemical compounds or bioactive components, often, termed phytochemicals (Greek word, phyto meaning plant) or, phytoconstituents, protect the plant against microbial infections o r infestations, by pests., Phytochemistry is the study of natural prod ucts. Phytoconstituents are isolated, and characterised from fruits (grapes and apples), vegetables (broccoli and, onion), spices (turmeric), beverages (green tea and red wine), and many, other, sources., , 7.1.2., , Classes of Phytoconstituents, , The phytoconstituents are categorised into the following classes:, 1) Alkaloids: These are the largest secondary chemical con stituents. They are, made up of ammonia compounds containing nitrogen bases synthesised f rom, amino acid building blocks with various radicals replac, ing one or more, hydrogen atoms in the peptide ring, most containing oxygen. They have basic, properties and are alkaline in nature, i.e., they turn red litmus paper blue., The plant-derived alkaloi ds which end with the, suffix –ine include, morphine and codeine (analgesics), (+), -tubocurarine (muscle relaxant),, sanguinarine and berberine (antibiotics), vinblastine (anticancer agent),, ajmaline (anti -arrhythmic), atropine (pupil dilator), scopolamine (se dative),, and caffeine, nicotine, codeine, morphine, ergotamine, cocaine, and, ephedrine (addictive stimulants)., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 207, , common a double hydroxylation at C, -l and C -8. Presence of free, anthraquinones in the plant sample can be detected by mixing the powdered, plant material with organic solvent; the mixture is filtered and an aqueous, solution of a base (NaOH or NH 4OH) is added; a pink or violet colour in the, base layer indicates the presence of anthraquinones., 9) Essential Oils: These are odorous volatile products of various plant and, animal species. They evaporate when exposed to air even at ambient, conditions, and thus are also termed volatile or ethereal oils. They are either, secreted directly from the plant protoplasm or are the hy drolysis products of, some glycosides. They can be prepared from various plant sources by direct, steam distillation, extraction, or enzymatic hydrolysis., 10) Steroids: Plant steroids (or steroid glycosides), also termed as, cardiac, glycosides, are one of the most naturally oc curring plant phytoconstituents., They are therapeutically used as arrow poisons or cardiac drugs. Diosgen in, and cevadine (from Veratrum viride) are examples of plant steroids. Animal, steroids (or anabolic steroids) promote nitrogen retention in osteoporosis., , 7.2. TERPENOIDS, 7.2.1., , Introduction, , Terpenoids are a group of naturally occurring compounds, which mostly occur in, leaves and fruits of higher plants, conifers, citrus, and eucalyptus; but a few of, them are obtained from other sources. They are volatile compounds that impart, fragrance to the plants and flowers., The term terpene was derived from terpentine, which i s a volatile liquid, obtained from pine trees. This term formerly represented a mixture of isomeric, hydrocarbons (molecular formula C 10H16) occurring in the essential oils obtained, from the sap and tissue of plants, and trees. The simpler, mono- and, sesquiterpenes are the chief constituents of essential oils obtained from sap and, tissues of some plants and trees. The di- and tri- terpenoids are not steam volatile,, and are obtained from plant and tree gums and resins. Tetraterpenoids form a, separate group of compounds called carotenoids., , 7.2.2., , Menthol, , Menthol is a 10 -carbon monocyclic terpene alcohol with a molecular weight of, 156 and molecular formula of C 10H20O. It is naturally produced in plants of, Mentha genus and Lamiaceae family. One such plant, is Mentha piperita, (peppermint plant), which is a hybrid cross between, Mentha aquatica, (watermint) and Mentha spicata (spearmint). Menthol imparts the mint odour and, taste to these plants and their essential oils., The (–)-menthol is the primary and characteristic monoterpene in mentha essential, oils. Seven more menthol stereoisomers exist, i.e., (+) -menthol, (+/–)-isomenthol,, (+/–)-neomenthol, and (+/ –)-neoisomenthol. These are obtained from the leaves, and stems of certain species of mentha by steam dist, illation just before flowering., *, , *
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208, , 7.2.2.1., , Pharmacognosy and Phytochemistry-II, , Isolation, , Mentha oil is obtained by hydrodistillation or steam distillation of fresh above ground plant parts just before flowering. For isolating (, −)-menthol from, peppermint plant, th e peppermint oil is subjected to cooling. Menthol, crystallises out from the oil and is separated by centrifugation., Corn mint oil is obtained by the steam distillation of the flowering herb, Mentha, arvensis. This oil contains 70-80% of free (−)-menthol which can be obtained by, cooling the oil. The menthol crystals obtained are separated by centrifugation., Pure ( −)-menthol is obtained by re -crystallisation using low boiling point, solvents. Dementholised corn mint oil from which ( −)-menthol is removed by, crystallisation and which contains 40 -50% free menthol can also be reused for, producing (−)-menthol., , 7.2.2.2., , Identification, , A few drops of menthol mixed with 5ml nitric acid solution (prepared by adding, 1ml nitric acid to 300ml glaci al acetic acid) are heated on a water bath. Within 5, minutes, a blue colour develops which deepens on further heating and produces, copper colour fluorescence that turns golden yellow after some time., , 7.2.2.3., , Analysis, , The steps followed for analysis of menthol are:, 1) Accurately weighed 10gm of test sample is taken in an extraction flask., 2) 10ml acetic anhydride and 2gm sodium acetate anhydrous are added to the, flask., 3) A reflux condenser is attached to the flask and boiled for an hour., 4) The cont ents are then cooled, 30ml water is added, and the flask is again, heated for 15 minutes in boiling water with occasional stirring., 5) The contents are transferred to a separating funnel to separate the oil layer,, which is washed with water until it becomes neutral., 6) 2gm of anhydrous sodium sulphate is added with vigorous shaking, and, allowed to stand for 30 minutes., 7) Then the mixture is filtered through a dried paper filter., 8) Accurately weighed 2gm of the obtained (acetylated) oil and 5gm of the test, sample (un-acetylated oil) are taken in separate extraction flasks., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 209, , 9) 5ml of ethanol and 2 drops of phenolphthalein solution are added to each flask., 10) 0.5mol/lml of potassium hydroxide -ethanol solution is added to both the, flasks for neutralising the solutions., 11) Then 25ml of 0.5mol/lml of potassium hydroxide-ethanol solution is added., 12) A reflux condenser is attached to the flasks and the contents are boiled for an, hour., 13) After boiling, 25ml of water is added immediately and stirred., 14) The solution is cooled and the excess amo unt of potassium hydrate is titrated, with 0.5mol/lml of hydrochloric acid., 15) A blank test is performed by the same procedure., , , 0.021 (c b), 7.814 (c a ) 1 , , unacetylated oil (gm) , %menthol , acetylated oil (gm) 0.021 (c a ), , Where, a and b = Titration volumes (ml) of 0.5mol/lml of hydrochloric acid, added to acetylated oil and un-acetylated oil, respectively., c = Titration volume (ml) of hydrochloric acid used in the blank test., , 7.2.3., , Citral, , Citral is an acyclic monoterpenoid, and a major constituent (60 -80%) of lemon, grass oil. It is pale yellow liquid with a strong lemon, -like odour. It can be, obtained from lemongrass oil by fractional distillation under reduced pressure., Two geometrical isomers of citral are naturally found:, , 7.2.3.1., , Isolation, , Citral can be isolated by following the below mentioned steps:, 1) It is isolated from lemon grass oil (contains 90% citral and 10% neral) by, steam distillation., 2) Steam distillation is carried out in a 5ml short necked round -bottomed flask, in which 0.5ml of lemon grass oil and 3ml of water is added., 3) Water is injected dropwise using a syringe through the septum in the flask to, maintain a constant volume., 4) The distillate is collected in an ice -cooled 15ml centrifuge tube, and then, 2.5ml of tertiary butyl methyl ether is added., *, , *
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210, , Pharmacognosy and Phytochemistry-II, , 5) The centrifuge tube is then clo sed and shaken to dissolve the citral into the, upper ether layer., 6) This ether layer is removed using a Pasteur pipette and placed in a clean, reaction tube., 7) The aqueous layer is extracted twice with 1.5ml of tertiary butyl methyl ether, to remove all the citral., 8) The ether extracts are combined and placed in a reaction tube., 9) The ether layer is treated with 2ml of saturated sodium chloride solution to, remove all traces of water, and then the aqueous layer is discarded., 10) The remaining traces of water are removed b y treating the ether layer with, anhydrous calcium chloride pellets., 11) The mixture is shaken for 5-10 minutes in the stoppered reaction tube., 12) The dry ether layer is placed in another clean, dry and tarred reaction tube, with a boiling stone., 13) Fresh ether along with a drying agent is added to the tube to remove all traces, of citral., 14) The combined ether extract is evaporated using water bath., 15) The reaction tube is connected to the water aspirator to remove the traces of, ether., 16) The percentage yield of citral is calcu lated from the product obtained (a, clear, fragrant oil)., , 7.2.3.2., , Identification, , The presence of citral can be detected by the following two tests:, 1) An alcoholic solution of Sudan III is added to the test sample. Red colour, obtained by the globules indicates the presence of citral in the volatile oil., 2) A drop of tincture alkane is added to the test sample. Red colour indicates the, presence of citral in the volatile oil., , 7.2.3.3., , Analysis, , Citral can be analysed in a Sh imadzu Gas Liquid Chromatograph (Model GC 14B) coupled with a non-polar DB-5 capillary column [30m × 0.250mm and 0.25, micron Internal Diameter (ID)] using a Flame Ionisation Detector (FID). The, instrument is connected to a computer having the CLASS GC 10 s oftware that is, used for data analysis. Retention Indices (RI), using n -alkanes (octane, nonane,, decane, dodecane, octadecane, eicosane, docosane, tetracosane, and hexacosane), are used as the basis., The percentage of each compound is calculated by dividi ng the peak area by the, total area of component peaks. The temperature range is maintained from 50, 250°C, with a temperature programme rate of 20°C/min, starting at 2 minutes, and finishing at 10 minutes. The detector and injection temperatures are, maintained at 250°C. The Retention Indices (RI) of standard compounds that, matches with the RI of the unknowns is detected by GC. By comparing the RI, match with a standard compound, citral is confirmed., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.2.4., , 211, , Artemisin, , Artemisin is a semi -synthetic derivative of a drug that possesses the most rapid, action against Plasmodium falciparum malaria. It is isolated from the plant,, Artemisia annua (or sweet wormwood). This plant was widely employed in, Chinese traditional medicine., , Artemisinin, , Chemically, artemisin is a sesquiterpene lactone containing an unusual peroxide, bridge, which is responsible for the drug‘s mechanism of action. Few other, natural compounds with such a peroxide bridge are known., Artemisin and its endoperoxide derivatives have been used for treating infections, related to P. falciparum. However, the low bioavailability, poor pharmacokinetic, properties, and high cost of the drugs are a major drawback., The WHO has, banned the use of this drug as a monotherapy, as some studies have revealed that, the malarial parasites are developing resistance to the drug., Therefore, therapies combining artemisin or its derivatives with other, antimalarial drug are preferred for malaria due to their effectiveness as well as, tolerability in patients., , 7.2.4.1., , Isolation, , Artemisin can be isolated by following the below mentioned steps:, 1) 100gm of powdered Artemisia annua L. is macerated with methanol in an, Erlenmeyer flask with a magnetic stirrer rotating at a speed of 700 rpm for 1, hour. This process is repeated till the methanol layer becomes colourless., 2) The extract obtained is evaporated in a rotavapor vacuum at 40°C, temperature till the extract volume reaches 100ml., 3) The extract solution is partitioned multiple times using 50ml of hexane to, obtain a colourless hexane layer., 4) Two layers, i.e., hexane extracts (non -polar fraction) and methanol extracts, are obtained from this process., 5) The methanol extract obtained is added with 10ml of distilled water and, again partitioned with 50ml of ethyl acetate., 6) Partitioning is done multiple times till the ethyl acetate layer becomes, colourless., 7) By this process, ethyl acetate extract (semi -polar fraction) and methanol water extract (polar fraction) is obtained., *, , *
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212, , Pharmacognosy and Phytochemistry-II, , 8) Each extract is concentrated using a rotavapor at 40ºC temperature., 9) The most viscous extract containing artemisin is fractionated by column, chromatography, using silica gel 60 as the stationary phase and a mixture of, ethyl acetate-hexane as the mobile phase., 10) Each fraction is collected, and the presence of artemisin is identified., , 7.2.4.2., , Identification, , 1gm of finely divided sample is boiled with 10ml of alcohol and filtered. The, filtrate is added with sodium hydroxide and again heated. The solution develops, red colour indicating the presence of artemisin., , 7.2.4.3., , Analysis, , Artemisin can be analysed by the following techniques:, 1) IR Spectrophotometer: 2mg of isolates are crushed and mixed with 98mg, of KBr (dried for 24 hours at 105 ºC temperature). The isolates are analysed, at wave number 4000 -400cm-1 using KBr as the baseline. The spectrum of, isolates obtained is compared with the spectrum of standard artemisin., 2) UV Spectrophotometer: 1mg of isolates is dissolved in 10ml of methanol, and analysed at wavelength of 200-400nm. The spectrum of isolates obtained, is compared with the spectrum of standard artemisin., 3) Thin Layer Chromatography (TLC): 1mg of isolates is dissolved in 5ml of, ethyl acetate. Isolates are spotted with a solution using the capillary tube on a, silica gel 60 F254 (the stationary phase). Ethyl acetate: hexane (3:97) and, ethyl acetate: hexane (7:93) are used as the mobile phase., 4) Liquid Chromatography-Mass Spectrometry (LC-MS): 1mg of sample is, dissolved in 1ml of methanol and then 20μl is injected and, eluted using, methanol:water (9:1). With a flow rate of 1ml/min, separation is done, through C18 column (RP 18). The samples are analysed separately as per, their retention times., 5) HNMR Spectroscopy: Isolates are analysed using CDCl3 as the solvent and, tetramethylsilane as the reference compound. The spectrum of isolates, obtained is compared with the spectrum of standard artemisin., , 7.3. GLYCOSIDES, 7.3.1., , Introduction, , Glycosides are the condensation products of sugars having different varieties of, organic hydroxy compound s in such a manner that the hemiacetal entity of the, carbohydrate should take part in the condensation. The glycosides are regarded as, internal acetate., The two series of stereoisomeric glycosides are termed as - and β-glycosides., Thus, by considering the example of methyl D-glucosides, its - and β-structures, are represented as:, *, , *
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d Analysis of Phytoconstituents (Chapter 7), , 213, , 6, CH2OH, H, , 1, , H —— OCH3, , 5, , H, , 2, , H —— OH, , O, H, , HOCH2, , HO, , H, , 3, HO ——, H, , O, , 4, H ——, OH, 5, H ——, 6 CH OH, 2, , 4, , HO, , 1, , H, , (a), , 2, , 3, , HO, , OCH3, , 2, , 1, , H, OCH3, , 1, , OCH3, H, , OH, , H, , H, , HO, , (b), , O, , 5, , H, , O, H3, , H, , 6, , 4, , (c), , Methyl--D-Glucoside, 6, , H, , CH2OH, 1, , H3CO —— H, , 5, , 2, , H —— OH, 3, , HO —— H, , O, , H, , 4, H ——, OH, 5, H ——, 6, CH2OH, , (d), , HO, , O, , 5, , H, HO, , H, 2, , 3, , O, 2, , 3, , 1, , OH, , H, , 4, , OCH3, , 4, , 6, , HOCH2, , H, , H, , H, , OH, , HO, H, , (e), , (f), , OH, Methyl--D-Glucoside, , hetinic Acid, , an oleanolic acid obtained from glycyrr, hiza, which possesses some anti -allergic, antibacteria, n allergic or infectious skin inflammation as a topical formulation, while it is used orally for its ald, n. It also plays the role of an immunomodulator., *
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214, , Pharmacognosy and Phytochemistry-II, , Glycyrrhetinic acid is a pentacyclic triterpenoid, a cyclic terpene ketone, and a, hydroxy monocarboxylic acid. It is a conjugate acid of a, glycyrrhetinate. It is, derived from a, hydride of an oleanane. It is, olean-12-ene substituted by, a hydroxy group at position 3, an oxo group at position 11, and a carboxy group, at position 30. It is a pentacyclic triterpenoid., , 7.3.2.1., , Isolation, , Glycyrrhetinic acid can be isolated by the following three methods:, 1) From a Glycyrrhizin Salt: In this method, glycyrrhizin is extracted from, liquorice root, purified, and then converted to glycyrrhetinic acid. There are, different forms of this method. In one type, concentrated aqueous extract of, liquorice root is treated with H2SO4 to precipitate a brown sediment, which is, washed with water and extracted with alcohol to isolate glycyrrhizin., The extract is added with KOH solution to precipitate the potassium salt of, glycyrrhizin, which is filtered and crystallised twice in acetic acid. The salt is, hydrolysed with H 2SO4 to obtain crude glycyrrhetinic acid that is acetylated, by acetic anhydride. The cake obtained is purified with CHCl 3 and CH 3OH,, and hydrolysed with NaOH solution to obtain white glycyrrhetinic acid., 2) By Enzymatic Reactions: In this method, glycyrrhetinic acid is produced by, the hydrolysis of glycyrrhizin or its salts with aeromonas species or their, cultured preparations in the presence of nitrogen, phosphate, and potassium., The yield of this method is 91% and the glycyrrhetinic acid obtained has an, acceptable purity. However, the method is highly expensive., 3) By Hydrolysis of Liquorice Root:, In this method, glycyrrhizin is, hydrolysed directly, i.e., without isolation from liquorice root. There a, re, different forms of this method. In one type, the liquorice root powder is, hydrolysed in tri -chloroacetic acid for 18 hours at 95°C temperature. The, cooled suspension is neutralised with lime solution and filtered. The cake, obtained is washed with water, and the released glycyrrhetinic acid is, extracted with ethanol. The extract is evaporated and residue crystals are, crystallised twice in acetic acid to produce glycyrrhetinic acid., , 7.3.2.2., , Identification, , Sulphuric acid (80%) is added to a thick section of the drug or powde r. A deep, yellow colour produced instantly indicating the presence of glycyrrhetinic acid., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.3.2.3., , 215, , Analysis, , The extract to be analysed is prepared by continuous hot extr action method and, dried using evaporating dish and heating mantle. Dilution is obtained to 5μg/ml, concentration and scanned in UV range (200, -400nm) in 10mm cell against, solvent blank. Glycyrrhetinic acid shows a well -defined λmax at 254nm; thus, this, wavelength is selected for spectrophotometric evaluation., Preparation of Standar d Stock Solution: 10mg of glycyrrhetinic acid is, dissolved in phosphate buffer (pH - 6.8):ethanol (70:30 proportion) in 10ml of, volumetric flask. The final volume is adjusted using the same solvent to get a, solution containing 1000μg/ml concentration of gly cyrrhetinic acid. Aliquots of, working stock solutions of glycyrrhetinic acid are prepared with the same solvent, to get 5 -35μg/ml concentration of glycyrrhetinic acid. The absorbance of, resulting solutions is measured at 254nm., Analysis of the Herbal Extra, ct: Accurately weighed 20mg of herbal, hydroalcoholic extract of liquorice is dissolved in phosphate buffer (pH, 6.8):ethanol (70: 30 proportion) in a 10ml volumetric flask. The final volume is, adjusted using the same solvent. The sample solution is filtered, through, Whatman filter paper no. 41. From the above solution, 0.1ml of solution is, withdrawn and diluted to 10ml with phosphate buffer (pH -6.8):ethanol (70:30, proportion) to get final concentration containing 20μg/ml of glycyrrhetinic acid., , 7.3.3., , Rutin, , Rutin is a bioflavonoid. Its pure form is yellow or yellow-green coloured, needleshaped crystal. It is a flavon, ol glycoside containing quercetin and the, disaccharide rutinose (rhamnose and glucose). Rutin is found in many plants,, fruits, and vegetables, and the chief source is buckwheat. It is also found in citrus, fruits, noni, black tea, and apple peel. Rutin most ly metabolises to quercetin (its, aglycone part) during digestion., , Rutin, , Rutin has strong antioxidant properties. It can also chelate metal ions, such as, iron, thereby reducing the Fenton reaction (production damaging oxygen, radicals). It can stabil ise vitamin C (ascorbic acid), thus on administering, together, vitamin C activity will be strengthened. Rutin strengthens the, *, , *
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216, , Pharmacognosy and Phytochemistry-II, , capillaries, thus is helpful in people who bruise or bleed easily. It has been, proved to be effective in preventing venous oedema (an early sign of chronic, venous disease of the leg). Rutin has anti -inflammatory and healing properties., Rutin can inhibit some cancerous and pre -cancerous conditions. It can prevent, atherogenesis and reduce the cytotoxicity of oxidised LDL-cholesterol., , 7.3.3.1., , Isolation, , Rutin can be isolated by following the below mentioned steps:, 1) 20gm of powdered Ginkgo biloba (leaves and stem bark) is extracted with, 250ml of 80% ethanol until exhaustion in a Soxhlet apparatus., 2) The obtained extract is filtered and evaporated under vacuum till the volume, comes down to 10ml., 3) To this residue, 25ml water is added., 4) The mixture is extracted with petroleum ether followed with chloroform., 5) After extraction, the aqueous layer is collected and left undisturbed in a cold, place for 3 days., 6) A yellow precipitate separates out of the solution, which is filtered and, washed with a combination of chloroform:ethyl acetate:ethanol (50:25:25)., 7) The un -dissolved part of the precipitate is dissolved in hot methanol and, filtered., 8) The filtrate is evaporated to dryness to obtain 100mg yellow coloured, powder (rutin), and its melting point is measured., , 7.3.3.2., , Identification, , Rutin can be identified by the following tests:, 1) On treating an aqueous solution of ruti n with ferric chloride solution, a dark, green colour appears., 2) On treating an aqueous solution of rutin with lead acetate, an orange -yellow, precipitate is formed., 3) On treating an aqueous solution of rutin with ammonium molybdate and, antimony trichloride, an orange-yellow coloured precipitate is formed., , 7.3.3.3., , Analysis, , Rutin can be analysed by the following two techniques:, 1) TLC: The isolated rutin is compared with standard rutin using TLC method., An aluminium sheet coated with silica gel G is used as the stationary phase, with ethyl acetate:butanone:formic acid:water (50:30:10:10), ethyl, acetate:formic acid:acetic acid:water (100:11:11:27) as the mobile phases., 2) Paper Chromatography: Whatman No.1 filter paper is used as a stationary, phase and ac etic acid:water (15:85) and isopropyl alcohol:water (60:40) are, used as mobile phases., 3) Spectrophotometric Analysis: The isolated rutin is dissolved in methanol, and its UV radiation absorption peaks are determined and compared with, standard rutin. The IR spectrum of the isolated rutin is determined using KBr, disk methodology., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 217, , 7.4. ALKALOIDS, 7.4.1., , Introduction, , Alkaloids are basic chemical compounds, obtained from plants. Chemically, one, nitrogen atom is always present in the 5, - or 6 -membered ring structure of, alkaloids. Their basic nature is due to this N -atom only. They are valuable to the, body‘s physiological system., W. Meissner (a pharmacist) named nitrogen -containing basic compounds, obtained from plants as alkaloids. Ladenburg defined alkaloids as, ‗compounds, originated from plants with a basic nature, having one or more nitrogen, atom in its ring structure‘., Development in the research work related to chemistry of alkaloids added two, significant characteristics to the definition of alkaloids:, 1) Their molecular structure is complex, and, 2) They produce a substantial pharmacological activity., Hence, based on the above findings, alkaloids are now defined as,, ‗physiologically active basic compounds of plant origin in which at least one, nitrogen atom forms part of a cyclic system‘., , 7.4.2., , Atropine, , Atropine is the alkaloid obtained from plants of Solanaceae family, such as, Atropa belladonna (deadly night shade), Datura stramonium (thorn apple), and, Hyoscyamus niger (henbane)., , Atropine, , Properties of atropine are:, 1) It has a melting point of 115-116°C., 2) It is optically inactive., 3) It is a strong poison and has a sharp bitter taste., 4) It is used in ophthalmology due to its dilating action on the eye pupils., 5) It stimulates and then depresses the CNS on internal administration., 6) It is the racemic modification of the laevo -rotatory hyoscyamine. Atropine, does not exist naturally but is formed during the isolation of hyoscyamine., Atropine and hyoscyamine have similar physiolo gical activity; however, the, activity of latter on the peripheral nerves is more than that of atropine., *, , *
