Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Ravindra Kumar Pandey Shiv Shankar Shukla Amber Vyas Vishal Jain Parag Jain Shailendra Saraf CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742

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Library of Congress Cataloging-in-Publication Data

Names: Pandey, Ravindra, author. Title: Fingerprinting analysis and quality control methods of herbal medicines / Ravindra Kumar Pandey [and five others]. Description: Boca Raton : CRC Press, Taylor & Francis Group, 2018. | Includes bibliographical references. Identifiers: LCCN 2018006941 | ISBN 9781138036949 (hardback) Subjects: LCSH: Herbs--Therapeutic use--Safety measures. | Materia medica, Vegetable--Analysis. | Materia medica, Vegetable--Safety measures. | Chromatographic analysis. | Drugs--Quality control. Classification: LCC SB293 .F56 2018 | DDC 615.3/21--dc23 LC record available at https://lccn.loc.gov/2018006941

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List of Figures...... xv List of Tables...... xvii Preface...... xix Authors...... xxi

Chapter 1 Introduction...... 1 1.1 Herbal Drugs...... 2 1.2 Terms Relating to Herbal Medicines...... 2 1.2.1 Herbal Medicines...... 2 1.2.2 Herbal Materials...... 3 1.2.3 Herbal Preparations...... 3 1.2.4 Finished Herbal Products or Herbal Medicinal Products...... 3 1.3 Indian System of Medicine (ISM)...... 3 1.4 Herbal Regulation in India...... 5 1.5 Risk Assessment...... 6 1.6 Quality Control of Herbal Drugs...... 6 1.6.1 Identity...... 6 1.6.2 Purity...... 6 1.6.3 Content or Assay...... 7 1.6.4 Several Problems Influence the Quality of Herbal Drugs...... 8 1.7 Standardization of Herbal Formulation...... 8 1.7.1 Specification...... 9 1.7.2 Specifications for Herbal Substances...... 9 1.7.3 Characterization...... 9 1.7.4 Pharmacopoeial Tests and Acceptance Criteria...... 10 1.7.5 In-Process Tests...... 10 1.7.6 Reference Standard...... 10 1.8 Drug Adulteration...... 11 1.9 Conclusion...... 11 References...... 12

Chapter 2 Method of Extraction...... 13 2.1 Introduction...... 13 2.2 Solvent for Extraction...... 13 2.3 Selection of the Solvents...... 14 2.4 Regeneration of the Solvent...... 14 2.5 Solutions (Solute and Solvent)...... 15 2.6 Factors Affecting Choice of Extraction Process...... 15

v vi Contents

2.6.1 Character of Drug...... 15 2.6.2 Therapeutic Value of the Drug...... 15 2.6.3 Stability of Drug...... 16 2.6.4 Cost of Drug...... 16 2.6.5 Solvent...... 16 2.6.6 Concentration of Product...... 16 2.6.7 Recovery of Solvent from the Marc...... 16 2.7 Procedures for Extraction of Herbal Drugs...... 16 2.7.1 Maceration...... 17 2.7.1.1 Modification of General Processes of Maceration...... 18 2.7.2 Vortical or Turbo Extraction...... 19 2.7.3 Ultrasound Extraction...... 19 2.7.4 Extractions by Electrical Energy...... 20 2.7.5 Percolation and Re-Percolation...... 20 2.7.5.1 Percolation Procedure...... 21 2.7.5.2 Modification of the General Process of Percolation...... 21 2.7.5.3 Reserved Percolation...... 22 2.7.6 Cover and Run Down Method...... 22 2.7.7 Small Scale or Laboratory Scale Extraction...... 23 2.7.7.1 Hot Continuous Extractions: Soxhletion.....23 2.7.7.2 Continuous Apparatus (Official Extractor)...... 23 2.7.8 Large Scale Extractor (Counter Current Extractions)...... 25 2.7.9 Infusion and Decoction...... 26 2.7.9.1 General Method for Preparing Fresh Infusion...... 26 2.7.10 Aqueous Alcoholic Extraction by Fermentation...... 26 2.7.11 Steam Distillation...... 27 2.7.12 Supercritical Fluid Extractions...... 27 2.7.13 Phytonics Process...... 28 2.7.14 High Pressure Extraction (HPE)...... 28 2.8 Conclusions...... 29 References...... 29

Chapter 3 Separation and Isolation of Plant Constituents...... 31 3.1 Introduction...... 31 3.2 Classes of Phyto-Constituents...... 31 3.2.1 Glycosides...... 31 3.2.2 Alkaloids...... 32 3.2.3 Flavonoids...... 33 3.2.4 Terpenes...... 34 3.2.5 Phenolics...... 35 Contents vii

3.2.6 Saponins...... 35 3.2.7 Tannins...... 36 3.2.8 Steroids...... 36 References...... 36

Chapter 4 Methods of Phyto-Constituent Detection...... 39 4.1 Introduction...... 39 4.2 Phytochemical Analysis...... 39 4.2.1 Phytochemical Analysis Tests for Alkaloids...... 39 4.2.2 Phytochemical Screening of Anthocyanin...... 40 4.2.3 Phytochemical Analysis Tests for Anthraquinone...... 40 4.2.4 Phytochemical Analysis Test for Cardiac Glycosides...... 40 4.2.5 Phytochemical Analysis Tests for Coumarins...... 40 4.2.6 Phytochemical Analysis Tests for Cynogenetic Glycosides...... 41 4.2.7 Phytochemical Analysis Tests for Phenolics and Flavonoids...... 41 4.2.8 Phytochemical Analysis Test for Saponins...... 41 4.2.9 Phytochemical Analysis Tests for Triterpenes...... 42 4.2.10 Phytochemical Analysis Tests for Steroids...... 42 4.2.11 Phytochemical Analysis Tests for Tannins...... 42 4.2.12 Phytochemical Analysis Tests for Fixed Oils and Fats...... 43 4.2.13 Phytochemical Analysis Test for Gums and Mucilages...... 43 4.2.14 Phytochemical Analysis Tests for Lactones...... 43 4.2.15 Phytochemical Analysis Test for Diterpenes...... 43 4.3 Conclusion...... 43 References...... 44

Chapter 5 Regulatory Aspects for Herbal Drugs...... 45 5.1 Introduction...... 45 5.2 Regulation...... 46 5.2.1 Aim of Regulatory Guidelines for Herbal Medicines...... 46 5.2.2 Regulation and Registration of Herbal Medicines.....46 5.3 WHO Regulatory Requirements...... 47 5.3.1 Objectives...... 48 5.3.2 Guidelines for the Regulation of Herbal Medicines in the Southeast Asia Region...... 48 5.3.3 WHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance Systems.....48 viii Contents

5.3.4 WHO Guidelines on Good Agricultural and Collection Practices (GACP) for Medicinal Plants....48 5.3.5 National Policy on Traditional Medicine (TM) and Regulation of Herbal Medicine...... 49 5.3.6 WHO Guidelines for Quality Control of Herbal Formulation...... 49 5.3.7 WHO Guidelines for Herbal Drug Standardization.... 49 5.4 Herbal Drug Regulations in India...... 50 5.5 Regulatory Aspects and Approval of Herbal Drugs in Different Countries...... 51 5.5.1 European Herbal Guidelines...... 52 5.5.1.1 European Medicines Agency—EMA...... 52 5.5.2 United States of America...... 52 5.5.3 Australia...... 53 References...... 53

Chapter 6 Ethnopharmacology of Medicinal Plants...... 55 6.1 Introduction...... 55 6.2 Phytotherapy...... 55 6.3 Practicing Herbal Medicine...... 56 6.4 Need of Documentation of Ethnopharmacological Plants...... 57 6.5 Ethnopharmacognostical Studies of Medicinal Plants of Chhattisgarh, India...... 57 6.6 Conclusion...... 59 References...... 59

Chapter 7 Quality Control of Herbal Medicine...... 61 7.1 Introduction...... 61 7.2 Quality Control: Present Scenario...... 61 7.3 Quality Control of Herbal Drugs...... 62 7.3.1 Identity...... 62 7.3.2 Purity...... 63 7.3.3 Content or Assay...... 63 7.4 Stability Studies of Herbal Medicines...... 63 7.4.1 Specific Characteristics of Herbal Medicinal Products...... 64 7.4.2 Analytical Methods for Herbal Products...... 64 7.4.3 Stability Study of Herbal Drugs...... 64 7.4.4 Shelf Life...... 65 7.4.5 Challenges in Stability Testing of Herbal Medicinal Products...... 65 7.4.6 Predictable Changes in Herbal Drug Material...... 65 7.4.7 Importance of Stability Testing...... 66 Contents ix

7.5 Biological Markers for Herbal Medicines...... 67 7.5.1 Markers are Categorized into Two Classes...... 67 7.5.1.1 DNA Markers...... 67 7.5.1.2 Chemical Markers...... 68 7.6 Conclusion...... 71 References...... 72

Chapter 8 Bioavailability of Herbal Drugs...... 75 8.1 Need for Bioavailability Enhancers...... 75 8.2 Drug Absorption Barriers...... 76 8.3 Mechanism of Action of Bioenhancers...... 76 8.4 Medicinal Plants and their Compounds as Drug Bioavailability Enhancers...... 77 References...... 78

Chapter 9 Thermal Analysis of Herbal Drugs...... 79 9.1 Introduction...... 79 9.1.1 Thermogravimetry (TG)...... 79 9.1.1.1 Characteristics...... 80 9.1.1.2 Applications of Thermogravimetric Analysis...... 80 9.1.2 Differential Thermal Analysis (DTA)...... 80 9.1.2.1 Characteristics...... 80 9.1.2.2 Applications...... 81 9.1.3 Dynamic Mechanical Analysis (DMA)...... 81 9.1.3.1 Principles of DMA...... 81 9.1.3.2 Instrument and Working of DMA...... 81 9.1.4 Thermomechanical Analysis (TMA)...... 82 9.1.4.1 Instrumentation of TMA...... 82 9.1.4.2 Applications of TMA...... 83 9.2 Conclusion...... 84 References...... 84

Chapter 10 Validation of Herbal Drugs...... 85 10.1 Introduction...... 85 10.2 Concept of Validation...... 86 10.3 Validation of Herbal Drugs...... 86 10.4 Process Validation...... 87 10.5 Validation According to WHO...... 87 10.6 Value Addition...... 87 10.7 Conclusion...... 88 References...... 88 x Contents

Chapter 11 Stability Study of Plant Products...... 89 11.1 Introduction...... 89 11.2 Role of Markers...... 90 11.3 Analytical Methods for Herbal Products...... 90 11.4 Shelf Life...... 91 11.5 Challenges in Stability Testing of Herbal Medicinal Products...... 91 11.6 Predictable Changes in Herbal Medicinal Products...... 92 11.7 Novel Approaches for Stability Improvements in Natural Medicines...... 93 11.8 Conclusion...... 93 References...... 94

Chapter 12 Fingerprinting Techniques for Herbal Drugs Standardization...... 95 12.1 Introduction...... 95 12.2 Phytoequivalence and Chromatographic Fingerprints of Herbal Medicines...... 95 12.2.1 Evaluation of Chemical Fingerprints of Herbal Medicines...... 96 12.2.1.1 Chromatographic Fingerprinting...... 97 12.2.1.2 DNA Fingerprinting...... 105 12.3 Summary...... 109 References...... 110

Chapter 13 Spectroscopic Techniques...... 113 13.1 Introduction...... 113 13.1.1 Ultraviolet Spectroscopy (UV)...... 113 13.1.2 Infrared Spectroscopy (IR)...... 114 13.1.3 Fourier Transform Infrared (FTIR) Spectroscopy...... 115 13.1.3.1 Principle...... 115 13.1.4 Mass Spectroscopy...... 116 13.1.5 NMR Spectroscopy...... 117 13.2 Conclusion...... 118 References...... 119

Chapter 14 Standardization of Herbal Drugs...... 121 14.1 Introduction...... 121 14.2 Different Techniques Involved in Standardization of Crude Drugs...... 123 14.2.1 Botanical Methods...... 123 14.2.1.1 Macroscopic Methods...... 123 14.2.2 Microscopic Methods...... 124 14.2.2.1 Microscopical Examination...... 125 Contents xi

14.2.3 Powder Studies...... 127 14.2.4 Histochemical Detection...... 128 14.2.4.1 Cellulose Cell Walls...... 129 14.2.4.2 Lignified Cell Walls...... 129 14.2.4.3 Suberized or Cuticular Cell Walls...... 129 14.2.4.4 Aleurone Grains...... 129 14.2.4.5 Calcium Carbonate...... 129 14.2.4.6 Calcium Oxalate...... 129 14.2.4.7 Fats, Fatty Oils, Volatile Oils, and Resins...... 129 14.2.4.8 Hydroxyanthraquinones...... 130 14.2.4.9 Inulin...... 130 14.2.4.10 Mucilage...... 130 14.2.4.11 Starch...... 130 14.2.4.12 Tannin...... 130 14.2.4.13 Leaf Stomata...... 130 14.2.5 Measurement of Specimen...... 130 14.2.5.1 Determination of the Stomatal Index...... 131 14.3 Physical Standardization of Herbal Drugs...... 131 14.3.1 Foreign Organic Matter...... 132 14.3.2 Viscosity...... 132 14.3.3 Melting Point...... 132 14.3.4 Solubility...... 132 14.3.5 Moisture Content and Volatile Matter...... 132 14.3.6 Optical Rotation...... 133 14.3.7 Refractive Index...... 133 14.3.8 Ash Values and Extractives...... 133 14.3.9 Total Ash...... 133 14.3.9.1 Determination of Total Ash...... 133 14.3.10 Acid Insoluble Ash...... 134 14.3.10.1 Determination of Acid Insoluble Ash...... 134 14.3.11 Water Soluble Ash...... 134 14.3.11.1 Determination of Water Soluble Ash...... 134 14.3.12 Bitterness Value...... 134 14.3.13 Hemolytic Activity...... 135 14.3.14 Swelling Index...... 135 14.3.15 Foaming Index...... 136 14.3.15.1 Procedure...... 136 14.3.16 Extractive Value...... 136 14.3.17 Total Solid Content...... 136 14.3.18 Water Content...... 137 14.3.19 Volatile Oil Content...... 137 14.3.20 Determination of Tannins...... 137 14.3.21 Loss on Drying (Volatile Matter)...... 138 14.4 Chemical Methods...... 138 14.4.1 Analytical Methods...... 138 xii Contents

14.4.2 Thin Layer Chromatography (TLC)...... 139 14.4.2.1 Equipment...... 140 14.4.2.2 Methodology...... 140 14.4.2.3 Determination of Rf Value...... 141 14.4.3 Chemical Examination of Herbal Drugs...... 141 14.4.3.1 Detection of Alkaloids...... 141 14.4.3.2 Detection of Carbohydrates and Glycosides...... 142 14.4.3.3 Detection of Phytosterols...... 142 14.4.3.4 Detection of Fixed Oils and Fats...... 142 14.4.3.5 Detection of Saponins...... 142 14.4.3.6 Detection of Phenolic Compounds and Tannins...... 142 14.4.3.7 Detection of Proteins and Free Amino Acids...... 142 14.4.3.8 Detection of Gums and Mucilages...... 142 14.4.3.9 Detection of Volatile Oil...... 142 14.4.4 Radioactive Contamination...... 143 14.5 Biological Methods...... 143 14.5.1 Bioassay...... 143 14.5.1.1 Types of Bioassays...... 144 14.5.2 Microbial Contamination...... 146 14.5.2.1 Total Viable Aerobic Count...... 146 14.5.2.2 Aflatoxins...... 147 14.5.3 Toxicological Standardization...... 147 14.5.3.1 Pesticides...... 148 14.5.3.2 Determination of Arsenic and Heavy Metals...... 148 14.6 Validation...... 149 14.7 Determination of Arsenic and Heavy Metals...... 149 14.8 Conclusion...... 150 References...... 150

Chapter 15 Omics Techniques...... 153 15.1 Introduction...... 153 15.2 Omics Techniques...... 153 15.2.1 Genomics and its Modified Techniques...... 153 15.2.2 Proteomics...... 153 15.2.3 Transcriptomics...... 154 15.2.4 Metabolomics...... 154 15.2.5 Application of Omics Techniques in the Context of Herbal Medicine...... 155 15.3 Conclusion...... 155 References...... 156 Contents xiii

Chapter 16 Toxicity Study of Plant Materials...... 157 16.1 Introduction...... 157 16.2 Need of Herbal Toxicity Testing...... 157 16.3 Toxicity of Herbs...... 161 16.4 Safety and Efficacy of Herbals...... 162 References...... 163

Chapter 17 Biological Markers for Quality Control of Herbal Medicines...... 169 17.1 Introduction...... 169 17.2 Markers are Categorized into Two Classes...... 169 17.2.1 DNA Markers...... 170 17.2.1.1 Applications of Biological Markers...... 170 17.2.2 Chemical Markers...... 171 17.2.2.1 Therapeutic Components...... 171 17.2.2.2 Bioactive Components...... 171 17.2.2.3 Synergistic Components...... 171 17.2.2.4 Characteristic Components...... 172 17.2.2.5 Main Components...... 172 17.2.2.6 Correlative Components...... 173 17.2.2.7 Toxic Components...... 173 17.2.2.8 Applications of Chemical Markers...... 173 17.3 Conclusion...... 174 References...... 174

Chapter 18 Pollutants for Herbal Drugs...... 177 18.1 Introduction...... 177 18.2 WHO Guidelines for Contaminants and Residues...... 177 18.2.1 Pesticides...... 178 18.2.2 Pesticide Residue...... 178 18.2.3 Pesticidal Toxicity...... 178 18.3 Heavy Metal Toxicity to Plants...... 178 18.4 Potentially Hazardous Contaminants and Residues in Herbal Medicines...... 179 18.5 Chemical Contaminants...... 179 18.5.1 Toxic Metals and Non-Metals...... 179 18.5.2 Persistent Organic Pollutants (POPs)...... 179 18.5.3 Residual Solvents...... 182 18.6 Radioactive Contamination...... 182 18.7 Mycotoxins and Endotoxins...... 182 18.8 Biological Contaminants...... 182 18.8.1 Microbiological Contaminants...... 182 18.8.2 Parasitic Contamination...... 183 18.8.3 Agrochemical Residues...... 183 xiv Contents

18.9 Pesticide Residues...... 183 18.10 Biochemical Changes in Medicinal Plant Leaves as a Biomarker of Pollution...... 183 18.10.1 APTI Factors...... 184 18.11 Conclusion...... 184 References...... 185

Index...... 187 List of Figures

Figure 1.1 India’s strength in herbal technology...... 2 Figure 1.2 Indian system of medicine...... 4 Figure 1.3 History of Indian system of medicine...... 5 Figure 1.4 Standardization of herbal drugs...... 8 Figure 2.1 Maceration process...... 18 Figure 2.2 Circulatory extractions...... 19 Figure 2.3 Commercial scale percolator...... 22 Figure 2.4 Soxhlet apparatus...... 24 Figure 2.5 Continuous apparatus...... 24 Figure 2.6 Large-scale extractor...... 25 Figure 3.1 Phyto-constituents of glycosides...... 32 Figure 3.2 Chemical constituents of alkaloids...... 33 Figure 3.3 Phyto-constituents of flavonoids...... 34 Figure 3.4 Phyto-constituents of terpenoids...... 35 Figure 5.1 WHO guidelines for herbals...... 47 Figure 7.1 Factors affecting stability of natural medicines...... 66 Figure 7.2 Chemical markers...... 70 Figure 9.1 Sinusoidal oscillation and response of a linear-viscoelastic material where, δ = phase angle...... 82 Figure 9.2 Block diagram of DMA...... 83 Figure 9.3 Block diagram of TMA...... 83 Figure 11.1 Goal of an analytical marker for analysis of HMPs...... 90 Figure 11.2 Factors affecting the stability of herbal medicines...... 92 Figure 12.1 Fingerprinting techniques in herbal drug standardization...... 96 Figure 12.2 Thin layer chromatography...... 98 Figure 12.3 Gas chromatography...... 100 Figure 12.4 HPLC chromatography...... 101 Figure 13.1 UV spectroscopy instrumentation...... 114

xv xvi List of Figures

Figure 13.2 IR spectroscopy instrumentation...... 114 Figure 13.3 Block diagram of an FTIR spectrometer...... 116 Figure 13.4 Mass spectroscopy instrumentation...... 117 Figure 13.5 NMR spectroscopy instrumentation...... 118 Figure 14.1 Standardization of herbal drugs...... 122 Figure 14.2 Graded response...... 145 Figure 14.3 Matching method...... 145 Figure 14.4 Four point assay...... 146 Figure 15.1 Workflow for a metabolomic experiment...... 155 Figure 16.1 Toxicity evaluation of herbal drugs...... 161 Figure 17.1 Synergistic components as chemical markers...... 172 Figure 17.2 Correlative components as chemical markers...... 173 List of Tables

Table 2.1 A Brief Summary of the Experimental Conditions for Various Methods of Extraction for Plant Materials...... 17 Table 6.1 Ethnopharmacological Plants of Chhattisgarh...... 58 Table 14.1 Macroscopic Characteristics of Herbal Drugs...... 124 Table 14.2 Classification of Powders...... 128 Table 14.3 Sieve Numbers and Specifications...... 128 Table 16.1 Toxicity Profile of Traditional Herbal Plants...... 158 Table 18.1 Classification of Major Contaminants and Residues in Herbal Medicines...... 180

xvii

Preface

Medicinal plants are the richest bioresource of drugs for traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, and chemical entities for synthetic drugs. Authentication and consistent quality are the basic requirements for traditional medicines and their commercial products, regardless of the kind of research conducted to modernize the traditional medicines. Due to the increase in the consumption of herbal medicine, there is a need to know which scientifically based methods are appropriate for assessing the quality of herbal medicines. Fingerprinting analysis is a rational option to meet the need for more effective and powerful quality assessment of herbal medicines. The phytoconstituents are identified at the molecular level using current analytical practices, which are unique characteristics termed as fingerprints. The fingerprints are used for assessment of quality consistency and stability by visible observation and comparison of the standardized fingerprint pattern. These fingerprints have the scientific potential to claim the authenticity and reliability of chemical constituents with total traceability. It starts from the proper identification, storage, stability during processing, and rationalizing the combination in the case of polyherbal drugs. Quality control is essential for natural products like natural medicine and related food products. Since the concentration varies for bioactive components in natural medicines or natural product extracts and is dependent on the place of collection, the season of its collection, the method of extraction, and subsequent treatment, the standardization of quality is necessary. Therefore, the methodology of quality control for natural medicines has been discussed in this book. The chromatographic (TLC, HPTLC, HPLC, GC) and spectral (UV-Vis., FTIR, MNR, MS, LC-MS, GC-MS, etc.) techniques have worldwide strong scientific approval as validated methods to generate the fingerprints of different chemical classes of active ingredients of herbal drugs. In this book, eighteen chapters have been included, discussing the various aspects of fingerprinting analysis, quality control, and standardization of herbal drugs.

Ravindra Kumar Pandey, Shiv Shankar Shukla, Amber Vyas, Vishal Jain, Parag Jain, and Shailendra Saraf

xix

Authors

Dr. Ravindra Kumar Pandey is currently working as a professor in Columbia Institute of Pharmacy, Raipur (Chhattisgarh). He has 15 years of teaching and industrial experience. He earned his MPharm from Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, Madhya Pradesh and PhD from Pandit Ravishankar Shukla University, Raipur (Chhattisgarh). His areas of interest are pharmacognosy and phytochemistry. He has successfully completed several research projects and a few are ongoing. He has several research papers published in national and international journals. Dr. Pandey is an author of books and book chapters. He is frequently invited as a resource person at various national and international conferences. He is an approved examiner and paper setter for different Indian universities, has attended several staff development programs, and has also participated in different conferences. He is a lifetime member of several national bodies and has attended career development programs organized by the government and institutes.

Dr. Shiv Shankar Shukla has 13 years of teaching experience and currently works as a professor at Columbia Institute of Pharmacy, Raipur (Chhattisgarh). He earned his BPharma from B. R. Nahata College of Pharmacy, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, Madhya Pradesh in 2002, MPharma from L. M. College of Science & Technology, Jodhpur, Jai Narain Vyas University, Jodhpur (Rajasthan) in 2005, and PhD from University Institute of Pharmacy, Pandit Ravi Shankar Shukla University, Raipur (Chhattisgarh) in 2012. His areas of research interest are analytical chemistry and quality control, and he heads the Department of Pharmaceutical Analysis of the institute. He has been recognized as “Young Scientist of Chhattisgarh” in 2009 by Chhattisgarh Council of Science and Technology and received the Dr. P. D. Sethi Annual Award in 2010 for his paper on TLC Densitometric Fingerprint Development and Validation of 6-Gingerol as Marker in Poly-herbal Ayurvedic Formulations. He is also an approved examiner and paper setter for different Indian universities. His research papers have been recognized on national and international levels, and he has organized and attended various workshops and conferences. He is a life member of the Indian Pharmaceutical Association.

Dr. Amber Vyas has 13 years of teaching and research experience. Dr. Vyas earned his master’s from K.L.E. Society’s College of Pharmacy, Belagavi, Karnataka. He was selected as an UGC Raman Fellow in 2016 and completed his post-doctoral studies from Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota, USA. He has 30 publications/research-poster presentations to his credit in national, international, and electronic journals/conferences. He has been recognized as “Young Scientist of Chhattisgarh” in 2011 by the Chhattisgarh Council of Science and Technology. He is an active member of Indian Pharmaceutical Association, Association of Pharmaceutical Teachers of India. His research interest extends from development of nanotechnology and targeted delivery-based newer sustained release dosage forms.

xxi xxii Authors

Dr. Vishal Jain has 15 years of teaching and research experience. Dr. Jain earned his master’s from the Department of Pharmaceutical Science, Dr. Hari Singh Gaur Vishwavidyalaya, Sagar, Madhya Pradesh. He has over 35 publications to his credit published in national and international journals and has presented several papers at various national and international conferences. He is an active member of the Indian Pharmaceutical Association and the Indian Society of Pharmacognosy (IST). His research interest focuses on phytochemical standardization of herbs.

Mr. Parag Jain has 5 years of teaching and research experience. He earned his BPharma from University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur and MPharma from Columbia Institute of Pharmacy, Chhattisgarh Swami Vivekanand Technical University, Bhilai (Chhattisgarh). He has been honored by the Indian Academy of Sciences (IASc), Bengaluru, for promotion toward research. He has conducted cutting-edge research at the Indian Institute of Integrative Medicine (IIIM), Council of Scientific & Industrial Research (CSIR), Jammu and All India Institute of Medical Sciences (AIIMS), New Delhi, where, during his master’s, he developed expertise in diabetes, toxicity testing, preclinical studies, pharmacokinetics, pharmacodynamics, with equal inclination to ethnopharmacology. His areas of interest include diabetes, neuropharmacology, toxicology, molecular biology, ethnopharmacology, and so on. He has recognized publications in national and international journals to his credit and a few more in the pipeline. He has been invited as a resource person in an ICMR- sponsored seminar and has contributed many books under national and international banners. His is currently exploring natural sources for the treatment of diabetes.

Professor Shailendra Saraf is a leading scientist and well-known academician in the field of pharmaceutical sciences. He is a vice-chancellor of Durg University, Chhattisgarh, India and has been the director of University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur (Chhattisgarh). Professor Saraf has over 30 years of research and teaching experience. He is an alumnus of the Department of Pharmaceutical Science, Dr. H. S. Gour University, Sagar, Madhya Pradesh. He has over 133 publications to his credit published in international and national journals of distinction and has also authored several books. He is on the editorial boards of many scientific journals. He has been a resource person and delivered invited lectures, chaired scientific sessions at several national conferences, congress, and symposia in India and abroad. Professor Saraf is the founder-president of the Association of Pharmaceutical Teachers of India (APTI) Chhattisgarh State Branch and founder-president, Indian Pharmaceutical Association (IPA) Chhattisgarh State Branch. He has been an executive member of the Indian Society of Pharmacognosy (ISP) and the IPA, Education Division. Professor Saraf is a permanent invitee to the Cost Engineering Committee (CEC), IPA. He is vice president of the Pharmacy Council of India (PCI) and a member of various educational bodies of India such as All India Council for Technical Education (AICTE), University Grant Commissions (UGC), National Assessment and Accreditation Council (NAAC) and National Board of Accreditation (NBA). He is a life member of IPA, ISP, APTI, Indian Society for Technical Education (ISTE), and MP Pharmacy Graduates Association (MPPGA). His research interest extends from herbal cosmetics to herbal drug standardization, modern analytical techniques, and new drug delivery systems. 1 Introduction

Traditional herbal medicines and their preparations have been widely used for thousands of years in developing and developed countries owing to their natural origins and lesser side effects or dissatisfaction with the results of synthetic drugs (Schmidt et al., 2008). Herbal drugs have been used since ancient times as medicines for the treatment of a range of diseases. Medicinal plants have played a key role in world health. In recent decades, in spite of the great advances observed in modern medicine, plants still make an important contribution to health care (WHO, 2005). This increased use of herbal medicines is due to several reasons; namely, the inefficiency of conventional medicines (e.g., side effects and ineffective therapy), abusive use of synthetic drugs resulting in side effects, a large percentage of the world’s population does not have access to conventional pharmacological treatment, and folk medicines and ecological awareness which suggest that natural products are harmless (Engebretson, 2002; Conboy, 2007). However, the quantity and quality of the safety and efficacy data on traditional medicines are far from sufficient to meet the criteria needed to support their use world wide. The reasons for the lack of research data are due to not only to health care policies, but also to a lack of adequate or accepted research methodology for evaluating traditional medicine (WHO, 2000, 2001). According to an estimate of the World Health Organization (WHO), about 80% of the world’s population still uses herbs and other traditional medicines for their primary health care needs. Of the 252 drugs considered as basic and essential by the WHO, 11% are exclusively of plant origin and a significant number of synthetic drugs are obtained from natural precursors. In olden days, vaidas (Kamboj, 2012) used to treat patients on an individual basis and prepare drugs according to the requirements of the patient, but now the scene has changed and herbal medicines are being manufactured on a large scale where manufacturers come across many problems, such as the availability of good quality raw material, authentication of raw material, availability of standards, proper standardization methodologies for single drugs and their formulation, quality control parameters, and so on; hence, the concept of quality from the very first step is a paramount factor that must receive much attention (Kokate et al., 2005; Raina, 2003). The chemistry of plants involves the presence of therapeutically important constituents usually associated with many inert substances (coloring agents, cellulose, lignin, etc.). The active constituents are extracted from the plants and purified for therapeutic utility for their selective pharmacological activity. Thus, quality control of herbal crude drugs and their constituents is of great importance in the modern system of medicine (Rishton, 2008). Lack of proper standard parameters for the standardization of herbal preparations and several instances of substandard herbs or adulterated herbs have come into existence. To meet the new thrust of inquisitiveness, standardization of herbals is mandatory (Chaudhry, 1999; Raven et al., 1999). Hence, every single herb needs to be quality checked to ascertain that it confirms to quality

1 2 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines requirements and consistently delivers the required properties. Standardization assures that products are reliable in terms of quality, efficacy, performance, and safety. It is, however, observed that the drugs in commerce are frequently adulterated and do not comply with the standards prescribed for authentic drugs (Kunle et al., 2012).

1.1 HERBAL DRUGS According to WHO, herbal medicines include herbs, herbal materials, herbal preparations, and finished herbal products. Herbs include crude plant materials, such as leaves, flowers, fruits, seeds, stems, woods, barks, roots, rhizomes or other plant parts, which may be entire, fragmented or powdered (WHO, 2007). Herbal materials, in addition to herbs, include fresh juices, gums, fixed oils, essential oils, resins, and dry powders of herbs. Various procedures, such as steaming, roasting or stir-baking with honey are carried out for their preparation. Herbal drugs are finished, labeled products that contain active ingredients such as aerial or underground parts of plants or other plant material or combinations thereof, whether in the crude state or as plant preparations. Herbal formulations are obtained by subjecting herbal substances to treatments such as extraction, distillation, expression, fractionation, purification, concentration or fermentation. These include powdered herbal substances, tinctures, extracts, essential oils, expressed juices, and processed exudates. Finished herbal products consist of herbal preparations made from one or more herbs which may contain excipients in addition to the active ingredients. India’s strength in herbal technology is shown in Figure 1.1.

1.2 TERMS RELATING TO HERBAL MEDICINES

1.2.1 Herbal Medicines Herbal medicine can also be termed as phytomedicine. Herbal drugs may contain one or more parts of plants as seeds, berries, roots, leaves, bark or flowers for medicinal

8000 Medicinal

Total 10,000 species

325

425

3500 Pesticides

Edible Fiber 550

1000 Gums, resins, and dyes

Others

FIGURE 1.1 India’s strength in herbal technology. Introduction 3 purposes. Herbal medicinal plants have a long tradition of use outside of conventional medicine. This is becoming more mainstream as improvements in analysis and quality control along with advances in clinical research show the value of herbal medicine in the treating and preventing of disease. People use herbal medicines to try to maintain or improve their health. Many people believe that products labeled “natural” are always safe and good for them. This is not necessarily true. Most of the time, herbal medicines do not have to go through testing for quality and safety (Moreira et al., 2014).

1.2.2 Herbal Materials Herbal materials are either whole plants or parts of medicinal plants in the crude state. They include herbs, fresh juices, gums, fixed oils, essential oils, resins, and dry powders of herbs. In some countries, these materials may be processed by various local procedures, such as steaming, roasting or stir-baking with honey, alcoholic beverages or other materials (Singh et al., 2016).

1.2.3 Herbal Preparations Herbal preparations are the basis for finished herbal products and may include comminuted or powdered herbal materials or extracts, tinctures, fatty oils, expressed juices, and processed exudates of herbal materials. They are produced with the aid of extraction, distillation, expression, fractionation, purification, concentration, fermentation or other physical or biological processes. They also include preparations made by steeping or heating herbal materials in alcoholic beverages and/or honey or in other materials.

1.2.4 Finished Herbal Products or Herbal Medicinal Products Herbal products contain active substances that are exclusively herbal drugs or herbal drug preparations. They may consist of herbal preparations made from one or more herbs. If more than one herb is used, the term mixed herbal product can also be used. They may contain excipients in addition to the active ingredients. In some countries, herbal medicines may contain, by tradition, natural organic or inorganic active ingredients which are not of plant origin (e.g., animal materials and mineral materials). Generally, however, finished products or mixed products to which chemically defined active substances have been added, including synthetic compounds and/or isolated constituents from herbal materials, are not considered to be herbal.

1.3 INDIAN SYSTEM OF MEDICINE (ISM) WHO defines traditional medicine as including diverse health practices, approaches, knowledge, and beliefs incorporating plant, animal, and/or mineral based medicines, spiritual therapies, manual techniques, and exercises applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness. It covers all the systems which originated in and outside of India, but were adopted 4 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

ies) s (oral) nitie ec mu m co l

a Indian system of me ib 2500 species r t y 900 sp. b Ayurveda a 17,500 sp

d (c

s 700 sp. u

Unani

t 600 sp.

n Siddha

p 250 sp.

l dicine

a Amchi

c i

e 30 sp. e Indian ﬂora M Modern

8000 species

FIGURE 1.2 Indian system of medicine. and adapted over the course of time. Herbal drug products constitute a major share of all the officially recognized systems of health in India, such as Ayurveda, Yoga, Unani, Siddha, Homeopathy, and Naturopathy (Figure 1.2). Most of the traditional medicine systems of India, including Ayurveda, have their roots in folk medicine. It is the science of life. Ayurveda was among the first medical systems to advocate an integrated approach toward matters of health and disease. It is the system that takes into consideration the physical, psychological, philosophical, ethical, and spiritual well-being of mankind. Unlike other medical systems, which developed their conceptual framework based on the results obtained with the use of drugs and therapy, it first provided a philosophical framework that determined the therapeutic practice with good effects. It laid great emphasis on the value of the evidence of senses and human reasoning (Prasad, 2002). The Siddha system has come to be closely identified with Tamil civilization. The term “Siddha” has come from “Siddhi,” which means achievement. The materia medica of the Siddha system of medicine depends to a large extent on drugs of metal and mineral origin in contrast to Ayurveda of the earlier period, which was mainly dependent upon drugs of vegetable origin. Diagnosis in the Siddha system is carried out by the well-known “ashtasthana pareeksha” (examination of eight sites), that encompasses examination of nadi (pulse), kan (eyes), swara (voice), sparisam (touch), varna (color), na (tongue), mala (feces), and neer (urine) (Narayanaswamy, 1975). Unani medicine originated in Greece. According to the basic principles of Unani, the body is made up of four basic elements, that is, Earth, Air, Water, and Introduction 5

Fire. In this system, prime importance is also given to the preservation of health. Examination of the pulse occupies a very important place in the disease diagnosis in Unani. The pulse is examined to record different features, such as size, strength, speed, consistency, fullness, rate, temperature, constancy, regularity, and rhythm (Khaleefathullah, 2002). A large number of studies have been carried out on a number of medicinal plants used in the ISM of medicine. However, one of the basic problems that still remains to be solved is related to proving the efficacy of the products used in these systems on the basis of controlled clinical trials and complementary pharmacological studies. It is difficult to ensure consistency in the results and components in the products. This is traced mainly to a lack of standardization of the inputs used and the processes adopted for preparation of the formulations.

1.4 HERBAL REGULATION IN INDIA The government of India has instituted Good Manufacturing Practices (GMPs) for the pharmacies manufacturing Ayurvedic, Siddha, and Unani medicines to improve the quality and standard of drugs. The Department of Indian Systems of Medicine and Homeopathy (ISM&H) is trying to frame safety and efficacy regulations for licensing new patent and proprietary botanical medicines. Indian Pharmacopoeia covers a few Ayurvedic medicines (Figure 1.3). Monographs have been given for various Ayurvedic drugs, such as clove, guggul, opium, menthe, senna, and so on. The Ayurvedic pharmacopoeia of India has given monographs for 258 different Ayurvedic drugs.

Ayurveda (900-800 BC)

Siddha Homeopathy (800-700 BC) (1850 AD)

ISM

Yoga and Unani naturopathy (460-377 BC) (500-800 AD)

FIGURE 1.3 History of Indian system of medicine. 6 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

1.5 RISK ASSESSMENT This is the determination of a qualitative or quantative estimate of risk related to a well defined situation and a recognized threat. The following definitions are established:

Hazard: This means a biological, chemical or physical agent in, or condition of, food or feed with the potential to cause an adverse health effect. Risk: Risk is a function of the probability of an adverse health effect and the severity of that effect, consequential to a hazard. Risk analysis: This is a process consisting of three interconnected components— risk assessment, risk management, and risk communication. Risk assessment: This is a scientifically based process consisting of four steps— hazard identification, hazard characterization, exposure assessment, and risk characterization; risk assessment is to be based on the available scientific evidence and undertaken in an independent, objective, and transparent manner. Risk management: This is the process, distinct from risk assessment, of weighing policy alternatives in consultation with interested parties, considering risk assessment and other legitimate factors, and, if need be, selecting appropriate prevention and control options; risk management is to take into account the results of the risk assessment and other factors legitimate to the matter under consideration and the precautionary principle. Risk communication: This is the interactive exchange of information and opinions throughout the risk analysis process as regards hazards and risks, risk-related factors, and risk perceptions, among risk assessors, risk managers, consumers, feed and food businesses, the academic community, and other interested parties, including the explanation of risk assessment findings and the basis of risk management decisions.

1.6 QUALITY CONTROL OF HERBAL DRUGS Quality control for the efficacy and safety of herbal products is of paramount importance. Quality can be defined as the status of a drug that is determined by identity, purity, content, and other chemical, physical or biological properties or by the manufacturing processes. Quality control is a term that refers to processes involved in maintaining the quality and validity of a manufactured product. Quality control is based on three important pharmacopoeial definitions, that is, identity, purity, and content or assay.

1.6.1 identity Identity can be achieved by macro- and microscopical examinations. Outbreaks of diseases among plants may result in changes to the physical appearance of the plant and lead to incorrect identification.

1.6.2 Purity Purity is closely linked with the safe use of drugs and deals with factors such as ash values, contaminants (e.g., foreign matter in the form of other herbs), and heavy Introduction 7 metals. However, due to the application of improved analytical methods, modern purity evaluation also includes microbial contamination, aflatoxins, radioactivity, and pesticide residues. Analytical methods such as photometric analysis (UV, IR, MS, and NMR), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography (GC) can be employed in order to establish the constant composition of herbal preparations.

1.6.3 content or Assay It is obvious that the content is the most difficult property to assess, since in most herbal drugs, the active constituents are unknown. Sometimes markers can be used which are, by definition, chemically defined constituents that are of interest for control purposes, independent of whether they have any therapeutic activity or not. To prove identity and purity, criteria such as type of preparation sensory properties, physical constants, adulteration, contaminants, moisture, ash content, and solvent residues have to be checked. The correct identity of the crude herbal material, or the botanical quality, is of prime importance in establishing the quality control of herbal drugs (WHO, 1992). Identity can be achieved by macro- and microscopical examinations. Voucher specimens are reliable reference sources. Outbreaks of diseases among plants may result in changes to the physical appearance of the plant and lead to incorrect identification. Purity is closely linked with the safe use of drugs and deals with factors such ash values, contaminants (e.g., foreign matter in the form of other herbs), and heavy metals. However, due to the application of improved analytical methods, modern purity evaluation includes microbial contamination, aflatoxins, radioactivity, and pesticide residues. Analytical methods such as photometric analysis (UV, IR, MS, and NMR), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography (GC) can be employed in order to establish the constant composition of herbal preparations. Quality assurance and control measures, such as national quality specification and standards for herbal materials, good manufacturing practices (GMP) for herbal medicines, labelling, and licensing schemes for manufacturing, imports, and marketing, should be in place in every country where herbal medicines are regulated. Content or assay is the most difficult area of quality control to perform, since in most herbal drugs, the active constituents are not known. Sometimes markers can be used. In all other cases, where no active constituent or marker can be defined for the herbal drug, the percentage extractable matter with a solvent may be used as a form of assay, an approach often seen in pharmacopeias. The choice of the extracting solvent depends on the nature of the compounds involved and might be deduced from the traditional uses. A special form of assay is the determination of essential oils by steam distillation. When the active constituents (e.g., sennosides in Senna) or markers (e.g., alkydamides in Echinacea) are known, a vast array of modern chemical analytical methods such as ultraviolet/visible spectroscopy (UV/VIS), TLC, HPLC, GC, mass spectrometry (MS) or a combination of GC and MS (GC/MS) can be employed (Booksh and Kowalski, 1994). 8 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

1.6.4 several Problems Influence the Quality of Herbal Drugs Herbal drugs are usually mixtures of many constituents. The active principle(s) is (are), in most cases, unknown. Selective analytical methods or reference compounds may not be available commercially. Plant materials are chemically and naturally variable. Chemo-varieties and chemo-cultivars exist. The source and quality of the raw materials are variable. The methods of harvesting, drying, storage, transportation, and processing, (e.g., mode of extraction and polarity of the extracting solvent, instability of constituents, etc.) have an effect. Strict guidelines have to be followed for the successful production of a quality herbal drug. Among them are proper botanical identification, phytochemical screening, and standardization. Quality control and the standardization of herbal medicines involve several steps. The source and quality of raw materials, good agricultural practices, and manufacturing processes are certainly essential steps for the quality control of herbal medicines and play a pivotal role in guaranteeing the quality and stability of herbal preparations.

1.7 STANDARDIZATION OF HERBAL FORMULATION Standardization is a process of evaluating the quality and purity of an herbal drug on the basis of various parameters like morphological, microscopical, and physical parameters such as moisture content, ash value, extractive value, and so on, identification, chemical, and biological parameters (Figure 1.4). Standardization of herbal raw drugs includes the passport data of raw plant drugs, botanical authentication, specification of chemical composition by various chromatographic techniques, and determination of biological activity of the whole plant. Herbal substances are a diverse range of botanical materials including leaves, herbs, roots, flowers, seeds, bark, and so on. A comprehensive specification must be developed for each herbal substance even if the starting material for the manufacture of the herbal

Moisture content, ash value, extractive value, viscosity, Physical density, bitterness value, solubility, swelling index, foaming index, speciﬁc gravity

Color, odor, taste, texture and Botanical fracture, qualitative, quantitative, Standardization of SEM studies, powder studies herbal drugs Chromatographic techniques, Chemical heavy metal, pesticide residue, mycotoxins

Microbial contamination, Biological pharmacological evaluation, toxicological studies

FIGURE 1.4 Standardization of herbal drugs. Introduction 9 medicinal product is an herbal preparation. In the case of fatty or essential oils used as the active substances of herbal medicinal products, a specification for the herbal substance is required unless it is justified.

1.7.1 specification A specification is defined as a list of tests, references to analytical or biological procedures, and appropriate acceptance criteria, which are numerical limits, ranges or other criteria for the tests described. It establishes the set of criteria to which an herbal substance, herbal preparation or herbal medicinal product should conform to be considered acceptable for its intended use. Specifications are legally binding quality standards that are proposed and justified by the manufacturer and approved by regulatory authorities. The setting of specifications for an herbal substance/ preparation and herbal medicinal product is part of an overall control strategy which includes control of the raw materials and excipients, in-process testing, process evaluation/validation, stability testing, and testing for consistency of batches. When combined in total, these elements provide assurance that the appropriate quality of the product will be maintained. Since specifications are chosen to confirm the quality rather than to characterize the product, the manufacturer should provide the rationale and justification for including and/or excluding testing for specific quality attributes. The following points should be taken into consideration when establishing scientifically justifiable specifications.

1.7.2 specifications for Herbal Substances • Botanical characteristics of the plant (genus, species, variety, chemotype; usage of genetically modified organisms), parts of the plants • Macroscopical and microscopical characterization, phytochemical characteristics of the plant part constituents with known therapeutic activity or markers, toxic constituents (identity, assay, limit tests) • Biological/geographical variation • Cultivation/harvesting/drying conditions (microbial levels, mycotoxins, aflatoxins, ochratoxin, toxic metals, etc.) • Pre-/post-harvest chemical treatments (pesticides, fumigants) • Profile and stability of the constituents • Quality of the herbal substance • Method of preparation from the herbal substance • Constituents—constituents with known therapeutic activity or active or analytical markers • Other constituents (identification, assay, limit tests) • Drying conditions (e.g., microbial levels, residual solvents in extracts)

1.7.3 characterization Characterization of an herbal substance/preparation or herbal medicinal product (which includes a detailed evaluation of the botanical and phytochemical aspects 10 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines of the plant, manufacture of the preparation, and the herbal medicinal product) is, therefore, essential to allow specifications to be established which are both comprehensive and relevant. Extensive characterization is usually performed only in the development phase and where a necessary significant process changes. If necessary, at the time of submission the manufacturer should have established appropriately characterized in-house reference materials (primary and working) which will serve for identification and determination of the content of production batches, such as macroscopic/microscopic characterization, phytochemical characterization, impurities’ identification, and biological variation.

1.7.4 Pharmacopoeial Tests and Acceptance Criteria Countries’ Pharmacopoeias contain important requirements pertaining to certain analytical procedures and acceptance criteria that are relevant to herbal substances, herbal preparations, and their herbal medicinal products. Wherever they are appropriate, pharmacopoeial methods should be utilized. Phytochemical standardization encompasses all the possible information generated with regard to the chemical constituents present in an herbal drug. Hence, the phytochemical evaluation for standardization purposes includes the following:

• Preliminary testing for the presence of different chemical groups. (e.g., total alkaloids, total phenolics, total triterpenic acids, total tannins, etc.). • Quantification of chemical groups of interest. • Establishment of fingerprint profiles based upon single or multiple markers.

1.7.5 in-Process Tests In-process tests are tests which may be performed during the manufacture of either the herbal preparation or herbal medicinal product. In-process tests, which are used for the purpose of adjusting process parameters within an operating range, for example, hardness and friability of tablet cores which will be coated, are not included in the specification. Certain tests conducted during the manufacturing process, where the acceptance criteria are identical to or tighter than the release requirement (e.g., pH of a solution), may be used to satisfy specification requirements when the test is included in the specification.

1.7.6 reference Standard In the case of herbal medicinal products, the reference standard may be a botanical sample of the herbal substance, a sample of the herbal preparation, for example, extract or tincture or a chemically defined substance, for example, a constituent with known therapeutic activity, an active marker or an analytical marker or a known impurity. The reference standard has a quality appropriate to its use. The composition of reference standards of herbal substances and herbal preparations intended for use in assays should be adequately controlled and the purity of a standard should be measured by validated quantitative procedures. Introduction 11

1.8 DRUG ADULTERATION The substitution of an original drug partially or wholly by other similar substances, which are inferior in chemical and biological activities, is termed drug adulteration. It is one of the major problems associated with the commercialization of herbal drugs. It is malpractice to mix crude drug material with other spurious, inferior, spoiled, harmful or useless substances, which are unauthentic and substandard. The substandard quality of drug products may become life threatening (Ahmed and Hasan, 2015; Kokate et al., 2005). Adulteration may be intentional or unintentional; direct or intentional adulteration is done intentionally, which usually includes practices in which an herbal drug is substituted for partially or fully by other inferior products. Due to morphological resemblance to the authentic herb, many different inferior commercial varieties are used as adulterants. These may or may not have any chemical or therapeutic potential. Substitution by “exhausted” drugs entails adulteration of the plant material with the same plant material devoid of the active constituents. This practice is most common in the case of volatile oil-containing materials, where the dried exhausted material resembles the original drug, but is free of the essential oils. Foreign matter such as other parts of the same plant with no active ingredients, sand and stones, manufactured artifacts, and synthetic inferior principles are used as substitutes. Unintentional or undeliberate adulteration sometimes occurs without any bad intention of the manufacturer or supplier. Sometimes in the absence of proper means of evaluation, an authentic drug partially or fully devoid of the active ingredients may enter the market. Factors such as geographical sources, growing conditions, processing, and storage are all factors that influence the quality of the drug. It may be due to faulty collection, imperfect preparation, incorrect storage, gross substitution with plant material, substitution with exhausted drugs, and so on.