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218, , 7.4.2.1., , Pharmacognosy and Phytochemistry-II, , Isolation, , Figure 7.1 represents the schematic arrangement of isolation of atropine:, , solution, , Oxalic, , Figure 7.1: Isolation of Atropine, , 7.4.2.2., , Identification, , Vitali-Morin Reaction: In this method, the tropane alkaloid is first treated with, fuming nitric acid and then evaporated to dryness. Methanolic potassium, hydroxide solution is added to an acetone solution, of nitrated residue. Violet, colour develops confirming the presence of tropane derivative., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.4.2.3., , 219, , Analysis, , Atropine can be analysed by the following techniques:, 1) GC/MS Analysis: 0.5ml sample, 5µl IS (Internal Standard) solution and, 1.0ml borate solution (0.1M, pH 9.3) are mixed in a test tube and poured into, an Extrelut column. After 15 minutes, the target compounds are eluted with, 4ml dichloromethane. The obtained eluate is evaporated to, dryness under, nitrogen stream., The residue is mixed with 50µl of N,O-bis(trimethylsilyl)trifluoroacetamide :, trimethylchlorosilane (BSTFA:TMCS) in 99:1 ratio and warmed at 45°C, temperature for 20 minutes for TMS derivatisation in a glass vial with a, teflon cap. The solution obtained is mixed with 100 µl dichloromethane, and, a 1µl aliquot of it is injected into GC/MS for analysis., 2) HPLC Analysis: 0.5ml sample is poured into an activated HLB cartridge,, which is washed with 1ml of 5% methanol. The target compounds are eluted, with 1ml methanol. The obtained, eluate is evaporated to dryness under, reduced pressure. The residue is dissolved in 100µl of the mobile phase, and, a 10µl aliquot of it is injected into HPLC for analysis., , 7.4.3., , Quinine, , Quinine alkaloid is obtained from the bark of the plants, Cinchona ledgeriana, and Cinchona officinalis of Rubiaceae family. The barks of these trees contain, about 8% quinine; several of the grafted varieties produce more than 15%, quinine (as the sulphate)., , Quinine, , Properties of quinine are:, 1) It crystallises with three moles of water, and melts at 177°C under anhydrous, conditions., 2) It is laevo-rotatory and is bitter in taste., 3) Its sulphate or dihydrochloride salt is used as the most effective anti -malarial, agent since many years., 4) Quinine and cinchonine have been used in the resolution of racemic mixtures, via formation of diastereoisomeric salts., , 7.4.3.1., , Isolation, , Figure 7.2 represents the schematic presentation for isolation of quinine from, cinchona bark:, *, , *
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220, , Pharmacognosy and Phytochemistry-II, , Figure 7.2: Isolation of Quinine from Cinchona Bark, , 7.4.3.2., , Identification, , Quinine can be identified by the following tests:, 1) Thalleioquin Test: A small amount of quinine is dissolved in dilute, sulphuric acid and 1 -2ml of water. To the resulting mixture, 2 -3 drops of, bromine water are added and shaken. A drop of strong ammonia is added to, the mixture which produces an emerald green colour., 2) Erythroquinine Test: A quinine solution in dilute acetic acid is added with, 1-2 drops of bromine water and a drop of 10% solution of potassium, ferricyanide. A drop of strong ammonia solution is added to the mixture, which produces a red colour., 3) Quinine gives a strong blue fluorescence in extremely dilute solutions of, sulphuric, acetic, phosphoric or tartaric acids., 4) On slightly moistening small quantity of quinine with glacial acetic acid and, heating in an ignition tube, blood-red drops condense on the sides of the tube., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.4.3.3., , 221, , Analysis, , Reagents, Dilute sulphuric acid ( 0.05M) prepared by adding 3.0ml concentrated sulphuric, acid to 100ml water, and diluting up to 1000ml with distilled water., Standard Solution of Quinine, Accurately weighed 0.1gm of quinine is dissolved in 1L 0.05M sulphuric acid in, a graduated flask. 10ml of this solution is diluted to 1000ml with 0.05M, sulphuric acid so th at the resulting solution contains 0.00100mg/ml quinine., Using a calibrated burette, 10.0, 17.0, 24.0, 31.0, 38.0, 45.0, 52.0 and 62.0ml of, the above dilute standard solution is run into separate 100ml graduated flasks and, each solution is diluted up to the mark with 0.05M sulphuric acid., Procedure, The fluorescence of each of the above solutions is measured at 445nm, using, 62ml of dilute quinine solution as a standard for the fluorimeter. LF2 or an, equivalent primary filter ( ex = 350nm) and gelatin are us ed as the secondary, filters in a simple fluorimeter. Test solutions containing 0.00025 and, 0.00045mg/ml quinine are prepared. Their concentrations are determined by, measuring the fluorescence on the instrument and with the calibration curve., , 7.4.4., , Reserpine, , Reserpine is the active principle of Rauwolfia species. Chemical investigation of, around 60 different Rauwolfia species led to the isolation of 50 -70 Rauwolfia, alkaloids; of which the most important ones are reserpine, yohimbine, ajmalicine,, and ajmaline. The snake -like roots of Rauwolfia are used for controlling, hypertension, epilepsy, insomnia, fevers, cholera, dizziness, and headaches., , Reserpine, , Properties of reserpine are:, 1) It has a melting point of 265ºC., 2) It is stable, but gradually darkens on exposure to light., 3) It is combustible., 4) It is incompatible with strong acids, reducing agents, and oxidising agents., 5) It exists as white or cream to slightly yellow crystals or crystalline powder., 6) It has no odour and has a bitter taste., *, , *
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222, , 7.4.4.1., , Pharmacognosy and Phytochemistry-II, , Isolation, , Reserpine can be isolated by following the below mentioned steps:, 1) Rauwolfia root powder is extracted with 90% alcohol by percolation., 2) The alcoholic extract is concentrated and dried under reduced pressure below, 60°C temperature to yield rauwolfia dry extract that contains 4% of total, alkaloids., 3) This dry extract is again extracted with proportions of ether:chloroform:alcohol, (20:8:2.5)., 4) The extract obtained is added with little dilute ammonia with alternating, shaking to convert the alkaloid into water-insoluble base., 5) Water is added to the above mixture and the drug is allowed to settle after a, few vigorous shakings., 6) The solution is filtered and the residue is extracted with 4 volumes of 0.5M, NH2SO4 in a separating funnel., 7) The total acid extract containing the alkaloidal salt is combined., 8) The extract is filtered, made alkaline with dilute ammonia to liberate the, alkaloid., 9) The resulting alkaline solution is extracted with chloroform., 10) The total chloroform extract is filtered and chlor, oform is removed by, distillation., 11) The total alkaloidal extract is dried under vacuum to yield total rauwolfia, alkaloids (consists of the mixture of over 30 different components)., 12) This is subjected to column chromatographic fractionation for separating, reserpine., , 7.4.4.2., , Identification, , Reserpine can be identified by the following tests:, 1) On treatment with solution of vanillin in acetic acid, reserpine shows violet, red colour., 2) Reserpine is colorimetrically determined by reaction bet ween acidic solution, of alkaloids and sodium nitrite., , 7.4.4.3., , Analysis, , HPLC Analysis: Water (0.1% CH 2O2 and 0.1% triethylamine; pH 5.0, adjusted, with NH 4OH and H 3PO4) as solvent A and acetonitrile (0.1% CH 2O2 and 0.1%, triethylamine) as solvent B are used as the mobile phase. Both the solvents are, filtered through a millipore PTFE 0.45μm membrane. Separations are carried out, using a linear gradient: 0 min 50% B, 3 min 50% B, 3.10 min 55% B, 6 min 55%, B, 6.10 min 100% B, 7 min 100% B, 7.10min 50% B, and 10 min 50% B. Flow rate, of mobile phase is 1.0ml/min, and the injection volume is 20μl. Chromatographic, runs are carried out at 25ºC. UV detection is performed at 268nm., , 7.4.5., , Caffeine, , Caffeine is a naturally occurring xanthine alkaloid. It is the component of tea, leaves (5%), coffee (1 -2%), and kolanuts (1 -2%). It is responsible for the, stimulating action these beverages produce on the nerves and heart, and thus is, employed in medicine., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 223, , Properties of caffeine are:, 1) It has a melting point of 235ºC., 2) It crystallises as silky needles., 3) It has a bitter taste., 4) It is sparingly soluble in water and alcohol., 5) It is a weak base., 6) It forms salts with strong acids, which get easily, decomposed by water., 7) Its citrate and hydrochloride salts are used as diuretics and heart and nerve, stimulants. However, its excessive use disturbs the digestion., , 7.4.5.1., , Isolation, , Caffeine can be isolated by the following methods:, 1) Damaged tea leaves are powdered and boiled with water, preferabl y alkaline,, and filtered hot. To the filtrate, basic lead acetate is added to precipitate out, the tannins and albuminoids. Sulphuric acid is used for removing the excess, of lead (as lead sulphate) from the filtrate., The filtrate is decolourised with anima l charcoal, and caffeine is extracted, with chloroform. The solvent is recovered by distillation and caffeine is, purified by re-crystallisation with water., 2) Finely or coarsely powdered tea leaves are extracted with ethanol in Soxhlet, extractor. The extract o f caffeine obtained is adsorbed on magnesium oxide., Caffeine is then desorbed after treatment with 10% H, 2SO4. It is then, extracted with chloroform and re-crystallised., 3) Caffeine is extracted from coffee beans by leaching with water. Yield of, about 90% can b e obtained by extracting the coarse coffee powder with, water at 75C. Extraction with water/coffee (9:1) takes about 90 minutes., 4) Decaffeination of coffee is done with supercritical fluid extraction. This, process was first developed by K. Zosel who used liquefied carbon dioxide., The supercritical medium in a pressure vessel is circulated through moist, coffee where it becomes charged with caffeine., It is then passed through second pressurised vessel containing an adsorbing, medium (such as activated carbon, r esin, or water). This medium adsorbs the, caffeine, which is then separated by extraction with chloroform., , 7.4.5.2., , Identification, , Caffeine can be identified by the following tests:, 1) Murexide Test: Caffeine is taken in a petri dish and added with hydrochloric, acid and potassium chlorate. The mixture is then heated to dryness. The, residue is exposed to vapours of dilute ammonia. A purple colour appears,, which disappears on adding fixed alkali., 2) On treatment with tannic acid solution , c affeine produces a white coloured, precipitate., *, , *
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224, , Pharmacognosy and Phytochemistry-II, , 7.4.5.3., , Analysis, , Caffeine can be analysed by the following three methods:, 1) By HPLC, Standard Solutions: Caffeine stock solution of 1000ppm is prepared by, taking accurately weighed 100mg of pure caffeine in a 100ml volumetric, flask and making the volume up to the mark with the mobile phase. Working, standards of 10, 20, 40, 60 and 80ppm are prepared by serial dilution of the, stock solution with the mobile phase., Sample Preparation and Analy te Determination : 2gm of tea and coffee, samples are weighed in triplicate and transferred into 250ml beakers. 100ml, of boiling distilled water is added and left undisturbed for 5 minutes with, stirring. The solution is cooled and filtered into conical flask s. 5ml of the, filtrate is pipetted into clean 50ml volumetric flasks and volume is made up, to the mark with the mobile phase. The standards and the samples are run in, the HPLC system. A calibration curve of peak areas is plotted against the, concentration o f standards. The caffeine level of various samples is, calculated using the regression equation of the best line of fit., 2) By UV/Vis Spectrophotometry, Calibration Standards: Caffeine stock solution (1000ppm) is prepared by, dissolving 100mg of pure caffeine i n 100ml of distilled water. Working, standards of 0, 10, 20, 40, 60 and 80ppm are prepared by serial dilution of, the stock in 25ml volumetric flasks with the addition of 1.0ml hydrochloric, acid before making up the volume up to the mark with distilled water., Sample Preparation and Analytic Determination: 0.25gm of accurately, weighed sample is dissolved in water and volume is made up to 20ml with, distilled water; this is the sample solution. 20ml of this solution is pipetted, into 250ml flask, and added with 10ml 0.01M hydrochloric acid and 2ml, basic lead acetate solution. The volume is made up to the mark with distilled, water, shaken, and filtered to clarify., 50ml of the filtered solution is pipetted into 100ml flask, and added with, 0.2ml sulphuric acid. T he volume is made up to the mark with distilled, water, shaken, and filtered. Absorbance of the working standards and, samples are measured on a UV/Vis spectrophotometer at 274nm wavelength, using 10mm quartz cuvette. The caffeine levels in the samples are ca lculated, from the regression equation of the best line of fit of the standards., 3) By FTIR Spectrophotometry, Preparation of Beverage Samples: Around 1.5gm of powdered coffee, samples are dissolved in 70ml of hot distilled water and boiled for 10, minutes. The solutions are diluted to 100ml and cooled. Solutions with fine, suspension are filtered through a Whatman No. 1 filter paper., 3ml of an aqueous coffee solution and 3ml of chloroform are mixed. After, phase separation, 1ml of caffeine solution in chloroform is diluted 10 -fold, with chloroform, and the final solution is used for FTIR analysis., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 225, , Data Collection: FTIR spectra are recorded using a MIDAC (M 2000), spectrometer equipped with a liquid nitrogen cooled mercury/cadmium, telluride detector and a 45° horizo ntal zinc selenide crystal as an ATR, accessory. Spectra are recorded at 4cm resolution with 512 co -added scans., To avoid IR signals from moisture, the instrument is purged with dry air for 4, hours before recording any interferogram., An open beam backgroun d spectrum is collected through the clean zinc, selenide crystal washed with chloroform. For collecting sample spectra,, 0.5ml of caffeine solution in chloroform is layered on the crystal, and the, solvent is dried before collecting the interferograms of the, caffeine film, formed on the ATR crystal., Each sample interferogram is ratioed against the background interferogram,, and the resulting transmission spectrum is transmuted into an absorption, spectrum. The caffeine film from zinc selenide crystal is removed, using, chloroform before the next sample is applied for spectral recording., Concentration of caffeine in a coffee extract is determined using the standard, curve and the absorbance of extracted caffeine., , 7.5. RESINS, 7.5.1., , Introduction, , Resins are complex chemical comp ounds which are amorphous in nature and, comprises of essential oils and oxygenated products of terpene and carboxylic, acids (found as exudations from the trunk of various trees). They contain organic, acids, alcohols, esters, and neutral resins., Resins ar e transparent or translucent solids, semi -solids or liquid substances, containing numerous carbon atoms. They are mostly heavier than water,, insoluble in water, but soluble in alcohol, volatile oils, fixed oils, chloral hydrate,, and non-polar organic solvents (like benzene or ether). They are electrically non conductive and combustible masses. They are hard, but soften and ultimately, melt upon heating. They are formed as end products of metabolism in, schizogenous or schizolysigenous cavities or ducts., , 7.5.2., , Podophyllotoxin, , Podophyllotoxin is a natural product isolated from Podophyllum peltatum and, Podophyllum emodi. It possesses many medicinal properties. Etoposide is a, podophyllotoxin derivative, and is used in the treatment of cancers, m ainly small, cell lung carcinoma and testicular cancer., Properties of podophyllotoxin are:, 1) It exists as solvated crystals., 2) It has a melting point of 183ºC., 3) It is insoluble in ethyl ether and ligroin ; slightly soluble in water; soluble in, acetone and benzene; and highly soluble in ethanol and chloroform., *, , *
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226, , Pharmacognosy and Phytochemistry-II, , Podophyllotoxin, , 7.5.2.1., , Isolation, , Podophyllotoxin can be isolated by following the below mentioned steps:, 1) The powdered rhizome or roots of P. emodi are extracted with methanol., 2) The extract obtained is reduced under vacuum., 3) The semi -solid mass obtained is put into acidulated water (10ml HCl in, 100ml water)., 4) The precipitate formed is allowed to settle and filtered., 5) The filtrate is decanted and washed with cold water., 6) The resin obtained is dried, which gives a dark brown amorphous powder of, podophyllin., 7) This powder is extracted with chloroform and purified by repeated re, crystallisation from benzene (either alone or a, mixture of alcohol and, benzene)., 8) The resultant is washed with petroleum ether or hexane to yield, podophyllotoxin., Another extraction method of pure podophyllotoxin involves the following steps:, 1) The chloroform-soluble fraction is dissolved in alcohol., 2) The resultant is refluxed with neutral aluminium oxide., 3) This makes the solution light yellow in colour., 4) To this alcoholic solution, benzene is added to obtain podophyllotoxin with a, yield of 95-98%., Another extraction method of podophyllotoxin involves the following steps:, 1) The roots or rhizomes of P. emodi are extracted over a bed of neutral alumina, with solvents like benzene, toluene, xylene, etc., for 1.5-4 hours., 2) The resultant is re -crystallised from organic solvents such as hot benzene,, toluene, and xylene to yield pure podophyllotoxin (95-97%)., Podophyllotoxin is a tetrahydronaphthalene derivative having hydroxy and, lactone groups. The attachment at cis-position is responsible for its purgative, property and the attachment at trans-position is responsible for its anti -cancer, property., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.5.2.2., , 227, , Identification, , Podophyllotoxin can be identified by the following tests:, 1) 0.5gm of the drug is macerated with 10ml of alcohol and filtered. The filtrate, is added with 0.5ml of strong c opper acetate solution. Formation of a brown, precipitate confirms the presence of podophyllotoxin., 2) 0.5gm of powdered resin is mixed with 5ml 60% alcohol and 5ml 1N, potassium hydroxide solution. If on shaking the mixture, a stiff jelly is, produced, the presence of Indian podophyllum resin is confirmed, while if no, precipitate is formed, the presence of American podophyllum resin is, confirmed., , 7.5.2.3., , Analysis, , HPLC Analysis: Podophyllotoxin and its glycosides are identified by HPLC in, which the retention time and UV spectrum of the test compound is compared, with that of the reference compound. HPLC analysis is performed on a Thermo, Finnigan HPLC machine with pump system equipped with a 966, -photodiodearray detector, with the detecti on wavelength set at 283nm. A satisfactory, separation is obtained with rever se phase column utilising an E . Merck RP -18, column with a diode array detector and auto -injector. Elution is done for 30, minutes using methanol:water (60:40) as the mobile phase at, a flow rate of, 0.8ml/min. A standardised mixture of two marker compounds with known, concentration of podophyllotoxin and podophyllotoxin -D glycoside is used to, create calibration curves (percentage area with respect to the quantity of pure, compounds). Bot h the marker compounds exhibit sufficient differences in their, retention times, thus can be quantified easily., , 7.5.3., , Curcumin, , Curcumin is a diarylheptanoid obtained from the dried and fresh rhizomes of, Curcuma longa. It is the principal curcuminoid of turmeric, and exists as several, tautomeric forms, including a 1,3 -diketo form and two equivalent enol forms ., The 1,3-diketo form is more stable in water, while the enol form is more stable in, the solid phase and in organic solvents., , Curcumin Keto Form, , Curcumin Enol Form, , *, , *