1.9 CONCLUSION Quality assurance of herbal medicinal products is the shared responsibility of manufacturers and regulatory bodies. All herbal-based medicinal products should meet requirements for safety, efficacy, and quality as per the categories of herbal medicines. Weak regulation and quality control may result in a high incidence of adverse reactions attributable to the poor quality of herbal medicines, in particular resulting from adulteration with undeclared potent substances and/or contamination with potentially hazardous substances and residues. As commercialization of herbal medicines has happened, assurance of safety, quality, and efficacy of medicinal plants and herbal products has become an important issue. Herbal raw materials are prone to a lot of variation due to several factors, the important ones being the identity of the plants and seasonal variation (which has a bearing on the time of collection), the ecotypic, genotypic, and chemotypic variations, drying and storage conditions, and the presence of a xenobiotic. Another one of the major problems faced by the herbal drug industry is the unavailability of rigid quality control profiles for herbal materials and their formulations. 12 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

REFERENCES Ahmed S and Hasan MM. Crude drug adulteration: A concise review, WJPPS 2015; 4(10): 274–283. Booksh KS and Kowalski BR. Theory of analytical chemistry. Anal. Chem 1994; 66: 782A–791A. Chaudhury RR. Herbal Medicine for Human Health. World Health Organization, Geneva, CBS Publishers and Distributors LTD, New Delhi, 1999. Conboy L, Kaptchuk TJ, Eisenberg DM, Gottlieb B, Acevedo-Garcia D. The relationship between social factors and attitudes toward conventional and CAM practitioners. Complement Ther Clin Pract 2007; 13: 146–157. Engebretson J. Culture and complementary therapies. Complement Ther Nurs Midwifery 2002; 8: 177–184. Kamboj A. Analytical evaluation of herbal drugs. In: Drug Discovery Research in Pharmacognosy, Vallisuta O and Olimat SM (Eds.), INTECH Publications, 2012, pp. 23–24. Khaleefathullah S. Unani medicine. In: Chaudhury RR, Rafei UM (Eds.), Traditional Medicine in Asia. WHO- Regional Office for South East Asia, New Delhi, 2002, pp. 31–46. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, 31st Edition. Nirali Prakshan, Pune, India, 2005, pp. 97–131. Kunle OF, Egharevba HO, Ahmadu PO. Standardization of herbal medicines—A review. Int J Biodiv Conserv. 2012; 4(3): 101–112. Moreira DL, Teixeira SS, Monteiro MHD, De-Oliveira AC, Paumgartten FJR. Traditional use and safety of herbal medicines. Rev. bras. farmacogn. 2014; 24(2): 248–257. Narayanaswamy V. Introduction to the Siddha System of Medicine. Director, Pandit S.S. Anandam Research Institute of Siddha Medicine, T. Nagar, Madras (Chennai), 1975. Prasad LV. In: Roy CR, Muchatar RU. (Eds.), Indian System of Medicine and Homoeopathy Traditional Medicine in Asia. WHO- Regional Office for South East Asia, New Delhi, 2002, 283–286. Raina MK. Quality control of herbal and herbo-mineral formulations. Indian J Nat Prod 2003; 19: 11–15. Raven PH, Evert RF, Eichhorn SE. Biology of Plants, 6th Edition. Freeman, New York, 1999. Rishton GM. Natural products as a robust source of new drugs and drug leads: Past successes and present day issues. Am J Cardiol 2008; 101: 43D–49D. Schmidt B, Ribnicky DM, Poulev A, Logendra S, Cefalu WT, Raskin I. A natural history of botanical therapeutics. Metabolism 2008; 57: S3–S9. Singh P, Mahmood T, Shameem A, Bagga P, Ahmad N. A review on Herbal Excipients and their pharmaceutical applications. Sch Acad J Pharm 2016; 5(3): 53–57. WHO. Quality Control Methods for Medicinal Plant Materials. World Health Organization, Geneva, 1992. WHO. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine. World Health Organization, Geneva, 2000. WHO. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines. World Health Organization, Geneva, 2001, p. 1. WHO. National Policy on Traditional Medicine and Regulation of Herbal Medicines. Report of WHO Global Survey, Geneva, 2005. WHO. WHO Guidelines for Assessing Quality of Herbal Medicines with Reference to Contaminants and Residues. World Health Organization, WHO Press, Geneva, 2007. 2 Method of Extraction

2.1 INTRODUCTION Phytochemicals are naturally occurring chemical compounds in plant-based foods. They are concerned with the enormous variety of organic substances that are elaborated and accumulated by plants and deals with the chemical structures of those substances, their natural distribution, and biological function (Samuelsson, 2004). In all these operations, methods are needed for separation, purification, and extraction of many different constituents present in plants. Extraction is the process of withdrawing active agents or waste substances from a solid or liquid mixture with a liquid solvent (Sasidharan et al., 2011). The products so obtained from plants are relatively impure liquids, semisolids or powders intended only for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts, and powdered extracts (Azwanida, 2015). The extract thus obtained may be ready for use as a medicinal agent in the form of tinctures and fluid extracts, it may be further processed to be incorporated in any dosage form such as tablets or capsules or it may be fractionated to isolate individual chemical entities such as ajmalicine, hyoscine, and vincristine, which are modem drugs (Porwal et al., 2012). Thus, standardization of extraction procedures contributes significantly to the final quality of the herbal drug. The solvent is not or is only partially miscible with the solid or the liquid. The active agents are transferred from the solid or liquid mixture (raffinate) into the solvent (extract) by intensive contact. After mixing, the two phases are separated either by gravity or centrifugal force. Recovery of the solvent is important to get the active agent in pure form, thus a further separation is necessary called rectification or re-extraction. Depending on the phases, the following types of extraction exists:

• Solid–liquid extraction • Liquid–liquid extraction • Gas–liquid extraction

The method of extraction is applied for hydrometallic processes, in the pharmaceutical industry for producing active agents, in the petroleum industry for production of monomers and aromates, and for the cleaning of waste water to separate solved compounds.

2.2 SOLVENT FOR EXTRACTION Solvents are, under normal conditions, volatile, usually organic liquids capable of dissolving other gaseous, liquid or solid substances without either themselves or the dissolved substances being chemically altered. Water, pure organic liquids,

13 14 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines and mixtures of organic liquids with water or with other organic liquids are used as extraction solvents. These organic liquids are nearly always hydrocarbons and their derivatives such as halogenated hydrocarbons, alcohol, ester, ketones, ethers, oils, and so on. The solvent or the extraction agents used in the preparation of phytopharmaceuticals must be suitable for dissolving the important therapeutic drug constituents and thus for separating them from the substances containing the drugs which are to be extracted (Sarker et al., 2005). In pharmaceutical technology, the extraction agent or solvent is known as the menstruum and the extract solution separated from the residual insoluble drug plant material is called the miscella. Selectivity, ease of handling, economy, protection of the environment, and safety are major factors to be considered in the choice of the suitable solvent or of a mixture of several solvents.

2.3 SELECTION OF THE SOLVENTS The solvents chosen for the extraction should be considered carefully. They should dissolve the secondary metabolites under study, be easy to remove, inert, non-toxic, and not easily flammable. Solvents should be distilled or even double distilled before use if they are of low or unknown quality. Chloroform, methylene chloride, and methanol are usually the solvents of choice in a preliminary extraction of a plant part. Extraction under acid or basic conditions is often conducted for specific separations, for example, anthocyanins are extracted by crushing fresh plant material with methanol containing 1% w/v hydrochloric acid and alkaloids may be extracted in either acid or basic media. It has been reported that acid-base treatment of extracts may produce artifacts because of rearrangements (Sasidharan et al., 2011). Out of all the solvents, water is the most important of all extraction solvents. It is used either alone or mixed with organic solvents, principally a lower alcohol. Water is a solvent of proteins, coloring matter, gums, anthraquinone derivatives, most alkaloidal salts, glycosides, sugars, and tannins. In addition, water will dissolve enzymes, many organic acids, most organic salts, and small properties of volatile oils. Ethanol, known as alcohol in the British Pharmacopoeia, is a solvent of alkaloids, alkaloidal salts, glycosides, volatile oils, and resins, together with many forms of coloring matter. Mixtures of organic solvents such as ether and ethanol or mixtures of organic solvents such as alcohol with water are used to produce certain effects. Azeotropic mixtures represent a special type of mixture.

2.4 REGENERATION OF THE SOLVENT For all extraction processes, the regeneration by further separation processes is necessary. In this way, pure products are produced and the solvent can be recycled in the extraction process. In many cases, the regeneration step is the most cost intensive part of the whole process. Rectification is the most common method. Evaporation is used if the active agent is very highly volatile. The solvent should have a low boiling temperature and a low heat of evaporation. Crystallization causes cooling of the solvent, results in crossing the solubility, and the active agent falls Method of Extraction 15 out and can be separated by mechanical separation processes. Extraction, a further extraction step with another solvent, can be used to separate the active agent from the first solvent and in this way, the produced extract has to be separated once again (Akinyemi et al., 2005).

2.5 SOLUTIONS (SOLUTE AND SOLVENT) A solution may be classified according to the states in which the solute and solvent occur, since three states of matter (gas, liquid, and crystalline solid) exist. When solids or liquids dissolve in a gas to form a gaseous solution, the molecules of the solute can be treated thermo-dynamically like a gas, similarly when gases or solids dissolve in liquids, the gases and the solids can be considered to exist in the liquid state. In the format of solid solutions, the atoms of the gas or liquid take up positions in the crystal lattice and behave like atoms or molecules of solids. The solutes (whether gases, liquids or solids) are divided into two main classes, non-electrolytes and electrolytes. Non-electrolytes are the substances that do not yield ions when dissolved in water, and therefore, do not conduct an electric current through the solution, for example, sucrose, glycerin, naphthalene, and urea. Electrolytes are substances that form some ions in solution, conduct the electric current, and show apparent “anomalous” colligative properties, that is, they produce a considerably greater freezing point depression and boiling point elevation than do non-electrolytes of the same concentration, for example, HCl, sodium sulfate, ephedrine, and phenobarbital. Electrolytes may be sublimed further into strong electrolytes and weak electrolytes which depend on whether the substance is completely or only partly ionized in water. Hydrochloric acid and sodium sulfate are strong electrolytes whereas ephedrine and phenobarbital are weak electrolytes.

2.6 FACTORS AFFECTING CHOICE OF EXTRACTION PROCESS The final choice of the process to be used for the extraction of a drug will depend on a number of factors, including:

2.6.1 character of Drug Different characters of drugs may affect the extraction process, that is, hard and tough (such as nuxvomica), soft and parenchymatous (such as gentian), unpowderable (such as squill), and unorganized drugs (such as benzoin). Thus, knowledge of the pharmacognosy of the drug is essential for selection of the extraction process that will give the best result.

2.6.2 therapeutic Value of the Drug When the drug has considerable therapeutic value, the maximum extraction is required so that percolation is used, as in belladonna. If the drug has little therapeutic value, however, the efficiency of extraction is unimportant and maceration is adequate; for example, flavors (lemon) or bitters (gentian). 16 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

2.6.3 stability of Drug Continuous extraction should be avoided when the constituents of the drug are thermo-labile.

2.6.4 cost of Drug From the economic point of view, it is desirable to obtain complete extraction of an expensive drug, so that percolation should be used; for example, ginger. For cheap drugs, the reduced efficiency of maceration is acceptable in view of the lower cost of the process. In particular, the cost of size reduction to a powdered state is avoided, whereas this is a significant part of the percolation process.

2.6.5 solvent When the desired constituents demand a solvent other than a pure boiling solvent or an azeotrope, continuous extraction should be used.

2.6.6 concentration of Product Dilute products such as tincture can be made by maceration or percolation, depending on the previous factors. For semi-concentrated preparations (e.g., concentrated infusions), unless the drug cannot be powdered or is not worth powdering, double or triple maceration is chosen. Concentrated preparations (e.g., liquid extracts or dry extracts) are made exclusively by percolation with the exception that continuous extraction can be used if the solvent is suitable and the constituents are thermo-stable.

2.6.7 recovery of Solvent from the Marc The residue of the drug after extraction (often known as the marc) is saturated with solvent and, if economic, the latter is recovered.

2.7 PROCEDURES FOR EXTRACTION OF HERBAL DRUGS During the extraction of the herbal drugs, two processes run parallel with each other in the extraction of drugs, that is, rinsing of extractive substances out of disintegrated plant cells, and dissolution of extractive substances out of intact plant cells by diffusion These require:

• Prior steeping and swelling of the drug plant material in order to increase the permeability of the cell walls. • Penetration of the solvent into the plant cells and swelling of the cells. • Dissolution of the extractive substances. • Diffusion of the dissolved extractive substances out of the plant cell. Method of Extraction 17

The various extraction processes employed may be classified as:

• Extraction with organic solvents: percolation, maceration • Extraction using a soxhlet apparatus • Extraction with water: infusion, decoction, and steam distillation

The most popular method of extraction is to use a liquid solvent at atmospheric pressure, possibly with the application of heat. Other methods include steam distillation, supercritical fluid extraction, and the use of liquefied gases under moderate pressure. Table 2.1 shows a brief summary of the experimental conditions for various methods of extraction for plant materials.

2.7.1 Maceration Maceration is the process of extraction of a drug with a solvent with several daily shakings or stirrings at room temperature. Compared with other methods of extraction, the intensity of movement is so low that we use the term stationary conditions. As prescribed by various pharmacopoeias, maceration can be carried out by the following methods. The quantity of extraction fluid prescribed in the monograph is poured onto the comminuted drug materials, the mixtures then being kept for 5 days in tightly sealed vessels at room temperature, protected from sunlight and shaken several times daily. After decanting or straining, the residual liquid is expressed from the solid and the combined extract is kept for 5 days at below 15°C, filtered, and if necessary, adjusted to the required concentration with the prescribed extraction liquid; losses due to evaporation are to be avoided during the preparation (Handa et al., 2008). The line chart of herbal drugs’ extraction through maceration is shown in Figure 2.1. British Pharmacopoeia permits maceration only for the following tinctures and extracts: Senna liquid extract, Squill tincture, compound benzoin tincture, Catechu tincture, and Opium tincture. Mother tinctures of the Homeopathic Pharmacopoeia

TABLE 2.1 A Brief Summary of the Experimental Conditions for Various Methods of Extraction for Plant Materials Characteristics Soxhlet Extraction Sonication Maceration Common solvents used Methanol, ethanol, or Methanol, ethanol, or Methanol, ethanol, or a a mixture of alcohol a mixture of alcohol mixture of alcohol and and water and water water Temperature (°C) Depending on solvent Can be heated Room temperature used Pressure applied Not applicable Not applicable Not applicable Time required 3–18 hr 1 hr 3–4 days Volume of solvent 150–200 50–100 Depending on the required (mL) sample size 18 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Plant material Whole of the (crushed or cut Placed in a closed selected solvent small or moderately vessel (menstruum) added coarse powder)

Allowed to stand Solid residue (mark) for ﬁve days Liquid strained oﬀ pressed (recover as shaking much as occluded occassionally solution)

Strained and Clariﬁed by Evaporation and expressed liquids subsidence or concentration mixed ﬁltration

FIGURE 2.1 Maceration process. are in most cases prepared by maceration and in a few cases by percolation with ethanol of varying concentration. Maceration is used for preparing aqueous extracts.

2.7.1.1 Modification of General Processes of Maceration Repeated maceration may be more efficient than a single maceration, since an appreciable amount of active principle may be left behind in the first pressing of the marc. The repeated maceration is more efficient in cases where active constituents are more valuable. Double maceration is used for concentrated infusions which contain volatile oil, for example, concentrated compound gentian infusion. Where the marc cannot be pressed, a process of triple maceration is sometimes employed. The total volume of solvent used is, however, large and the second and third macerates are usually mixed and evaporated before adding to the first maceration. This precludes the use of the process for preparations containing volatile ingredients (Evans, 1998). The efficiency of extraction in a maceration process can be improved by arranging for the solvent to be continuously circulated through the drug as indicated in Figure 2.2. Solvent is pumped from the bottom of the vessel to the inlet where it is distributed through spray nozzles over the surface of the drug. The movement of the solvent reduces boundary layers, and the uniform distribution minimizes local concentration in a shorter time. In a few cases, it is desirable to change the physicochemical nature of the solvent during a single maceration process. Opium tincture is prepared by using a change of the physicochemical nature of the solvent as indicated below:

• First boiling water is poured over the sliced opium to disintegrate it. • Then, after macerating for six hours, 90% alcohols are added to the cold mixture and maceration is continued for a further 24 hours. • Adding alcohol during the second period of maceration reduces much of the gummy material in the final tincture. Method of Extraction 19

Spray nozzles

Drug

Pump

Product

FIGURE 2.2 Circulatory extractions.

2.7.2 vortical or Turbo Extraction Simple maceration is a very slow extraction process. There has, therefore, been no lack of endeavors to reduce the length of time involved. In addition to kinetic maceration with shaking and stirring, which gives the same yield but attains the concentration equilibrium in a shorter time, in vortical or turbo-extraction, the drug to be extracted is stirred in the menstruum with a high-speed mixer or homogenizer. The shredding and shearing forces break down the drug material to a particle size which is smaller than that of the material when it is first put in the mixer. The cells become highly disintegrated. The diffusion of extractive substances through the cell membranes is largely replaced by washing out from the destroyed cellular tissues which results in substantially faster establishment of the maceration equilibrium and hence in a considerable saving of time. The energy supplied for the high-speed stirring and comminution of the drug material raises the temperature during the extraction, which is undesirable because of the risk of decomposition of thermo-labile constituents. The temperature rise must, therefore, be kept as small as possible. This is achieved either by stopping the process from time to time or by cooling the vessel. The further comminution of the drug favors rapid establishment of the equilibrium, but makes separation of drug residues from the miscella more difficult. The separation can be carried out by filtration, sedimentation or centrifugation (Martinsa et al., 2017).

2.7.3 ultrasound Extraction Ultrasound is defined as frequencies above 20,000 Hz. In this extraction process, sound waves are forced to accelerate the extraction. In pharmaceutical practice, ultrasound 20 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines is usually produced with magnetostrictive or piezoelectric ultrasonic transmitters. In the magnetostrictive transmitter, the change of length undergone by ferromagnetic substances upon magnetization (magnetostriction) is used. In practice, nickel steels are used which are composed of numerous nickel foil discs isolated from each other to prevent eddy currents. The rod begins to vibrate in an alternating magnetic field produced by a high frequency alternating current. The amplitude is at its greatest when the frequency of the alternating current is the same as a natural frequency of the vibrating rod. Frequencies up to 200 KHz can thus be produced. Higher frequencies are obtained with piezoelectric ultrasound transmitters, which make use of the so-called reciprocal piezoelectric effect produced when a quartz crystal undergoes a change of length when an electric current is applied. When such a crystal is placed between the plates of a condenser to which an alternating electric current is being applied, it begins to vibrate at the frequency of the applied current (Wang et al., 2012). The main effects of ultrasound extraction can be summarized as:

• To increase the permeability of the cell walls • To produce cavitation (i.e., the spontaneous formation of bubbles in a liquid below its boiling point resulting from strong dynamic stressing) • To increase mechanical stressing of the cells

Treatment of herbal drugs with the ultrasound plays a major role in its extraction, for example, decomposition of the alkaloids in jaborandi leaves is observed after 30 s ultrasound treatment on the laboratory scale at 20 KHz. but in the case of foxglove leaves, the content of digitalis glycosides fell when an ultrasound output representing the optimum formation of hydrogen peroxide was used during the extraction.

2.7.4 extractions by Electrical Energy In this method, electrical energy is used in the form of an electric field, an electromagnetic field, and as electric discharges to accelerate extraction and improve the yield of the extraction. Extraction of scopolamine from the seeds and capsules of Indian thorn apple has been reported by this process with the aid of a steel plate as a cathode at the bottom of the extraction vessel and several carbon electrodes as anodes at the top. The alkaloid yield was significantly increased by application of a current. The extraction of valerianic acid from valerian root was performed by Rakhman- Zade with this method by surrounding an extraction column with an electric coil producing an alternating electromagnetic field of 50 Hz which was more effective than simple maceration.

2.7.5 Percolation and Re-Percolation Exhaustive extraction is defined as the complete removal of the desired extractive substances from the drug material. The skeletal material of the drug plant remains behind. The objective is a quantitative extraction which can be achieved in various ways. In percolation, the drug plant material is exhaustively extracted by fresh solvent. Only fresh solvent is used and the extraction consumes a large quantity of Method of Extraction 21 it and takes a long time. We speak of re-percolation when the drug is first extracted with fresh solvent and then some of the percolate is used for exhaustive extraction by stage wise concentration in another percolator. Continuous countercurrent extraction is a process in which fresh drug plant material is brought into contact with loaded/ charged solvent at the same time as fresh solvent is being brought into contact with already pre-extracted drug (Sasidharan, 2011). Percolation is as an effective tool for the extraction of herbals as follows:

• Quantity of menstruum • Diffusion constant of the drug into menstruum • Diffusion constant of menstruum into drug

Different in situ parameters are to be considered while proceeding with percolation of herbal drugs as follows:

• Pre-swelling of drug • Intermediate maceration • Percolation rate • Expression of drug residue • Total quantity of the menstruum

2.7.5.1 Percolation Procedure Percolation is usually one of the most widespread methods employed for plant extraction since it does not require much manipulation or time. The equipment used is a conical glass container with a tap at the base of the apparatus used to set the rate of the solvent elution. Hot or cold solvent may be used. Very fine powders, resins, and powders that swey or give a viscous eluent cannot be extracted by this method since percolation would be disrupted. The sample should be coarsely fragmented, and particles that pass through a 3 mm sieve would be adequate. Particles of too large a size may produce a high elution rate, precluding the necessary equilibrium for the dissolution of the metabolites, and the menstruum (solvent) would percolate unsaturated. Percolation is more efficient than maceration since it is a continuous process in which the saturated solvent is constantly being displaced by fresh menstruum. Normally, percolation is not used as a continuous method because the sample is kept in solvent in the percolator for 24 h and then the extracted materials are collected and pooled. The construction of percolation is shown in Figure 2.3.

2.7.5.2 Modification of the General Process of Percolation In general, in the process of percolation, particularly in the manufacture of concentrated preparations like liquid extracts, the following problems may arise:

a. If the active substances are thermo-labile, evaporation of a large volume of dilute percolate may result in partial loss of the active constituents. b. In the case of an alcohol-water mixture, evaporation results in preferential vaporization of alcohol leaving behind an almost aqueous concentrate which may not be able to retain the extracted matter in solution and hence gets precipitated. 22 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Port for charging drug and running in menstruum

Drug

Sacking or Straw

Percolator metal plate

Connections for removing percolate or for sparging steam

FIGURE 2.3 Commercial scale percolator. In such cases, a modification in the general process of percolation is required as given below.

2.7.5.3 Reserved Percolation In this case, the extraction is done through the general percolation procedure. At the last, the evaporation is done under reduced pressure in equipment like a climbing evaporator to the consistency of a soft extract (semi-solid) such that all the water is removed. This is then dissolved in the reserved portion which is strongly alcoholic and easily dissolves the evaporated portion without any risk of precipitation.

2.7.6 cover and Run Down Method This is the process which combines the maceration and percolation techniques. This process cannot be used for materials which contain volatile principles or those that undergo change during the evaporation stage. This procedure is advantageous because industrial methylated spirit may be used for extraction instead of the costly rectified spirit. After the imbibition stage, the material is packed in a percolator. After being macerated for a few hours with a suitable diluted industrial methylated spirit, liquid is run off and the bed is covered with more of the menstruum. It is macerated as before and the second volume of the extract is collected. This process is repeated several times with the later weaker extracts used for extraction of a fresh batch of the drug. More concentrated fractions are evaporated under reduced pressure to free from the toxic methanol. The concentrate is diluted with water and ethanol to produce the correct concentration of alcohol and the active principle. Method of Extraction 23

2.7.7 small Scale or Laboratory Scale Extraction The processes for the manufacture of concentrated preparations by maceration and percolation are involved in extraction followed by the evaporation of solvents. The two operations are combined in a continuous extraction process.

a. Soxhlet apparatus b. Continuous apparatus

2.7.7.1 Hot Continuous Extractions: Soxhletion The use of a commercially available Soxhlet extractor is a convenient way to prepare crude plant extracts. This procedure is used mainly with pure solvents. The main advantage of extraction using a Soxhlet apparatus is that it is an automatic, continuous method that does not require further manipulation other than concentration of the extractive and it saves the solvent by recycling it over the sample.

2.7.7.1.1 Met ho dolog y In this method, the material to be extracted is placed in a “thimble” made of cellulose or cloth in a central compartment with a siphoning device and side-arm, both of which are connected to a lower compartment. The solvent is placed in a lower compartment and a reflux condenser is attached above the central sample compartment. Note that each component of the set up (solvent container, sample compartment, and reflux condenser) is a separate item of glass ware which is assembled together with the appropriate contents to make the complete apparatus. The solvent in the lower container (usually a round-bottomed flask) is heated to boiling, and the vapor passes through the side-arm up into the reflux condenser. Here, the vapor liquefies and drips into the thimble containing the material to be extracted. The warm solvent percolates through the material and the wall of the thimble and the extract gradually collects in the central compartment. Once the height of the extract reaches the top of the siphon, the entire liquid in the central compartment flows through this and back into the lower solvent container (Figure 2.4). The process is then repeated (Subramanian et al., 2016).