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228, , Pharmacognosy and Phytochemistry-II, , Curcumin exhibits potent anti -inflammatory properties, which are also quite, protective against some form of cancer progression. However, it‘s some anti, cancer properties are independent of its anti -inflammatory effects, thus is under, research for prevention and treatment of cancer., The poor oral bioavailability of curcumin should be enhanced with piperine, (black pepper extract). Curcumin has antioxidant properties which can prevent, rancidity of foods and provide foodstuffs containing less oxidised fa, t or free, radicals. The anti -oxidation property of curcumin plays an important role in, keeping curry for a long time without making it rancid., , 7.5.3.1., , Isolation, , Curcumin can be isolated by following the below mentioned steps:, 1) 25gm of powdered dried turmeric rhizome is taken in a large beaker and, mixed with 1000ml of distilled n-hexane using a magnetic stirring rod., 2) The resultant suspension is stirred for 3 days, then filtered and placed in a, porous bag or ―thimble‖ of strong filter paper placed in the chamber of, Soxhlet apparatus., 3) 310ml of methanol is heated in a flask, and its vapours are condensed in a, condenser., 4) The condensed extractor drips into the thimble containing powdered turmeric, rhizome, and extracts it., 5) When the liquid level in chamber rises to the top of siphon tube, the liquid, contents flood into the flask. This process is continuously carried out till a, drop of solvent from the siphon tube does not leave residue when evaporated., 6) After 3 days, the solvent is evaporated in a, rotary apparatus, and then, dissolved in 100ml of toluene., 7) The solution obtained is poured into a separatory funnel, added with 100ml, 0.2M NaOH, and shaken for a few minutes., 8) The aqueous phase is collected and acidified to pH 3 using 0.2M HCl. The, brown extract turns yellow as it undergoes clarification., 9) The filtrate is extracted with diethyl ether (3×100 ml)., 10) In the final extraction, the ether turns pale yellow coloured indicating the end, of extraction., 11) The combined ethereal phases are washed with 30ml wat er and dried over, MgSO4., 12) Ether is removed completely under vacuum to leave a yellow coloured solid, crude curcuminoid, which is purified by thin layer chromatography., , 7.5.3.2., , Identification, , Curcumin can be identified by the following tests:, 1) On treating the powdered drug with sulphuric acid, a crimson colour appears., 2) On treating the aqueous solution of turmeric with boric acid, a reddish-brown, colour appears which turns greenish-blue on adding alkali., 3) On treating the powdered drug, with acetic anhydride and concentrated, sulphuric acid, a violet colour appears. A red fluorescence appears when this, test is observed under UV light., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 7.5.3.3., , 229, , Analysis, , Curcumin can be analysed by the following three methods:, 1) TLC Analysi s: The extracted curcumin can be analysed with thin layer, chromatography. Glass plates (20×20) coated with silica gel are used as the, stationary phase. Sample is applied on these plates at definite distance using, standard borosilicate capillary. These spots are allowed to dry., Mixture of chloroform:ethanol:glacial acetic acid (94:5:1) is used as the, mobile phase in the development chamber and allowed to saturate, . The, prepared glass is placed in the development chamber for curcumin, separation. The amount o f curcumin is measured with the aid of calibration, curve of standard curcumin., 2) UV/Visible Spectroscopy: 0.1gm of dried extract is dissolved in 25ml of, ethanol. The resultant solution is filtered and volume is made up to 100ml., Thereafter, 10ml of this solu tion is taken in a volumetric flask and volume is, made up to 100ml with ethanol. The absorbance is measured at 425nm. The, percentage yield of curcumin and its colour value are determined., A standard curcumin 0.25gm/litre gives absorbance at 425nm = 0.42, Absorptivity of curcumin (A) = 0.42 / 1 × 0.025, = 16.8, % Curcumin, = a × 100/L × A × W, Where, a = absorbance of sample at 425nm, L = Path length (1cm), A = Absorptivity, Colour value = a × 1000, 3) HPLC Analysis: This analysis is performed using a LC -100 with L C-UV100 UV detector. A CAPCELL (C -18) HPLC-packed column (4.6mm I.D. ×, 250mm), type MG 5 µm, number AKAD/05245 is used for chromatographic, separations. Methanol is used as the mobile phase. Separation is carried out, by isocratic elution for around 15 minut es at a flow rate of 1.2ml/min and, column temperature of 25°C. The injection volume is 25µl, and UV detection, is effected at 254nm. After phytochemical analysis, the ethanolic extract, (10µg/ml) is subjected to HPLC column and the obtained record is, superimposed on the retention time value of this extract., , 7.6. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Natural products are secondary metabolites of plant or animal origin., 2) Non-nutrient plant chemical compounds or bioactive components,, often, termed phytochemicals (Greek word, phyto meaning plant) or, phytoconstituents, protect the plant against microbial infections or infesta tions by pests., 3) Phytochemistry is the study of natural products., 4) Alkaloids have basic properties and are alkalin e in nature, i.e., they turn red, litmus paper blue., *, , *
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230, , Pharmacognosy and Phytochemistry-II, , 5) Glycosides are the condensation products of sugars (including, polysaccharides) with different varieties of organic hydroxy and thiol, (occasionally) compounds (invariably monohydrate in character)., 6) Chemically, the bitter principles contain diterpene lactones (e.g.,, andrographolide) or triterpenoids (e.g., amarogentin)., 7) Flavonoids are polyphenols and are found extensively distributed among the, plant flora., 8) Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical, compounds, which occur as natural colour pigments imparting colour to the, fruits., 9) Saponins are abundantly found in the plant, Saponaria vaccaria (Quillaja, saponaria)., 10) Tannins are high molecular weight phenolic compounds, found in the root,, bark, stem, and outer layers of plant tissue., 11) Anthraquinones are phenolic and glycosidic derivatives., 12) Terpenoids are a group of naturally occurring compounds, which mostly, occur in leaves and fruits of higher plants, conifers, citrus, and eucalyptus;, but a few of them are obtained from other sources., 13) Tetraterpenoids form a separate group of compounds called carotenoids., 14) Menthol is a 10-carbon monocyclic terpene alcohol with a molecular weight, of 156 and molecular formula of C10H20O., 15) Citral is an acyclic monoterpenoid, and a major constituent (60, -80%) of, lemon grass oil. It is pale yellow liquid with a strong lemon-like odour., 16) Artemisin is a semi-synthetic derivative of a drug that possesses the most, rapid action against Plasmodium falciparum malaria. It is isolated from the, plant, Artemisia annua (or sweet wormwood)., 17) Glycyrrhetinic acid is an oleanolic acid obtained from glycyrrhiza, which, possesses some anti-allergic, antibacterial and antiviral properties., 18) Rutin is a bioflavonoid. Its pure form is yellow or yellow -green coloured,, needle-shaped crystal., 19) Alkaloids are now defined as, ‗physiologically active basic compounds of plant, origin in which at least one nitrogen atom forms part of a cyclic system, ‘., 20) Atropine is the alkaloid obtained from plants of Solanaceae family, such as, Atropa belladonna (deadly night shade), Datura stramonium (thorn apple),, and Hyoscyamus niger (henbane)., 21) Quinine alkaloid is obtained from the bark of the plants,, Cinchona, ledgeriana and Cinchona officinalis of Rubiaceae family., 22) Caffeine is a naturally occurring xanthine alkaloid. It is the component of tea, leaves (5%), coffee (1-2%), and kolanuts (1-2%)., 23) Podophyllotoxin is a natural product isolated from Podophyllum peltatum, and Podophyllum emodi., 24) Curcumin is a diaryl heptanoid obtained from the dried and fresh rhizomes, of Curcuma longa., *, , *
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(Isolation, Identification and Analysis of Phytoconstituents (Chapter 7), , 231, , 7.7. EXERCISE, 7.7.1., 1), 2), 3), 4), 5), , 7.7.2., 6), 7), 8), 9), 10), , True or False, , Phytochemistry is the study of natural products., Podophyllotoxin is a xanthine alkaloid., Curcumin is obtained from Curcuma longa., Rutin is a bioflavonoid., Quinine is obtained from Hyoscyamus niger., , Fill in the Blanks, , __________ is a naturally occurring xanthine alkaloid., Atropa belladonna is the source of ___________., ___________ are phenolic and glycosidic derivatives., ___________ is an oleanolic acid obtained from glycyrrhiza., ____________ is the study of natural products., , Answers, 1) True, 5) False, 9) Glycyrrhetinic acid, , 7.7.3., 1), 2), 3), 4), 5), , 3) True, 7) Atropine, , 4) True, 8) Anthraquinones, , Very Short Answer Type Questions, , Write a short note on flavonoids., Discuss tannins., Write a short note on glycosides., Give the properties of caffeine., Give the identification methods for quinine., , 7.7.4., 1), 2), 3), 4), , 2) False, 6) Caffeine, 10) Phytochemistry, , Short Answer Type Questions, , Give the isolation and identification procedure of curcumin., Write a short note on menthol., How quinine is isolated?, Discuss the isolation and analysis of podophyllotoxin., , 7.7.5., , Long Answer Type Questions, , 1) Give the isolation and identification of resins., 2) Discuss the isolation procedure of:, i) Atropine, ii) Reserpine, iii) Rutin, 3) Write the isolation and identification procedure of menthol and artemisin., , *, , *
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232, , Pharmacognosy and Phytochemistry-II, , CHAPTER, 8, , Industrial Production, Estimation, and Utilisation of Phytoconstituents, , 8.1. INDUSTRIAL, PRODUCTION, PHYTOCONSTITUENTS, 8.1.1., , OF, , Introduction, , Before coming to the consumer market, a drug goes through, a number of, procedures for production in industries. Industrial production of a drug involves, common procedures like isolation, extraction, production, and development of the, product. Nowadays, some new procedures are also involved that are more, compliant and useful in analysing the chemical complexity of tissues and their, biogenetic behaviour.Industrial production of a drug involvesthe following steps:, 1) Herbs or their parts to be studied are selected and collected., 2) The genuine raw material is isolated from the bulk., 3) The desired active constituents are extracted from the raw material using, various extraction processes., 4) The e xtracted active constituents are incorporated into desired dosage form, and large scale manufacturing or production. For the pr oduction of active, constituents, some recently discovered methods of industrial production, e.g.,, tissue culture and biogenetic pathways are also used., 5) The p repared dosage form goes through quality control and quality, assurance, where the prepared drug is analysed to guarantee that it fulfils all, the essential parameters., 6) The product is packaged and stored., 7) Finally, the product is delivered to the consumer market., , 8.1.2., , Forskolin, , Chemically, forskolin is a labdane diterpene that is extracted from pulverised, dried plant material of Coleus forskohlii, b elonging to Labiatae family. Coleus, forskohlii wildly grows in arid and semi-arid parts of India, Nepal, and Thailand., It is a member of the mint family. Its roots have long been used in Ayurvedic, medicine to treat heart and lung disease, intestinal spasms, insomnia, and, convulsions., , *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 8.1.2.1., , 233, , Industrial Production, , The industrial production of forskolin involves the following steps:, 1) Tubers of C. forskohlii are collected, washed,dried, and pulverised into granules., 2) The whole forskolin is extracted (utilising methanol as a solvent) in crude, form by the traditional methods., 3) The obtained methanol extract is concentrated, and the resultant concentrate, is added with chloroform., 4) Equal volume of water is added to the separating funnel., 5) The resulting mixture is shaken thoroughly, then allowed to sett, le, after, which the chloroform layer is separated., 6) The mixture is treated with water for 2 -3 times and the chloroform layer is, separated and concentrated., 7) A precipitate of forskolin is obtained by using ice cold n-hexane., 8) Forskolin is obtained as a reddish brown to brown coloured powder., , 8.1.2.2., , Estimation, , HPLC is used to estimate forskol in content in the raw material, which should, contain more than 1% of forskolin on a dry basis. The raw material is analysed, by isocratic liquid chr omatography using a HPLC system, equipped with a, dual/quaternary pump, a manual/auto injector, and a photodiode array or UV, detector supported by a suitable software. The details are given in the table 8.1:, Table 8.1: Estimation of Forskolin by HPLC, Chromatographic Conditions, Mobile phase, Degassed mixture of 55 volumes of water and 45, volumes of acetonitrile., Column, Stainless steel column (250 ×4.6mm) packed with, octadecylsilane bonded to porous silica., Detector, Photodiode array or UV detector., Wavelength, 220nm, Flow rate, 1.8ml/min, Run time, 60 minutes, Injection volume, 20l, , Standard Preparation, Accurately weighed 10mg of forskolin reference standard is transferred to a 10ml, volumetric flask and dissolved in 5ml of acetonitrile by, gently warming on a, water bath. The obtained solution is cooled and added with 10ml of acetonitrile, to make up the volume up to 10ml., Sample Preparation, Accurately weighed 3gm of coarse powder sample is transferred to a 250ml beaker, and extracted with 50ml of acetonitrile by boiling on a water bath for 20 minutes., The obtained extract is transferred to a 250ml beaker and the process is repeated, for 4-5 times until a colourless extract is obtained. Thefiltrates and concentrate are, pooled to 80 -90ml, cooled to room tem perature, and then transferred to a 100ml, volumetric flask. Acetonitrile is used to make up the volume up to 100ml and, mixed thoroughly. Finally, the solution is filtered througha 0.45 membrane., *, , *
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234, , Pharmacognosy and Phytochemistry-II, , Procedure, The standard preparation is chromatographed by i njecting 20 l. The Relative, Standard Deviati on (RSD, not more than 2%) is calculated for at least three, replicate injections and a tailing factor , which should not be more than 1.5., Further, the sample preparation is chromatographed and the forskolin percentage, is calculated from the peak responses., A s Wstd Sd Pstd, , , , 100, A std Std d S wt 100, Where,, A = Area of the sample, Astd = Area of the standard, Wstd = Weight of the standard (in mg), Stdd = Standard dilution, Sd = Sample dilution, Swt = Sample weight in milligrams, Pstd = Purity of standard, , 8.1.2.3., , Utilisation, , Forskolin has the following uses:, 1) In Ayurvedic systems of medicine,, C. forskohlii has been used in the, treatment of heart diseases, abdominal colic, respiratory disorder s, insomnia,, convulsions, asthma, bronchitis, intestinal disorders, burning sensation,, constipation, epilepsy, and angina., 2) Its roots are used to treat infections caused by worms and to, relieve the, burning sensation in festering boils., 3) The root extract mixed with mustard oil is topically used in eczema and skin, infections., 4) Forskolin is added in the medicinal preparations used to prevent hair graying, and to restore normal hair colour., 5) It is also used to treat diseases in which reduced intracellu lar cAMP level is, the main factor responsible for the development of the diseases like eczema,, asthma, psoriasis, cardiovascular disorders, and hypertension., 6) It is used as an immunomodulator as it activates the macrophages and, lymphocytes., , 8.1.3., , Sennoside, , Sennoside is obtaine d from the dried leaves of Cassia angustifolia Vahl, of, Leguminosae family. It contains more than 2.0% of anthracene derivatives, calculated as calcium sennoside B., Senna leaves are examined for sennoside content before and after the grinding, process, and then extracted using methanol. The resultant methanolic extract is, added with calcium salt solution to obtain a precipitate of c alcium sennosides., The precipitate is centrifuged, washed with methanol, and dried under vacuum., The dried calcium sennoside crystals are grounded, tested, and packed., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 8.1.3.1., , 235, , Industrial Production, , There are two methods for industrial production of sennoside:, Method 1, 1) The leaves are dried, powdered, and extracted with benzene for 2 hours on an, electronic shaker., 2) The extract is filtered and the solvent is distilled off., 3) The resultant mark is dried, extracted with 70% methanol for 4 -6 hours, and, separated by filtration., 4) The extraction process is repeated using 70% methanol for 2 hours, and the, extract is filtered again., 5) The metabolic extracts are combined and concentrated till 1/8th of its original, volume remains., 6) The resultant concentrate is acidified with hydrochloric acid to obtain pH 3.2., 7) The extract is filtered and alcoholic anhydrou s calcium chloride is added, with continuous stirring., 8) Ammonia i s added to maintain the pH at 8,, and then the mixture is left, undisturbed for 2 hours., 9) The solution is filtered and the calcium sennoside precipitate is collected., 10) The precipitate is dried over phosphorous pentaoxide., 11) The percentage yield of calcium sennoside is calculated., Method 2, 1) The leaves of senna are dried, powdered , and extracted with chloroformethanol (93:7) mixture for 30 minutes., 2) The obtained extract is filtered., 3) The resultant mark is again extracted using acidic methanol., 4) Both the extracts are combined and concentrated., 5) The resultant mixture is left undisturbed at room temperature for 12 hours., 6) The precipitate of sennoside A obtained is recrystallised using triethylamine., 7) The sennoside B present in the solution is precipitated b y 10 % methanolic, calcium chloride solution., 8) The p recipitate of sennoside B is purified, using a mixture of ammonia methanol (40:60)., 9) The obtained precipitate is dried, washed with water, kept for, a day, and, recrystallised with glycomonoethylether., *, , *
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236, , 8.1.3.2., , Pharmacognosy and Phytochemistry-II, , Estimation, , Various spectrophotometric, spectrofluorimetric and chromatographic techniques, like HPLC, HPTLC, etc. may be used for the estimation of sennoside s. Calcium, sennosides are ext racted with boiling water and oxidised by treating with ferric, chloride. Subsequent acid hydrolysis produces anthraquinones from glycosides, that are extracted, with ether. The anthraquinone residue obtained from, evaporated ether solution forms a pink-coloured complex with 1N KOH which is, estimated spectrophotometrically., The steps involved are mentioned below:, 1) Accurately weighed 10mg of calc ium sennosides is transferred to a conical, flask, and added with 25ml of distilled water using a pipette., 2) The solution is heated on a boiling water bath for 20 minutes., 3) 2 to 3 drops of water are added to the above solution and filtered., 4) 20ml of 10% aqueous ferric chloride is added to the 10ml of obtained filtrate,, and the solution is heated for 15-20 minutes on a water bath., 5) 1ml concentrated HCl is added to the solution and heated till the precipitate, dissolves., 6) The solution is cooled and extracted with ether (4 × 20ml)., 7) The resultant ether layers are collected in a 100ml standard flask and volume, is made up with ether by continuous stirring to mix well., 8) 10ml of this solution is pipetted out in a clean China dish and evaporated., 9) 10ml of 1N KOH is uniformly mixed with the residue and the absorbance at, 500nm is determined using 1M KOH as blank., 10) The percentage of sennoside content is calculated on the basis of the extinction, value of sennosides in terms of free anthraquinon, es which is 200 at 500nm., , 8.1.3.3., , Utilisation, , Senna and its preparations are used as purgative to treat constipation. The, anthraquinone glycosides of senna are absorbed in the intestinal tract after which, the aglycone part is separated and released, in colon. The anthraquinone, molecules irritate and stimulate the colon , thus peristaltic movement increases, due to local action. The increased peristalsis also decreases water absorption in, colon, thus resulting in soft and bulky faeces., It is thought that the grip ping effect of senna results due to its resin or emodin, content. To reduce the gripping effect, the drug is often given wi th carminatives., The drug administered parenterally is secreted in colon and produces the, therapeutic action., , 8.1.4., , Artemisinin, , Artemisinin is a semi-synthetic derivative of a group of drugs that show the most, rapid action against malarial infection caused by Plasmodium falciparum ., Artemisinin is obtained from the plant Artemisia annua (sweet wormwood) of, Compositae/Asteraceae family. This herb is used in Chinese traditional medicine., A precursor molecule can be obtained by using genetically engineered yeast., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 8.1.4.1., , 237, , Industrial Production, , Artemisia annua is mainly grown in the temperate regions. Artemisinin is present, in the leaves and flowering tops of the plant. The, yield of art emisinin may be, enhanced by 30% using the growth regulator (e.g., chlormequat) before harvesting., For in vitro production, Artemisia annua is grown and propagated in a hormonefree medium by the process of micro cutting. If used for research purposes, the, plant is dried by lyophi lisation before extraction, thus the moisture content of, samples can be controlled easily., Artemisinin is extracted from air dried plants using ethyl ether, petroleum ether ,, and even gasoline as a solvent. Petroleum ether (30-60C) is the most satisfactory, solvent among various non -protic solvents. Extraction with hexane at room, temperature for several days is also effective., , 8.1.4.2., , Estimation, , Artemisinin and its derivative s are estimated by TLC with recovery rates of 80 90% and low sensitivity (1g for artemether and 0.06-0.2mg for artemisinin). For, the separation of epimers of artemisinin, reverse -phase liquid chromatography, using UV detection at 210-220nm range is preferred., These compounds lack physicochemical properties; therefore, detection by using, direct UV does not provide the required sensitivity and selectivity for the analysis, of trace amounts of these compounds., Chromatographic column treated with alkalis is used to detect a stable product of, artemisinin with maximum absorption at 289nm. It is used for metabolites having, peroxide linkages. The sensitivity of various detection methods can be enhanced, by chromogenic derivatisation methods., On exposing the TLC plates to ammonia and iodine vapour the d erivatives such, as p-nitrobenzyl ester of artesunic acid and Diacetyl, Dihydro Fluorescein, (DADF) esters of artemisinin dihydro artemisinin, deoxy artemisinin, appear as, deep-red spots which can be detected., Artemisinin is electrochemically active, therefore HPLC with r, eductive, electrochemical detection (HPLCFC) is used to estimate, the concentration of, artemisinin in plant extracts., The sensitivity of artemisinin can be measured by:, 1) Pulse polarography method (0.6g/ml), and, 2) Radioimmune assay method (2.3ng/ml)., , 8.1.4.3., , Utilisation, , Following are the uses of artemisinin:, 1) It has been used in clinical trials performed for study ing the treatment of, schizophrenia, malaria, and Falciparum and Plasmodium infections., *, , *
Page 239 :
238, , Pharmacognosy and Phytochemistry-II, , 2) Many of its derivatives are clinic ally used to prevent and treat malarial, infection. It has been observed that some derivatives of artemisinin are linked, to rare instances of acute liver injury., 3) An isomer of it, i.e., (+)-artemisinin, is a sesquiterpene lactone, and used in, the treatment of multi-drug resistant strains of Falciparum malaria., , 8.1.5., , Diosgenin, , Naturally, diosgenin is extracted fro m the dried tubers of the plants Dioscorea, deltoidea, D. composita , and other species of Dioscorea, belonging to, Dioscoreaceae family. Generally, it exists in combined form as saponin, glycoside; however, it may also occur in free state. Diosgenin is f, ound as, rhamno-rhamno-glucoside, known as dioscin, in the tubers of commercial ly, important species of Dioscorea., , 8.1.5.1., , Industrial Production, , Industrial production of diosgenin involves the following methods:, 1) Alcoholic Extraction Method, Dioscorea tubers are cut into small pieces and dried under sun, , Dried tubers are powdered, extracted with ethanol/methanol, twice for 6-8 hours, , Filtered and filtrate is concentrated to a syrupy liquid, , Concentrated liquid is hydrolysed using an acid (HCl or, H2SO4) for 2-12 hours, , 85% of diosgenin is precipitated, , Precipitate is filtered and washed with water, , Purification with alcohol, *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 239, , 2) Acid Hydrolysis Method, Dried rhizomes are powdered and subjected to hydrolysis by, refluxing with 5% HCl for 2 hours, The hydrolysed mass is filtered, washed twice with water and, then twice with 5% sodium bicarbonate solution, , Finally washed with water till the washing are neutral. The, residue obtained is dried and extracted with toluene for 8 hours, , Toluene extract is concentrated during which diosgenin gets, precipitated, , Diosgenin is filtered, washed with little hexane and dried (4060°C) to yield about 95% pure product, , 3) Fermentation cum Acid Hydrolysis Method, Fresh green roots are collected and smashed in a hammer mill., The mesh is placed in the fermentation bin and allowed for, fermentation for 2 days, The fermented mesh is dried in sun to reduce the moisture, content to 7-8 %. It is then subjected to hydrolysis with a, mineral acid at reduced temperature, , Resulting solution is extracted with heptane to obtain, diosgenin, , 4) Incubation cum Acid Hydrolysis Method, Fresh plant material is incubated in water at 37°C for few days., It is later subjected to acid hydrolysis, The hydrolysed liquid is concentrated and extracted with, hydrocarbon solvent to obtain diosgenin, , 8.1.5.2., , Estimation, , Diosgenin can be isolated by the following analytical methods:, 1) By TLC Method: The TLC profile is given:, i) Mixture of chloroform: ethanol (95:5) or chloroform:acetone (3:1) is the, solvent system., ii) Silica gel plates are used as the stationary phase., iii) Antimony trichloride (SbCl3) in chloroform is the detecting agent used., 2) By HPTLC Method: A stock solution of diosgenin (100µg/ml concentration), is prepared by dissolving1mg of diosgenin in 1ml of chloroform. A calibration, curve from 1000 -6000ng/spot is prepared and analysed for reproducibility,, *, , *