2.7.7.2 Continuous Apparatus (Official Extractor) Such a type of extraction is described in the official monographs (British Pharmacopoeia [BP], Indian Pharmacopoeia [IP], etc.). In such cases, the extraction is a continuous percolation extraction procedure. In this apparatus, vapor rises through the extraction chamber passing the drug container; the vapor condenses in the reflux condenser and returns through the drug, taking the soluble constituents to the flask (Figure 2.5) (Anonymous, 1980). The limitations of this process are:

• It is not useful when the raw materials contain thermo-labile active constituents, because the extraction is carried out at an elevated temperature, and the extract in the flask is also maintained in the hot condition until the process is complete. • It can be used only with pure solvents or with solvent mixtures forming azeotropes. • If an ordinary binary mixture is used as the menstruum, the composition of the vapor will be different from the liquid composition. 24 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Water in Condenser tube Water out

Bypass sidearm Extraction tube Reﬂux sidearm Cellulose thimble Glass wool Sample and sodium sulfate

Flask Organic solvent

Heat source

FIGURE 2.4 Soxhlet apparatus.

To condenser

imble

Drug

Solvent

Burner

FIGURE 2.5 Continuous apparatus. Method of Extraction 25

2.7.8 large Scale Extractor (Counter Current Extractions) In continuous counter current extraction (CCE), a solution, emulsion, suspension or solid mass is extracted by a liquid phase flowing against it. To proceed with this, the starting material for the drug is put in the extraction apparatus where it first comes into contact with the extraction solvent already containing the extract. Then, the further the starting material is moved into the extraction apparatus, the less concentrated is the extract in the solvent with which it is coming into contact until at the end of the apparatus, it eventually meets fresh solvent. In this way, a complete extraction is possible with the correct choice of quantity and velocity of flow. The theoretical relationships were first established for liquid-liquid countercurrent extraction and subsequently applied to solid-solid counter current extraction (Hewitson et al., 2009). Figure 2.6 given below shows a type of percolator used at the industrial scale. This extraction process has significant advantages:

• A unit quantity of the plant material can be extracted with a much smaller volume of solvent as compared to other methods like maceration, decoction, and percolation. • CCE is commonly done at room temperature, which spares the thermo- labile constituents from exposure to heat which is employed in most of the other techniques. • As the pulverization of the drug is done under wet conditions, the heat generated during comminution is neutralized by water. This again spares the thermo-labile constituents from exposure to heat. • The extraction procedure has been rated to be more efficient and effective than continuous hot extraction.

Solvent distribution nozzle Condenser

Drug charge

Drug discharge

Solution return pipe Heating coil

Product

FIGURE 2.6 Large-scale extractor. 26 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

2.7.9 infusion and Decoction Infusion and decoction are simple methods of extraction with water. Infusion is the method in which hot or cold water is added to the milled drug and decoction allows the sample to be boiled for about 15 min in water. It is applicable for soft drugs containing water-soluble constituents only. However, decoction is applicable for water soluble and heat stable drugs obtained from hard and woody sources. Extraction with water as the sole solvent is seldom used for plant material although some plant constituents are water-soluble, such as carbohydrates, flavonoid polyglycosides, quaternary alkaloids, saponins, and tannins. For example, taxifolin or dihydroquercetin is believed to undergo certain enzymatic reductions forming water-soluble oligomeric flavonoids that give adhesive properties to Douglas fir (Pseudotsuga menziesii) bark extracts. Water soluble compounds are usually extracted using mixtures of methanol-water or ethanol-water by one of the methods mentioned previously for organic solvents. lnfusions are prepared by leaving the plant material to soak in the solvent (generally at room temperature) for a period of time with or without intermittent shaking, followed by filtration to separate away the plant debris. If the plant material has settled, then the upper solvent extract can be decanted off and replaced if necessary with fresh solvent. It is possible to use pre-heated solvents (as in the preparation of a tea), but these will cool down during the extraction process.

Drug +→HO2 Filtrate

2.7.9.1 General Method for Preparing Fresh Infusion The drug is usually coarsely powdered, very fine powder being avoided (50 gm). Moisten the drug in a suitable vessel provided with a cover with 50 mL of cold water. Allow to stand for 15 minutes. Then add 900 mL of boiling water and cover the vessel tightly. Allow it to stand for 30 minutes. Then strain the mixture, pass enough water to make the infusion measure 1000 mL. Some drugs are supplied accurately weighed in muslin bags for preparing specific amounts of infusion. If the activity of the infusion is affected by the temperature of boiling water, cold water should be used. As the fresh infusions do not keep well, they should be made extemporaneously and in small quantities. The official monographs also recognize certain “concentrated infusions” in which 25% alcohol is added during or subsequent to the infusion process. Concentrated infusions are especially prepared in which the active and desirable principles of the drug are equally soluble in water or in the menstruum used for both concentrate and infusions.

2.7.10 aqueous Alcoholic Extraction by Fermentation Ayurvedic preparations like “asava and arista” adopt the technique of fermentation for extracting the active principles. The extraction procedure involves soaking the crude drug, in the form of either a powder or a decoction (kasaya), for a specified period of time, during which it undergoes fermentation and generates alcohol in situ; this facilitates the extraction of the active constituents contained in the plant Method of Extraction 27 material. The alcohol thus generated also serves as a preservative. If the fermentation is to be carried out in an earthen vessel water, it should first be boiled in the vessel. In large-scale manufacture, wooden vats, porcelain jars or metal vessels are used in place of earthen vessels. Some examples of such preparations are karpurasava, kanakasava, and dasmularista. In Ayurveda, this method is not yet standardized, but with the extraordinarily high degree of advancement in fermentation technology, it should not be difficult to standardize this technique of extraction for the production of herbal drug extracts.

2.7.11 steam Distillation Steam distillation is a popular method for the extraction of volatile oils (essential oils) from plant material. This can be carried out in a number of ways. One of the methods is to mix the plant material with water and heat up to boiling (distillation with water). The vapors are collected and allowed to condense, then oil gets separated from the water. However, prolonged boiling of this is to be avoided by separating the steam either through plant material which is suspended in water but not boiled (hydro-steam distillation) or directly through the plant material which is laid out in a mesh arrangement between the steam inlet and the condenser (direct steam distillation). Once the oil and steam have condensed, the two layers may be separated by physical means or the oil may be the upper or lower layer, depending on its density relative to water. Better yields will be obtained by solvent extraction of the aqueous layer. Oils with a density equal to or greater than water or an organic solvent, such as, xylene, which is less dense than water, are passed in the collection vessel. The volatile oil dissolves in this upper layer as it condenses. Precautions must be taken to ensure efficient condensation of the steam and vaporized oil and collection of the condensate in such a way as to prevent loss of the volatile material and increase the yield of oil. On the other hand, to avoid risk of explosion, a completely closed system must not be used.

2.7.12 supercritical Fluid Extractions Supercritical fluid extraction (SFE) is the technique of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent. SFE can be used as a sample preparation step for analytical purposes or on a larger scale to either strip unwanted material from a product (e.g., decaffeination) or collect a desired product (e.g., essential oils). SCF technology is making in-roads in several pharmaceutical industrial operations including crystallization, medium for particle design and engineering, particle size reduction, preparation of drug delivery systems, coating, and product sterilization. It has also been shown to be a viable option in the formulation of particulate drug delivery systems, such as micro particles and nanoparticles, liposomes, and inclusion complexes, which control drug delivery and/or enhance the drug stability. Carbon dioxide (CO2) is the most used supercritical fluid, occasionally modified by co-solvents such as ethanol or methanol. Working conditions for supercritical CO2 are above the critical temperature of 31°C and 28 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines critical pressure of 74 bars. Addition of modifiers may slightly modify the operational condition (Patil and Shettigar, 2010).

There are many advantages to the use of CO2 as the extracting fluid. In addition to its favorable physical properties, carbon dioxide is inexpensive, safe, and abundant. But while carbon dioxide is the preferred fluid for SFE, it possesses several polarity limitations. Solvent polarity is important when extracting polar solutes and when strong analyte-matrix interactions are present. Organic solvents are frequently added to the carbon dioxide extracting fluid to alleviate the polarity limitations. Of late, instead of carbon dioxide, argon is being used because it is inexpensive and more inert. The component recovery rates generally increase with increasing pressure or temperature: the highest recovery rates in the case of argon are obtained at 500 atm and 150°C. The largest area of growth in the development of SFE has been the rapid expansion of its applications. SFE finds extensive applications in the extraction of pesticides, environmental samples, foods and fragrances, essential oils, polymers, and natural products. The major deterrent in the commercial application of the extraction process is its prohibitive capital investment.

2.7.13 Phytonics Process A new solvent based on hydrofluorocarbon-134a and a new technology to optimize its remarkable properties in the extraction of plant materials offer significant environmental advantages and health and safety benefits over traditional processes for the production of high quality natural fragrant oils, flavors, and biological extracts. This technique was developed by Advanced Phytonics Limited (Manchester, UK) and this patented technology is termed a “phytonics process.” The products mostly extracted by this process are fragrant components of essential oils and biological or phytopharmacological extracts which can be used directly without further physical or chemical treatment.

2.7.14 High Pressure Extraction (HPE) For high pressure extraction, a densified gas is used as solvent. The pressure of the gas has to always be higher than the critical pressure. The density of a supercritical fluid is comparable to the density of liquids, but the viscosity of the fluid is like a gas and the diffusion coefficient is one to two orders higher than that of a liquid. Therefore, supercritical fluids are able to penetrate into a solid, solve substances, and transport them from the inner part to the surface. The advantage of supercritical fluid is that heavy volatile substances show a high solubility in supercritical fluids and the solubility can be influenced by variations of pressure and temperature, which have different influences on the solubility. The gas which is most used for high pressure extraction processes is carbon dioxide (CO2) because it is inflammable, non-toxic, and available in large amounts at a low price. The advantage of this method is that it is an isobaric process and the disadvantage is that no total separation can be achieved, therefore, preloaded fluid enters the extractor (Kaufmann and Christen, 2002). Method of Extraction 29

2.8 CONCLUSIONS The spectrum of constituents obtained by steady state extractions (simple macerations) differs from the spectrum obtained by exhaustive extractions (percolation). By the use of motive extraction methods, the aid of stirring and shearing forces, changes of temperature, and the quality of extraction solvent may lead to extracts with a spectrum of constituents similar (equivalent) to one obtained by percolation. Different manufacturing procedures have to be assessed as equivalent if the critical quality parameters of the specification are conformed to and if compliance with standards is proven by the results of a number of production batches.

REFERENCES Akinyemi KO, Oladapo O, Okwara CE, Ibe CC, Fasure KA. Screening of crude extracts of six medicinal plants used in South-West Nigerian unorthodox medicine for anti-methicilin resistant Staphylococcus aureus activity. BMC Complement Altern Med. 2005; 5: 6. Anonymous. British Pharmacopoeia, Vol. II. University Press, Cambridge, London, 1980, p. 576. Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Azwanida Med Aromat Plants. 2015; 4: 3. Evans WC. Trease and Evan’s Pharmacognosy, 14th Edition. W. B. Saunders Company Limited, London, 1998, p. 119. Handa SS, Khanuja SP, Longo G, Rakesh DD. Extraction Technologies for Medicinal and Aromatic Plants. International Centre for Science and High Technology, Trieste, Italy, 2008, pp. 67–81. Hewitson P, Ignatova S, Ye H, Chen L, Sutherland IA. Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal medicine. J Chromatogr A. 2009; 1216(19): 4187–4192. Kaufmann B, Christen P. Recent extraction techniques for natural products: Microwave- assisted extraction and pressurized solvent extraction. Phytochem Anal. 2002; 13: 105–113. Martinsa PM, Lanchotec AD, Thoratb BN, Freitasc AP. Turbo-extraction of glycosides from Stevia rebaudiana using a fractional factorial design. Revista Brasileira de Farmacognosia. 2017; 27(4): 1–9. Patil, PS, Shettigar R. An advancement of analytical techniques in herbal research. J Adv Sci Res. 2010; 1(1): 08–14. Porwal V, Singh P, Gurjar D. A comprehensive study on different methods of extraction from guajava leaves for curing various health problem. IJERA. 2012; 2(6): 490–496. Samuelsson G. A Textbook of Pharmacognosy, Swedish Pharmaceutical Society, 4th Edition. Swedish Pharmaceutical Press, Stockholm, Sweden, 2004. Sarker SD, Latif Z, Gray AI. Natural Products Isolation Book, 2nd Edition. Springer Publications, Totowa, New Jersey, 2005. Sasidharan S, Chen Y, Saravanan D, Sundram KM, Latha LY. Extraction, isolation and characterization of bioactive compounds from plants’ extracts. Afr J Tradit Complement Altern Med. 2011; 8(1): 1–10. Subramanian R, Subbramaniyan P, Ameen JN, Raj V. Double bypasses soxhlet apparatus for extraction of piperine from Piper nigrum. Arab J Chem. 2016; 9: S537–S540. Wang J, Zhao YM, Guo CY, Zhang SM, Liu CL, Zhang DS, Bai XM. Ultrasound-assisted extraction of total flavonoids from Inula helenium. Pharmacogn Mag. 2012 Apr–Jun; 8(30): 166–170.

Separation and Isolation 3 of Plant Constituents

3.1 INTRODUCTION Natural products are secondary metabolites which are derived from herb or animal sources. These are chemical compounds found in nature and they have pharmacological and biological activity. Natural products are generally used in drug discovery and drug design. Separation of a single molecular entity is very difficult from complex mixtures containing fats, oils, alkaloids, tannins, and glycosides. Medicinal plants have been the mainstay of traditional herbal medicine among rural dwellers worldwide since antiquity to date. Hippocrates (460–377 bc), one of the ancient authors who described medicinal natural products of plant and animal origins, listed approximately 400 different plant species for medicinal purposes. Over the years, they have assumed a very central stage in modern civilization as a natural source of chemotherapy as well as among scientists in search for alternative sources of drugs (Chopra, 1985). According to World Health Organization (WHO), a medicinal plant is any plant of which is one or more of its organs contain substances that can be used for therapeutic purposes or which are precursors for chemo-pharmaceutical semi synthesis. Such a plant will have its parts, including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of a disease condition, and will, therefore, contain chemical components that are medically active. These non-nutrient plant chemical compounds or biological active components are often referred to as phytochemicals or phyto-constituents and are responsible for protecting the plant against microbial infections or infestations by pests. Phytochemicals have been isolated and characterized from fruits, vegetables, spices, beverages, and so on. The plants are applied in different forms such as poultices, concoctions of different plant mixtures, infusions as teas or tinctures or as component mixtures in porridges and soups administered in different ways including oral, nasal, rectal, topical, and so on (Yalavarthi et al., 2013).

3.2 CLASSES OF PHYTO-CONSTITUENTS Physiologically active plant constituents are usually classified by their chemical structure rather than specific actions. These have been divided into different groups: Alkaloids, Glycosides, Flavonoids, Phenols, Saponins, Steroids, and Tannins.

3.2.1 glycosides Glycosides are colorless, crystalline carbon, hydrogen, and oxygen-containing, water- soluble phyto-constituents found in the cell sap. Chemically, glycosides contain a

31 32 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

OH O HO OH HO O H O OH O HO O HO O O OH O O HO O O O Gentiopicrin OH Amarogentin OH HO O HO HO O H C O 3 OH O OCH3 O HO O O HO OH HO H HO Hesperidin OH O Andrographolide

FIGURE 3.1 Phyto-constituents of glycosides. carbohydrate (glucose) and a non-carbohydrate part (aglycone or part). Glycosides are neutral in reaction and can be readily hydrolyzed into their components with ferments or mineral acids. Glycosides are classified on the basis of the type of sugar component and chemical nature of the aglycone or pharmacological action. Glycosides are purely bitter principles that are commonly found in plants of the Genitiaceae family, and though they are chemically unrelated, they possess the common property of an intensely bitter taste. The bitters act on gustatory nerves, which results in increased flow of saliva and gastric juices. Chemically, the bitter principles contain the lactone group that may be diterpene lactones (e.g., andrographolide) or triterpenoids (e.g., amarogentin). Some of the bitter principles are either used as astringents due to the presence of tannic acid as antiprotozoan or to reduce throxine and metabolism (Figure 3.1). Examples include cardiac glycosides (purgative and for treatment of skin diseases), chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, and ailanthone. It has been reported that extracts of plants that contain cyanogenic glycosides are used as flavoring agents in many pharmaceutical preparations (Kokate et al., 2010). Excessive ingestion of cyanogenic glycosides can be fatal. Some foodstuffs containing cyanogenic glycosides can cause poisoning (severe gastric irritations and damage) if not properly handled.

3.2.2 alkaloids These are the largest group of secondary chemical constituents made largely of ammonia compounds comprised basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, with most containing oxygen. The compounds have basic properties Separation and Isolation of Plant Constituents 33

OH N N OH CH O N 3 H H N HO H OCH H O O 3 H O H O CO OC OCH3 O OH O OCH3 OCH3 OCH3 Reserpine Hirsutine Chromanone

OMe H H O O HN N N H Me N O N O H O O Me Oximatrine Canthin-6-one OMe HO Me CH Taspine 3 O OH N Me OH HO CH3 H Me OH OO O OH H O O HO O OH O N HO O Senecionine O OH HO OH O HO OH OH O Naringin Hydrocotyline

FIGURE 3.2 Chemical constituents of alkaloids. and are alkaline in reaction, turning red litmus paper blue. The degree of basicity varies considerably, depending on the structure of the molecule and the presence and location of the functional groups. They react with acids to form crystalline salts without the production of water. Most alkaloids are readily soluble in alcohol though they are sparingly soluble in water. The solutions of alkaloids are intensely bitter. These nitrogenous compounds function in the defense of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to their potent biological activities. In nature, the alkaloids exist in large proportions in the seeds and roots of plants and often in combination with vegetable acids. The names of alkaloids end with the suffix “ine” and plant-derived alkaloids in clinical use include the analgesics morphine and codeine, the muscle relaxant (+) tubocururine, the antibiotics sanguinarine and berberine, the anticancer agent vinblastine, the antiarrhythmic ajmaline, the pupil dilator atropine, and the sedative scopolamine. Other important alkaloids of plant origin include the addictive stimulants caffeine, nicotine, codeine, atropine, morphine, ergotamine, cocaine, nicotine, and ephedrine. Amino acids act as precursors for biosynthesis of alkaloids with ornithine and lysine commonly used as starting materials (Kokate et al., 2010). Figure 3.2 represents the chemical structure of alkaloidal phyto-constituents.

3.2.3 Flavonoids Flavonoids are an important group of polyphenols widely distributed among the plant flora. Structurally, they are made of more than one benzene ring in structure 34 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

OH OH OH OH OH OH HO O HO O HO O OH OH OOH OH OOH OH O Quercetin Luteolin Myricetin

OH OH OH OH HO O HO O OH HO O OH OH O OH O OH Taxifolin Naringenin OH OH Leucocyanidin OH O HO O OH O HO O HO OH OH O O HO H C OH 3 OH Wogonin Mangiferin

FIGURE 3.3 Phyto-constituents of flavonoids. and numerous reports support their use as antioxidants or free radical scavengers. The compounds are derived from parent compounds known as flavans. Over four thousand flavonoids are known to exist and some of them are pigments in higher plants. Quercetin, kaempferol, and quercitrin are common flavonoids present in nearly 70% of plants. Other groups of flavonoids include flavones, dihydroflavons, flavanflavonols, anthocyanidins, proanthocyanidins, calchones and catechin, and leuco anthocyanidins (Galan-Vidal, 2009). Figure 3.3 represents the chemical structure of flavanoidal phyto-constituents.

3.2.4 terpenes Terpenes are among the most widespread and chemically diverse groups of natural products. They are flammable, unsaturated hydrocarbons, existing in liquid form commonly found in essential oils, resins or oleo-resins. Terpenoids include hydrocarbons of plant origin of the general formula (C5H8)n and are classified as mono, di, tri, and sesquiterpenoids depending on the number of carbon atoms. Examples of commonly important monoterpenes include terpinen-4-ol, thujone, camphor, eugenol, and menthol. Diterpenes (C20) are classically considered to be resins and taxol, the anticancer agent, is the common example. The triterpenes (C30) include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic activity. Sesquiterpenes (C15) are major components of many essential oils. They act as irritants when applied externally and when consumed internally their action resembles that of a gastrointestinal tract irritant. Terpenoids Separation and Isolation of Plant Constituents 35

O O O O OH H H H O H CO H 2 H O HO H O O HO O Betulic acid Oleanolic acid Nibmin O CH 3 OH CH3

H CH3 H C C 3 CH3 H H3CCH2 β-himachalene Ferruginol Limonene

CH3 OH O O 1,8-cineole

Carvacrol Nootkatone H3C CH3

FIGURE 3.4 Phyto-constituents of terpenoids. are classified according to the number of isoprene units involved in the formation of these compounds. Figure 3.4 represents the chemical structure of terpenoidal phyto-constituents.

3.2.5 Phenolics Phenolics, phenols or polyphenolics are chemical components that occur ubiquitously as natural color pigments responsible for the color of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, found in apples, green tea, and red wine, for their enormous ability to combat cancer, are also thought to prevent heart ailments to an appreciable degree, and sometimes are anti-inflammatory agents (Heinrich et al., 2004).

3.2.6 saponins The term spaonin is derived from Saponaria vaccaria (Quillaja saponaria), a plant which abounds in saponins and was once used as soap. Saponins, therefore, possess “soaplike” behavior in water, that is, they produce foam. On hydrolysis, an aglycone is produced which is called sapogenin. Sapogenins are of two types: steroidal and triterpenoidal. Quillaja saponaria is known to contain toxic glycosides quillajic acid and the sapogenin senegin. Saponins are regarded as high molecular weight compounds in which a sugar molecule is combined with triterpene of steroid aglycone. There are two major groups of saponins: steroid and triterpene saponins (Oakenfull, 1981). Saponins are 36 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines soluble in water and insoluble in ether. They give aglycones on hydrolysis like glycosides. Saponins are extremely poisonous as they cause hemolysis of the blood and are known to cause cattle poisoning. They possess a bitter and acrid taste besides causing irritation to mucous membranes. They are mostly amorphous in nature, soluble in alcohol and water, but insoluble in non-polar organic solvents like benzene and n-hexane. Saponins are also important therapeutically as they are shown to have hypolipidemic and anticancer activity. Saponins are also necessary for the activity of cardiac glycosides. The two major types of steroidal sapogenins are diosgenin and hecogenin (Podolak et al., 2010). Steroidal saponins are used in the commercial production of sex hormones for clinical use. For example, progesterone is derived from diosgenin.

3.2.7 tannins Tannins are widely distributed in plant flora. They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem, and outer layers of plant tissue. Tannins have a characteristic ability to tan, that is, to convert things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or a carboxylic group. They form complexes with proteins, carbohydrates, gelatins, and alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins (Chung et al., 1998). Hydrolysable tannins, upon hydrolysis, produce gallic acid and ellagic acid and depending on the type of acid produced, the hydrolysable tannins are called gallotannins or egallitannins. On heating, they form pyrogallic acid. Tannins are used as antiseptics and this activity is due to presence of the phenolic group (Frutos et al., 2004). Common examples of hydrolysable tannins include theaflavins, daidezein, genistein, and glycitein. Tannin-rich medicinal plants are used as healing agents in a number of diseases.

3.2.8 steroids Steroidal glycosides are also referred to as “cardiac glycosides” and are one of the most naturally occurring plant phyto-constituents that have found therapeutic applications as arrow poisons or cardiac drugs. The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action, mainly on the cardiac muscle when administered through injection into man or animal (Patel and Savjani, 2015). Anabolic steroids have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness. Caution should be taken when using steroidal glycosides as small amounts would exhibit the much needed stimulation on a diseased heart, whereas an excessive dose may cause even death.

REFERENCES Galan-Vidal CA. Chemical studies of anthocyanins: A review. Food Chem. 2009; 113: 859–871. Kokate CK, Purohit AP, Gokhale SB. Analytical Pharmacognosy, 45th Edition. Nirali Prakashan, Pune, 2010, pp. 6–22. Separation and Isolation of Plant Constituents 37

Chopra NR, Chopra IC, Handa KL, Kapoor LD. Anonymous, Pharmacogonosy of Indigenous Drugs, Vol. 1, Published by central council for research in Ayurveda and Siddha, New Delhi, 1985. Yalavarthi C, Thiruvengadarajan VS. A review on identification strategy of phyto constituents present in herbal plants. Int J Res Pharm Sci. 2013; 4(2): 123–140. Heinrich M, Barnes J, Gibbons S, Williamson EM. Fundamentals of Pharmacognosy and Phytotherapy, Second Edition, Churchill Livingstone, United Kingdom, 2004, pp. 245–252. Podolak I, Galanty A, Sobolewska D. Saponins as cytotoxic agents: A review. Phytochem Rev. 2010; 9(3): 425–474. Oakenfull D. Saponins in food—A review. Food Chem. 1981; 7(1): 19–40. Chung KT, Wong TY, Wei CI, Huang YW, Lin Y. Tannins and human health: A review. Crit Rev Food Sci Nutr. 1998; 38(6): 421–464. Frutos P, Hervás G, Giráldez FJ, Mantecón AR. Review: Tannins and ruminant nutrition. Span J Agric Res. 2004; 2(2): 191–202. Patel SS, Savjani JK. Systematic review of plant steroids as potential anti-inflammatory agents: Current status and future perspectives. J Phytopharmacology. 2015; 4(2): 121–125.