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240, , Pharmacognosy and Phytochemistry-II, , linearity, and validating the proposed method. The correlation coefficient (r),, coefficient of variance (CV%), and the linearity of r esults are also measured., To perform TLC of diosgenin , silica gel 60 F254 pre -coated plates (10 × 10, cm) are used with hexane:ethyl acetate (7.2:2.5)., By using Linomat 5 spotter, the pre -coated TLC plates are spotted with the, sample. The TLC plates are d eveloped up to 10 cm in the ascending mode., After this, the plates are sprayed with 20% antimony chloride in chloroform,, and dried in a chromatographic oven at 105ºC for 10 minutes. The diosgenin, concentration in the given sample is estimated by comparing, the area of, chromatogram of standard diosgenin., , 8.1.5.3., , Utilisation, , Diosgenin is used due to the following properties:, 1) It is used in the semi-synthesis of progesterone., 2) It is frequently used as the main ingredient in the production of early oral, contraceptive agents., 3) It acts as an unmodified steroid which shows estrogenic activity., 4) It helps in reducing the serum cholesterol level., , 8.1.6., , Digoxin, , Digoxin is obtained from Digitalis lanata of Plantaginaceae family, and is u sed, as a cardiotonic agent. Chemically, it is a glycoside having three sugars and a, aglycone part, digoxigenin., It shows positive inotropic and negative chronotropic activity. It helps, in, controlling the ventricular rate during atrial fibrillation. It i s also used in the, management of congestive heart failure with atrial fibrillation. However, its use, in congestive heart failure and sinus rhythm is not clearly understood. Digoxin, shows very small therapeutic window so the margin between toxic and, therapeutic doses is small., , Properties of digoxin are:, 1) It occurs as clear to white coloured crystals or crystalline powder., 2) It tastes bitter and has no odour., 3) It starts melting at 235ºC., 4) It is soluble in dilute alcohol, pyridine, or mixture of chloroform and alcohol., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 241, , 5) It is insoluble in ether, acetone, ethyl acetate, chloroform, and water., 6) It is slightly soluble in diluted alcohol, and very slightly soluble in, 40% propylene glycol., 7) On heating, it gets decomposed and emits acrid smoke and irritating fumes., , 8.1.6.1., , Industrial Production, , Industrial production of digoxin involves the following steps:, 1) Initial Extraction and Removal of Phenolics by Precipitation, i) The powdered drug (20ml) is extracted with 100ml of 70% etha nol and, heated on a hot plate for 20 minutes., ii) The extract is cool ed and filtered or centrifuged to remove solid plant, debris., iii) 150ml of water and 20ml of strong lead sub -acetate solution is added to, the filtrate or supernatant., iv) A precipitate of phenolic compounds is obtained in the form of insoluble, lead complexes., v) The mixture is centrifuged, the supernatant is pipette d out, and 10%, H2SO4 is added drop wise till no precipitate forms., vi) This treatment removes excess amount of lead ions in the form of, insoluble lead sulphate., 2) Solvent Partition to Remove Cardenolides from Aqueous Layer, i) The mixture is centrifuged, the supernatant is pipetted out , and further, extracted with 50ml of chloroform for four times., ii) The chloroform extract is combined and washed with 20 ml of water to, remove any residual lead ions., iii) The chloroform extract is separated and dried u sing anhydrous sodium, sulphate., iv) The obtained chloroform solution is filtered into a round bottom flask., v) 5ml aliquots of chloroform are, evaporated to dryness in separa te, evaporating dishes., vi) In the final step , two tests are performed on the residue; first is Kedde, test for cardenolides and the second is iron (III) chloride test for phenolic, compounds., To isolate digitoxin and digoxin, TLC is performed and the reference solution for, digoxin is 5%w/v solution in ethanol., , 8.1.6.2., , Estimation, , Concentration of digoxin in plant extracts, therapeutic preparation s, body fluids,, etc. may be measured by various methods based on the principal of colorimetry,, fluorimetry, gas liquid chromatography, HPLC, and radioimmunoassay., Method I (IP Method), 1) Accurately weighed 40mg of drug sample is dissolved in 95% ethanol, solution to produce 50ml., 2) 5ml of this solution is transferred to a 100ml standard flask and volume is, made up with the same solvent., *, , *
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242, , Pharmacognosy and Phytochemistry-II, , 3) 5ml of this solution is added with 3ml of alkaline picric acid solution., 4) The resultant solution is left undisturbed in the dark for 30 minutes., 5) Steps (1) to (3) are repeated on a standard sample of digoxin to make, the, standard solution., 6) The absorbances of test and standard solutions, are calculated at the, maximum at 495nm against a blank made up to 5ml 95% ethanol and 3ml, alkaline picric acid solution., 7) The digoxin content is calculated from the absorbance of the standar d of, known quantity., Method II - Paper Chromatography Process, 1) The sample solution [2mg in 5ml of CHCl 3 and CH 3OH (1:1)] and standar d, solution of same concentration are compared by placing 10 l aliquots of, each as separate spots on a filter paper strip (10cm from one end)., 2) Chromatogram is developed in a closed chamber according to the descending, technique, in which the end of the strip which is nearest to the sample is kept, in a t rough with equilibrated solvent and the trough is located in the upper, part of chamber. Thus, the solvent runs down through the paper., 3) A saturated amount is placed by placing small amount of both phases of the, equilibrated solvent in the bottom of the chromatographic chamber., 4) The chromatogram is removed from the chamber and air, dried when t he, solvent reaches a point within 5cm of the bottom edge of the strip., 5) Location of glycosidic zones in the sample and standard is determined by, spraying the strip with a 25% solution of trichloroacetic acid in methanol, followed by heating at 100C for a minute., 6) Presence of greyish green spot indicates the presence of digoxin in the, sample, having R1 value same as in standard solution:, i) On paper chromatograms, dig oxin is determined by using Baljet p icric, reagent,, ii) Both digoxin and isodigoxin can be determined by using Keller -Killiani, reagent (ferric chloride with sulphuric and glacial acetic acids)., TLC densitometry is used as an alternative method to HPLC and GLC for the, standardisation of crude medicinal plant extracts of Digitalis lanata., , 8.1.6.3., , Utilisation, , Digoxin is used along with other medications for treating heart failure. It is also, used to treat certain types of irregular heartbeat (such as chronic, atrial, fibrillation). Treatment of heart failure helps to regul ate the irregular heartbeat, and also decreases the risk of blood clots, thus reducing the chances of having a, heart attack or stroke. Digoxin is a cardiac glycoside that acts by affecting, some minerals (sodium and potassium) of the heart cells. This reduces the strain, on heart and also helps in maintaining a normal, steady, and strong heartbeat., , 8.1.7., , Atropine, , Atropine is obtained from the dried leaves and flowering tops of plant Atropa, belladonna of Solanaceae family . Total alkaloid al content in atropine as, calculated for hyoscyamine should not be less than 0.30%., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 243, , Properties of atropine are:, 1) It has a melting point of 115-116°C., 2) It is optically inactive., 3) It is a strong poison and has a sharp bitter taste., 4) It is used in ophthaquinology due to its dialating action on the eye pupils., 5) It stimulates and then depresses the CNS on internal administration., 6) It is the racemic modification of the laevo -rotatory hyoscyamine. Atropine, does not exist naturally but is formed during the isolation of hyoscyamine ., Atropine and hyoscyamine have similar physiological activity; however, the, activity of latter on the peripheral nerves is more than that of atropine., , 8.1.7.1., , Industrial Production, , Industrial production of atropine involves the following methods:, Method 1, 1) The powdered crude drug is moistened by adding sodium carbonate solution., 2) Then the powder is extracted with benzene or ether., 3) The alkaloidal bases are extracted with acidified water., 4) The acidified extract is treated with, solvent ether to remove the colouring matter., 5) Alkaloids are precipitated by sodium carbonate., 6) The obtained residue is filtered, washed, and dried., 7) The dry mass is dissolved in solvent ether and dehydrated with sodium, sulphate., 8) The obtained extract is filtered., 9) The filtrate is concentrated and cooled to obtain a mixture of closely related, alkaloids (majorly atropine and hyoscyamine)., 10) After filtration , the crude crystalline mass is separated and dissolved in, alcohol., 11) To convert hyoscyamine into atropine, sodium hydroxide solution is added to, the above mixture., 12) Acetone is added to crude atropine for re-crystallisation., 13) The obtained product is converted to atropine sulphate that occurs as white, crystalline powder., Method 2, 1) The plant leaves are powdered and extracted with 95% ethanol., 2) Then the ethanol is distilled off., 3) The resinous matter is removed by treating the obtained syrupy mass with, 1% hydrochloric acid., 4) This acidified solution is tre ated with light petroleum ether, followed by the, addition of ammonia to make it alkaline., 5) The alkaline solution is then treated with chloroform., 6) The chloroform layer is treated with dilute acid., 7) The chloroform layer is separated, made alkaline, and re -extracted with, chloroform., 8) This chloroform layer is evaporated, and oxalic acid is added to get oxalates, of atropine and hyoscyamine., 9) Atropine is separated from hyosyamine by fractional crystallisation., *, , *
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244, , Pharmacognosy and Phytochemistry-II, , Method 3, 1) Isolation of atropine is done from roots of belladonna plant or juice of datura, plant., 2) 95% alcohol is used to extract atropine from the powdered drug., 3) To convert hyoscyamine to atropine, the alcoholic extract obtained in the, above step is heated with potassium carbonate., 4) The obtained extract is re-extracted with chloroform., 5) The chloroform layer is evaporated, and the resi due is treated with dilute, sulphuric acid., 6) Atropine is precipitated by treating the above solution with po tassium, carbonate., 7) This atropine is further extracted with ether, and purified by adding oxalic, acid to yield crystals of atropine oxalate., , 8.1.7.2., , Estimation, , Atropine can be estimated by the following methods:, 1) USP Assay for Belladonna Leaf, i) It is useful for quantitative estimation of total alkaloids., ii) Alkaloids are extracted with ether and purified by re -extracting with, 0.5N H2SO4 (as sulphates) and then with chloroform (as free bases)., iii) The chloroform extract is evaporated to dryness., iv) The alkaloidal residue is taken up with a definite quantity of standardised, H2SO4 solution in slight excess of the quantity of stand, ard H2SO4 in, slight excess of the quantity of acid required to form sulphate salts with, the alkaloids present., v) The amount of un -reacted acid is estimated through titration with, standard alkali., vi) The quantity of alkaloids can be determined from the molar quantity of, acid that reacted with alkaloids producing atropine salts., 2) Assay for Atropine Methonitrate, i) Accurately weighed 0.5gm of atropine methonitrate is dissolved in 50ml, of acetic anhydride., ii) The resultant solution is titrated with 0.1N HClO4 solution., iii) The endpoint is determined potentiometrically., iv) A blank determination is done and necessary corrections are made., v) Perform a blank determination with necessary corrections., vi) Each ml of 0.1N HClO4 acid used ≡ 0.036640gm of atropine methonitrate., 3) Assay of Atropine Sulphate, i) Accurately weighed 0.5gm of atropine is dissolved in 50ml of glacial, acetic acid solution., ii) The resultant solution is titrated with 0.1N HClO4 solution., iii) The end point can be determined potentiometrically., iv) A blank determination is performed and the requiredcorrections are made., v) Each ml of 0.1N HClO4 solution ≡ 0.06770gm of atropine sulphate., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 8.1.7.3., , 245, , Utilisation, , Atropine is a parasympatholytic drug . It has stimulant act, ion on central,, medullary and higher nerve centres. It has various therapeutic uses:, 1) It acts as an antidote for poisoning by pilocarpine, physostigmine, and other, choline esters., 2) It is used in asthma to relieve bronchial spasms, 3) It is used in the treatment of peptic ulcer as it suppresses gastric secretions., 4) In is used in ophthalmic practice, since it has dilatory effects on eye pupil., 5) In Parkinsonism, it reduces tremor and rigidity., , 8.1.8., , Podophyllotoxin, , Podophyllotoxin occur s naturally and is obtained from Podophyllum peltatum, and Podophyllum emodi. It bears medicinal properties. A podophyllotoxin, derivative, etoposide has usefulness in the treatment of many cancers, especially, small cell lung carcinoma and testicular cancer., Properties of podophyllotoxin are:, 1) It occurs as solvated crystals., 2) It has a melting point of 183ºC., 3) It is highly soluble in ethanol and chloroform; soluble in acetone and benzene;, slightly soluble in water; and insoluble in ethyl ether and ligroin., , 8.1.8.1., , Industrial Production, , Industrial production of podophyllotoxin involves the following methods:, Method 1, 1) Accurately weighed powdered drug is extracted with methanol., 2) The extract is filtered and concentrated., 3) The obtained semisolid concentrate is dissolved in acidified water., 4) The resultant precipitate is allowed to settle for 2 hours., 5) The filtrate is decanted and washed with cold water., 6) The residue is collect ed, washed with acidified water, and dried to obtain, dark brown amorphous powder of podophyllin., 7) This powder is treated with hot alcohol, filtered, and evaporated to dryness., 8) The residue is re-crystallised with benzene or mixture of alcohol and benzene, followed by washing with petroleum ether., Method 2, 1) The chloroform soluble fraction of powder is dissolved in alcohol., 2) The resultant mixture is refluxed with neutral aluminium oxide to obtain light, yellow coloured solution., 3) The alcoholic solution is added with benzene to obtain podophyllotoxin., Method 3, 1) This method involves extraction over a bed of neutral alumina with benzene,, toluene etc. solvents for 1-4 hours., 2) Pure podophyllotox in is obtained by re-crystallisation using h ot benzene,, toluene or xylene., *, , *
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246, , 8.1.8.2., , Pharmacognosy and Phytochemistry-II, , Estimation, , Podophyllotoxin can be estimated by the following methods:, Method 1: Thin Layer Chromatography Method, Test Solution:, Reference Solution, 1mg podophyllotoxin is dissolved in 2ml methanol., Solvent System, Toluene:Ethyl acetate (5:7)., Procedure, 5ml of each test and reference solution s are taken in separate tracks on a plate, previously coated with silica gel G (5815cm) of uniform thicknes s (0.2mm). The, plate is developed in a solvent system up to a distance of 10 cm., Scanning, Scanning is done with densitometer at 280mm for reference and test solution, tracks and finger print profiles are recorded. The test solution profile is compared, with that of the sample by spraying with sulphuric acid reagent heated at 120°C, for 10 minutes., Evaluation, In day light , a violet coloured spot (R f = 0.39) in reference and test solution, tracks indicates podophyllotoxin. A dark brown spot is visible at application site., Method 2 (Used for Absence of Peltatins), Test Solution, 1gm of the powdered drug is heated under reflux for 10 minutes with, methanol and filtered. The filtrate is evaporated up to 3ml., , 10ml, , Solvent System Chloroform, Methanol (90:10) for 6cm and then toluene:acetone (65:35) for 1.5cm distance., Procedure, On a pre -coated silica gel plate, 20 -30ml of test solution is applied uniformly., The solvent system is made to run on the plate., Visualisation, For indication, fast blue salt reagent is sprayed over the plate., Evaluation, 1) Red brown tailing zones indicate tannins (RF value up to 0.6 or above)., 2) No red brown zones indicate absence of peltatins., 3) Red brown zones at R f = 0.65 and R f= 0.80 will be visible if and are, present (Podophyllum peltatum)., Method 3: HPLC Determination of Podophyllotoxin, Column, cis-symmetry 4.6 × 150mm of 5., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 247, , Mobile Phase, Methanol:water (62:38)., Flow Rate, 1.0ml/min., UV detection at 280 mm., Standard Preparation, Solution of p odophyllotoxin (of known concentration) is prepared in methanol, within the linearity range (20g–2g)., Sample Preparation, 1) The powdered sample (2gm) is refluxed with 50ml methanol for an hour,, cooled, and filtered., 2) The marc is refluxed for another 1 hour with 50ml methanol., 3) The filtrates of both the cycles of refluxes are combined and evaporated to, dryness under vacuum., 4) The residue is dissolved in 20ml methanol and volume is made up to 50ml, with the same solvent., Procedure, 5ml each of standard and sample preparations are injected to HPLC on triplicate., Then the peak areas for podophyllotoxin are recorded, and the yield % in the, sample is calculated., , 8.1.8.3., , Utilisation, , Podophyllum resin has the following uses:, 1) It produces cytotoxic action, hence is used for removing soft venereal and, other warts., 2) Etoposide is used as an antineoplastic agent., 3) It is a drastic but slow-acting purgative., 4) Podophyllotoxin shows anti -tumour (antineoplastic) properties , therefore is, used in cancer treatment., 5) It is prescribed with other pur gatives like henbane or belladonna to avoid, gripping in infants., , 8.1.9., , Caffeine, , Caffeine is obtained from the dried ripe seeds of, Coffea a rabica Linne or C., liberica Hiern (deprived of most of the seed coat). Another source is the prepared, leaves and leaf buds of Thea sinensis. Properties of caffeine are:, 1) It has a melting point of 235ºC., 2) It crystallises as silky needles., 3) It has a bitter taste., 4) It is sparingly soluble in water and alcohol., 5) It is a weak base., 6) It forms salts with strong acids, which get easily decomposed by water., 7) Its citrate and hydrochloride salts are used as diuretics and heart and nerve, stimulants. However, its excessive use disturbs the digestion., *, , *
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248, , 8.1.9.1., , Pharmacognosy and Phytochemistry-II, , Industrial Production, , Industrial production of caffeine involves the following methods:, Method 1, 1) The moistened coffee beans are fed to the extractor. For 10 hours, CO 2 from, CO2 holding tank is passed through the bed of beans., 2) This process extracts around 97% of caffeine from the beans. The CO, 2containing caffeine is stored in the holding tank till the extraction process, ends. Thereafter, the exhausted material is removed from the extractor and, fresh material is fed., 3) Then the caffeine -containing CO 2 stored in the holding tank is passed at a, steady rate through the water wash column., 4) Here the supercritical CO 2 comes in contact with a steam of water. This, removes 99.5% of caffeine from the CO2., 5) The regenerated supercritical CO 2 leaves the water wash column. At this, point, CO2 is at a lower pressure due to pressure drop through the system., 6) The regenerated CO2 accumulates in the holding tank after 2 hours CO2 is fed, back to the extractor, to start decaffeination of a new batch of coffee., 7) The caffeine-rich water in the process vessel is added with, water. The, caffeine extract is concentrated in the reverse osmosis unit, and is sent to a, drying section to obtain solid caffeine., 8) Caffeine is extracted from green tea by supercritical fluid extraction under, the following conditions:, i) 15ml ethanol is used as co-solvent, and, ii) Extraction is carried out at 80°C temperature., 9) The flow rate and pressure of SC -CO2 are maintained at 1.5 l/min and 300, bars, respectively. The extraction duration is 2 hours. This method produce s, 70.2% caffeine and 6.2% catechins., Method 2, 1) Caffeine is isolated from instant coffee granules by supercritical CO, 2 methanol., 2) The apparatus has two reciprocating pumps, column oven, back, -pressure, regulator, a circulating water bath with ethylene glycol mixture, an extraction, cell, and a collection cell., 3) SFE is done under the conditions stated below:, i) The flow rate of liquid carbon dioxide and methanol is maintained at, 1.8ml/min and 0.02ml/min, respectively., ii) The oven temperature is set at 60°C and pressure, is maintained at, 2, 250kg/cm ., , 8.1.9.2., , Estimation, , Caffeine can be estimated by the following methods:, 1) Determination of Caffeine by HPLC, Standard Solutions: Stock solution of caffeine (1000 ppm) is prepared by, weighing 100mg of pure caffeine and taking it in a 100 ml volumetric flas k., The volume is made up to the mark with the mobile phase. Through serial, dilution of the stock solution with the mobile phase, working standards of 10,, 20, 40, 60 and 80 ppm are made., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 249, , Sample Preparation and Analyte Determination: 2gm of tea and coffee, samples are weighed in triplicate , taken in 250ml beakers, and added with, 100ml of boiling distilled water. The beakers are left undisturbed, for 5, minutes and stirred. This solution is cooled and filtered in conical flasks. 5ml, of the obtained filtrate is pipetted out in clean 50 ml volumetric flasks and, volume is made up to the mark with the mobile phase. The standards and the, samples are run in the HPLC. The calibration curve of peak areas versus, concentration of the standards is plotted, and the caffeine level in samples is, calculated from the regression equation of the line of best fit., 2) Determination of Caffeine by UV/Vis Spectrophotometry, Calibration Standards: Stock solution of caffeine (1000 ppm) is prepared, by d issolving 100mg of pure caffeine in 10 0ml distilled water . Caffeine, working solutions of 0, 10, 20, 40, 60 and 80 ppm, strengths are made by, serial dilution of the stock in 25ml volumetric flasks. 1ml of hydrochloric, acid is added and volume is made up to the mark with distilled water., Sample Preparation and Analytic al Determination: 0.25gm of sample is, dissolved in water. Volume is made up to 20ml with distilled water as sample, solution. 20ml sample solution is pipetted out in a 250ml flask, and added, with 10 ml 0.01M hydrochloric acid and 2 ml basic lead acetate solution., Volume is made up to the mark with distilled water, s haken, and filtered to, clarify. 50ml of this filtered solution is t ransferred to a 100ml flask, and, added with 0.2ml sulphuric acid. Volume is made, with distilled water,, shaken, and filtered. Absorbance of the working standards and samples is, measured with UV/Vis spectrophotometer at 274nm wavelength using 10mm, quartz cuvette. The caffeine levels in the samples are calculated from the, regression equation for the best line of fit of the standards., 3) Determination of Caffeine by FTIR Spectrophotometry, Preparation of Beverage Samples : 1.5gm of coffee powder is dissolved in, 70ml of hot distilled water, and boiled for 10 minutes. This solution is diluted, to 100ml and cool ed. Fine sus pensions are filtered through W hatman No. 1, filter paper. In 3 ml of aqueous coffee solution, 3 ml of chloroform is added., Upon phase separation, 1ml of caffeine solution is diluted to 10-fold with, chloroform, and the final solution is used for FTIR analysis., Data Collection: FTIR spectra is recorded using, MIDAC (M 2000), spectrometer carrying liquid nitrogen cooled mercury/cadmium telluride, detector and a 45° horizontal zinc selenide crystal as an ATR accessory. The, spectra are recorded at 4cm resolution with 512 co-added scans. The moisture, may give rise to some unwanted IR signals, which can be avoided by purging, the instrument with dry air for 4hours prior to interferogram recording., Firstly, an open beam background spectrum is collected using clean zinc, selenide crystal that has been washed with chloroform. Sample spectra are, collected by applying 0.5 ml caf feine solution in chloroform on the crystal., The solvent is allowed to dry before collecting the interferograms of caffeine, film on the ATR crystal . Each sample interferogram is ratioed against the, background interferogram. The resulting transmission spectrum is converted, *, , *
Page 251 :
250, , Pharmacognosy and Phytochemistry-II, , into an absorption spectrum. Before, the application of new sample for, spectral recording, the caffeine film from the zinc selenide crystal is removed, using chloroform. Caffeine concentration in a coffee extract is estimated, from the standard curve and absorbance of the extracted caffeine., , 8.1.9.3., , Utilisation, , Caffeine is known to be, a CNS stimulant, given in combi nation with other, therapeutic agent; as in case of analgesic preparations. It presents pharmacological, effects on CNS, heart, peripheral and central vasculature, renal, gastrointestinal and, respiratory system.It releases adrenaline and increases cyclic AMP by competitively, inhibiting phosphodiesterase. This results in CNS stimulation, bronchial smooth, muscle relaxation, and diuresis. Products including caffeine are being consumed, from several years due to its pleasant flavour and stimulating effects., , 8.1.10. Taxol, Taxol (or paclitaxel) is a natural diterpenoid. It is isolated from the barks of the, plants Taxus bravifolia , Taxus c uspidata, and Taxus canadensis of Taxaceae, family. Taxol has the following characteristics:, 1) It has a taxane ring system., 2) It has a 4-membered octane ring., 3) At C -13 of the taxane ring , an ester side chain is present, which is, responsible for the cytotoxic activity of taxol., 4) At C -2 of the ester side chain, a h ydroxyl moiety is present, which causes, enhancement of the cytotoxic activity., , 8.1.10.1. Industrial Production, Industrial production of taxol involves the following steps:, 1) The dried ground bark is extracted with methanol or ethanol , and alcohol is, removed by concentrating the combined extract., 2) The obtained concentrate is re -extracted with dichloromethane and the, solvent extract is concentrated to a powder., 3) This powder is stirred with a mixture of acetone and ligroin (1:1) and filtered, to remove the insoluble matter., 4) The filtrate containing taxol is concentrated, dissolved in 30% acetone in, ligroin, and applied to a column of Florisil., *, , *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 251, , 5) The taxol fraction from the column is twice purified by crystallisation., 6) The crystalline taxol is subjected to chromatography on a silica column. The, closely related analogue, cephalomannine, separates out from taxol., 7) The purified taxol obtained from the column is crystallised twice., 8) The unseparated mixtures and mother liquors are recycled through the silica, column to obtain more pure taxol., , 8.1.10.2. Estimation, Taxol can be estimated by the following steps:, 1) In a 250ml conical flask, 0.125gm of drug + 25ml water + 50ml of 0.83M, KMnO4 + 100ml of H2SO4 are taken., 2) The solution is heated up to the boiling point, cooled , and transfer red to a, volumetric flask., 3) The solution is diluted with distilled water up to 250ml., 4) From the abovesolution, 50ml is treated with 1M Fe(NH4)2SO4, ferroin sulphate, solution (an indicator) is added drop wise, and the blank is performed., 5) Each ml of 0.1M ferrous ammonium sulphate ≡ 0.000675gm of cellulose., Taxol can be estimated by the following three methods:, Method 1: Immunological Assay, In this method, immune-affinity is developed to be applied in the extraction of, various samples having taxanes. Polyalcohol antibodies (isolated from animals ), acting against taxanes are immobilised on an application support. Samples having, less taxane content are extracted and analysed by HPLC diode assay by, the, previously developed methods. HPLC analysis is simplified and more informative,, as many contaminants would get discarded duringthe immune-assay method., Method 2: Liquid Chromatography-Mass Spectrometer (LC/MS), The t raditional structure analysis protocols involve scale -up extraction and, fractionation. Then individual fraction spectroscop ic analysis is done using, LC/MS instrument., Method 3: High Performance Liquid Chromatography -Mass Spectrometry, (HPLC-MS), In this method, taxol concentration is determined against the authentic standard., , 8.1.10.3. Utilisation, Taxol is in dicated in the treatment of bladder, prostate, melanoma, oesophageal,, and other solid tumour cancers. Taxol is a prescription medicine used against the, following forms of cancer:, 1) Advanced ovarian cancer., 2) Breast cancer; upon failure of combination chemother, apy for metastatic, disease or relapse within 6 months of adjuvant chemotherapy, taxol is the, preferred choice., 3) Non-small cell lung cancer in patients who cannot undergo curative surgery, and/or radiation therapy., 4) Kaposi’s sarcoma forms patches of abnormal tissue growth under the skin, in, the nose, in the mouth lining, in the throat, or other organs., *, , *
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Pharmacognosy and Phytochemistry-II, , ncluding:, n the treatment and management of metastatic carcinoma of the ovarian glandson failure of follow-u, eat breast cancer when combination chemotherapy for metastatic disease fails., c in nature, its injectable concentrate (for intravenous infusion ) is solubilised in polyoxyethylated, saline or dextrose solution or combination, and then injected., , ine and Vinblastine, , stine are isolated from the dried whole plant of Catharanthus roseus or Vinca rosea of Apocynacea, , l Production, , of vincristine and vinblastine involves the following steps:, erial is extracted with a solution of hot ethanol-water-acetic acid (9:1:1)., d and the residue is added with hot 2% hydrochloric acid solution., is adjusted to pH 4 for precipitating the non, -alkaloidal components that are separated by, *
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Industrial Production, Estimation and Utilisation of Phytoconstituents (Chapter 8), , 253, , 4) Now the aqueous acidic oslution pH is adjusted to 7 andextracted with benzene., 5) The benzene layer is evaporated to obtain vinca alkaloids., , 8.1.11.2. Estimation, Vincristine and vinblastine can be estimated as follows:, 1) Chromatographic Method, i) Detection:, a) UV = 254nm, b) DRG No. 13B = visible range, ii) Drug Sample: Vinca (fresh leaves)., iii) Reference Compounds: T1 = v incamine, T 2 = vincaminine, T, vincine, T4 = vincamajine, T5 minouincine, and T6 = reserpinine., iv) Solvent System: Ethyl acetate:methanol (90:10)., , 3, , =, , 2) USP NF-1995, i) Diethyl Amine Solution: 5ml of diethylamine is mixed with 295ml of, water, and the pH is adjusted to 7.5 with phosphoric acid., ii) Mobile Phase: A filtered and degassed mixture of methanol and diethyl, amine solution (70:30) is prepared., iii) Standard Preparation: Accurately weighed quantity of USP vincristine, sulphate is dissolved in water to achieve 1mg/ml concentration., iv) Assay Preparation, a) A portion of vincristine is equilibrated for 30 minutes with an, ambient humidity., b) About 10mg if transferred to 10ml of volumetric flask, and dissolved, in water to make up the required volume., v) System Suitability Preparation, a) 5mg each of USP vincristine sulphate and vinblastine sulphate are, transferred in a 5ml volumetric flask, and dissolved in water., b) The resultant s olution is further diluted with water to make up the, volume., Chromatographic System, Column, 4.6mm* 25cm analytical containing L 7 packing 2.5cm guard column containing, L1 packing., Flow rate, 1.5ml/min., Detector, 297nm., Procedure, 1) 10ml each of standard an d assay preparations are fed into the chromatograph, and the peak responses are recorded., 2) The quantity of vincristine sulphate (in mg) is calculated using:, 10C(ru/rs)., *, , *
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256, , Pharmacognosy and Phytochemistry-II, , CHAPTER, 9, , Basics of Phytochemistry, , 9.1. PHYTOCHEMISTRY, 9.1.1., , Introduction, , Phytochemistry involves the study of chemicals (mainly the secondary, metabolites) the plants produce as a measure to protect themselves from insects,, pests, pathogens, herbivores, UV exposure, and, environmental hazards., Phytochemistry includes the structural compositions, the biosynthetic pathways,, functions, mechanism of actions in the living systems, and the medicinal,, industrial, and commercial applications of secondary metabolites., Phytochemicals (derived from the Greek word, phyto meaning plant) are, naturally occurring and biologically active chemical compounds present in, plants. They provide health benefits for humans in addition to those provided by, the macronutrients and micronutrients., , 9.1.2., , Extraction, , The process of separating medicinally active constituents of plant and animal, tissues with the help of selective solvents and standard procedures is termed, extraction. The extracted products of plant tissues obtained in liquid or semisolid, state (after removing the solvent) or in dry powdered form are complex mixtures, of metabolites; these products are meant for oral or external use. The extracted, preparations include decoctions, infusions, fluid extracts, tinctures, pilular, (semisolid) extracts, or powdered extracts; these preparations are named as, galenicals after Galen (a Greek physician of 2nd century)., The standardised extraction procedures involve treatment with a selective solvent, (menstruum) to yield the therapeutically active constituents of crude drugs,, removing the inactive ones. The undissolved residue left behind is termed marc., The extract obtained by this process, after standardisation is either used as a, medicinal agent in the form of tinctures or fluid extracts, or is further processed, for incorporation in tablet and capsule forms., The drug extraction process is divided into the following four steps:, 1) The solvent penetrates the drug,, 2) The drug constituents dissolve in the solvent,, 3) The solution within the cells diffuses out, and, 4) The dissolved portion separates from the exhausted drug., The efficiency of extraction process depends on the following factors:, 1) Nature and properties of drug and its extractable constituents,, *, , *
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Basics of Phytochemistry (Chapter 9), , 257, , 2) Particle size of the powdered drug,, 3) Solvent nature, and, 4) The state of contact between the solvent and drug particles., Drug extraction process is also influenced by various, physical factors , like, gravitation, diffusion, osmosis, adhesion, capillarity, convection, solubility, and, surface tension. A solvent’s wetting property can be improved by displacing the, air entrapped within the capillaries and by using a surfactant, which also, increases the solubility of cellular contents by solubilisation in the menstruum., With the help of agitation, equilibrium bet ween the outer and inner cellular, contents can be attained fast; this in turn facilitates dissolution of the desired, constituents in the menstruum., Extraction at elevated temperatures has the following advantages:, 1) Decreased viscosity reduces the boundary layers., 2) Convection currents act similar to agitation during the extraction process., 3) Diffusion coefficients are directly proportional to absolute temperature and, inversely proportional to viscosity, thus diffusion rate is influ, enced by, increased temperature., 4) Solubility of the constituents in the menstruum increases with increased, temperature., The menstruum used for extraction should have the following properties:, 1) Chemically and physically inert,, 2) Non-toxic,, 3) Inexpensive, and, 4) Selective, i.e., it should disso lve the desired active constituents with a, minimum of the inert material., Water, ethanol, and their mixtures are most commonly used as they fulfil the, above mentioned considerations., , 9.1.2.1., , Principles of Extraction, , The principles and mechanisms of extracting cr, ude drugs by maceration,, percolation, and infusion are similar to those of extracting soluble constituents, from solid materials using solvent (referred to as leaching which involves simple, physical solution or dissolution)., Extraction procedures get affe cted by the transport rate of solvent into the mass,, the solubilisation rate of the soluble constituents by the solvent, and the transport, rate of solution out of the insoluble material. Extraction of crude drugs gets, facilitated by increase in the surface area of the material to be extracted and, decrease in the radial distances travelled between the crude drug particles., The theory of mass transfer states that maximum surface area is obtained by, size reductions that involve reduction of materials into in dividual cells; however,, this theory is not acceptable in many cases of vegetable material. It has been, proved that hundreds of unbroken cells having intact cell wall are present in 200, mesh particles. Therefore, these unbroken cells are used to carry out extraction so, *, , *
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258, , Pharmacognosy and Phytochemistry-II, , that an extract with a high degree of purity can be obtained. This also provides, sufficient time to the solvent to diffuse through the cell wall for dissolution of the, desired solute (or groups of constituents) and to the solution (or the ext ract) to, diffuse through the cell wall surface., , 9.1.2.2., , Choice of Solvents, , The solvent used for extraction process is significantly important for determining, the biologically active compounds from plant materials. The choice of solvent is, affected by the quantity of phytochemicals to be extracted, the extraction rate,, diversity of inhibitory compounds extracted, ease of handling of the extracts,, toxicity of the solvent in the bioassay, and the potential health hazards of the, extractants. Since traces of residual solvent are always present in the end product,, the solvent used should be non-toxic and should not interfere with the bioassay., An ideal solvent used for extraction should have the following properties:, 1) It should be economical., 2) It should be non-toxic., 3) It should be stable, i.e., physically and chemically inert., 4) It should not be too volatile or inflammable., 5) It should be selective in nature, i.e., the desired amount of active ingredient, can be extracted using minimum amount of inert material., The different solvents used for the extraction process are discussed below:, 1) Water: It is a universal solvent that is used for extracting plant products, having antimicrobial properties. The traditional healers use water extract, but, the plant extracts from organic solvent s provide more reliable antimicrobial, action. On the other hand, the water, -soluble flavonoids (mostly, anthocyanins) have no antimicrobial activity and water -soluble phenolics are, only important as antioxidant., 2) Acetone: It is miscible with water, is volatil e, has a low toxicity, and is a, very useful extractant for antimicrobial studies in which more phenolic, compounds are to be extracted. Acetone dissolves many hydrophilic and, lipophilic components from plants. In a study it was revealed that tannins and, other phenolics can be better extracted using aqueous acetone than the, aqueous methanol. However, acetone and methanol are both useful in, extracting saponins having antimicrobial activity., 3) Alcohol: The activity of ethanolic extracts is much higher than that o f the, aqueous extracts due to the presence of higher amounts of polyphenols. It, means that the ethanolic extracts more efficiently degrade the non -polar cell, walls and seeds to release polyphenols from the cells. The decreased activity, of aqueous extract c an also be due to the polyphenol oxidase enzyme, which, degrades polyphenols only in water extracts, while it becomes inactive in, methanolic and ethanolic extracts. Besides, water also provides a better, medium for microbial growth in comparison to ethanol. The concentration of, bioactive flavonoids was found to be more with 70% ethanol due to its higher, polarity than the pure ethanol. However, the polarity of pure ethanol can be, increased by adding water to it up to 30% for preparing 70% ethanol., *, , *
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Basics of Phytochemistry (Chapter 9), , 259, , Moreover, e thanol can also easily penetrate the plant cellular membrane to, extract the intracellular ingredients. Almost all the known plant components, that are active against microorganisms are aromatic or saturated organic, compounds, thus they are often obtained th, rough primary ethanol or, methanol extraction. Methanol is although polar than ethanol, but its, cytotoxic nature makes it inappropriate to be used for extraction a s it may, give improper results., 4) Chloroform: Terpenoid lactones can be obtained by successive e xtractions, of dried barks using hexane, chloroform, and methanol with activity, concentrating in chloroform fraction. Occasionally tannins and terpenoids are, found in the aqueous phase, but they are more often obtained by extraction, using less polar solvents., 5) Ether: It is commonly used for extracting coumarins and fatty acids., 6) Dichloromethanol: It is mainly used for the selective extraction of, terpenoids., , 9.1.2.3., , Modern Methods of Extraction, , Extraction of crude drugs can be carried out by various processes, and th, selection of process depends on the chemical properties of the drug’s active, constituents., , e, , Factors Affecting Choice of an Extraction Process, 1) Nature of the Drug: The detailed pharmacognosy of the drug to be extracted, helps in selecting the extraction pr ocess with optimum results. For extracting, hard and tough drugs ( e.g., nux vomica), percolation method is used; while, for soft and parenchymatous drugs ( e.g., gelatin), drugs which cannot be, easily powdered ( e.g., squill), and for unorganised drugs ( e.g., benzoin),, maceration method is preferred., 2) Therapeutic Value of the Drug:, For extracting less therapeutically, important drugs [e.g., flavours (lemon), bitters (gentian)], maceration method, is used; while for drugs with significant therapeutic value ( e.g., belladonna),, percolation method is preferred., 3) Stability of the Drug:, For drugs whose constituents are thermolabile,, continuous extraction method should be avoided., 4) Cost of Drug: For costly drugs (e.g., ginger), percolation method is used (as, it is economical); while for cheaper drugs, maceration method is preferred., 5) Solvent: For drugs whose desired constituents require a solvent other than a, simple boiling solvent or an azeotrope, reserved percolation method (and not, continuous extraction) is used., 6) Concentration of the Product: Dilute products (like tinctures) can be, prepared by maceration or percolation method. Percolation is used for, preparing semi -concentrated products ( e.g., concentrated infusions) and, concentrated products ( e.g., liquid extracts or dry extracts). In case the, solvent is suitable and the desired constituents are heat -stable, continuous, extraction method is used., *, , *
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260, , Pharmacognosy and Phytochemistry-II, , Various extraction methods employed are:, 1) Maceration,, 2) Digestion,, 3) Percolation,, 4) Continuous hot extraction (Soxhlet extraction),, 5) Supercritical fluid extraction,, 6) Counter current extraction,, 7) Microwave assisted extraction,, 8) Ultrasonic assisted extraction,, 9) Infusion and decoction,, 10) Pressure cooker extraction,, 11) Extraction by passage through a colloid mill,, 12) Use of surface active agents in drug extraction, and, 13) Expression and diacolation., , 9.1.2.4., , Maceration, , The word maceration denotes softening. The maceration process (or Process, M) is used for producing tinctures, extracts, and concentrated infusions. It is the, simplest method of crude drug extraction, which was official in I.P., 1966., Classification, 1) Simple Maceration: It is a method for preparing tinctures from organised, drugs, e.g., roots, stems, leaves, etc., 2) Modified Maceration: It is a method for, preparing tinctures from, unorganised drugs, e.g., oleo-resins and gum resins., 3) Multiple Maceration: It is a method for, This method includes:, i) Double maceration, and, ii) Triple maceration., , preparing concentrated extracts., , Simple Maceration, Simple macerati on involves extraction of organised drugs having specific cell, structures, e.g., roots, stems, leaves, flowers, etc. It is a very simple method,, which does not require trained operators. Tincture of myrrh and compound, tincture of benzoin are examples of products prepared by simple maceration., Principle, In simple maceration, solid ingredients and the solvent are taken in a stoppered, container, and left undisturbed for at least 3-7 days with frequent agitation. When, the soluble matter dissolves in the solvent, the resultant mixture is passed through, sieves or nets. The marc retained in the sieves is pressed, the liquids are, combined, and filtered or decanted after standing., Apparatus, Simple maceration is performed using a wide mouthed bottle or any other, container which can be closed tightly to prevent evaporation of the menstruum, (figure 9.1)., *, , *