Methods of Phyto- 4 Constituent Detection

4.1 INTRODUCTION Phytochemicals are chemical compounds responsible for organoleptic properties that also have biological significance. Plant chemistry includes the miracle of photosynthesis, plant respiration, structure, growth, development, and reproduction. Much of the chemical basis of life is common to both plants and animals. From a holistic perspective, the whole of the plant must be respected as an integrated biologically evolved unit that is beyond the analytical comprehension of science. The active constituents in plants are the chemicals that have a medicinal effect on the body. These are the active ingredients of the plant, the chemicals that have a marked, definable physiological, and therefore, possibly medical activity upon the body (Al-Daihan et al., 2013). Phytochemistry is mainly concerned with enormous varieties of secondary plant metabolites which are biosynthesized by plants. Most of the best plant medicines are the sum of their constituents. The beneficial physiological and therapeutic effects of plant materials typically result from the combinations of these secondary products present in the plant. The information on the constituents of the plant clarifies the uses of the plants, but only a small percentage have been investigated for their phytochemicals and only a fraction has undergone biological or pharmacological screening. As more phyto-constituents are being identified and tested, traditional uses of the plants are being verified (Tapia et al., 2004).

4.2 PHYTOCHEMICAL ANALYSIS In phytochemical evaluation, the powdered leaves are subjected to phytochemical screening for the detection of various plant constituents, characterized for their possible bioactive compounds which have been separated, and subjected to detailed structural analysis.

4.2.1 Phytochemical Analysis Tests for Alkaloids Alkaloids are a chemically heterogenous group of natural substances and pharmacologically active compounds. They compose more than 6000 basic nitrogen- containing organic compounds which occur in about 15% of all vascular terrestrial plants and in more than 150 different plant families (Kokate et al., 2002).

a. Dragendorff’s test: A few drops of Dragendorff’s reagent is added in one tube and the occurrence of an orange-red precipitate is taken to signify the presence of alkaloids (Davies, 2004).

39 40 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

b. Mayer’s test: Mayer’s reagent is added to a test tube and the appearance of a buff-colored precipitate is taken as a positive test for the presence of alkaloids. c. Wagner’s test: Alkaloids give a reddish-brown precipitate with Wagner’s reagent (solution of iodine in potassium iodide). d. Hager’s test: Alkaloids produce a yellow colored precipitate with Hager’s reagent (saturated solution of picric acid). e. Tannic acid test: Alkaloids give a buff-colored precipitate with 10% tannic acid solution.

4.2.2 Phytochemical Screening of Anthocyanin Glycosides are molecules in which a sugar is bound to a non-carbohydrate moiety, usually a small organic molecule (Wu et al., 2006).

Test: The presence of anthocyanins should be confirmed by adding 2 mL of the test drug to 2 mL of 2 N HCl. The appearance of a pink-red color that turns purplish-blue after the addition of ammonia indicates the presence of anthocyanins.

4.2.3 Phytochemical Analysis Tests for Anthraquinone a. Borntragor’s Test: To 1 g of test drug, add 5–10 mL of dilute HCl or

dilute H2SO4; boil on a water bath for 10 minutes and filter (Ivana et al., 2008). Filtrate the extract with CCl4/benzene, add an equal amount of ammonia solution to the filtrate, and shake. The formation of a pink or red color in the ammonical layer shows the presence of the anthraquinone moiety. b. Modified Borntragor’s Test: To 1 g of test drug, add 5 mL dilute HCl followed by 5 mL ferric chloride (5% w/v). Boil for 10 minutes on a water bath, cool, and filter, then filtrate the extract with carbon tetrachloride or benzene and add an equal volume of ammonia solution. Formation of a pink to red color signifies the presence of the anthraquinone moiety.

4.2.4 Phytochemical Analysis Test for Cardiac Glycosides a. Keller–Killani test: To 2 mL of test drug, add 1 mL of glacial acetic acid and one drop 5% ferric chloride, then add concentrated sulfuric acid (Prassas et al., 2008). The appearance of a reddish-brown color at the junction of the two liquid layers indicates the presence of cardiac glycosides.

4.2.5 Phytochemical Analysis Tests for Coumarins

a. FeCl3 test: A few drops of alcoholic FeCl3 solution are added to the test drug. The formation of a deep green color, which turns yellow on addition

of concentrated HNO3, indicates the presence of coumarins. Methods of Phyto-Constituent Detection 41

b. Fluorescence test: Mix the test drug with 1N NaOH solution. The development of a blue-green fluorescence indicates the presence of coumarins.

4.2.6 Phytochemical Analysis Tests for Cynogenetic Glycosides a. Ferriferrocyanide test: Macerate 1 g of the test drug with 5 mL of alcoholic KOH for 5 min (Ilza et al., 2000). Transfer it to an aqueous solution

containing FeSO4 and FeCl3, and maintain at 60–70°C for 10 minutes. Now transfer the contents to HC1 (20%) where the appearance of a distinct Prussian blue color will confirm the presence of cynogenetic glycosides.

b. Precipitation of Hg from HgNO3: The reduction of aqueous mercurous nitrite solution to metallic Hg by HCN is observed by an instant formation of black metallic Hg in the cells. c. Cuprocyanate test: Saturate pieces of filter paper in a freshly prepared solution of guaiac resin dissolved in absolute ethanol and allow them to dry completely in air. Now, carefully moisten a piece of the above paper with a

very dilute solution of CuSO4 and place it into contact with a freshly exposed surface of the drug. When HCN is generated, it gives rise to a distinct stain on the paper.

4.2.7 Phytochemical Analysis Tests for Phenolics and Flavonoids Flavonoids are a large group of naturally occurring phenolic compounds found in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine. a. Shinoda’s test for flavonoids: Dissolve 500 mg of test drug in 5 mL of ethanol, warm slightly, and then filter. Add a few pieces of magnesium chips to the filtrate followed by the addition of a few drops of concentrated HCl. A pink, orange or red to purple coloration is taken as a confirmation for the presence of flavonoids. b. Ferric chloride test: Mix an alcoholic solution of the test drug with a few drops of neutral ferric chloride solution to produce a green color. c. Lead acetate tests: Mix an alcoholic solution of the test drug with a few drops of 10% lead acetate to produce a yellow precipitate.

4.2.8 Phytochemical Analysis Test for Saponins Saponins are a heterogeneous group of natural products found in many plant- derived foods and medicinal plants. There are two types of saponins: triterpenoids and steroidal saponins. Many plants containing steroidal saponins have a marked hormonal activity while in triterpenoids, saponins are often strong expectorants and aid in the absorption of nutrients.

Test: Boil 1 g of test drug in 10 mL of distilled water and then filter. Add 3 mL of distilled water to the filtrate and shake vigorously for about 5 min. The formation of a foam after shaking is taken as a confirmation for the presence of saponins. 42 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

4.2.9 Phytochemical Analysis Tests for Triterpenes a. Salkowski test: When shaken with concentrated sulfuric acid, the lower layer of a chloroform solution of the test drug will turn yellow on standing. b. Lieberman–Burchard test: A chloroform solution of the test drug with a few drops of acetic acid and one mL of concentrated sulfuric acid produces a deep red at the junction of the 2 layers. c. Tschugajen test: A chloroform solution of the test drug with an excess of acetyl chloride and a pinch of zinc chloride, when warmed in a water bath, produces an Eosin red color.

4.2.10 Phytochemical Analysis Tests for Steroids a. Salkowski tests: Shake a chloroform solution of the test drug with concentrated sulfuric acid, which produces a red color. b. Lieberman–Burchard test: Mix a chloroform solution of the test drug with a few drops of acetic anhydride and one mL of concentrated sulfuric acid from the sides to produce a reddish ring at the junction of the two layers.

4.2.11 Phytochemical Analysis Tests for Tannins a. Gelatin test: To a solution of tannin, add an aqueous solution of gelatin and sodium chloride (Sofawora, 1982). A white buff color precipitates, indicating the presence of tannins. b. Goldbeater’s skin test: Soak a small piece of Goldbeater skin in 20% hydrochloric acid, rinse with distilled water, and place in a solution of tannin for 5 minutes. Wash the skin piece with distilled water and keep in a solution of ferrous sulfate. A brown or black color produced on the skin indicates the presence of tannins. c. Phenazone test: Take a mixture of the test drug and sodium phosphate and heat, cool, and filter. Add a solution of phenazone to the filtrate. A bulky and colored precipitate shows the presence of tannins. d. Matchstick test (Catechin test): Take a matchstick dipped in the test drug, dry it near a burner, and moisten with concentrated hydrochloric acid. On warming near a flame, the matchstick wood turns pink or red due to formation of phloroglucinol. e. Chlorogenic acid test: Treat an extract of chlorogenic acid containing the test drug with aqueous ammonia. A green color forms on exposure to air. f. Vanillin-hydrochloric acid test: In a sample solution, add vanillin- hydrochloric acid reagent. A pink or red color forms due to formation of phloroglucinol. g. Ferric chloride test: Mix the test drug mix with 1% ferric chloride solution which gives a blue, green, or brownish-green color. Methods of Phyto-Constituent Detection 43

4.2.12 Phytochemical Analysis Tests for Fixed Oils and Fats a. Press a small quantity of the test drug between two filter papers. The appearance of an oil stain on the paper indicates the presence of fixed oil. b. Add a few drops of 0.5N alcoholic potassium hydroxide to a small quantity of various extracts along with a drop of phenolphthalein. Heat the mixture in a water bath for 1–2 h. The formation of a soap or partial neutralization of the alkali indicates the presence of fixed oil and fats.

4.2.13 Phytochemical Analysis Test for Gums and Mucilages About 10 mL of test drug is added separately to 25 mL of absolute alcohol with constant stirring and filter. The precipitate dries in air. Examine it for its swelling properties and for the presence of carbohydrates.

4.2.14 Phytochemical Analysis Tests for Lactones a. Legal’s test: Mix the test drug with a mixture of sodium nitroprusside and pyridine. Treatment with methanol alkali to produce a deep red color. b. Feigel’s test: Shake the acidified test drug with solvent ether and add few drops of saturated alcoholic solution of potassium hydroxide in a porcelain crucible heated over a flame until cooling. It produces a light pink color with a 1% ferric chloride solution. c. Baljel’s tests: Mix the test drug with a solution of sodium picrate to give it a yellow-orange color.

4.2.15 Phytochemical Analysis Test for Diterpenes a. Copper acetate test: Mix the test drug with a solution of copper acetate to give it a green color.

4.3 CONCLUSION It is believed that there may be about 4000 phytochemicals contained in plants that can be used to prevent, minimize or remedy medical conditions such as strokes, cancer or metabolic syndrome. Some of the bioactive substances that can be derived from plants are flavonoids, alkaloids, carotenoids, tannin, antioxidants, and phenolic compounds. Although the knowledge of how these substances provide medicinal value to humans reflects a relatively recent scientific understanding, the use of plants and plant extracts to heal, relieve pain, and promote good health dates back to before the beginnings of medical science. It is very necessary to detect the phyto-constituents present in plants for the preparation of medicines against disease. Thus , phytochemical analysis opens the path for new drugs and development. 44 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

REFERENCES Al-Daihan S et al. Antibacterial: Activity and phytochemical screening of some medicinal plants commonly used in saudi arabia against selected pathogenic microorganisms. J King Saud Univ Sci. 2013; 25: 115–120. Davies KM. Plant Pigments and Their Manipulation. Annual Plant Reviews, Vol. 14. Blackwell Publishing Ltd., 2004, pp. 95–97, 251–256. Ilza A. Francisco and maria helena pimentapinotti. Cyanogenic glycosides in plants. Braz Arch Biol Technol. 2000; 43(5): 487–492. Ivana R et al. Glucosinolates and their potential role in plant. Period Biol. 2008; 110(4): 297–309. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, 20th Edition. Nirali Prakashan, Pune, 2002, pp. 108–109. Prassas I, Eleftherios P, Diamandis EP. Novel therapeutic applications of cardiac glycosides. Nat Rev Drug Discov. 2008; 7: 926–935. Sofawora EA. Medicinal Plants and Traditional Medicine in Africa, Wiley, Chichester, 1982, p. 256. Tapia A, Rodríguez J, Theoduloz C, Lopez S, Feresin GE, Schmeda-Hirschmann G. Preliminary phytochemical activity of the pluchea species. J Ethnopharmacol. 2004; 95: 155. Wu X, Beecher G, Holden JM. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Food Chem. 2006; 54: 4069–4075. Regulatory Aspects 5 for Herbal Drugs

5.1 INTRODUCTION Although modern medicine is well developed in most of the world, large sections of the population in developing countries still rely on the traditional practitioners, medicinal plants, and herbal medicines for their primary care. Moreover, during the past decades, public interest in natural therapies has increased greatly in industrialized countries with expanding use of medicinal plants and herbal medicines (Sharma and Arora, 2006). The evaluation of these products is based on ensuring their safety through registration and regulation of the present important challenges. Medicinal plants are important for pharmacological research and drug development, not only when plant constituents are used directly as therapeutic agents, but also as starting materials for the synthesis of drugs or as models for pharmacologically active compounds. Regulation of exploitation and exportation is, therefore, essential together with international cooperation and coordination for their conservation so as to ensure their availability for the future (Chaudhary, 1996). According to European Union definitions, herbal medicines are the herbal products containing active ingredients that are exclusively plant material and/or vegetable drug preparations. Herbal drug technology includes all the steps that are involved in converting botanical materials into medicines, where standardization and quality control with proper integration of modern scientific techniques and traditional knowledge will remain important. All the countries using medicinal plants and traditional medicines are aware of the need for regulating the use of these medicinal substances. There is a need for countries to regulate the use of medicinal plants because there is a growing interest in herbal medicines in the population of these countries (Warude and Patwardhan, 2005). The aim of such national policies would be to develop regulatory and legal reforms to ensure good practice and to extend primary health care coverage, while ensuring the authenticity, safety, and efficacy of these medicines. Main objectives include the recognition of traditional medicine as an integral part of national health care systems, cooperation between modern and traditional medicine, promotion of the rational use of products, the introduction of quality assurance systems, the guarantee of regular supplies, and the promotion of research and development of regulatory measures. Basic scientific principles and special requirements related to their use in traditional practice are incorporated into these guidelines, the main objectives of which are to ensure their safety and efficacy, to promote their rational use, and to provide research criteria for their evaluation. The guidelines provide a basis for member states to develop their own research guidelines, and for the exchange of research experience and other information so that a body of reliable data for the validation of herbal medicines may be built up (Verpoorte and Mukherjee, 2003).

45 46 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

There is a widespread misconception that “natural” always means “safe,” and a common belief that remedies from natural origins are harmless and carry no risk. However, some medicinal plants are inherently toxic. Further, as with all medicines, herbal medicines are expected to have side effects which may be of an adverse nature. Some adverse events reported in association with herbal products are attributable to problems of quality. Major causes of such events are adulteration of herbal products with undeclared other medicines and potent pharmaceutical substances, such as corticosteroids and non-steroidal anti-inflammatory agents. Adverse events may also arise from the mistaken use of the wrong species of medicinal plants, incorrect dosing, and errors in the use of herbal medicines both by health care providers and consumers, interactions with other medicines, and the use of products contaminated with potentially hazardous substances, such as toxic metals, pathogenic microorganisms, and agrochemical residues (Patel et al., 2011).

5.2 REGULATION National regulation and registration of herbal medicines vary from country to country. Where herbal medicines are regulated, they may be categorized as either prescription or nonprescription medicines. Herbal products may also be categorized other than as medicines. Moreover, the regulatory status of a particular herbal product may differ in different countries. The national regulatory framework usually also includes involved qualified providers and distributors of the respective substances. Regulatory status consequently determines the access to or distribution route of these products (Anonymous, 2000a; WHO, 1998b).

5.2.1 aim of Regulatory Guidelines for Herbal Medicines The quality of herbal substances, herbal preparations, and herbal medicinal products is determined by the quality of the starting plant material, development, in-process controls, GMP controls, and process validation, and by specifications applied to them throughout development and manufacture. This guideline addresses specifications, that is, those tests, procedures, and acceptance criteria used to assure the quality of the herbal substances/preparations and herbal medicinal products at release and during the shelf life (Verpoorte and Mukherjee, 2003).

5.2.2 regulation and Registration of Herbal Medicines The legal situation regarding herbal preparations varies from country to country. Developing countries, however, often have a great number of traditionally used herbal medicines and much folk knowledge about them, but have hardly any legislative criteria to establish these traditionally used herbal medicines as part of the drug legislation. For the classification of herbal or traditional medicinal products, factors applied in regulatory systems include: description in a pharmacopoeia monograph, prescription status, claim of a therapeutic effect, scheduled or regulated ingredients or substances or periods of use (WHO, 2000). Regulatory Aspects for Herbal Drugs 47

5.3 WHO REGULATORY REQUIREMENTS Traditional Medicines (TM) are indigenous medicines existent in the region either recognized or ethnic as in Chinese medicine, Indian Ayurveda, Arabic Unani medicine, African, and Latin American practices. Complementary/Alternative Medicines (CAM) are added or used alternatively to dominant health care systems of allopathic medicine as in the US, Canada, and Europe. It is essential to know what regulatory and legislative controls on the manufacture and sale of such herbal medicines exist or are required to be implemented in various places around the world (Anonymous, 2002). World Health Organization (WHO) has tried to establish internationally recognizable regulatory guidelines to define basic criteria for the evaluation of quality, safety, and efficacy of botanical medicines. For assessing the quality of botanical materials, the need to ensure the quality of medicinal plant products by using modern techniques and applying suitable standards is mainly emphasized (Figure 5.1). In 1997, WHO developed a draft guideline for methodology on research and evaluation of traditional medicine (TM). Its major focus is on current major debates on the safety and efficacy of traditional medicine. Purity and quality of herbs is a critical determinant of safety. The first stage in assuring the quality, safety, and efficacy of medicinal herbs is identification and selection of the correct plant species

Good Agricultural and Collection Guidelines on Practice Non-clinical (GACP) Good Documentation Manufacturing for Herbal Practice (GMP) Medicinal Products

Guidelines on Guidelines on Guidelines Quality of Quantitative and for Herbal Qualitative Herbals Medicinal Analysis Products

Guidelines on Guidelines on Test Procedure and Quality of Acceptance Combination Criteria for Guidelines on Herbal Medicinal Herbal Declaration Products Substances of Herbal Substances and Herbal Preparations

FIGURE 5.1 WHO guidelines for herbals. 48 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

(Anonymous, 2000b). Regulatory authorities for control of raw materials have suggested various methods. The objectives of this guideline are to provide:

• Guiding principles for assessing the quality in relation to the safety of herbal medicines, with specific reference to contaminants and residues • Model criteria for use in identifying possible contaminants and residues • Measures for controlling the quality of finished herbal products

5.3.1 objectives The objective of these guidelines is to propose to member states a framework for facilitating the regulation of herbal medicines/products used in traditional medicine (TM). The proposed framework, which has a regional perspective, should help accelerate the establishment of appropriate mechanisms for registration and regulation of herbal medicines, based on criteria for safety of use, therapeutic efficacy, quality control, and pharmacovigilance. Traditional medicine involves not only the use of herbal medicines, but also the use of animal parts and minerals. As herbal medicines are the most widely used of the three, and as the other types of materials involve other complex factors, this document will concentrate on herbal medicines (Anonymous, 2000c).

5.3.2 guidelines for the Regulation of Herbal Medicines in the Southeast Asia Region These guidelines aim to propose to member states a framework for facilitating the regulation of herbal medicines/products used in traditional medicine. They cover issues like classification of herbal medicines, minimum requirements for assessment of the safety of herbal medicine, minimum requirements for assessment of the efficacy of herbal medicines, quality assurance of herbal medicinal products, pharmacovigilance of herbal medicinal products, and control of advertisements of herbal medicinal products (Anonymous, 2001).

5.3.3 wHO Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance Systems The safety of herbal medicine is an important public health issue. The guidelines stress the importance and process of monitoring the safety of herbal medicines within the pharmacovigilance system (WHO, 2002). Standard definitions of terms related to pharmacovigilance and safety monitoring of herbal medicine are used (Bowdler, 1997). Challenges in monitoring the safety of herbal medicine and the need for good communications for ensuring successful safety monitoring are also stressed.

5.3.4 wHO Guidelines on Good Agricultural and Collection Practices (GACP) for Medicinal Plants These guidelines are intended to provide technical knowledge on obtaining medicinal plant materials of good quality for the sustainable production of herbal products Regulatory Aspects for Herbal Drugs 49 classified as medicines (WHO, 2003b). They apply to the identification, authentication, cultivation, and harvest of medicinal plants, good collection practices for medicinal plants, common technical aspects of good agricultural practices for medicinal plants in terms of personnel, packaging, storage, and transportation, and relevant issues of ethical/legal considerations and research.

5.3.5 national Policy on Traditional Medicine (TM) and Regulation of Herbal Medicine There is a lack of common standards and appropriate methods for evaluating traditional medicine to ensure the safety, efficacy, and quality control of traditional medicine (TM) and complementary/alternative medicine (CAM) (WHO, 2001). In 2001, WHO developed a global survey questionnaire which was focused on a general review of the policies and regulations of TM/CAM, regulation of herbal medicines, and countries’ needs for future WHO support and guidance.

5.3.6 wHO Guidelines for Quality Control of Herbal Formulation These WHO guidelines present general consideration for potentially hazardous contaminants and residues in herbal medicines and include guiding principles of assessing quality of herbal medicines in terms of major contaminants and residues. It also recommends analytical methods for qualitative and quantitative determination of such contaminants and residues. Within the overall context of quality assurance, these guidelines intended to provide general technical guidance to member states in assessing quality relating to the safety of herbal materials and products classified as medicines with regards to major and common contaminants and residues (WHO, 1998a; WHO, 2010).

• Quality control of crude drugs’ material, plant preparations, and finished products • Stability assessment and shelf life • Safety assessment; documentation of safety based on experience or toxicological studies • Assessment of efficacy by pharmacological information and biological activity evaluations

5.3.7 wHO Guidelines for Herbal Drug Standardization The subject of herbal drug standardization is massively wide and deep. The guidelines set by WHO can be summarized as follows:

• Reference for the identity of the drug: Botanical evaluation-sensory characters, foreign organic matter, microscopic, histological, histochemical evaluation, quantitative measurements, and so on (WHO, 1995). • Refers to the physicochemical character of the drug: Physical and chemical identity, chromatographic fingerprints, ash values, extractive values, 50 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

moisture content, volatile oil, and alkaloidal assays, quantitative estimation protocols, and so on (WHO, 1996). • A reference to the pharmacological parameters: Biological activity profiles, bitterness values, hemolytic index, astringency, swelling factor, foaming index, and so on (WHO, 2003a). • Toxicity details: Pesticide residues, heavy metals, microbial contamination like total viable count, pathogens such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, and members of the family Enterobacteriaceae, to name a few. • Microbial contamination. • Radioactive contamination.

5.4 HERBAL DRUG REGULATIONS IN INDIA Recognizing the global demand, the government of India has realized Good Manufacturing Practices (GMPs) for the pharmacies manufacturing Ayurveda, Siddha, and Unani medicines to improve the quality and standard of drugs. The Department of Indian Systems of Medicine and Homeopathy (ISMH) is trying to frame safety and efficacy regulations for licensing new patents and proprietary botanical medicines. Indian Pharmacopoeia covers few ayurvedic medicines; monographs have been given for some ayurvedic drugs such as clove, guggul, opium, menthe, and senna. The ayurvedic pharmacopoeia of India gives monographs for 258 different ayurvedic drugs. The standards mentioned are quite inadequate to build the quality of the botanicals materials. Indian Drug Manufacturers Association (IDMA) has published the Indian Herbal Pharmacopoeia with 52 monographs of widely used medicinal plants found in India. In the case of herbal medicinal products, specifications are generally applied to the herbal substance, to the herbal preparation, and to the herbal medicinal product. Specifications are primarily intended to define the quality of the herbal substance/preparation and herbal medicinal product rather than to establish full characterization, and should focus on those characteristics found to be useful in ensuring the safety and efficacy of the herbal substance/preparation and herbal medicinal product. For quality control of herbal medicines, separate chapters for Ayurveda, Siddha, and Unani (ASU) medicine have been introduced in Chapters IV-A of the Drugs and Cosmetics Act, 1940, and Rules, 1945:

• 33 C—Separate Drug Technical Advisory Board (SDTAB) under the Drugs and Cosmetics Act, 1940, for Indian systems of medicines to advise the government on all aspects related to quality control and drug standardization. • 33 D—Separate Drugs Consultative Committee (SDCC) comprising state drugs licensing authorities set up under the act for securing uniformity in the administration of the act throughout India. • 33 E—Misbranded drugs, 33 EE—Adulterated drugs, 33EEA—Spurious drugs. • 33 EEB—Regulation of manufacture for sale of ayurvedic drugs through drug license system. Regulatory Aspects for Herbal Drugs 51

• 33 EEC—Prohibition of manufacture and sale of certain drugs. • 33 EED—Power of central government to prohibit manufacture and so on of drugs in the public interest. • 33 F—Provision for government analysts. • 33 G—Provision for inspectors to visit factory. • 33 H—Penalty for manufacture and sale of drugs in contravention of the Act. • 33 J—Penalty for subsequent offences.