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Basics of Phytochemistry (Chapter 9), , Procedure, In simple maceration, the crude drug to be, extracted and the menstruum are placed in close, contact in a closed vessel for 7 days with, frequent agitation. After 7 days, the resultant, mixture is strained through sieves, the marc, retained is pressed, and the liquids are combined, and filtered. The drug, should be properly, comminuted., , 261, , Stopper, Maceration, bottle, Solvent layer, Drug, , The menstruum penetrates the cellular structure, of drug, and softens and ultim ately dissolves the, soluble portions. A closed vessel is used for, Figure 9.1: Maceration Bottle, preventing menstruum loss by evaporation., Occasional shaking is recommended to maintain a rapid equilibrium between the, intra- and extra -cellular fluids. The degree of pressing the marc may vary, thus, the final product is not adjusted to a fixed volume. Simple maceration process, may take around 14 days to complete. The ratio of drug to menstruum should be, 1:10. When the drug and menstruum are left undisturbed, sediment may form,, which can be avoided by standing the fluid product for a few days prior to use., Simple maceration method cannot be used for extracting all the drugs, thus other, maceration processes have to be employed., Modified Maceration, Modified maceration is used for extraction of drugs having no cellular or tissue, structure, i.e., unorganised drugs ( e.g., gums, resins, gum -resins, and oleo gum resins). During the extraction of unorganised drugs, the marc forms a compact, mass and retains no macerate; thus pressing the marc is not required. In modified, maceration, the final product is adjusted to a definite volume for extracting, unorganised drugs, because complete extraction of constituents occurs within a, short time period. Since the unorganised drugs lack cellular structure, the soluble, components are in direct contact with the menstruum, thus quickening the, extraction process. Modified maceration occurs in the following steps:, 1) The unorganised drug to be extracted is reduced to minute particl, es and, placed in a closed vessel with the menstruum (equal to 80% of the bulk of the, finished product) for 2-7 days., 2) This mixture is frequently agitated., 3) After a week, the mixture is strained and the strained liquid is filtered., 4) The marc left behind is was hed with fresh menstruum (less than 20% of the, finished bulk) and the washings obtained are passed through the strainer and, filter used previously (in step 3)., 5) Menstruum is passed through the filter in amount sufficient to make up the, desired volume., 6) A dry receiver or a receiver washed with the menstruum, is used for, collecting the filtrate; this is because unorganised drugs contain water, insoluble resinous matter which gets precipitated, thus affecting the clarity of, the finished product., *, , *
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262, , Pharmacognosy and Phytochemistry-II, , Multiple Maceration, Multiple maceration process achieves maximum extraction by utilising the, menstruum in portions for successive maceration. However, allowance is made, for the volume of menstruum retained by the marc at the end of the process. The, ratio of drug to menstruum is low . The menstruum volume for each maceration, process can be calculated as:, Double Maceration, Vol.of menstruum for first maceration , , Total vol. – Vol.retained by drug, Vol.retained by drug, 2, , If 200gm of drug is to be extracted with 1100ml of the total volume of, menstruum by double maceration, the menstruum volume to be used for first, maceration would be:, , , 1100 100, 100 600ml, 2, , It is assumed that after pressing, each 2gm of drug retains 1ml of menstruum., Hence, the menstruum volume to be used for second maceration would be:, 1100 – 600 = 500ml., Triple Maceration, Vol.of menstruum for first maceration , , Total vol. Vol.retained by drug, Vol.retained by drug, 3, , If 1000gm of drug is to be extracted with 9000ml of the menstruum by triple, maceration, the menstruum volume to be used for first maceration would be:, 9000 3000, , 3000 5000 ml, 3, It is assumed that each gm of unpressed drug retains 3ml of menstruum. Hence, the menstruum volume to be used for second and third maceration would be:, 9000 5000, , 2000ml each, 2, If 200gm of drug is to be extracted with 1100ml of the menstruum by single,, double, and triple maceration, the extractives will approximately be 90%, 96.5%,, and 98.5%, respectively. Thus, the extraction effectiveness increases with the, increase in number of macerations in multiple operations. Multiple maceration,, although is not in use at the present time., , 9.1.2.5., , Digestion, , Digestion is a modified maceration process . It involves extraction at such a, high temperature which does not put adverse effects on the active ingredients., Higher temperature enhances the solvent action of menstruum and constant, mechanical agitation of the system speed up the attainment of equilibrium. If at, the used temperature the menstruum gets volatilised easily, a reflux condenser, *, , *
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Basics of Phytochemistry (Chapter 9), , 263, , should be attached to the vessel in which the digestion process is being, performed; this facili tates the condensation of menstruum, so that it can be, recovered and returned back to the container., , 9.1.2.6., , Percolation, , The term percolation has been derived from the Greek word percolare which, means to pass through . Percolation (or Process P) is also termed lixivation. It, involves extracting the constituents of granulated or powdered drug by slowly, passing down through it a suitable menstruum. The menstruum while travelling, down the drug column under the influence of gravity, extracts the dru g particles, layer-wise, which are further replaced with the layers above as it moves, downwards. Percolation method achieves complete drug extraction., Principle, Percolation is a short successive maceration or extraction by displacement, method. The powdered drug to be extracted is placed in a percolator (cylindrical, or conical vessel) with a diaphragm at the base. The menstruum is passed at a, regulated rate through the drug column. While passing down, the menstruum, dissolves the active constituents and becom es saturated. The gravitational force, and the force of the above liquid column together make the saturated menstruum, to move downward. This movement is however opposed by the capillarity force., Gravitational force, viscosity, adhesion, friction, osmosis, c, apillarity, surface, tension, and dissolution rate are the physical factors important in percolation., Procedure, Step 1: Size Reduction of the Drug to a Suitable Degree: The degree of size, reduction of the drug to be extracted should be established by conducting, experiments, because this is not always necessary that finest grade of the, powdered drug will only undergo better extraction; sometimes, optimum grade of, powdered drug also achieves efficient extraction., Step 2: Moistening or Imbibition: If the drug to be extracted, is moistened, initially, air contained within the cells is removed, which facilitates menstruum, penetration. The drug should be moistened with the menstruum for around 4 hours., If the powdered drug is packed in the percolator in dry form, uniform packing will, not occur; also the drug particles will swell and block the column when menstruum, will be added, thus the entire extraction process is hindered. The menstruum used, for moistening the drug should be used in amount sufficient to swell the drug, particles, before being packed in the percolator. Initial moistening of some, unorganised drugs (e.g., oleo gum-resins and in case of cold percolation of sugar in, making syrups) may bring about opposing effects to the extraction process., Step 3: Packing: After completing t he moistening of drug, it should be packed, layer-wise in the percolator. Each consecutive layer should be packed uniformly, as per the requirement (either moderately or firmly). Incomplete extraction of the, drug will occur if the packing is non -uniform, as the menstruum will flow, through the loosely packed portion and the tightly packed portion will remain as, such. The extraction rate will slow down if the column is packed very tightly ,, *, , *
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264, , Pharmacognosy and Phytochemistry-II, , as the menstruum will not flow properly; in the same manner, extraction will not, occur adequately if the column is packed very loosely , as the menstruum will, flow freely and will be required in large quantity. The drug occupies 2/3 rd of the, column. The exit of the percolator is plugged with jute tow. The top of the drug is, spread with a layer of washed sand, which keeps the drug intact when the, menstruum is added. Packing of a percolator is shown in figure 9.2., Bottle-containing menstruum, Layer of sand, Filter paper, Drug, Jute tow, Graduated receiver, , Figure 9.2: Packing Arrangement in a Percolator, , Step 4: Maceration: The menstruum is added in the percolator leaving atleast, 2cm layer at the top. The menstruum, amount should be sufficient enough to, saturate the column. When the menstruum is being added, the stopcock at the exit, should be kept open to allow air displacement by the menstruum. If not done so,, the displaced air will be forced upward in the column, a nd the packing will be, disturbed. Menstruum is again added leaving a layer at the top so that the upper, layers of drug do not dry up. Thereafter, the percolator is closed for 24 hours,, during which preliminary maceration occurs, the menstruum penetrates th e cells,, and the soluble constituents get dissolved. This step makes the drug exhausted by, a small volume of the menstruum., Step 5: Percolation: With the completion of maceration step, percolation is, initiated by opening the lower stopcock. The percolation rate should be kept, moderate (i.e., neither too fast nor too slow). If the rate is too fast , menstruum, will be required in large quantity; and in case of slow rate of percolation , the, extraction process will slow down. During percolation, the drug column in the, percolator should always be moist; this can be achieved by inverting a, menstruum bottle with a delivery tube over the percolator, so that the tube dips, into the menstruum layer on the drug column (this also avoids the need to, constantly keep an eye over th e percolation process). This process is continued, till three quarters of the volume of the finished product has been collected. The, extraction is considered to be completed by performing chemical tests for active, ingredients collecting a few drops towards the end of percolation., Step 6: Pressing the Marc: The marc left behind in the percolator is removed, and pressed using a tincture press or a hydraulic press. This aids in recovering the, liquid remaining in the marc. However, marc expression does not achieve, complete removal of menstruum, thus it is distilled using specially designed, solvent recovery stills., *, , *
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Basics of Phytochemistry (Chapter 9), , 265, , Step 7: Adjustment of Volume: The liquid obtained after marc expression is, mixed with the percolate, and the mixture is added with more menstruum to, make up the desired volume., Step 8: Clarification: It is carried out by subsidence or filtration to obtain a, clear finished preparation., Modified Percolation, The different methods of modified percolation are discussed below:, 1) Intermittent Percolation: In this method, percolation is followed by, maceration so that the menstruum and the drug particles remain in contact for, a prolonged period. This results in greater extraction using small quantity of, menstruum., 2) Re-Percolation: In this method of modified percolation, the same volume of, menstruum is used in divided quantities for drug extraction. The active, constituents of drug are completely extracted with the first portion of the, menstruum. The percolate from the first lot is used as the men struum for the, second lot, and this continues. The drug powder to be extracted is divided, into 4 or 5 lots. The re, -percolation method aims to extract maximum, constituents using minimum quantity of menstruum. This method although is, not used widely , as the drug in the last percolator undergoes incomplete, extraction by the time the final percolate is obtained in desired volume., 3) Hot Percolation: This method modifies the efficiency of percolation by, applying higher temperatures, which can be tolerated by the ac, tive, constituents and the menstruum, and also the finished product remains, unaffected. Hot percolation (performed at a higher temperature using a hot, water jacketed percolator) achieves complete extraction with little volume of, the menstruum. Only water ca n be used as a menstruum in this method. Hot, percolation cannot be used for drugs whose active constituents are, thermolabile, and if a volatile menstruum is used., 4) Reserved Percolation: This method involves reserving the first part of the, percolate (which measures to almost three quarters of the finished, preparation) and continuing percolation till complete extraction is achieved., The first percolate is comparatively more concentrated and contains soluble, ingredients in maximum amounts. The succeeding perco, lates are less, concentrated although fresh menstruum with maximum solvent action is, employed, but the drug is exhausted and contains ingredients in small, amounts., Reserved percolation is used when the drug is prepared in 1:1 or 2:1 ratio,, and when the pr oduct is fluid. On completion of extraction, the marc is, pressed, the expressed liquid is mixed with the succeeding percolate, and the, th, mixture is then concentrated to a volume less than 1/4, of the finished, preparation by vacuum distillation. The concentr, ate is mixed with the, reserved percolate (i.e., the first portion) and the final volume is adjusted, with fresh menstruum after assay., *, , *
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266, , Pharmacognosy and Phytochemistry-II, , Advantages, i) The reserved percolate with maximum amount of ingredients does not, require heat treatment., ii) The method is economic as the complete percolate is not distilled., iii) The reserved percolate forms no precipitate due to solvent fractionation ,, which would occur if the complete percolate was distilled., Disadvantage, This method cannot be used if the concentrated preparation is a dry product., , 9.1.2.7., , Continuous Hot Extraction (Soxhlet Extraction), , The apparatus for continuous hot extraction, used on a laboratory scale (figure 9.3), consists, Water condenser, of a flask, a Soxhlet extractor, and a reflux, condenser. The ra w material is placed in a, thimble (of filter paper) inserted in the wide, Thimble, Vapour tube, central tube of the extractor. The drug after, Drug, Syphon, getting moistened with the menstruum is packed, into the extractor in a way that the extract outlet, present at the bottom is not blocked., The, Solvent, menstruum is placed in the flask and boiled at, Heat, its boiling point. The resultant vapours rise up, the larger right tube in the upper part of the drug Figure 9.3: Soxhlet Apparatus, and then enter the condenser, where it condenses and drops back on to the drug., During percolation, the menstruum extracts the soluble constituents of the drug., When the extract reaches to the top level of syphon tube, the complete percolate, syphons over into the flask. The suction effect of syphoning allows the menstruum, to penetrate the drug. Thus, the men struum is required in a limited amount for, repeated percolation through the drug. This process is continued till the drug gets, completely extracted, and the final extract obtained in the flask is further processed., A simpler design of, continuous hot ext raction, To condenser, apparatus, which is also official in I.P. (figure 9.4),, has an advantage that the hot vapours which rise up, the tube encircle the drug material being extracted., Thus, this apparatus will use a slightly higher, Thimble, extraction temperature than the Soxhlet in which a, Drug, small fraction of heat from the material bed is lost in, the surroundings. However, the condensed menstruum, percolates through the drug and drops back into the, Solvent, flask without collecting until syphoning (as in the, Soxhlet). Consequently, the macer ation period of the, Heat, drug with the hot menstruum is lost in this apparatus., Figure 9.4: Apparatus for, Continuous Hot Extraction, This process has the following limitations:, 1) It is not suitable for drugs having th, ermolabile active constituents, e.g.,, enzymes, alkaloids, anthraquinone derivatives, esters, etc.,, because this, extraction process requires a high temperature and also the extract in the, flask is maintained in hot condition during the entire process., *, , *
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Basics of Phytochemistry (Chapter 9), , 267, , 2) It is suitable only for pure solvents , constant boiling mixtures (like alcohol water), or solvent mixture forming azeotropes., 3) If the menstruum is an ordinary binary mixture, the vapour composition and, the liquid composition will be different., 4) It is not used if the drug to be extracted is of such physical nature that it, would block the Soxhlet apparatus, e.g., opium, gum, resin, orange peel, etc., , 9.1.2.8., , Supercritical Fluid Extraction (SFE), , The process of SFE involves separating one component (i.e., the extractant) from, another (i.e., the matrix) using supercritical fluids ( i.e., the extracting solvent)., The extraction is generally from a solid matrix or from liquids. SFE can be used, as a sample preparation step for analytical purposes, or it can be used on a larger, scale to strip unwanted materials from a product (e.g., decaffeination) or collect a, desirable product (e.g., essential oils)., Supercritical fluid is a substance at temperature and pressure above its critical, point. It can diffuse through solids like a gas and dissolve materials like a liquid., It can be suitably used as a substitute for organic solvents in various industrial, and laboratory processes. The most commonly used supercritical fluids are, carbon dioxide (CO2) and water, which are used for decaffeination and power, generation, respectively. However, modified co -solvents such as ethanol or, methanol can also be used. CO 2 is used as an extraction solvent for botanicals, it, does not leave behind any toxic residues, and its extraction properties can be, manipulated by making subtle changes in pressure and temperature., An ideal supercritical fluid has the following properties:, 1) It has highly compressed gases, which combine the properties of gases and, liquids., 2) It can lead to reactions, which are difficult to achieve using conventional solvents., 3) It has a solvent power similar to light hydrocarbons for most of the solutes., However, the solubility of fluorinated compounds is more in supercritical, CO2 than in hydrocarbons; and this increased solubility is important for, polymerisation., 4) It increases solubility with increasi ng density (i.e., with increasing pressure)., Rapid expansion of supercritical fluid leads to precipitation of a finely, divided solid (this is a key feature of flow reactors)., 5) Since it is miscible with permanent gases ( e.g., N2 or H2), this leads to much, higher concentrations of dissolved gases than can be achieved using, conventional solvents., Process, 1) The mixture to be fractionated is passed in the extraction column having a, heater along its length., 2) CO2 is purged through the column., 3) Once the extraction colu mn is pressurised, the supercritical fluid moving, along the column length saturates the drug material., 4) The operating conditions (i.e., pressure and temperature) are selected., *, , *
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268, , Pharmacognosy and Phytochemistry-II, , 5) In the pressure-controlled extraction, the solution is expanded in the separation, stage to precipitate the extract and the gass iagain recompressed for recycling, ., 6) In the temperature -controlled extraction, the solution is heated which lowers, the solvent density and precipitates the extract. The density is then increased, by isobaric cooling for recycling., 7) The working of supercritical fluid extraction system is controlled from a PC,, which sets the operating conditions like pressure, temperature, and flow rate., 8) The PC is programmed to shut down the unit in case of overpressure or over, temperature (figure 9.5)., Heated exit line, , Helium, , PSE, , TIC, , A, P1, P1, , High, pressure, piston, pump, F1, , Collection, chamber, , B, , C, , UV, , D, , Volum, e callb, vessel E, , PCV, , FIV, , Fluid injection, , SIV, , Soil, bed Extractor, , G, , F1, , PES, , P1, P1, F1, , Liquid CO2, T1, , Sample, chamber, , F, , CO2, purge, , PCV, , Circulation pump, Temperature controlled, oven, , TIC, , Methanol, sample, pump, , Figure 9.5: Diagrammatic Representation of Supercritical Extraction Unit, , Advantages, 1) SFE determines the rate at which the extraction can be performed., 2) The SFE process completes within 20-60 minutes., 3) Many steps have been eliminated from SFE, thus the accu, racy and, reproducibility of the extraction is increased., 4) SFE produces less waste solvents and also offers less exposure of labo ratory, personnel to toxic solvents., 5) SFE yields quantitative recovery of target analytes without loss or, degradation during extraction., 6) SFE offers selective extraction by selecting the fluid polarity and density., 7) SFE avoids purification by adsorp tion chromatography and keeps the other, active ingredients intact in the matrix., 8) Supercritical fluids have solute diffusivities in order of magnitude higher and, solute viscosities in order of magnitude lower than liquid sol vents, and this, increases the extraction efficiency., 9) The solvent strength of a supercritical fluid can be easily controlled, while, the solvent strength of a liquid is constant., 10) The supercritical fluids are mostly gasses at ambient conditions., Disadvantages, 1) Carbon dioxide (the most commonly used solvent in SFE) cannot be used for, extracting polar compounds due to its low polarity., *, , *
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Basics of Phytochemistry (Chapter 9), , 269, , 2) Presence of water in SFE process may cause problems., 3) In SFE, the matrix effect is unpredictable., 4) SFE process requires specialised/expensive equipment., , 9.1.2.9., , Counter Current Extraction (CCE), , In CCE, toothed disc disintegrators are used to pulverise the wet raw material to, be extracted. A fine slurry is obtained which is moved in one direction within a, cylindrical extractor. Here the slurry comes in contact with the extraction solvent., The farther the slurry moves, the more concentrated the extract becomes. When, the quantities of solvent and material and their flow rates are optimised, complete, extraction is achieved. Lastly, an appropriately concentrated extract is received at, one end of the extractor while the marc (which is free of visible solvent) falls out, from the other end. This entire process is highly efficient, is less time-consuming,, and poses no risk from high temperature. The process of CCE has the following, advantages:, 1) A unit quantity of the plant material can be extracted using a small volume of, solvent., 2) It is carried out at room te mperature, thus the thermolabile constituents are, not exposed to heat., 3) The drug is pulverised under wet conditions, so the heat generated during, comminution is neutralised with water. Thus, the thermolabile constituents, are not exposed to heat., 4) The CCE ext raction procedure is more efficient and effective than the, continuous hot extraction method., , 9.1.2.10. Microwave Assisted Extraction (MAE), Microwaves are non, -ionising electromagnetic waves present in the, electromagnetic spectrum between X-rays and infrared rays. They are made up of, the electric and magnetic field (two oscillating perpendicular fields), of which the, former is responsible for heating., Principle, Mostly the dried plant materials are used for extraction; however, th e plant cells, containing microscopic traces of moisture also serve as the target for microwave, heating. When these plant cells are exposed to microwave heat, the moisture, within gets heated up and evaporates. As a result, the plant cells swell up and, exert pressure on the cell wall. Under the influence of this pressure, the cells, stretch and ultimately rupture, thus leaching out the active constituents into the, surrounding solvent and improving the yield of phytoconstituents., This phenomenon can be more st rengthened by saturating the plant matrix with, solvents having higher heating efficiency under microwave. The higher, temperature achieved by microwave radiation hydrolyses the ether linkages of, cellulose (the main plant cell wall constituent) and converts, them into soluble, fractions within 1 -2 minutes. The higher temperature achieved by the cell wall, during MAE facilitates cellulose dehydration, reduces its mechanical strength,, and thus helps the solvent to easily enter the cellular components., *, , *
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270, , Pharmacognosy and Phytochemistry-II, , Instruments, The closed extraction vessels and focused microwave ovens (or Focused, Microwave Assisted Soxhlet or Solvent Extraction, FMASE) are the two types, of commercially available MAE systems. The former is used for extraction under, controlled pressure and temperature, and in the latter only a part of the extraction, vessel containing the sample is irradiated with microwave. However, both the, systems are available as multimode and single-mode or focused systems., , Reflux, system, Diffused microwaves, Magnetron, Closed bomb, , Magnetron, Vessel, Wave guide, , Solvent, , Focused microwaves, , Solvent, , Sediment, , Sediment, , (a) Focused Microwave Oven, , (b) Multimode Microwave Oven, , Figure 9.6: Monomode (a) versus Multimode Systems (b), , In single -mode or focused systems, the microwave radiation is focused on a, restricted zone wherein the sample is subjected to a relatively stronger electric field, (figure 9.6). In the multimode system, the microwave radiation randomly disperses, in the microwave cavity, therefore each zone, in the cavity and the sample it, contains is uniformly irradiated ( figure 9.6). A modified multi mode domestic, microwave oven also operates as an open vessel extraction systemfigure, (, 9.7)., To drain, Condensing coil, Energy attenuator, Connecting, tube, , Cooling water, , 250ml, boiling flask, Microwave, cavity, , Shielded, thermistor, Temperature, recorder, Time presenting, , Time controller, Magnetic surface, , Figure 9.7: Scheme of a Modified Multimode Domestic, Microwave Oven (Open Vessel Extraction), *, , *
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Basics of Phytochemistry (Chapter 9), , 271, , The focused and multimode microwave devices are made up of the following, four components:, 1) Magnetron is the microwave generator used for generating microwave, energy., 2) Wave guide is used for propagating the microwave from the source to the, microwave cavity., 3) Applicator contains the sample., 4) Circulator allows the microwave to move forward., Plant Material, Pre-leaching time to, be Optimised, No, Immerse in a suitable, extracting solvent and, allow for pre-leaching, , Powdered to different, size, , Particle size to be optimised., Particles screened beyond sieve 60, not recommendable, , Sample pre-treated with, high microwave absorbing, solvent, Yes, Is the analyte highly heat, sensitive?, , Yes, Choose a microwave transparent solvent or a, combination of microwave transparent and, microwave absorbing solvent. Solubility to be, checked., , Microwave irradiation – Irradiate with, intermittent cooling step in between to, avoid super heating and thermal, degradation, , Required to bring about selective, heating of plant matrix, Solvent composition and volume to, be optimised, No, A combination of methanol water or, ethanol water may be a wise choice., Solubility to be checked, , Needs optimisation for, microwave power and, irradiation time, , Centrifugation for 15-20 minutes or, filtration to separate the matrix from solvent., Subjected to chromatography, Figure 9.8: Extraction Scheme for Open Vessel MAE System, Note: Thicker arrows indicate the steps which needs proper optimisation to determine the, optimum extraction conditions to attain the maximum yield., , Figure 9.8 represents a brief flow chart for open vessel extraction systems, performing MAE under atmospheric pressure., Advantages, MAE is a much potential substitute to the traditional solid, -liquid e xtraction, method due to the following advantages over Soxhlet:, 1) It is less time -consuming as it takes only a few seconds to few minutes, (15-20 minutes) to complete the extraction process., 2) It requires less solvent (only a few milliliters)., 3) It improves the extraction yield., 4) Since it is an automatic process, it provides better accuracy and precision., 5) It can be used for the extraction of thermolabile constituents., *, , *
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272, , Pharmacognosy and Phytochemistry-II, , 6) It can be used for extracting minute traces of constituents including heavy, metals and pesticide residues from a few milligrams of plant sample., 7) It provides agitation during extraction, which improves the phenomenon of, mass transfer., 8) Since its instrumental set up (like Soxwave) combines the features of Soxhlet, as well as benefits of microwave, it makes extraction even more attractive., , 9.1.2.11. Ultrasonic Assisted Extraction (UAE), The range of human hearing is from 16 -18KHz. Ultrasound is the sound waves, whose frequen cies are higher than those to which the human ear can r, espond., There are many natural products which are thermally unstable and which degrade, during thermal extraction. Due to this reason, recently UAE is considered as an, efficient alternative to various conventional thermal extraction processes., Many recen tly performed studies have established UAE as the process which, enhances the extraction efficien cy of secondary metabolites from various plant, tissues by increasing the yield and reducing the extraction time. Various studies, have also shown that ultrasoun d can even be used for extracting organic, compounds present in the plant tissues by disrupting the cell walls and enhancing, the mass transfer of cell contents. This phe nomenon of modifying the extraction, efficiency of organic compounds using ultrasound wa, ves is called acoustic, cavitation., The ultrasound wave on passing through a liquid causes the expansion cycles to, exert negative pressure on the liquid, thus pulling the molecules apart. If the, ultrasound wave is of sufficient intensity, the expansion cyc, le even creates, cavities or micro-bubbles in the liquid. This occurs when the negative pressure is, more than the local tensile strength of the liquid, which however varies with the, type and purity of the liquid. The micro -bubbles formed ab sorb the energy from, the sound waves, grow during the expansion cycles, and recompress during the, compression cycle., The potential en ergy of such expanded micro -bubbles converts into ki netic, energy in the form of a liquid jet that moves inside the bubbles (at a speed o f, around 400km/h) penetrates their op posite wall. This liquid jet puts a strong, impact on the solid surface, and can cause serious damage to impact zones and, produce newly exposed highly reactive surfaces., With the help of scanning electron mi, crographs, the mechanical effects of, ultrasound, especially the destruction of cell walls and release of cell contents can, be easily shown. Unlike the conventional extractions, the ultrasound waves allow, the plant extracts to diffuse across the cell walls, causing cell rupture within a, shorter time period, and ultimately increasing the mass transfer phenomenon., The UAE process has the following advantages over conventional Soxhlet, extraction:, 1) Cavitation enhances the polarity of the extractants, analytes, and matrix; thus,, increasing the extraction efficiency., *, , *
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Basics of Phytochemistry (Chapter 9), , 273, , 2) Ultrasound-assisted leaching allows the ad, dition of a co -extractant to, enhance the polarity of liquid phase., 3) It also allows the leaching of thermolabile analytes, which generally gets, altered during Soxhlet extraction., 4) The operating time of UAE is comparatively shorter with faster kinetics. The, average time of ultrasonic extraction ranges from a few to 30 minutes;, however, it can be as long as 70 minutes., 5) The extraction conditions of UAE can be op timised wi th respect to time,, polarity amount of solvent, and the amount and kind of sample., 6) It is performed at room temperature, thus making it suitable for the extraction, of thermolabile analytes., The need for separation of the extract from the sample after the, process is the only disadvantage of UAE., , extraction, , 9.1.2.12. Infusion and Decoction, These methods are now rarely used. Infusions were prepared from vegetable drugs, with water-soluble and easily extractable constituents; and decoction process was, used for extracting vegetable drugs with water, -soluble and heat -soluble, constituents. In the infusion process, the drug was moistened with water, macerated, with boiling water, the liquid was strained, and desired volume was made. In the, decoction process, the drug was boiled with water, cooled, expressed, the liquid, was strained, and desired volume was made. Concentrated chirata infusion and, compound chirata infusion arethe examples of infusions official in the I.P., 1966., , 9.1.2.13. Pressure Cooker Extraction, In this method, the drug is initially macerated with the menstruum and then is, held for 5 -15 minutes in a pressure cooker at 15lb/sq. inch pressure. The cooker, is then cooled and the extract is removed by straining and pressing the marc. This, method achieves complete drug extraction in comparatively less time. However,, it cannot be used for drugs with thermolabile constituents., , 9.1.2.14. Extraction by Passage through a Colloid Mill, In this method, a drug suspension is prepared in the menstruum, macerated, and, then passed through a colloid mill running at 1500rpm speed. For achieving, efficient extraction, re -circulation through the mill is required. On passing the, drug particles through the colloid mill, direct dissolution of drug constituents is, not enhanced; however permeability of the cell walls when they are passed, through narrow clearance of a colloid mill is increased., , 9.1.2.15. Use of Surface Active Agents in Drug Extraction, By using surface active agents (e.g., PEG 400-monooleate, PEG 600-monolaurate,, PEG 400-dilaurate, sorbitol laurate, and polyoxyethylene sorbitol monolaurate) in, small amounts, the extraction speed of i ngredients of some drugs ( e.g., cinchona,, hyoscyamus, and belladonna) is increased. In this method, the mixture of surfactant, and menstruum is added to the powdered drug, the entire mixture is agitated in a, mechanical shaker, and the extract obtained is separated from the marc., *, , *
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274, , Pharmacognosy and Phytochemistry-II, , 9.1.2.16. Expression and Diacolation, In this method, screw press (for small scale operations) and hydraulic press (for, large scale operations and applying pressures up to 30 tonnes) are used. In this, method, the menstruum is allowed to flow from the bottom of the drug -charged, cylinder under the influence of positive pressure of compressed air. Diacolation, method is best suited for the extraction of those drugs which offer resistance to, the upward flow of menstruum under hydraulic pressure., , 9.1.3. Techniques Used in the Isolation, Purification, and, Identification of Crude Drugs, The progress in the isolation and analysis techniques has led to the identification, of many unknown compounds. For isolating a particular compound from its plant, material, various processes are involved. These isolation processes depend on the, nature of the active constituents present in the crude drug., For example, trapping of the components is done for volatile chemicals, while, extraction of non-volatile compounds is done using organic solvents. Isolation of, components is done for both known and unknown constituents, and this demands, various processes for the separation, purification and identification of compounds, along with biological scre ening. When the required crude extract has been, isolated from the plant, the marker component needs to be isolated and identified, for its study with respect to chemical nature or for the development of newer, formulations. The advances in the field of chrom atography have allowed the, separation and purification of compounds., After isolation, identification, and purification of a plant constituent, the class of, the compounds and the particular substance within that class should also be, determined. Complete identification depends on measuring other properties (such, as melting point, boiling point, optical rotation, and retention factor) and, comparing the data with those in standard literature with techniques of, Ultraviolet (UV), Infrared (IR), Nuclear Magnetic Resonance (NMR), and Mass, Spectral (MS) measurements., For the final confirmation of a known plant compound that has been identified on, the above basis is compared with the standard sample; while the identity of new, compounds is confirmed by chemical degra, dation or by the synthesis of, compound in laboratory., , 9.1.3.1., , Spectroscopy, , Spectroscopy involves the study of interaction of electromagnetic radiation with, matter. The various spectroscopic methods that are utilised for identification of, natural compounds are:, Ultraviolet (UV) and Visible Spectroscopy, Ultraviolet (UV) and visible absorption techniques involve the analytical, methods which measure the light absorption lying in the wavelength region from, 190-900nm by different substances. The w avelength region from 190 -380nm is, *, , *
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Basics of Phytochemistry (Chapter 9), , 275, , the UV region and that from 380, -900nm is the visible region of the, electromagnetic spectrum. Absorption in the UV -visible region results from the, electronic transitions within the molecule., Many instruments are available for measuring light absorption in the UV -visible, region of the spectrum. In a single beam instrument , light passing through a, monochromator enters the sample and then the detector. In a, dual beam, instrument, the ratio of the intensity of beam coming throu gh the sample cell to, a second beam which does not pass through the sample is measured., Multichannel detectors are used in a rapid scanning spectrophotometer., The separation step and absorption measurement in UV-visible region is the most, widely used anal ytical procedure in the field of pharmacy. The UV -spectrum, along with other analytical data often serves as a tool for identity confirmation, and is also used as a detector in HPLC. The absorption spectra of every plant, constituent are different in specific range of visible light specimen. A very dilute, solution should be used, and the wavelength should be recorded in nanometres, with maximum and minimum of the peak., Table 9.1 enlists the spectral ranges of some important groups of plant constituents:, Classes of Compounds, Anthocyanins, Betacyanins, Carotenes, Anthraquinone and its derivatives, Flavonoids, Chlorophylls, , UV Range, 275-330, 250-275, 400-500, 425-400, 250-275, Very short range, , Visible Range, 475-555, 540-554, 400-525, 220-290, 350-390, 430-470 and 650-660, , Infrared (IR) Spectroscopy, The IR spectroscopy is used for the identification of many functional, groups, present in the compound, as different functional groups have specific absorbance, (in the form of radiant energy) in the electromagnetic spectrum. The I, R, spectroscopy can be measured on spectrophotometer. The, IR spectrum is, divided in the following three regions:, 1) Near region ranging from 1250-4000cm–1,, 2) Mid region ranging from 4000-400cm–1, and, 3) Far region ranging from 400-20cm–1., The IR spectrum can be measured by the following two methods:, 1) In the form of chloroform or carbon tetrachloride solution, and, 2) In the form of potassium bromide pellets., The IR spectroscopy is being widely used for identification of drugs, polymorphic, modifications, and excipients, and raw materials used in pharmaceutical, manufacturing. This is because it is highly sensitive and the spectra can be easily, obtained on any type of sample including insoluble solids, polymers, solutions, and, *, , *
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276, , Pharmacognosy and Phytochemistry-II, , gases. Obtaining super -imposable spectra (ident ical IR spectra of two samples), proves that the two samples are similar in their chemical structure. The IR, spectrometric analysis helps in the structural elucidation of functional groups of, bio-molecules. It also aids in the quantitative analysis of antib iotics, alkaloids,, quinine and strychnine (6.2 and 6.06), steroidal sapogenins (11.11), etc., Table 9.2 represents the IR frequencies of some important groups of plant, constituents:, Table 9.2: IR Frequencies of the Important Group of Plant Constituents, Groups of Constituents, Characteristic Bands (Approx.), Alkanes, 2860cm–1, Alkenes, 1650cm–1, Acetylenes, 2150cm–1, Alcohols, 3600cm–1, Phenols, 1410cm–1, Aldehydes, 2750cm–1, Ketones, 1420cm–1, Esters, 1680cm–1, Carboxylic acids, 2500cm–1, Amines, 3400cm–1, , The I R is also used for finger printing analysis for identifying the compounds, qualitatively., Nuclear Magnetic Resonance Spectroscopy (NMR), The NMR spectroscopy involves absorption of radioactive constituents at, limited radio frequency in a particular magnetic, field, which differs from, compound to compound., The two types of NMR techniques are:, 1) 1H NMR Spectroscopy (Proton NMR): This type of NMR spectroscopy, measures the magnetic moments of different hydrogen atoms. This helps in, determining the number of hydro gen atoms in a particular compound and/or, a particular group, thus establishing the compound structure., 2), , 13, , C NMR Spectroscopy, (Carbon-Spectroscopy): This type of NMR, spectroscopy measures the nature of carbon skeleton of a parti, cular compound., , For NMR analysis, the compound is dissolved in a, standard inert solvent,, known as TMS (Trimethylsilane), which is subjected to a very strong magnetic, field. The different types of proton depending on their molecular nature undergo, various chemical shifts, which are represented by delta (). NMR is used as an, important tool in the elucidation of molecular structure, especially the, stereochemistry and configuration. It determines the positions of protons in a, complex molecule. It is used for identification test in pharmaceutical analysis. It, is also used in quantitative analysis, in the sense that a reference standard is not, required for the substance being analysed., *, , *
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Basics of Phytochemistry (Chapter 9), , 277, , NMR is widely used for determining impurities and minor components in the, mixture due to its ease, speed , and specificity. NMR can also be coupled with, mass spectrometer., Mass Spectroscopy (MS), The technique of MS measures the mass -to-charge ratio and abundance of gas phase ions to identify the amount and type of chemicals a samp le contains. The, information recorded using small amounts of sample and contained in a single, mass spectrum helps in the identification or structural elucidation of an unknown, compound. In many newly developed ionisation techniques, there is no need to, vaporise the sample prior to ionisation. This has made various compounds of, biomedical and biochemical importance responsive to mass spectrometry., A gas chromatograph is used as the mass spectrometer inlet to enhance the high, sensitivity and specificity of, the mass spectrometer; and combined, Gas, Chromatography-Mass Spectrometry (GCMS) is currently a powerful, analytical technique. Even the highly sensitive immunological techniques cannot, offer the high sensitivity and specificity of GCMS., Various major devel opments have been made recently in GCMS methodology,, and these include the direct coupling of capillary gas chromatographic columns, to mass spectrometers, the availability of ion sources for chemical ionisation, and, the increasing use of isotopic ally labe lled internal standards in assays using, Quantitative Selected Ion Monitoring (QSIM)., MS relies on the basic principle of utilising an appropriate method to generate, ions either from inorganic or organic compounds, then separating these ions on, the basis o f their mass -to-charge ratio (m/z), and finally detecting them, qualitatively and quantitatively by their respective m/z and abunda, nce of gas, phase ions. The analyte may be thermally ionised by electric fields or by, impacting energetic electrons, ions, or p hotons. The ions can be single ionised, atoms, clusters, molecules or their fragments. Static or dynamic electric or, magnetic fields affect the ion separation. The mass spectrum obtained is a 2D, representation of signals’ intensity (on the ordinate) versus m/z (on the abscissa)., Fluorescent Analysis, Different compounds show different fl uorescence in different organic solvents,, because the light absorption through compound molecules takes place in a, definite wavelength range of the light. As a result, differ ent radiations are re emitted from the molecules and different colours are seen in the UV light., Many natural products that do not visibly fluoresce in day light produce, fluorescence using UV light obtained from mercury vapour lamp or tungsten. The, fluorescence lamps are fitted with a filter which removes visible radiation from the, lamp and transmits UV radiation of definite wavelength. For example, cinchona, bark shows many luminous yellow and a few light blue patches under fluorescent, light. The other qua litatively evaluated drugs are calumba (shows yellow, fluorescence, but the phloem and cambium show dark green fluorescence), hydrastis, *, , *
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278, , Pharmacognosy and Phytochemistry-II, , rhizome (shows golden yellow fluorescence),, Rheum emodi (shows brown, fluorescence), Rheum rhaponticum (shows violet fluor escence), ergot powder, (shows red fluorescence), olive oil (shows deep golden yellow fluorescence),, arachis and sesame oils (show blue fluorescence), berberis (shows yellow, fluorescence), gentian and aconitine (show light blue fluorescence), emetine (shows, orange fluorescence), precipitated chalk (shows greyish, -violet fluorescence), etc., The resolved components can be located on the developed TLC or paper, chromatogram by observing under UV lamp or by Guruvich method on TLC, plates. Quantitative fluorescence analysis using the fluorescence produced by a, compound in UV light can be done with fluorimeter or spectrofluorimetry. For, example, sennosides extracts of rhubarb and berberis, and cinchona alkaloids can, be evaluated using this technique., Table 9.3 represents the fluorescence analysis of different constituents:, Table 9.3: Fluorescent Analysis of Different Constituents, Constituents, Solvents, Colour of Fluorescence, Triterpenoids, Chloroform in UV light, Reddish brown, Anthraquinone types, Methanol in UV light, Brown, Tropane types, Chloroform in UV light, Violet, Indole alkaloids, Chloroform in UV light, Yellowish red, , 9.1.3.2., , Chromatography, , Chromatography is the most versatile separation technique and involves, separation of two or more substances, by distribution between a fixed (or, stationary) phase and a moving (or mobile) phase. Chromatography is used for, separation, isolation, purification, and identification of components in a mixture., Plant materials can be separated and purified using variou, s chromatographic, techniques. Herbal medicine is a complex system of mixtures. Thus, the, identification methods for botanical drugs obtain a characteristic fingerprint of a, specific plant that shows the presence of a particular quality defining chemical, constituent. Chromatographic techniques like High Performance Liquid, Chromatography (HPLC), Gas Chromatography (GC), Gas Chromatography, Mass Spectrometry (GC -MS), and Thin Layer Chromatography (TLC) are, widely used for such purposes., Techniques of Chromatography, 1) Adsorption Column Chromatography: In this chromatography technique,, the solid support ( e.g., activated alumina, powdered cellulose, silicic acid, or, kieselguhr) is packed as a dry solid or as a slurry into a tube (of glass, plastic,, or other suitable material) having a restricted orifice (protected by a sintered, glass disc) for efflux of the mobile phase. The solution of material to be, subjected to chromatography is added to the column top to flow into the, adsorbent. Then the solvent constituting the mobile phase is added to the, column top to flow downwards either under gravity or by applying positive, pressure. The column top during this process should not become dry., *, , *