5.5 REGULATORY ASPECTS AND APPROVAL OF HERBAL DRUGS IN DIFFERENT COUNTRIES The legal process of regulation and legislation of herbal medicines changes from country to country. The WHO has published guidelines in order to define basic criteria for evaluating the quality, safety, and efficacy of herbal medicines aimed at assisting national regulatory authorities, scientific organizations, and manufacturers in this particular area. Furthermore, the WHO has prepared monographs on herbal medicines and the basis of guidelines for the assessment of herbal drugs. Thus, the need to establish global and/or regional regulatory mechanisms for regulating herbal drugs seems obvious. The European agency of evaluation of medicinal products provides general guidelines for setting a uniform set of specifications for herbal preparations manufactured and sold in Europe. Preclinical and clinical studies are proposed if a completely new indication is requested for the herbal product that has been already marketed for a different use. In Australia, complementary medicines including herbal medicines are regulated under therapeutic good legislation. Registered medicines are individually evaluated for safety, quality, and efficacy before they are released into the market. An important feature of risk management in Australia is that early market access for low risk complementary medicines is supported by appropriate post-market regulatory activity. In Chinese markets, the importation of herbal crude drugs needs the approval of the provincial department of public health. The Republic of China has a section on “Standard for Processing of Chinese Materia Medica.” If Chinese herbal medicines are produced in factories either for export or for local use in other parts of the country, they have to undergo quality control tests before being released. There is a rigid criterion for assessing patented traditional Chinese medicines. Only the herbal products which conform to the Chinese traditional system of medicines, that are safe, and in which the ingredients are compatible with each other are allowed to be released into the market. In Brazil, the legal requirements for registration of herbal medicines needs complete documentation of the efficacy, safety, and well-defined quality control. However, the Canadian regulatory system is consistent with WHO guidelines for the assessment of herbal medicines. Germany’s Commission E for phytotherapy and herbal substances was established in 1978. It is an independent division of the German Federal Health Agency that collects information on herbal medicines and evaluates them for safety and efficacy. The German market exists for monographs of standardized marketing authorization and temporary marking authorization for old herbal drugs until they are evaluated for safety and efficacy. 52 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Although considerable progress has been made in characterizing herbal medicine, there is need for global harmonization of the herbal quality and health claims. International Conference on Harmonization (ICH) has tried to harmonize the technical requirements for registration of pharmaceuticals for human use by setting specific guidelines. These guidelines may be applicable to globally uplift the quality of botanicals.

5.5.1 european Herbal Guidelines 5.5.1.1 European Medicines Agency—EMA Herbal medicinal products fall within the scope of the European Directive 2001/83/ EC (European Communities) that foresees the marketing of each medicinal product and requires an ad hoc authorization to be granted on the basis of the results of tests and experimentations concerning quality, safety, and efficacy. The main features of Directive 2001/EC are a traditional herbal medicine definition, simplified registration procedure, provisions for community herbal monographs, community list of herbal substances and preparations, and establishment of the Committee for Herbal Medicinal Products (HMPC). Most individual herbal medicinal products are licensed nationally by member states, and the process for the licensing, information of herbal substances, and preparations is harmonized across the European Union. In the United Kingdom, to get a product registered, companies have to submit a dossier to the Medicines and Healthcare products Regulatory Agency (MHRA) demonstrating that it meets the requirements of quality, safety, and patient information as per the Traditional Herbal Registration Scheme. The HMPC scientifically evaluates all available information, including non- clinical and clinical data, but also documented long-standing use and experience in the community. Community monographs are divided into two columns: well- established use (marketing authorization) and traditional use (simplified registration). The well-established use section describes the safety and efficacy data, while the traditional use section is accepted on the basis of sufficient safety data and plausible efficacy. The Committee on Herbal Medicinal Products (HMPC) has developed a procedure to invite the public to submit scientific data on herbal substances and preparations. The provided information may then be used by the committee in the development of community monographs and for community list entries.

5.5.2 united States of America In the United States, the term complementary/alternative medicines (CAM) are most commonly used for traditional medicine systems. Complementary medicine refers to use of CAM together with conventional medicine, such as using acupuncture in addition to the usual care to help lessen pain. The Food and Drug Administration (FDA) in its draft guidance; “Guidance for industry on complementary and alternative medicine products and regulation by the food and drug administration,” clarified different categories of Complementary Alternative Medicines (CAM) products into cosmetics; devices; dietary supplements; and drugs, as well as “new drugs” and “new animal drugs;” foods; and food additives. The Office of Dietary Supplements, an Regulatory Aspects for Herbal Drugs 53 office within the National Institutes of Health, was established in 1995 to explore the potential role of supplements for improving health care in the United States. Along with herbs, supplements also include vitamins, minerals, amino acids, homeopathic remedies, concentrates, extracts, and various combinations of these ingredients.

5.5.3 australia Complementary medicines including botanical medicines in Australia are regulated under therapeutic goods legislation. The listed medicines are considered to be of lower risk than registered medicines. Most but not all complementary medicines are listed medicines which are individually assessed by the therapeutic goods administration for compliance with legislation. They may only be formulated from ingredients that have undergone pre-market evaluation for safety and quality and are considered a low risk. Listed complementary medicines may only carry indications and claims for the symptomatic relief of non-serious conditions, health maintenance, health enhancement, and risk reduction. Registered medicines are individually evaluated for safety, quality, and efficacy before they are released into the market. An important feature of risk management in Australia is early market access for low risk management medicines which is supported by appropriate post-market regulatory activity.

REFERENCES Anonymous. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine, World Health Organization, Geneva, 2000a (WHO/EDM/TRM/2000.1). Anonymous. Safety Monitoring of Medicinal Products: Guidelines for Setting Up and Running a Pharmacovigilance Center, Uppsala Monitoring Centre, Uppsala, 2000b (reproduced in Part II of this publication). Anonymous. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine, World Health Organization, Geneva, 2000c (WHO/EDM/TRM/2000.1). Anonymous. Current challenges in pharmacovigilance: Pragmatic approaches. In: Report of CIOMS Working Group V. The Council for International Organizations of Medical Sciences, Geneva, 2001. Anonymous. US report calls for tighter controls on complementary medicine. Br Med J. 2002; 324: 870. Bowdler J. Effective Communications in Pharmacovigilance: The Erice Report, W Lake, Birmingham, 1997. Chaudhary RD. Regulatory Requirements. Herbal Drug Industry—A Practical Approach to Industrial Pharmacognosy, 1st Edition. Eastern Publishers, New Delhi, 1996, pp. 537–546. Patel P, Patel NM, Patel PM. WHO guidelines on quality control of herbal medicines. IJRAP. 2011; 2(4): 1148–1154. Sharma RK, Arora R. Herbal Drugs: Regulation Across the Globe. Herbal Drugs—A Twenty First Century Perspective, 1st Edition. Jaypee Brothers Medical Publishers Pvt. Ltd., New Delhi, 2006, pp. 625–627. Verpoorte R, Mukherjee PK. Overview of Global Regulatory Status. GMP for Botanicals— Regulatory and Quality Issues on Phytomedicines, 1st Edition. Business Horizons Pharmaceutical Publishers, New Delhi, 2003, pp. 27–41. 54 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Warude D, Patwardhan B. Botanicals: Quality and regulatory issues. J Sci Ind Res. 2005; 64: 83–92. WHO. Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. In: The Use of Essential Drugs. Sixth Report of the WHO Expert Committee. World Health Organization, Geneva, 1995, Annex 3 (WHO Technical Report Series, No. 850). WHO. Guidelines for assessment of herbal medicines. In: WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-Fourth Report. World Health Organization, Geneva, 1996, Annex 11 (WHO Technical Report Series, No. 863). (These guidelines are also included in: Quality assurance of Pharmaceuticals: A compendium of guidelines and related materials, Vol. 1. Geneva, World Health Organization, 1997). WHO. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1998a. WHO. Regulatory Situation of Herbal Medicines: A Worldwide Review, World Health Organization, Geneva, 1998b (WHO/TRM/98.1). WHO. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines, World Health Organization, Geneva, 2000 (WHO/EDM/TRM/2000.1). WHO. Legal Status of Traditional Medicines and Complementary/Alternative Medicines: A Worldwide Review, World Health Organization, Geneva, 2001 (WHO/EDM/ TRM/2001.2). WHO. The Importance of Pharmacovigilance: Safety Monitoring of Medicinal Products, World Health Organization, Geneva, 2002. WHO. Good manufacturing practices for pharmaceutical products: Main principles. In: WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-Seventh Report. World Health Organization, Geneva, 2003a, Annex 4 (WHO Technical Report Series, No. 908). WHO. WHO Guidelines on Good Agricultural and Collection Practices (GACP) for Medicinal Plants, World Health Organization, Geneva, 2003b. WHO. WHO Guidelines for Assessing Safety and Quality of Herbal Medicines with Reference to Contaminants and Residues, World Health Organization, Geneva, 2010. Ethnopharmacology 6 of Medicinal Plants

6.1 INTRODUCTION Ethnopharmacology is the scientific study of traditional medicines, which continue to provide new drugs and lead molecules for the pharmaceutical industry. A large amount of information still awaits disclosure to the scientific community. Despite the fact that modern medicine is well developed in most parts of the world, the World Health Organization (WHO) through its Traditional Medicine Program recommends its Member States to formulate and develop policies for the use of complementary and alternative medicine (CAM) in their national health care programs (Bieski et al., 2012). The World Health Organization (WHO) acknowledges the value of traditional medicine and the preservation and protection of this knowledge is one of their objectives. Ethnobotany is the study of plants used by indigenous societies for food, medicine, building materials, economic application or ceremony (Giday et al., 2002). The majority of the people still rely on traditional medicine (TM) for their everyday healthcare needs. People who use traditional remedies may not understand the scientific rationale behind their medicines, but they know from personal experience that some medicinal plants can be highly effective if used at therapeutic doses (Anonymous, 2001). People believe that plant remedies used for mediation are less toxic than modern medicines. Traditional medicine (TM) is composed of a number of skills such as the use of plants, animal products, and minerals as well as magic and suppression. The indigenous knowledge about many medicinal plants has justified its existence by the biomedical benefits that have been established through observations of generations of people (Gurib Fakin, 2006). This has been demonstrated by the history of modern drug discoveries from plants which were employed in TM in other countries such as China and India. Medicinal plants and knowledge of their use provide a vital contribution to human and livestock health needs throughout the world (Gedif and Han, 2003). As is happening elsewhere in the world, both the traditional knowledge and plants utilized by these people are under threat due to the aforementioned reasons. The modes of therapy of these herbal remedies are based on empirical findings. Natural products and their derivatives represent more than 50% of all the drugs clinically used in the world and higher plants contribute no less than 25% of the total natural products (Pramon, 2002).

6.2 PHYTOTHERAPY Phytotherapy has been practiced by the greater percentage of the world’s population through the use of plants or their derivatives, and occupies a significant and unique position. Plants have always played a major role in the treatment of human traumas and diseases worldwide. They have been used as sources of modern drugs, either

55 56 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines by providing pure compounds, starting materials for partial synthesis of useful compounds or models for synthesis of new drugs. Ethnopharmacological information is an important tool in drug discovery. In this sense, documentation of the indigenous knowledge through ethnobotanical studies is important in the conservation and utilization of biological resources (Agbovic et al., 2002). The preservation of local culture and the practice of traditional medicinal plant species themselves represent important strategies for the sustenance of popular knowledge of CAM in the local systems of health care and environmental education (Mesfin et al., 2005). Moreover, ethnobotanical and pharmacological studies provide information essential for guidance in bioprospecting for new drugs of plant origin in the consolidation of therapeutic practices of the community. Medicinal plants have been used to prevent and treat various health problems. Several African and Asian nations are now encouraging traditional medicines as an internal component of their public health care programs (Abera, 2003). Indigenous medicines are relatively inexpensive, locally available, and readily accepted by the local population. The extensive knowledge of the traditional uses of these plants has not been fully documented and most of the knowledge is conveyed from one generation to the next generation by word of mouth (Wolde and Gebre-Mariam, 2002).

6.3 PRACTICING HERBAL MEDICINE Herbal medicines are the sources of therapeutics from the experiences of practicing physicians of indigenous systems of medicine for more than hundreds of year. Medicinal plants are a source of raw materials for both traditional systems of medicine and modern medicine. Plants have been used as a medicinal agent since ancient times (Lee, 2004). These medicines are also in great demand in the developed world for primary health care because of their efficacy, safety, and lesser side effects. Drugs derived from natural sources have also served as “drug leads” suitable for optimization (by synthetic means) into potent pharmaceuticals (Bhutani, 2008). They also offer therapeutics for age-related disorders like memory loss, osteoporosis, immune disorders, and so on. The herb or crude drug used in the traditional system medicine is a complex potpourri of compounds, some beneficial, some harmful, and some toxic, but all integrated under certain natural rules to make the crude fraction into a single chemical agent (Choudhury et al., 2012). The herbal drug preparation in its entirety is regarded as the active substance and the constituents are either of known therapeutic activity or are chemically defined substances or groups of substances generally accepted to contribute substantially to the therapeutic activity of the drug. The therapeutic potential of herbal drugs depends on their form: whether parts of a plant, simple extracts or isolated active constituents. Herbal medicine is still the mainstay of about 75%–80% of the world’s population, mainly in the developing countries, for primary health care because of better cultural acceptability and better compatibility with the human body. Traditionally, herbs and herbal products have been considered to be nontoxic and have been used by the general public and traditional medicinal doctors worldwide to treat a range of ailments. The fact that something is natural does not necessarily make it safe or effective. The active ingredients of plant extracts are chemicals that are similar to those in purified medications, and they have the same potential to cause serious adverse effects. While the literature documents Ethnopharmacology of Medicinal Plants 57 show severe toxicity resulting from the use of herbs, on many occasions, the potential toxicity of herbs and herbal products has not been recognized. Most herbal remedies when used as directed and under the supervision of knowledgeable individuals are safe, but the potential for adverse effects certainly exists. The high demand of the cultivation, conservation, and export of medicinal plants is an important segment of the medicinal plants fields (Patel, 2012).

6.4 NEED OF DOCUMENTATION OF ETHNOPHARMACOLOGICAL PLANTS Ethnopharmacology involves the observation, description, and experimental investigation of indigenous medicines and their biological activities as an approach to drug discovery. Information about medicinal plants is still passing from one generation to another by oral communication, posing the danger of losing some knowledge. There is, therefore, a need to document medicinal plants before the information disappears. Meanwhile, most of these plants have already been endangered by arid/ semi-arid climatic conditions and man-made activities (Inngjerdingen et al., 2004).

6.5 ETHNOPHARMACOGNOSTICAL STUDIES OF MEDICINAL PLANTS OF CHHATTISGARH, INDIA Herbal plants play a dynamic role in human life. Chhattisgarh is the herbal state of India. The state contains numerous medicinal plants and has been used by the ethnic people for several decades. Jashpur is one of the 27 districts of Chhattisgarh. It covers an area of 196,338 square miles. The tribal population in the Jashpur district amounts to approximately 55%–60%. The north-south length of this district is about 150 km, and its east-west breadth is about 85 km. Its total area is 6205 km2. It lies between 22° 17 and 23° 15 North latitude and 83° 30 and 84° 24 East longitude. It has been geographically divided into two parts. The northern hilly belt is called the Upper Ghat. The remaining, southern part is called Nichghat. The Upper Ghat runs from Loroghat Kastura, Narayanpur, Bagicha, up to the Surguja district. This belt is a forest area and contains a reserve forest. It covers the Sanna, Bagicha, and Narayanpur (Kujur and Ahirwar, 2015). This paper deals with some ethnomedicinal plants used by various tribal communities of the district of Jashpur, Chhattisgarh, India. Tribes concentrated in the rural area are Oraon, Kawar, Gond, Virhore, Ahir, and Korwa; these are the main tribal communities of this area. They depend upon traditional agricultural activities and cattle rearing. The remarkable thing about these communities living in hilly and plain regions is that for their healthcare, they use only herbal-based medicines (Addis et al., 2001). As they are not very civilized because of living away from any educational field, allopathic medicines are unknown to them. This is the reason for their using herbal medicines alone (Ekka and Dixit, 2007). The literature available is about the medicinal plants belonging to different families found in this region. Plants have been used to treat common illnesses such as paralysis, epilepsy, tuberculosis, tetanus, snakebite, cancer, arthritis, and so on. Table 6.1 shows the list of ethopharmacological plants found in this region of Chhattisgarh (Alam et al., 1990). 58 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

TABLE 6.1 Ethnopharmacological Plants of Chhattisgarh Disease Botanical Name Family Local Name Plant Part Treatment Acacia leucophloea Leguminosae Bambary Bark Diabetes Acorus calamus Araceae Ghod bach Tuber Phallic agent Adhatoda vasica Acanthaceae Adusa Leaf Tuberculosis Aegle marmelos Rutaceae Khotta Leaf Jaundice Ageratum conyzoides Compositae Chawlattee Leaf Cuts and wound Aloe barbadensis Liliaceae Ghikuwire Leaf Headache Andrographis Paniculata Acanthaceae Bhui neem Whole plant Malaria Argemone mexicana Papaveraceae Raangainee Root Tooth ache Asparagus racemosus Liliaceae Satawari Root Dysentery Abrus precatorius Fabaceae Ghumchi Root Whooping cough Achyranthes aspera Amaranthaceae Chirchita Root Asthma Adhatoda vasica Acanthaaceace Adusa Leaf Asthma Azadirachta indica Meliaceae Neem Whole plant Eczema Bauhina variegate Leguminosae Koynar Bark Body pain Bauhinia vahlii Caesalpiniaceae Mohlain Root Syphilis Boerhaavia diffusa Nyctagin aceae Khapra sag Root Dysentery Buchanania Lanzan Anacardiaceae Kitti Root Asthma Butea monospera Fabaceae Palas Bark Dysentery Calotropis procera Asclepiadaceae Aak Flower Whooping cough Capsicum annum Solanaceae Mirch Fruit Paralysis Carica papaya Caricaceae Pavitar Root Tetanus Carissa spinarum Apocynaceae Karonda Root Abdominal pain Caesalpinia bonducella Caesalpiniaceae Gataran Leaf Asthma Cassia fistula Leguminosae Sonarkhi Bark Hydrocele Catharanthus roseus Apocynaceae Sadabahar Leaf Dysentery Centella asiatica Umbelliferae Mukha adkha Whole plant Epilepsy Chlorophytma Liliaceace Safedmusli Tuber Nocturnal rundinacuem emission Cissampelo spareira Menispermaceae Pathar Root Diuretic Cocculus hirsutus Menispermaceae Nappa kand Tuber Stomach ache Coleus aromaticus Labiatae Pathorcur Leaf Cough cold Costus specious Costaceace Keokand Rhizome Jaundice Curculigo orchioides Hypoxidaceae Kalimusli Tuber Impotency Cuscutare flexa Cuscutaceae Amarbel Leaf Skin disease Cyperus rotundus Cyperaceae Kissi lattee Tuber Stomach ache Dalbergia latifolia Leguminosae Seesum Leaf Spermatorrhoea Daturastra monium Solanaceae Dhatura Leaf Asthma Dentrocalamus Strictus Gramineae Parta bans Stem Eczema Desmodium gangeticum Fabaceace Balraj Whole plant Headache Diospyrus Melanoxylon Ebenaceae Tela Root Blood clotting Elephantopus scaber Compositae Meejur chundi Tuber Abdominal pain (Continued) Ethnopharmacology of Medicinal Plants 59

TABLE 6.1 (Continued) Ethnopharmacological Plants of Chhattisgarh Disease Botanical Name Family Local Name Plant Part Treatment Emblica officinalis Euphorbiaceae Amia Fruit Tuberculosis Euphorbia hirta Euphorbiaceae Dudhia grass Whole plant Snake bite Ficus bengalensis Moraceae Bara Latex Cataract Ficus racemosa Moraceae Dumer Latex Dysentery Euphorbia prostata Euphorbiaceae Chotta dudhia grass Whole plant Lactogogue Glorio sasuperba Liliaceae Kalihari Root Dysentery Gymnema sylvestre Asclepiadaceae Gaychemak Root Vomiting Jatropha curcas Euphorbiaceae Bhakranda Stem Toothache Madhu calatifolia Sapotaceae Mahua Flowers Analgesic Mimods apudicea Mimosaceae Chunimui Seed Veneral diseases Mucuna puriens Fabaceae Kemach Root Gout Pongamia pinnata Fabaceae Karanj Seed Toothache Shorearo busta Dipterocarpaceae Sarai Fruits Dysentery Syzgium cuminii Myrtaceae Jamun Seed Diabetes Terminalia arjuna Combretaceae Arjun Fruit Purgative

6.6 CONCLUSION Medicinal plants have been available for human consumption since time immemorial. The remote areas of Chhattisgarh have been the traditional sources of medicinal herbs. During the past decade, a dramatic increase in exports of valuable plants attests to the worldwide interest in traditional health systems. Most of these plants are being taking from the wild, and hundreds of species are now threatened with extinction because of over exploitation.

REFERENCES Abera B. Medicinal plants used in traditional medicine Jimma Zone. Ethiop J Hlth Sci. 2003; 13(2): 86–90. Addis G, Abebe D, Urga K. Survey of traditional medicinal plants in Shirka district, Arisi Zone, Ethiopia. Ethio Pharm J. 2001; 19: 30–34. Agbovic T, Dennis F, Amponsah K. Conservation and sustainable use of medicinal plants, in Ghana; ethinopharmacology survey, Org/species/plants/Ghana, 2002. Alam MM, Siddiqui MB, Husain W. Treatment of diabetes through herbal drugs in rural India. Fitoterapia. 1990; LXI: 240–242. Anonymous. Reconstruction and development Traditional medicine and the bridge to better health, IK notes, World Bank No 35, 2001, 113–115. Bhutani KK. Herbal Wealth of North-East India- A Pictorial and Herbaria Guide, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab, India, 2008. Bieski IG, Santos FR, Oliveira RM et al. Ethnopharmacology of medicinal plants of the Pantanal Region (Mato Grosso, Brazil). Evid Based Complement Alternat Med. 2012; 2012: 1–36. 60 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Choudhury S, Sharma P, Choudhury MD, Sharma GD. Ethnomedicinal plants used by Chorei Assam, North India. Asian Pac J Trop Dis. 2012; S141–S147. Ekka NR, Dixit VK. Ethno-pharmacognostical studies of medicinal plants of jashpur district (chhattisgarh). Int J Green Econ Pharm. 2007; 1(1): 1–4. Gedif T, Han HJ. Use of medicinal plants for self care in Butagra ceteral Ethiopia. J Ethnopharmacol. 2003; 87: 155–161. Giday M, Asfaw Z, Woldu Z. An ethnobotanical study of medicinal plants used by the Zay people in Ethiopia. J Ethnopharmacol. 2002; 85: 43–52. Gurib Fakin A. Medicinal plants, traditions of yesterday and drugs of tomorrow. Molecular Aspects Med. 2006; 27: 5–13. Inngjerdingen K, Nergard C, Dillo D, Mounkore PJ. An ethnopharmacological survey of plants used for wound healing in Dogonland, Mali, West Africa. J Ethnopharmacol. 2004; 92(2–3): 233–244. Kujur M, Ahirwar RK. Folklore claims on some ethno medicinal plants used by various tribes of district Jashpur, Chhattisgarh, India. Int J Curr Microbiol App Sci. 2015; 4(9): 860–867. Lee KH. Current developments in the drug discovery and design of new drug candidates from plant natural leads. J Nat Prod. 2004; 67(2): 273–283. Mesfin T, Hunde O, Getachew Y, Tadesse M. Survey of medicinal plants used for treatment of human diseases in Seka Chekorsa, Jimma Zone Ethiopia. Ethiop J Hlth Sci. 2005; 15(2): 90–95. Patel DK. Medicinal plants in G.G.V. Campus, Bilaspur, Chhattisgarh in central India. Int J Med Arom Plants. 2012; 2(2): 292–300. Pramon E. The commercial use of traditional knowledge and medicinal plants in Indonesia, www.eisecier/locate/jeb pharm, 2002, 73–75. Wolde B, Gebre-Mariam T. Household herbal remedies for self-case in Addis Ababa. Ethiop Pharm J. 2002; 20(1): 61–67. Quality Control of 7 Herbal Medicine

7.1 INTRODUCTION Herbal medicines (HMs) and their preparations have been widely used for thousands of years in many oriental countries, such as India, China, Korea, Japan, and so on, (Liang et al., 2004) and they are attracting more and more attention from all over the world. Individual countries are also giving increasing emphasis to promote their use under the direction of the World Health Organization (WHO). However, in African and Asian countries, traditional medicines are the only affordable option. On the other hand, the same medicines are the option of choice in developed nations like Japan and the United States and in the European States. Despite being the more common medical option in Africa, use of traditional medicines has not matured to the expected level. However, some countries in Asia, especially India and China, have developed them to a level that has benefited all countries of the world. These medicines are affordable, safer, and better tolerated by the biological system. This has led to an increased consumption and cross-country movement of the raw materials of medicinal plants. But the uncontrollable quality of HMs is the obstacle for internationalization and modernization. Because of uncertainty and complexity, there is great difficulty in establishing a specific method of quality control for HMs.

7.2 QUALITY CONTROL: PRESENT SCENARIO The quality control of HMs has become more challenging and demanding. The quality considerations of drugs are the most stringent among all consumer products. The purity of active pharmaceutical ingredients has been stretched to an all-time high with more and more restrictions on the level of the impurities. The situation is very different in the case of plant-derived medicine, where we are still striving to define specifications to ensure consistency and safety. Therefore, the standards of plant drugs are more relaxed and are in the process of development (Huang et al., 2002). The inherent problems of plant drugs are obvious because they are combinations of infinite chemical molecules, known and unknown; inadequate knowledge about active constituents, huge variations in the content and quality of active constituents, and complete chemical profiling of plant drugs is beyond the present scope. Therefore, standardization and quality control for such drugs is not an easy task and a comprehensive system of standards cannot be laid down for such drugs. Morphological identification and microscopic identification are utilized to determine the authenticity of HMs, and the physical and chemical characteristics are used to evaluate the quality of herbs in the existing quality standards. However, chromatographic fingerprinting analysis features the fundamental attributions of

61 62 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

“integrity” and “fuzziness” or “sameness” and “difference,” which can chemically represent the characteristics of the HM investigated. Fingerprint analysis has been internationally accepted as one of the efficient methods to control the quality of herbal medicines (Liang et al., 2004). Two key issues are involved in the development of a fingerprint method: (i) how to gain more effective and stable information and (ii) how to evaluate the similarities and differences with a chemometric method (Liang et al., 2009). The fingerprinting of herbal medicines is really an interdisciplinary and comprehensive research which is based on the chemical composition of a traditional herbal medicinal system. It needs the crossover of herbal medicine, separation science, analytical science, and bioinformatics to provide a platform for the quality control of traditional herbal medicines. Quality control of herbal medicines can be performed using a “component-based” approach and “pattern-based” approach (Mok and Chau, 2006). The compound- oriented approach includes the marker approach and the multi-compound approach. The pattern-oriented approach, namely fingerprint analysis, is more popular now, because most HMs’ chemical ingredients have been studied, but it is difficult to determine which compound is effective (Kong et al., 2009; Zhou et al., 2008). The research and establishment of fingerprints contributed much to solving the quality control problem of herbal medicines. The fingerprint analysis has been internationally accepted as one of the efficient methods to control the quality of herbal medicines as it can evaluate the integrative and holistic properties of herbal medicines by comparing the similarity and correlation of the analytes. This technique is effective throughout the whole producing process, such as manufacture, processing, storage of raw materials for preparation, intermediate products, finished products, and distribution of products (Liang et al., 2004). The Food and Drug Administration has also started to accept fingerprinting, because the fingerprint method can be utilized for the quality control of the Botanical Drug Substances and Botanical Drug Products in application materials named Chemistry, Manufacture, and Control (CMC) of Investigations of New Drugs (IND). Furthermore, France, Germany, Britain, India, and the WHO adopted fingerprinting to evaluate the quality of medicinal plants.

7.3 QUALITY CONTROL OF HERBAL DRUGS Quality control for the efficacy and safety of herbal products is of paramount importance. Quality can be defined as the status of a drug that is determined by identity, purity, content, and other chemical, physical or biological properties, or by the manufacturing processes. Quality control is a term that refers to processes involved in maintaining the quality and validity of a manufactured product. Quality control is based on three important pharmacopoeial definitions, that is, identity, purity, and content or assay.

7.3.1 identity Identity can be achieved by macro- and microscopical examinations. Outbreaks of diseases among plants may result in changes to the physical appearance of the plant and lead to incorrect identification. Quality Control of Herbal Medicine 63

7.3.2 Purity Purity is closely linked with the safe use of drugs and deals with factors such as ash values, contaminants (e.g. foreign matter in the form of other herbs), and heavy metals. However, due to the application of improved analytical methods, modern purity evaluation also includes microbial contamination, aflatoxins, radioactivity, and pesticide residues. Analytical methods such as photometric analysis (UV, IR, MS, and NMR), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography (GC) can be employed in order to establish the constant composition of herbal preparations.

7.3.3 content or Assay It is obvious that the content is the most difficult property to assess, since in most herbal drugs, the active constituents are unknown. Sometimes markers can be used which are, by definition, chemically defined constituents that are of interest for control purposes, independent of whether they have any therapeutic activity or not. To prove the identity and purity, criteria such as the type of preparation, sensory properties, physical constants, adulteration, contaminants, moisture, ash content, and solvent residues have to be checked. The correct identity of the crude herbal material, or the botanical quality, is of prime importance in establishing the quality control of herbal drugs (EMEA, 2001; Sharma, 1995; WHO, 2009).

7.4 STABILITY STUDIES OF HERBAL MEDICINES Herbal medicinal drugs may have a single active constituent or the entire herb source may be considered as the medicinal product. Most of herbal drug products used are groups of constituents. A stable drug product maintains its identity, strength, and therapeutic effect within given specifications throughout the shelf life. Herbal medicinal products are of different natures, from thermo-labile to volatile. Stability testing is an obligatory requirement in the registration process for all medicinal products, including Herbal Medicinal Products (HMPs). Stability testing of herbal products is a complicated issue because the entire herb or herbal product is regarded as the active substance, regardless of whether the constituents with defined therapeutic activity are known. The stability testing of herbal products checks the quality of herbal products which varies with time under the influence of environmental factors, such as temperature, humidity, light, oxygen, and moisture, other ingredients or excipients in the dosage form, particle size of drug, microbial contamination, trace metal contamination, leaching from the container, and so on, and also provide statistics for the determination of shelf-lives (Sachan and Kumar, 2015). Therefore, evaluation of the parameters based upon chemical, physical, microbiological, therapeutic, and toxicological studies can serve as an important tool in stability studies. The tests are performed to define storage conditions and the product’s shelf life (Pingale et al., 2008). Stability studies should be performed on at least three production batches of the herbal products for the proposed shelf life, which is normally denoted as long term stability and is performed under natural atmospheric conditions. With the help 64 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines of modern analytical techniques like spectrophotometry, HPLC, and HPTLC and by employing proper guidelines, it is possible to generate sound stability data of herbal products and predict their shelf life, which will help in improving the global acceptability of herbal products (Thakur et al., 2011). In many aspects, stability testing of HMPs follows the same requirements as stability testing of chemically defined substances. However, some specific characteristics have to be taken into consideration:

• Herbal drugs and preparations (extracts) are the active pharmaceutical ingredient. • HMPs are complex in nature due to their high numbers of constituents. • Constituents belong to different chemical classes with different analytical behaviors. • Constituents sometimes have very low concentrations in the finished product.

7.4.1 specific Characteristics of Herbal Medicinal Products Herbal drugs and preparations are classified in their entirety, such as the active pharmaceutical ingredient (API) in the HMP. From the chemical and analytical point of view, herbal drugs, herbal preparations, and HMPs are complex in nature due to the high numbers of constituents belonging to different chemical classes and having different analytical behaviors (for example, flavonoids versus essential oils). The stability testing of HMPs, in view of the complex and natural composition of their constituents, should take account of the particular requirements and conditions. Although studies are generally comparable with those for products containing chemically defined substances, the specific features for herbals are as follows; the herbal drug substance should be at only 25°C/60% relative humidity with no requirement for intermediate/accelerated testing.

7.4.2 analytical Methods for Herbal Products The analysis of herbal preparations is mostly done by running high performance liquid chromatography (HPLC) or gas chromatography (GC) and thin layer chromatography (TLC) methods, quantitative determinations by UV-Visible spectroscopy or combinations of these. HPLC and GC methods can be used for identification and purity testing, as well as the detection of single compounds for assay and are possible during one analysis. LC and GC mass coupling are also tools for determination, but they are highly sophisticated and expensive methods.

7.4.3 stability Study of Herbal Drugs The stability is aimed at assuring that the drug and drug product remains within the specifications established to ensure its identity, strength, quality, and purity. It can be interpreted as the length of time under specific conditions and storage that a product will remain within the predefined limits for all its important characteristics. Each ingredient, whether therapeutically active or inactive, can affect stability. Stability Quality Control of Herbal Medicine 65 testing on typical natural extracts such as flavonoids containing herbal drugs has been reported by researchers to understand the stability criteria for natural products (Heigl et al., 2003; Poetsch et al., 2006).

7.4.4 shelf Life The determination of shelf life of herbal medicinal drug products is the same as chemically defined APIs, but the special nature of herbal products should be taken into consideration. It is recommended that in case of a herbal medicinal product containing a natural product or a herbal drug preparation with constituents of known therapeutic activity, the variation in components during the proposed shelf life should not exceed ±5% of the initial assay value, unless it is justified to widen the range up to ±10% or even higher. The low marker concentration in the finished product justifies the wider range.

7.4.5 challenges in Stability Testing of Herbal Medicinal Products Evaluating the stability of HMPs presents a number of challenges when compared to chemically defined substances. In particular, active substances in HMPs consist of complex mixtures of constituents and in most cases, the constituents responsible for the therapeutic effects are unknown. The situation is further complicated when two or more herbal substances and/or herbal preparations are combined in an HMP. In addition, many herbal substances/herbal preparations are known to be unstable. As part of a total control strategy for herbal substances, herbal preparations, and HMPs, a set of test criteria including qualitative and quantitative parameters has been recognized as indicating the quality of the substances, preparations, and HMPs. With regard to stability tests, chromatographic fingerprints as well as appropriate methods of assay via marker substances represent the fundamental part of this concept, laid down in shelf life specifications. Notwithstanding the appropriateness of this approach, its realization is often associated with analytical problems and high costs. In summary, HMPs have a number of characteristics that clearly differentiate them from chemically defined medicinal products, therefore, specific stability guidance needs to be established, which covers particular aspects that existing specific herbal guidelines and general guidelines on stability do not address.

7.4.6 Predictable Changes in Herbal Drug Material The following predictable changes may occur in an herbal medicinal product during storage and in shelf life determination: hydrolysis, oxidation, racemization, geometric isomerization, temperature, moisture, and light. Environmental factors such as temperature, light, air (specifically oxygen, carbon dioxide, and water vapor), and humidity can affect stability. Similarly, factors such as particle size, pH, the properties of water and other solvents employed, the nature of container, and the presence of other chemicals resulting from contamination or from the intentional mixing of different products can influence stability (Figure 7.1). Physical instability is one of the major problems related to the stability of herbal products. This is due to 66 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Storage conditions Drug interaction Content variability

Moisture content Packaging interaction

Stability

Mold growth Insect attack

Container Microorganisms Environment factors

FIGURE 7.1 Factors affecting stability of natural medicines. the presence of impurities and reactions with the container. Volatility is the problem related to the active components of natural medicine and their decreasing activity during storage for a long time. The rate of chemical reaction increases with an increase in temperature and this leads to degradation of quality. Thus, this tropical area must be taken into consideration during preparation of the formula of the herbal substance. Moisture absorbed on the surface of a solid drug will often increase the rate of decomposition if it is susceptible to hydrolysis. Many types of chemical reactions are induced by exposure to light of high energy. Autoxidation of volatile oil/fixed oil takes place and the substance becomes colored.

7.4.7 importance of Stability Testing This evaluates the efficacy of a drug. Stability studies are used to develop suitable packaging information for quality, strength, purity, and integrity of the product during its shelf life. It is used for determination of the shelf life. Depending on the type of preparation, sensory properties, physical constants, moisture, ash content, solvent residues, and adulterations have to be checked to prove identity and purity. Microbiological contamination and foreign materials such as heavy metals, pesticide residues, aflatoxins, and radioactivity also need to be tested for. Impurities in herbal products are also a big concern related to their stability. This can be traced by the analytical methods such as high performance liquid chromatography (HPLC), capillary electrophoresis, spectrophotometry, gas chromatography-mass spectrometry (GC-MS), thin layer chromatography (TLC), and so on (Mukherjee et al., 2007). Deterioration of herbal products can also be protected against by using airtight containers made of materials that will not interact physically or chemically with the material being stored. Storage in a ventilated cool, dry area and periodic spraying of the stored area with insecticides will help to prevent the spread of infestation (Thakur et al., 2008). A few advanced techniques are also available, which deal with instability problems related to natural medicines such as nanoparticles, that is, nanospheres and nanocapsules, liposomes, proliposomes, solid lipid nanoparticles, nanoemulsions, and so on. Nanocoating of active components of an herbal formulation is effective in Quality Control of Herbal Medicine 67 protecting the active drug molecule from oxidative, hydrolytic, and environmental degradation processes and hence enhances the shelf life of the herbal products (Musthaba et al., 2004). A supercritical carbon dioxide technique has been found to effectively increase the stability of the herbal preparation with a high amount of active principle.

7.5 BIOLOGICAL MARKERS FOR HERBAL MEDICINES The term marker compounds can be defined as standard reference compounds used for the purpose of comparison and quality control purposes. Development of a marker provides a suitable and important parameter for quality control of plants and herbal formulations. The selection of biological markers is crucial for the quality control of herbal medicines, including authentication of genuine species, harvesting the best quality raw materials, evaluation of post-harvesting handling, assessment of intermediates and finished products, and detection of harmful or toxic ingredients. Marker assisted selection of desirable chemotypes along with authentication of species identity and prediction of the concentration of active phytochemicals may be required for quality control in the use of plant materials for pharmaceutical purposes. Identification of DNA markers that can correlate DNA fingerprinting data with a quantity of selected phytochemical markers associated with that particular plant would have wide applications in quality control of raw materials. Ideal chemical markers should be the therapeutic components of herbal medicines. However, for most herbal medicines, the therapeutic components have not been fully elucidated or easily monitored. Bioactive, characteristic, main, synergistic, correlative, toxic, and general components may be selected. Quality control of herbal medicines aims to ensure their consistency, safety, and efficacy. Chemical fingerprinting has been demonstrated to be a powerful technique for the quality control of herbal medicines. A chemical fingerprint is a unique pattern that indicates the presence of multiple chemical markers within a sample.

7.5.1 Markers are Categorized into Two Classes • DNA markers are reliable for informative polymorphisms as the genetic composition is unique for each species and is not affected by age, physiological conditions or environmental factors. DNA can be extracted from fresh or dried organic tissue of the botanical material; hence the physical form of the sample for assessment does not restrict detection. • Chemical markers generally refer to biochemical constituents, including primary and secondary metabolites and other macromolecules such as nucleic acids.

7.5.1.1 DNA Markers DNA markers as new pharmacognostic tools. Various types of DNA-based molecular techniques are utilized to evaluate DNA polymorphism. These are hybridization- based methods, Polymerase Chain Reaction (PCR)-based methods, and sequencing- based methods. 68 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

7.5.1.1.1 Applications of DNA Markers DNA-based molecular markers have proved their utility in fields such as taxonomy, physiology, embryology, genetics, and so on.

• Genetic variation/genotyping: RAPD-based molecular markers have been found to be useful in differentiating different accessions of herbal drugs collected from different geographical regions. Interspecies variation has been studied using RFLP and RAPD in different genera such as Glycerrhiza, Echinacea, Curcuma, and Arabidopsis. RAPD has served as a tool for the detection of variability in Jojoba (Simmondsia chinensis L. Schneider), Vitis vinifera L., and tea (Camellia sinesis). • Authentication of medicinal plants: Sequence Characterized Amplified Region (SCAR), arbitrarily primed polymerase chain reaction (AP–PCR), RAPD, and RFLP have been successfully applied for differentiation of these plants and to detect substitution by other closely related species. Certain rare and expensive medicinal plant species are often adulterated or substituted by morphologically similar, easily available or less expensive species. For example, Swertia chirata is frequently adulterated or substituted by the cheaper Andrographis paniculata. • Marker assisted selection of desirable chemo types: Amplified fragment length polymorphism (AFLP) analysis has been found to be useful in predicting phytochemical markers in cultivated Echinacea purpurea germplasm and some related wild species DNA profiling has been used to detect the phylogenetic relationship among Acorus calamus chemotypes differing in their essential oil composition. • Medicinal plant breeding: Molecular markers have been used as a tool to verify sexual and apomictic offspring of intraspecific crosses in Hypericum perforatum, a well-known antihelminthic and diuretic. • Applications in foods and nutraceuticals: Roundup ready soybeans, maize, cecropin, and capsicum have been successfully discriminated from non- genetically modified (GM) products using primers specific for inserted genes and crop endogenous genes.

These markers have shown remarkable utility in quality control of commercially important botanicals like Ginseng, Echinacea, and Atractylodes. Although DNA analysis is currently considered to be cutting-edge technology, it has certain limitations due to which its use has been limited to academia. Another important issue is that DNA fingerprints will remain the same irrespective of the plant part used, while the phytochemical content will vary with the plant part used, the physiology, and the environment.

7.5.1.2 Chemical Markers Selection of chemical markers is crucial for the quality control of herbal medicines, including authentication of genuine species, harvesting the best quality raw materials, evaluation of post-harvesting handling, assessment of intermediates and finished products, and detection of harmful or toxic ingredients. Chemical markers as chemically defined constituents or groups of constituents of an herbal medicinal Quality Control of Herbal Medicine 69 product are of interest for quality control purposes regardless of whether they possess any therapeutic activity as defined by European Medicines Agency (EMEA). Chemical markers can be further categorized as therapeutic components, bioactive components, synergistic components, characteristic components, main components, correlative components, toxic components, and general components used with fingerprint spectra. All markers may contribute to the evaluation, standardization, and safety assessment of herbal medicines (Figure 7.2).

7.5.1.2.1 Therapeutic Components Therapeutic components possess the direct therapeutic effects of an herbal medicine. They may be used as chemical markers for both qualitative and quantitative assessments. Therapeutic components were originated from bulbs of Bulbus fritillariae, which is commonly prescribed as an antitussive and expectorant. Verticine, verticinone, and imperialine were identified as the major therapeutic components that account for the antitussive effect (Li et al., 2006). Therefore, isosteroidal alkaloids were selected as the chemical markers for the quality assessment of Bulbus Fritillariae using a series of chromatographic techniques such as pre-column derivatizing gas chromatography–flame ionization detection (GC-FID), direct GC-FID, gas chromatography–mass spectrometry (GC-MS), pre- column derivatizing high performance liquid chromatography–ultraviolet detection (HPLC-UV), high performance liquid chromatography–evaporative light scattering detection (HPLC-ELSD), and high performance liquid chromatography–mass spectrometry (HPLC-MS) methods (Lin et al., 2001; Wang et al., 2010).

7.5.1.2.2 Bioactive Components Bioactive components are structurally different chemicals within an herbal medicine; while individual components may not have direct therapeutic effects, the combination of their bioactivities does contribute to the therapeutic effects. Bioactive components may be used as chemical markers for qualitative and quantitative assessment. Bioactive components, including isoflavonoids and saponins, were simultaneously used in the evaluation of the quality of Radix Astragali (Song et al., 2007; Yu et al., 2007).

7.5.1.2.3 Synergistic Components Synergistic components do not directly contribute to the therapeutic effects or related bioactivities. However, they act synergistically to reinforce the bioactivities of other components, thereby modulating the therapeutic effects of the herbal medicine. Synergistic components may be used as chemical markers for qualitative and quantitative assessment. Naphthodianthrone, hypericin, and hyperforin (a phloroglucinol derivative) were identified as the major components that contribute to the pharmacological activities of St John’s wort (Butterweck et al., 2007). Rutin is a ubiquitous flavonoid that demonstrated synergistic antidepressant actions in St John’s wort (Noldner and Schota, 2002).

7.5.1.2.4 Characteristic Components While characteristic components may contribute to the therapeutic effects, they must be specific and unique ingredients of an herbal medicine. Valerenic acids, the 70 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

HO CH3 HH

H H H N N H H H OH O H CH3 H H CH3 H H

HH HO HO Imperialine H Verticinone O OH

OH OHO HO O OH OH HO O OH HO OH O CH3 HO O O OH

HO OHO HO O Naphthodianthrone Rutin OH

CO H O 2

O O NO2

OO Isopsoralen OCH3 Aristolochic acids O HO O O O

HO OH O O O O OH OH O OH O Psoralen Icarrin OH OH

FIGURE 7.2 Chemical markers. Quality Control of Herbal Medicine 71 characteristic components of valerian derived from the roots of Valeriana officinalis L., have sedative effects and improve sleep quality (Bent et al., 2006). Valerenic acids are used as chemical markers to evaluate the quality of valerian preparations although their sedative effects have not been fully elucidated.

7.5.1.2.5 Main Components Main components are the most abundant components in an herbal medicine (or are significantly more abundant than other components). They are not characteristic components and their bioactivities may not be known. Main components may be used for both qualitative and quantitative analysis of herbal medicines, especially for differentiation and stability evaluation. Flavonoids, including epimedin A, B, C, and icariin are the main components of Herba Epimedii. Total flavonoids and icariin are used as chemical markers for Herba Epimedii (Pei and Guo, 2007).

7.5.1.2.6 Correlative Components Correlative components in herbal medicines have a close relationship with one another. Correlative components can be used as chemical markers to evaluate the quality of herbal medicines originated from different geographical regions and stored for different periods of time. Psoralen and isopsoralen are used as chemical markers for assessing the quality of Fructus Psoraleae (Qiao et al., 2006).

7.5.1.2.7 Toxic Components Traditional Chinese medicine literature and modern toxicological studies have documented some toxic components of medicinal herbs. For instance, aristolochic acids (AAs) and pyrrolizidine alkaloids (PAs) may cause nephrotoxicity and hepatotoxicity, respectively (Cosyns, 2003).

7.5.1.2.8 Applications of Chemical Markers The applications of chemical markers include identification of adulterants, differentiation of herbal medicines with multiple sources, determination of the best harvesting time, confirmation of collection sites, assessment of processing methods, quality evaluation of herbal parts, identification and quantitative determination of proprietary products, stability test of proprietary products, diagnosis of herbal intoxication and lead compounds for new drug discovery.

7.6 CONCLUSION Quality control of herbal medicines aims to ensure their quality, safety, and efficacy. The use of chromatographic techniques and marker compounds to standardize botanical preparations has limitations because of their variable sources and chemical complexity. Markers can have a vital role in various applications such as applications of molecular markers in herbal drug technology for authentication, detection of adulteration/substitution of medicinal plants, marker assisted selection of desirable chemotypes, DNA markers as new pharmacognostic tools, marker applications in foods and nutraceuticals, and for the purposes of safety and efficacy of the drugs. DNA-based molecular markers have utility in the fields such as taxonomy, physiology, 72 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines embryology, genetics, and so on. Chemical markers are pivotal in the current practice of quality control. Chemical markers should be used at various stages of the development and manufacturing of an herbal medicine, such as authentication and differentiation of species, collecting and harvesting, quality evaluation and stability assessment, diagnosis of intoxication, and discovery of lead compounds. Lack of chemical markers remains a major problem for the quality control of herbal medicines. Furthermore, there are many technical challenges in the production of chemical markers. For example, temperature, light, and solvents often cause degradation and/ or transformation of purified components; isomers and conformations may also cause confusion to chemical markers. The fingerprinting profile of the marker compounds in plant drugs which show the presence/percentage of the active principle along with the closely related bioactive principles is necessary for all herbal formulations.