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Basics of Phytochemistry (Chapter 9), , 279, , The effluent solution (or the eluate) is monitored either continuously (, for, example, with a flow -through UV absorption cell) or serially ( for example,, collecting fractions at intervals determined by time, volume, or weight of, eluate and subsequent examination of each fraction for the separated, component)., 2) Partition Column Chromatography: In this chromatography technique,, one liquid phase is held on the surface of stationary phase (i.e., a, chromatographic support), while the other liquid (i.e., the mobile phase) is, passed through the packed column; thus, maintaining an intimate contact, between the two phases. This is the stage where distribution of the sample, components occurs., Paper Chromatography (PC), In PC, a coarse paper is used instead of a packed column; therefore, the solid, support of silica gel for the polar phase is replaced with a filter paper. A partially, water-miscible organic solvent, e.g., butanol or collidine, is preferably used., Paper chromatography technique involves the analysis of an unknown compound, by the flow of solvent on a specially designed filter paper. One solvent, is, immiscible or partially miscible in the other solvent. The separation of, components occurs due to the differences in partition coefficients, and is affected, by the differential migration of the mixture of substances., The speed of an ion in a given sys tem (for the given phases and support) and, under the given conditions (temperature), i.e., the, retardation factor or, retention ratio (Rf) should be measured. This is the ratio of the distance, travelled by the solute (desired component) from the original li ne to the distance, travelled by the solvent front from the original line. Thus,, Rf , , Distance Moved by the Solute from the Original Line, Distance Moved by the Solvent from the Original Line, , The apparatus for PC should be selected depending on the amount of laboratory, space available. Horizontal or circular PC demands a larger space than a standard, TLC tank; however, it has a good resolution and is used for separating, carotenoids., On a laboratory scale, PC is carried out in tanks containing Whatman filter, papers (46 57cm). Descending PC is mostly preferred since the solvent can be, more easily over -run, and it is also more convenient for two, -dimensional, separations., In the technique of PC, the compounds react with a chromogenic reagent (used as, a spray or a dip) and are then detected as coloured or UV, -fluorescent spots., Dipping is easier for large sheets, but the solvent content of the spray should be, modified to achieve quick drying and avoid diffusion during the dipping. The, paper is then heated to develop the colours., *, , *
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280, , Pharmacognosy and Phytochemistry-II, , Applications of Paper Chromatography, Paper chromatography is widely used for the separation of complex mixtures of, amino acids, peptides, carbohydrates, steroids, purines, various simple organic, compounds, and even inorganic ions. Some examples are:, 1) It is used for determining indoles in urine., 2) It is used in the study of barbiturates, antibiotics, phosphates, and amino acids., 3) It is also used in the study of inorganic metal salts and complex ions., Thin Layer Chromatography (TLC), TLC is a simple yet significant chromatographic technique used for separation of, compounds. It is widely used in the phytochemical evaluation of herbal drugs, due to the following reasons:, 1) It involves rapid analysis of herbal extracts using minimum sample clean -up, requirement., 2) It provides qualitative and semi -quantitative information of the resolved, compounds., 3) It also allows the quantification of chemical constituents., The data in TLC fingerprinting is recorded using a High, -Performance TLC, (HPTLC) scanner that includes the chromatogram, retardation factor (R f) values,, and the colour of separated ban ds, their absorption spectra, λmax and shoulder, inflection/s of all the resolved bands., The TLC fingerprint profile of the sample can be represented by the above, mentioned data and also the profiles on derivatisation with different reagents., The obtained information facilitates the identification of an authentic drug and, the adulterants, and also maintains the drug quality and consistency. Long before, the instrumental chromatography methods (like GC and HPLC) were established,, TLC was the preferred method for herbal analysis., Applications of TLC, 1) It enables qualitative identification of compounds based on their flow rate, along the plate. Various qualitative applications of TLC are:, 2) It enables quantitative analysis of compounds by estimating the spot area and, the amount of material in the spot. Different techniques for quantitative assay, of substances using TLC are:, Gas-Liquid Chromatography (GLC), The technique of GLC is considered ideal for separating and purifying volatile, substances and fatty acids. The volatile substance to be analysed is injected in the, apparatus, vaporised, and percolated through the long column filled with fine, granules of inert material. These granules are coated with suitable substances., The bands of compounds travel through the co lumn and are combusted in the, combustion part at the outlet. A carrier gas ( e.g., nitrogen, helium, and argon) is, selected based on the sample nature and is passed th rough the column. The two, commonly used detectors in GLC are, Flame Ionisation Detector (FID) and, *, , *
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Basics of Phytochemistry (Chapter 9), , 281, , Thermal Conductivity Detector (TCD). The obtained results are recorded, graphically indicating different peaks, whose area can be calculated for, quantitative estimation., Applications of GLC, 1) It is used for the assay of starting materials and drug substances., 2) It is used for the quantification of drug to formulations., 3) It is used for the assay of impurities and/or solvents in the raw material or in, drug substances., 4) It is used for examining many volatile oils; plant acids; alkaloids of opium,, tobacco, con ium and belladonna; resins of cannabis; steroidal compounds;, cardioactive glycosides and aglycones; sugars and amino acids, etc., Gas-Solid Chromatography (GSC), The separation technique of GSC utilises a carrier gas (helium or nitrogen) as the, mobile phase and an adsorbent (silica gel, alumina, or carbon) as the stationary, phase. Solute distribution occurs between the gaseous mobile phase and the, adsorbent surface. The solute’s distribution isotherm between a gas and solid, surface occurs as the Langmuir or, the Freundlich isotherm, and thus, the, isotherms approach linearity only at very low concentrations of solute., Applications of GSC, 1) It is used for separating permanent gases or the low molecular weight, hydrocarbons., 2) With the help of special phase systems, it is used for separating halogens and, other highly corrosive gases and vapours., High Performance Liquid Chromatography (HPLC), The HPLC is a high resolution chromatographic technique, which is widely used, in pharmaceutical industries for process developme nt, and identification and, detection of adulterants in herbal products. It also facilitates the identification of, pesticide content and mycotoxins. It has been reported that a number of samples, can be simultaneously run using a smaller quantity of mobile p, hase than in, HPLC. It has also been reported that mobile phases of pH 8 and above can be, used for HPTLC, which also offers an advantage of repeated detection (scanning), of the chromatogram with the same or different conditions. Therefore, HPTLC is, used for simultaneous assay of several components in a multi, -component, formulation., Applications of HPLC, 1) It is used for separating various structurally similar components in plant, extracts, e.g., analysis of digitalis, cinchona, liquorice, and ergot extracts., 2) It is used for determining the stability of various pharmaceuticals,, e.g.,, stability studies of atropine., 3) It is used for the analysis of complex molecules (like antibiotics and peptide, hormones), whose bioassays are expe, nsive, time -consuming, require, replicates, and result in poor precision., *, , *
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282, , 9.1.3.3., , Pharmacognosy and Phytochemistry-II, , Electrophoresis, , The technique of electrophoresis involves migra tion of a charged particle under, the influence of electric field (electro -charged particle and phoresis -movement)., There are many imp ortant biologi cal molecules (like amino acids, peptides,, proteins, nucleotides, and nucleic acids) that possess ionisable groups and exist in, solution as electrically charged species (cations or anions) at a given pH. These, charged particles under the influence of an electric field migrate to either cathode, or anode based on the nature of their net charge., For separating mixtures by electrophoresis, a filter paper strip saturated with an, electrolyte solution (a buffer solution) is supported in the centr e and its two ends, are dipped into solutions having immersed electrodes. After placing a spot of the, material to be fractionated on the paper , the whole apparatus is sealed and a, potential difference of 2 -10volts/cm is applied along the paper. Some mixture s, are separated at higher voltages. The solutes move towards the anode or cathode, based on the nature of charge on the ions of solute mixture. Thus, the amino, acids can be separated into either groups (acidic, neutral or basic group) or, individual amino ac ids. A substance’s migration velocity depends on the, magnitude of ionic charge and the size and shape of the particular molecule., The filter paper used can also be replaced with thin layers of gels, and the process, is then termed gel filtration. Many alka loidal mixtures, plant acids, component, sugars of cardiac glycosides, and anthraquinone derivatives have been separated, by this method. Polyacrylamide gradient gel electrophoresis combines the, advantages of both gel filtration and electrophoresis. It is a, two-dimensional, electrophoresis technique that separates the solutes according to their mobility in, one direction and size in the other., Capillary electrophoresis is a technique that provides separation efficiencies of, the order of 4 ×105 theoretical plat es. It provides a rapid analysis than gel, electrophoresis and utilises detector systems (such as the laser, -induced, fluorescence detector) along with a high resolution, thus increasing sensitivity, 500-times over UV detection. This method is used for the ana lysis of flavonoids., One of the major advantages of capillary electrophoresis over other separation, technique is its ability to separate charged and uncharged molecules., The analyte ions in capillary electrophoresis are separated in an electrolyte, solution (background electrolyte) present in a narrow fused -silica capillary. The, capillary ends are immersed in vials (inlet and outlet) filled with electrolyte, solution and also electrodes connected to a high voltage supply ( figure 9.9). The, sample solution is injected in the capillary as a small plug by applying pressure, (hydrodynamic injection) or voltage (electrokinetic injection)., By applying high voltage (5-30kV) across the capillary, analyte zones are formed, due to different electrophoretic mobilities of ionic species. These zones migrate, towards the outlet side of the capillary. Different ions can be separated based on, their different charge/size ratio. The separated analyte bands are detected directly, through the capillary wall (i.e., before they reach the capillary end)., *, , *
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Basics of Phytochemistry (Chapter 9), , 283, Capillary, , Detector, , Sample injection, system, High voltage, Electrodes, Sample Inlet, , Outlet, , Figure 9.9: Instrumentation of Capillary Electrophoresis, , Advantages, 1) It provides a high separation efficiency., 2) It takes a short time for analysis., 3) It requires less amount of sample and electrolyte., 4) It generates less waste., 5) It is easy to operate., Disadvantages, Since the capillary tube has a small diameter, heat is dissipated and causes, increased diffusion, thus resulting in inaccurate resolution., , 9.2. SUMMARY, The details given in the chapter can be summarised as follows:, 1) Phytochemistry involves the study of chemicals (mainly the secondary, metabolites) the plants produce as a measure to protect themselves from insects,, pests, pathogens, herbivores, UV exposure, and environmental hazards., 2) Phytochemicals (derived from the Greek word phyto meaning plant) are, naturally occurring and biologically active chemical compounds present in, plants., 3) The process of separating medicinally active constituents of plant and animal, tissues with the help of selectiv e solvents and standard procedures is termed, extraction., 4) The extracted preparations include decoctions, infusions, fluid extracts,, tinctures, pilular (semisolid) extracts, or powdered extracts; these preparations, are named as galenicals after Galen (a Greek physician of 2nd century)., 5) Drug extraction process is also influenced by various physical factors, like, gravitation, diffusion, osmosis, adhesion, capillarity, convection, solubility,, and surface tension., 6) The theory of mass transfer states that maximum surface area is obtained, by size reductions that involve reduction of materials into individual cells;, however, this theory is not acceptable in many cases of vegetable material., 7) Water is a universal solvent that is us ed for extracting plant products having, antimicrobial properties., 8) The activity of ethanolic extracts is much higher than that of the aqueous, extracts due to the presence of higher amounts of polyphenols., 9) The word maceration denotes softening. The maceration process (or Process, M) is used for producing tinctures, extracts, and concentrated infusions., *, , *
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284, , Pharmacognosy and Phytochemistry-II, , 10) Simple Maceration is a method for preparing tinctures from organised, drugs, e.g., roots, stems, leaves, etc., 11) Modified maceration is a method for preparing tinc tures from unorganised, drugs, e.g., oleo-resins and gum resins., 12) Multiple maceration is a method for preparing concentrated extracts., 13) Digestion is a modified maceration process. It involves extraction at such a, high temperature which does not put adverse effects on the active ingredients., 14) The term percolation has been derived from the Greek word, percolare, which means to pass through . Percolation (or Process P ) is also termed, lixivation., 15) Percolation involves extracting the constituents of granulated or powder ed, drug by slowly passing down through it a suitable menstruum., 16) The process of SFE involves separating one component (i.e., the extractant), from another (i.e., the matrix) using supercritical fluids (i.e., the extracting, solvent)., 17) In counter current extrac tion, toothed disc disintegrators are used to, pulverise the wet raw material to be extracted., 18) Microwaves are non -ionising electromagnetic waves present in the, electromagnetic spectrum between X-rays and infrared rays., 19) The phe nomenon of modifying the extra, ction efficiency of organic, compounds using ultrasound waves is called acoustic cavitation., 20) Spectroscopy involves the study of interaction of electromagnetic radiation, with matter., 21) The IR spectroscopy is used for the identification of many functional groups, present in the compound, as different functional groups have specific, absorbance (in the form of radiant energy) in the electromagnetic spectrum., 22) The NMR spectroscopy involves absorption of radioactive constituents at, limited radio frequency in a particu lar magnetic field, which differs from, compound to compound., 23) For NMR analysis, the compound is dissolved in a standard inert solvent,, known as TMS (Trimethylsilane), which is subjected to a very strong, magnetic field., 24) The technique of mass spectroscopy measures the mass -to-charge ratio and, abundance of gas -phase ions to identify the amount and type of chemicals a, sample contains., 25) Chromatography is the most versatile separation technique and involves, separation of two or more substanc es by distribution between a fixed (or, stationary) phase and a moving (or mobile) phase., 26) Chromatography is used for separation, isolation, purification, and, identification of components in a mixture., 27) Retardation factor is the ratio of the distance travelle, d by the solute, (desired component) from the original line to the distance travelled by the, solvent front from the original line., 28) The technique of electrophoresis involves migra tion of a charged particle, under the influence of electric field (electro -charged particle and phoresis movement)., *, , *
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Basics of Phytochemistry (Chapter 9), , 285, , 9.3. EXERCISE, 9.3.1., , True or False, , 1), 2), 3), 4), , Paper chromatography is used for volatile liquids., Thermal conductivity detector is used in gas chromatography., A standard inert solvent called trimethylsilane (TMS) is used in NMR analysis., Microwaves are ionising electromagnetic waves., 5) Counter current extraction involves supercritical fluids., , 9.3.2., , Fill in the Blanks, , 6) ____________ involves migra tion of a charged particle under the influence of, electric field., 7) ___________ measures the mass-to-charge ratio and abundance of gas-phase ions., 8) ______________ involves the study of interaction of electromagnetic radiation with, matter., 9) Percolation is also termed as __________., 10) ___________ is a method for preparing tinctures from organised drugs., 11) ____________ is a method for preparing tinctures from unorganised drugs., 12) ____________ is a modified maceration process., Answers, 1) False, 2) True, 3) True, 4) False, 5) False, 6) Electrophoresis, 7) Mass spectroscopy, 8) Spectroscopy, 9) Lixivation 10) Simple Maceration 11) Modified Maceration 12) Digestion, , 9.3.3., 1), 2), 3), 4), 5), 6), , 9.3.4., 1), 2), 3), 4), 5), , Very Short Answer Type Questions, , Write a short note on phytochemistry., What are the properties of an ideal solvent?, What is maceration? Classify., Write a short note on percolation., Discuss pressure cooker extraction., Discuss digestion., , Short Answer Type Questions, , Write a short note on counter current extraction., Discuss extraction by passage through a colloid mill., Explain the procedure of paper chromatography., Discuss ultrasonic assisted extraction., Write a short note on criteria for selection of solven ts and various solvents used for, extraction., , 9.3.5., , Long Answer Type Questions, , 1) Discuss microwave assisted extraction. Also give its instrumentation., 2) Give a detailed account on chromatography techniques., 3) Write an exhaustive note on spectroscopy., *, , *
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286, , Pharmacognosy and Phytochemistry-II, , Index, A, aglycone, 202, Alkaloids, 42, Biogenesis, 48, Biosources, 57, Chemical Classes, 54, Ammonia Test, 162, Anthraquinones, 230, Antimony Trichloride Test, 161, Applications of Tracer Technique, 38, Analysis, 212, Identification, 212, Isolation, 211, Artemisinin, 236, Estimation, 237, Industrial Production, 237, Utilisation, 237, , B, Baljet Test, 90, 162, Belladonna, 65, Benzoin, 144, Bitter Almond, 174, Black Catechu, 133, Borntrager’s Test, 161, Bromine Test, 173, Bufadienolides, 88, , C, Caffeine, 222, 230, 231, 247, Analysis, 224, Estimation, 248, Cardenolides, 88, Cardiac Glycosides, 88, Biosources, 92, Biosynthesis, 88, Chromatography, 278, 284, Cinnamon, 118, Citral, 209, Analysis, 210, Identification, 210, Isolation, 209, , D, Digestion, 262, Digitalis, 97, Digitalis purpurea, 97, 105, Digoxin, 240, Estimation, 241, Industrial Production, 241, Utilisation, 242, , E, Electrophoresis, 282, *, , Ellagitannins, 127, Expression and Diacolation, 274, Extraction, 256, 283, , F, FeCl3 Test, 162, Fennel, 120, Flavonoids, 77, 230, Biosources, 80, Biosynthesis, 78, Chemical Classes, 78, Commercial Applications, 81, Therapeutic Uses, 80, , G, Galenicals, 283, Gallitannins, 127, Gelatin Test, 126, Gentian, 190, Gentiana lutea, 202, Ginger, 149, Glycone, 202, , H, Hager’s Reagent, 49, Hemolysis Test, 161, Hydrolysable Tannins, 127, , I, Infusion and Decoction, 273, Iridoids, 186, Isolated Organs/ Tissues, 33, Isoprene Rule, 176, 202, , K, Keller-Kiliani Test, 89, 161, Klunge’s Isobarbaloin Test, 174, , L, Legal Test, 90, 162, Libermann Burchard Test, 161, Lignans, 81, Liquorice, 93, 105, , M, Maceration, 260, Modified Maceration, 261, Multiple Maceration, 262, Simple Maceration, 260, Matchstick Test, 126, Mayer’s Reagent, 49, Mentha, 113, Mentha piperita, 156, Menthol, 207, *
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Index, Analysis, 208, Identification, 208, Isolation, 208, , N, Naphthoquinones, 197, Nitric Acid Test, 173, Nitrous Acid Test, 173, , O, Opium, 68, , P, Pale Catechu, 130, Percolation, 263, Modified Percolation, 265, Phenazone Test, 126, Phenolics, 230, Phenylpropanoids, 74, Biosources, 74, Biosynthesis, 75, Chemical Classes, 74, Pressure Cooker Extraction, 273, Proto Alkaloids, 54, Pseudo Alkaloids, 55, Pseudotannins, 128, Pterocarpus, 135, , Q, Quinine, 219, Analysis, 221, Identification, 220, Isolation, 219, , R, Rauwolfia, 61, Reserpine, 221, Analysis, 222, Identification, 222, Isolation, 222, Ruta, 84, Rutin, 215, Analysis, 216, Identification, 216, Isolation, 216, , S, Salkowaski Test, 161, Saponins, 230, Schonteten’s Test, 173, Secondary Metabolites, 19, Biogenesis of Alkaloids, 20, Biogenesis of Glycosides, 23, Biogenesis of Steroids, 27, , *, , 287, Steps Involved in Tracer Technique, 35, Steroids, 86, Biosources, 87, Chemical Classes, 87, Supercritical Fluid Extraction, 267, , T, Tannins, 125, 230, Biosources, 128, Chemical Classes, 127, Chemistry, 125, Commercial Applications, 129, Taxol, 250, Estimation, 251, Industrial Production, 250, Utilisation, 251, Terpenoids, 176, Biogenesis, 177, Biosurces, 185, Chemical Classes, 183, Tetranitro Methane Test, 161, Trichloro Acetic Acid Test, 161, Biosources, 103, Chemical Classes, 101, True Alkaloids, 54, , U, Ultrasonic Assisted Extraction, 272, Use of Surface Active Agents in Drug, Extraction, 273, Utilisation of Radioactive Isotopes, 34, , V, Vanillin HCl Test, 162, Vanillin-Hydrochloric Acid Test, 126, Vinca, 59, Vincristine and Vinblastine, 252, Estimation, 253, Industrial Production, 252, Utilisation, 254, Volatile oils, 107, 112, Biosources, 112, Chemical Classes, 110, Chemistry, 107, Isolation, 110, Qualitative Analysis, 109, , W, Wagner’s Reagent, 50, , Z, Zimmermann Test, 161, , *