REFERENCES Bent S, Padula A, Moore D, Patterson M, Mehling W. Valerian for sleep: A systematic review and meta-analysis. Am J Med. 2006; 119: 1005–1012. Butterweck V, Schmidt M. St. John’s wort: Role of active compounds for its mechanism of action and efficacy. Wien Med Wochenschr. 2007; 157: 356–361. Cosyns JP. Aristolochic acid and “Chinese herbs nephropathy”: A review of the evidence to date. Drug Safety. 2003; 26: 33–48. EMEA. Note for Guidance on Quality of Herbal Medicinal Products. The European Agency for the Evaluation of Medicinal Products, London, UK. 2001. Heigl D, Franz G. Stability testing on typical flavonoid containing herbal drugs. Pharmazie Govi-Verlag Pharmazeutischer Verlag GmbH. 2003; 58: 881–885. Huang Y, Qin MJ, Yang G, Xu LS, Zhou KY. Chinese traditional and herbal drugs. 2002; 33(10): 935–937. Kong WJ, Zhao YL, Xiao XH, Jin CZLL. Quantitative and chemical fingerprint analysis for quality control of Rhizoma Coptidis chinensis based on UPLC-PAD combined with chemometrics methods. Phytomedicine. 2009; 16: 950–959, 0944–7113. Li HJ, Jiang Y, Li P. Chemistry, bioactivity and geographical diversity of steroidal alkaloids from the Liliaceae family. Nat Prod Rep. 2006; 23: 735–752. Liang XM, Jin Y, Wang YP, Jin GW, Fu Q, Xiao YS. Qualitative and quantitative analysis in quality control of traditional Chinese medicines. J Chromatogr A. 2009; 1216(2008) 2033–2044, 0021–9673. Liang YZ, Xie PS, Chan K. Quality control of herbal medicines. Chromatogr. B 812, 2004; 53–70, 1570–0232. Lin G, Li P, Li SL, Chan SW. Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb. J Chromatogr A. 2001; 935: 321–338. Mok DKW, Chau FT. Chemical information of Chinese medicines: A challenge to chemist. J. Chemometrics and Intelligent Laboratory Systems 2006; 82(2005): 210–217, 0269–3879. Mukherjee PK. Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals. First edition. Business Horizons, India, 2002, pp. 113–119. Musthaba SM, Ahmad S, Ahuja A, Ali J, Baboota S. Nano approaches to enhance pharmacokinetic of stable nanoparticulate drugs have the improved stability than conventional dosage forms. PCT Int Appl. 2004; 68. Noldner M, Schota K. Rutin is essential for the antidepressant activity of Hypericum perforatum extracts in the forced swimming test. Planta Med. 2002; 68: 577–580. Quality Control of Herbal Medicine 73

Pei LK, Guo BL. A review on research of raw material and cut crude drug of Herba Epimedii in last ten years (Chinese). Zhongguo Zhongyao Zazhi, 2007; 32: 466–471. Pingale SS, Pokharkar RD, Pingale MS. Stability study of a herbal drug. Pharmacologyonline. University of Salerno. 2008; 1: 20–3. Poetsch FA, Steinhoff B. Cantor Verlag. Stability testing of herbal medicinal products: A report on problematic cases from practice with discussion of possible resolution approache. Pharmazeutische Industrie. 2006; 68: 476–483. Qiao CF, Han QB, Song JZ, Mo SF, Kong LD, Kung HF, Xu HX. Quality assessment of Fructus Psoraleae. Chem Pharm Bull. 2006; 54: 887–890. Sachan AK, Kumar A. Stability testing of herbal products. J Chem Pharm Res. 2015; 7(12): 511–514. Sharma PP. How to Practice GMPs. Vandana Publications, Lucknow. 1995. Song JZ, Mo SF, Yip YK, Qiao CF, Han QB, Xu HX. Development of microwave assisted extraction for the simultaneous determination of isoflavonoids and saponins in Radix Astragali by high performance liquid chromatography. J Sep Sci. 2007; 30: 819–824. Thakur AK, Prasad NA, Laddha KS. Stability testing of herbal products. The Pharma Review. 2008; 4: 109–112. Thakur L, Ghodasra U, Patel N, Dabhi M. Novel approaches for stability improvement in natural medicines. Pharmacogn Rev. 2011; 5(9): 48–54. Wang HL, Yao WF, Zhu DN, Hu YZ. Chemical Fingerprinting by HPLC-DADELSD and Principal Component Analysis of Polygala japonica from Different Locations in China. J.Chinese Journal of Natural Medicines. 2010; 8(sep.2010): 343−348. WHO. Stability testing of active pharmaceutical ingredients and finished pharmaceutical products. WHO Technical Report Series, 2009; No. 953, Annex 2. Yu QT, Qi LW, Li P, Yi L, Zhao J, Bi Z. Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi (Radix Astragali) by HPLC-DAD-ELSD. J Sep Sci. 2007; 30: 1292–1299. Zhou FR, Zhao MB, Tu PF. Qualitative evaluation and quantitative determination of 10 major active components in Carthamus tinctorius L. by high-performance liquid chromatography coupled with diode array detector. J Chromatogr A. 2008; 1216(2009): 2063–2070, 0021–9673.

Bioavailability of 8 Herbal Drugs

Bioavailability is the rate and extent to which a substance enters systemic circulation and becomes available at the required site of action (Brahmankar and Jaiswal 1995). Thus, there is need for molecules which themselves have no therapeutic activity, but when combined with other drugs/molecules, enhance their bioavailability. The impact of bioavailability in drug discovery and development is even more pronounced with products intended for oral use, whereby gastro-intestinal (GI) absorption constitutes the primary barrier between an active ingredient and systemic circulation. Thus, bioavailability should also be considered when the efficacy of herbal dietary supplements is evaluated in animal models and/or human clinical trials. Many natural compounds from medicinal plants have the capacity to augment bioavailability when co-administered with another drug. Thus, bioenhancers are chemical entities which promote and augment the bioavailability of the drugs which are mixed with them and do not exhibit synergistic effects with the drug (Drabu et al., 2011). Bioenhancers are such agents which by themselves are not therapeutic entities, but when combined with an active drug lead to the potentiation of the pharmacologic effect of the drug. Such formulations have been found to increase the bioavailability/ bioefficacy of a number of drugs, even when reduced doses of drugs are present in such formulations (Randhawa et al., 2011). Evidence has been obtained for such classes of drugs which are (a) poorly bioavailable and/or efficacious, (b) require prolonged therapy, and (c) are highly toxic and expensive. These are phytomolecules, the development of which is based on the ancient knowledge of Ayurveda. They augment the bioavailability or biological activity of drugs when administered at low doses. They reduce the dose and shorten the treatment period thus reducing drug- resistance problems. The treatment also is made cost effective, minimizing drug toxicity and adverse reactions. When used in combination with a number of drug classes such as antibiotics, antituberculosis, antiviral, antifungal, and anticancerous drugs, they are quite effective. Oral absorption of vitamins, minerals, herbal extracts, amino acids, and other nutrients are improved by them. They act through several mechanisms which may mainly affect the absorption process, drug metabolism or action on the drug target.

8.1 NEED FOR BIOAVAILABILITY ENHANCERS Lipid solubility and molecular size are the major limiting factors for molecules to pass the biological membrane and to be absorbed systematically following oral or topical administration. Several plant extracts and phytoconstituents, despite having excellent bioactivity in vitro, demonstrate less or no in vivo action due to their poor lipid solubility or improper molecular size or both, resulting in poor absorption and

75 76 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines poor bioavailability. It is often found that when individual constituents are isolated from the plant extract, there is a loss of specific bio-activity (Kesarwani and Gupta 2013). Bioenhancers should have novel properties:

• Should be nontoxic to humans or animals • Should be effective at a very low concentration in a combination • Should be easy to formulate • Should complement uptake/absorption and activity of the drug molecules

8.2 DRUG ABSORPTION BARRIERS The drug must cross the epithelial barrier of the intestinal mucosa for it to be transported from the lumen of the gut into the systemic circulation and exert its biological actions. There are many anatomical and biological barriers that must be overcome for the oral drug delivery system to penetrate the epithelial membrane. There are many structures in the intestinal epithelium which serve as barriers to the transfer of drugs from the gastrointestinal track to the systemic circulation. The hydrophilic nature of an aqueous stagnant layer is a potential barrier to the absorption of drugs. The membranes around cells are lipid bilayers containing proteins such as receptors and carrier molecules. Drugs cross the lipid membrane by passive diffusion or carrier mediated transport which involves the spending of energy (Viega et al., 2000). For the passage of small water soluble molecules such as ethanol, there are aqueous channels within the proteins. P-glycoprotein inhibits drug entry into the systemic circulation. P-glycoprotein is a type of ATPase and an energy dependent transmembrane drug efflux pump, thus it belongs to members of ATP-binding cassette (ABC) transporters. Drug molecules larger than about 0.4 nm face difficulty in passing through these aqueous channels (Juliano and Ling 1976).

8.3 MECHANISM OF ACTION OF BIOENHANCERS The following are the chief mechanisms via which the various bioenhancers exert their bioavailability by enhancing properties on the drug molecules:

1. By enhancing the absorption of orally administered drugs from the gastrointestinal tract by an increase in blood supply (Dudhatra et al., 2012). 2. By modulating the active transporters located in various locations, for example: P-glycoprotein (P-gp) is an efflux pump which pumps out a drug and prevents it from reaching the target site. Bioenhancers in such a case act by inhibiting the P-gp. 3. Decreasing the elimination process thereby extending the sojourn of a drug in the body. a. Inhibiting the drug metabolizing enzymes such as CYP 3A4, CYP1A1, CYP1B2, and CYP2E1 in the liver, gut, lungs, and various other locations. This will, in addition, help to overcome the first pass effect of administered drugs. Bioavailability of Herbal Drugs 77

b. Inhibiting the renal clearance by preventing glomerular filtration and active tubular secretion by inhibiting P-gp and facilitating passive tubular reabsorption. Sometimes biliary clearance is also affected by inhibiting the uridine diphosphate (UDP) glucuronyl transferase enzyme which conjugates and inactivates the drug (Kang et al., 2009).

In addition to the above mentioned mechanisms, a few other postulated theories for herbal bioenhancers are also known such as a reduction in hydrochloric acid secretion and increase in gastrointestinal blood supply, inhibition of gastrointestinal transit, gastric emptying time and intestinal motility, modifications in gastrointestinal tract (GIT) epithelial cell membrane permeability, cholagogous effect, bioenergetics and thermogenic properties, suppression of first pass metabolism, inhibition of drug metabolizing enzymes, and stimulation of gamma glutamyl transpeptidase (GGT) activity which enhances the uptake of amino acids (Tatiraju et al., 2013).

8.4 MEDICINAL PLANTS AND THEIR COMPOUNDS AS DRUG BIOAVAILABILITY ENHANCERS Piperine (1-piperoyl piperidine) is a pioneer alkaloidal component of Piper nigrum Linn, which increases the bioavailability, blood levels, and efficacy of a number of drugs including ingredients of vasaka leaves, vasicine, sparteine, rifampicin, phenytoin, sulfadiazine, and propranolol (Patil et al., 2011). Ginger (Zingiber officinale) has a powerful effect on the GIT mucous membrane. It increases the bioavailability of different antibiotics such as azithromycin (85%), erythromycin (105%), cephalexin (85%), cefadroxil (65%), amoxycillin (90%), and cloxacillin (90%) (Drabu et al., 2011). Niaziridin, a nitrile glycoside, is isolated from the pods of Moringa oleifera which enhances the bioactivity of commonly used antibiotics against gram-positive bacteria like myobacterium smegmatis, bacillus subtilis, and gram-negative bacteria such as escherichia coli. Liquorice (Glycyrrhiza glabra) containing glycyrrhizin as an active constituent enhances cell division inhibitory activity of the anticancerous drug “Taxol” by five-fold against the growth and multiplication of breast cancer cell lines. Black cumin (Cuminum cyminum); its bioactive fraction enhances the bioavailability of erythromycin, cephalexin, amoxycillin, fluconazole, ketoconazole, zidovudine, and 5-fluorouracil. The bioavailability/bioefficacy activity of Cuminum cyminum was attributed to various volatile oils, luteolin, and other flavonoids.Garlic (Allium sativum); allicin, the active bioenhancer phytomolecule in garlic, enhances the fungicidal activity of amphotericin B against pathogenic fungi such as candida albicans, aspergillus fumigatus, and the yeast saccharomyces cerevisiae. In developing countries like India, the cost of treatment is a major concern for modern medicines. The drug discovery process has been highly aided by Ayurveda through reverse pharmacology with new means of identifying active compounds and an accompanying reduction of drug development cost. The researchers are now aimed at methods of reduction of drug dosage and drug treatment cost, making treatment available to a wider section of society, including the financially challenged. It has taken its lead from the use of “trikatu” as a bioenhancer from Ayurveda and successfully applied it to various modern medicines to enhance 78 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines their bioavailability. The Ayurvedic concepts of anupaan and sehpaan should also be incorporated into modern medicine. Modern medicine can take a cue from the Ayurvedic lead in developing more efficacious and safe medicines with safer routes of drug administration in the future.

REFERENCES Brahmankar DB, Jaiswal S. Biopharmaceutics and Pharmacokinetics: A Treatise, 1st Edition. Vallabh Prakashan, 1995, 24–26. Drabu S, Khatri S, Babu S, Lohani P. Review article use of herbal bioenhancers to increase the bioavailability of drugs. Res J Pharm, Biol Chem Sci. 2011; 2(4): 107. Dudhatra GB, Modi SK, Awale MM, Patel HB, Modi CM, Kumar A et al. A Comprehensive review on pharmacotherapeutics of herbal bioenhancers. Sci World J. 2012; 1–33. Juliano RL, Ling L. P-glycoprotein, a type of ATPase and an energy dependent transmembrane drug efflux pump. Biochem Biophys Acta. 1976; 555: 152–162. Kang MJ, Cho JY, Shim BH, Kim DK, Lee J. Bioavailability. J Med Plants Res. 2009; 3(13): 1204–1211. Kesarwani K, Gupta R. Bioavailability enhancers of herbal origin: An overview. Asian Pac J Trop Biomed. 2013; 3(4): 253–266. Patil UM, Singh A, Chakraborty AK. Role of piperine as a bioavailability enhancer. Int J Rec Adv Pharm Res. 2011; 1(4): 16–23. Randhawa GK, Kullar JS, Rajkumar. Bioenhancers from mother nature and their applicability in modern medicine. Int J App Basic Med Res. 2011; 1(1): 5–10. Tatiraju DV, Bagade VB, Karambelkar PJ, Jadhav VM, Kadam V. Natural bioenhancers: An overview. J Pharmacogn Phytochem. 2013; 2(3): 55–60. Viega F, Fernandes C, Teixeira F. Oral bioavailbility and hypoglycemic activity of tolbutamide/ cyclodextrin inclusion complexes. Int J Pharm. 2000; 202: 165–171. Thermal Analysis 9 of Herbal Drugs

9.1 INTRODUCTION Herbal medicines widely used in health care in both developed and developing countries are complex chemical mixtures prepared from plants and are limited in their effectiveness because they are poorly absorbed when taken orally. Thermal analysis is a term used to describe the analytical techniques that measure the physical and chemical properties of a sample as a function of temperature while the substance is subjected to a controlled temperature program. It includes thermal gravimetry (TG), differential thermal analysis (DTA), derivative thermogravimetry (DTG), thermo-mechanical analysis (TMA), and dynamic mechanical analysis (DMA) (Gomathinayagam and Venkataraman, 2015). According to an estimate of the World Health Organization (WHO), about 80% of the world’s population still uses herbs and other traditional medicines for their primary health care needs. Thermal analysis is used to characterize the materials, the physical or chemical changes in various products including herbal drugs, and to study the pre-formulation or drug excipient compatibility. These methods are widely used in the quality control of natural and synthetic drugs, because they can quickly provide data on the stability of the analyzed material in the presence of its thermal behavior. Thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimetry (DSC) have been employed to study any physical or chemical changes in various products including herbal drugs and are also used to study pre-formulation or drug excipient compatibility (Silva et al., 2011). TGA may be operated under subambient conditions to analyze ethanol in herbal formulations such as asavas and arista (Yongyu et al., 2011).

9.1.1 thermogravimetry (TG) Thermogravimetric analysis (TGA) is the most widely used thermal method. It is based on the measurement of the mass loss of material as a function of temperature. In thermogravimetry, a continuous graph of mass change against temperature is obtained when a substance is heated at a uniform rate or kept at a constant temperature. A plot of mass change versus temperature (T) is referred to as the thermogravimetric curve (TG curve). For the TG curve, we generally plot mass (m) decreasing downward on the y axis (ordinate), and temperature (T) increasing to the right on the x axis (abscissa). Sometimes we may plot time (t) in place of T. A TG Curve helps in revealing the extent of the purity of analytical samples and

79 80 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines in determining the mode of their transformations within a specified range of temperature (Ma et al., 2017).

9.1.1.1 Characteristics In a TG curve of single stage decomposition, there are two characteristic temperatures; the initial Ti and the final temperature Tf. Ti is defined as the lowest temperature at which the onset of a mass change can be detected by a thermobalance operating under particular conditions. Tf is defined as the final temperature at which the particular decomposition appears to be complete. The difference between Tf and Ti is termed as the reaction interval. In a dynamic thermogravimetry, a sample is subjected to a continuous increase in temperature which is usually linear with time, whereas in isothermal or static thermogravimetry, the sample is maintained at a constant temperature for a period of time during which any change in mass is noted.

9.1.1.2 Applications of Thermogravimetric Analysis • Purity and thermal stability • Solid state reactions • Decomposition of inorganic and organic compounds • Determining composition of the mixture • Corrosion of metals in various atmospheres • Pyrolysis of coal, petroleum, and wood • Roasting and calcinations of minerals • Reaction kinetics studies • Evaluation of gravimetric precipitates • Oxidative and reductive stability • Determining moisture, volatility, and ash contents • Desolvation, sublimation, vaporizations, sorption, and chemisorptions

9.1.2 differential Thermal Analysis (DTA) In Differential Thermal Analysis, the temperature difference that develops between a sample and an inert reference material is measured when both are subjected to identical heat treatments. The related technique of Differential Scanning Calorimetry relies on differences in energy required to maintain the sample and reference at an identical temperature. The analytical method for recording the difference in temperature (T) of a substance and an inert reference material as a function of temperature or time and any transformation change in specific heat or an enthalpy of transition can be detected by DTA (Brandão et al., 2016).

9.1.2.1 Characteristics A DTA curve can be used as a fingerprint for identification purposes. DTA detects the release or absorption of heat, which is associated with chemical and physical changes in materials as they are heated or cooled. Such information is essential for understanding the thermal properties of materials, including analysis of decomposition of glass batch materials, crystalline phase changes, chemical reactions, and glass transition Thermal Analysis of Herbal Drugs 81 temperature. DTA shows two types of heat changes, in which one is endothermic and the other is exothermic. They are classified as,

• Sharp Endothermic: changes in crystallanity or fusion • Physical changes: usually result in endothermic curves • Chemical reactions: exothermic or endothermic

9.1.2.2 Applications • DTA is used for the determination of phase diagrams, heat change measurements, and decomposition in various atmospheres. • DTA is widely used in the pharmaceutical and food industries. • DTA may be used in cement chemistry, mineralogical research, and in environmental studies. • DTA curves may also be used to date bone remains or to study archaeological materials.

9.1.3 dynamic Mechanical Analysis (DMA) Dynamic Mechanical Analysis is a technique where a small deformation is applied to a sample in a cyclic manner. This allows the material’s response to stress, temperature, frequency, and other values to be studied. The term is also used to refer to the analyzer that performs the test. DMA is also called DMTA for Dynamic Mechanical Thermal Analysis. It is one of the most flexible and cost-effective instruments available today. With a fully rotational sample compartment and accessories, you can test samples by simulating real world scenarios easily and effectively. It is widely used to characterize a material’s properties as a function of temperature, time, frequency, stress, atmosphere or a combination of these parameters.

9.1.3.1 Principles of DMA Dynamic mechanical analysis (DMA) yields information about the mechanical properties of a specimen placed in minor, usually sinusoidal, oscillation as a function of time and temperature by subjecting it to a small, usually sinusidal, oscillating force (Figure 9.1). The complex modulus E is the ratio of the stress amplitude to the strain amplitude and represents the stiffness of the material. The magnitude of the complex modulus is:

σA E = εA

9.1.3.2 Instrument and Working of DMA DMA works by applying a sinusoidal deformation to a sample of known geometry. The sample can be subjected to a controlled stress or a controlled strain. For a known stress, the sample will then deform a certain amount. In DMA, this is done sinusoidally. How much it deforms is related to its stiffness. A force motor is used to generate the sinusoidal wave and this is transmitted to the sample via a drive shaft. 82 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Stress σ Strain ε σA

εA

FIGURE 9.1 Sinusoidal oscillation and response of a linear-viscoelastic material where, δ = phase angle.

One concern has always been the compliance of this drive shaft and the effect of any stabilizing bearings to hold it in position (Kevin, 2008; Paul, 2008). The sample is clamped in the measurement head of the DMA instrument. During measurement, a sinusoidal force is applied to the sample via the probe. Deformation caused by the sinusoidal force is detected and the relation between the deformation and the applied force is measured. Properties such as elasticity and viscosity are calculated from the applied stress and strain and are plotted as a function of temperature or time. The block diagram of DMA is shown in Figure 9.2.

9.1.4 thermomechanical Analysis (TMA) A technique in which a deformation of the sample under a non-oscillating stress is monitored against time or temperature, while the temperature of the sample in a specified atmosphere is programmed. The stress may be compression, tension, flexure or torsion. The basis of TMA is the change in the dimensions of a sample as a function of temperature (Menard, 1999).

9.1.4.1 Instrumentation of TMA The sample is inserted into the furnace and is touched by the probe, which is connected with the length detector and the force generator. The thermocouple for temperature measurement is located near the sample. The sample temperature is changed in the furnace by applying the force onto the sample from the force generator via a probe. The sample deformation such as thermal expansion and softening with changing temperature is measured as the probe displacement by the length detector. A Linear Variable Differential Transformer (LVDT) is used as a length detection sensor. The block diagram of DMA is shown in Figure 9.3. Thermal Analysis of Herbal Drugs 83

Force generator

Detector (LVDT)

Probe ermocouple

Furnace

Sample

FIGURE 9.2 Block diagram of DMA.

Force generator

Detector (LVDT) Probe

ermocouple Sample cylinder

Furnace

Sample

FIGURE 9.3 Block diagram of TMA.

9.1.4.2 Applications of TMA TMA applications are, in many ways, the simplest of the thermal techniques. TMA merely measures the change in the height of the sample. The resultant information is useful in supplying information needed to design and process everything from chips to food products to motors. Because of the sensitivity of modern TMA, it is often 84 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines used to measure Tgs that are difficult to obtain by DSC, for example, those of highly cross-linked thermosets.

9.2 CONCLUSION In recent years, plant derived products are increasingly being sought out as medicinal products, nutraceuticals, and cosmetics and are available in health food shops and pharmacies over the counter as self-medication or also as drugs prescribed in non- allopathic systems. Therefore, these technologies have been gradually applied to traditional medicine research, such as thermal analysis of the chemical compositions, decomposition, and identification of product origin or relatives.

REFERENCES Brandão DO, Guimarães GP, Santos RL et al. Model analytical development for physical, chemical, and biological characterization of momordica charantia vegetable drug. J Anal Meth Chem. 2016; 2016: 1–15. Gomathinayagam V, Venkataraman R. Standardization of Centella Asiatica (L.) urban with market potential using thermal methods of analysis. J Pharmacogn Phytochem. 2015; 3(5): 32–34. Kevin M. Dynamic Mechanical Analysis: A Practical Introduction, 2nd Edition, CRC Press, Boca Raton, 2008. Ma J, Meng X, Guo X, Lan Y, Zhang S. Thermal analysis during partial carbonizing process of rhubarb, moutan and burnet. PLoS ONE. 2017; 12(3): e0173946. Menard KP, Dynamic Mechanical Analysis; A Practical Introduction, CRC Press, Boca Raton, 1999. Paul G. Principles and Applications of Thermal Analysis, Blackwell Publishing, Oxford, UK, 2008. Silva JOC, Costa RMR, Teixeira FM, Barbosa WLR. Processing and quality control of herbal drugs and their derivatives. J Herbal Med. 2011; 14(1): 115–114. Yongyu Z, Shujun S, Jianye D et al. Quality control method for herbal medicine—Chemical fingerprint analysis. In: Quality Control of Herbal Medicines and Related Areas, Shoyama Y (ed.) InTech, Rijeka, Croatia, 2011, Chapter 10, pp. 171–194. 10 Validation of Herbal Drugs

10.1 INTRODUCTION Herbal drugs have been used world wide, particularly in developing countries, to treat and reduce the suffering of humans and animals and to improve their productivity since antiquity. India has a rich heritage of using medicinal plants in Ayurveda and Unani, besides other folk uses. The development of synthetic drugs has greatly affected the usage of herbal drugs. However, the importance of herbal drugs has not beaten all the potential adverse effects. Synthetic drugs are comprised with several toxicities and have adverse effects. Recognizing these reasons, there has been a great scientific interest in the evaluation of indigenous medicinal plants for the treatment of various diseases. The search for safe and efficacious herbal drugs may overcome some of the problems associated with synthetic drugs. The plethora of chemical and biological tests, including high-throughput screening, makes the development of herbal-based novel drugs an active area and highlights an appropriate time for research (Cragg et al., 1997). The majority of plant species has not been investigated chemically or biologically, and bioassay-guided fractionation, dereplication techniques, and powerful methods of structure determination will continue to help this research in the future. It may be predicted that there will be a continued demand for high quality, safe, and effective herbal medicinal products that also require continued scientific investigation (Phillipson, 2003). The Indian systems of medicine have identified 1500 medicinal plants, of which 500 species are mostly used in the preparation of drugs. The medicinal plants contribute to 80% of the raw materials used in the preparation of drugs. The effectiveness of these drugs mainly depends on the proper use and sustained availability of genuine raw materials, followed by evaluation using modern scientific methods and tools by chemical, biochemical, biotechnological, pharmacological, toxicological, and pathological means for rational drug development. India has great potential in the trade of herbal-based drugs. Thus, it is time that well validated and value added products are used in the indigenous and foreign markets. Validation is a must as there is lack of data on the reproducibility of effects, lack of data on controlled clinical trials, lack of recognition of active fraction/active principles, and lack of data on interactions with food and synthetic drugs and toxicity. Validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. It can be used to judge the quality, reliability, and consistency of analytical results; it is an integral part of any good analytical practice. It is the process of defining an analytical requirement, and confirms that the method under consideration has performance capabilities consistent with what the application requires. The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples and should be routinely analyzed. The preparation and execution should follow a validation protocol, preferably written in a step-by-step instruction

85 86 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines format (Desai et al., 2016). The validation of herbal products is a major public health concern. In this regard, there is no control by government agencies despite the existence of certain guidelines in some individual countries and those outlined by the World Health Organization (WHO) (Patel and Patel, 2011). If the herbal products are marketed as therapeutic agents, and irrespective of whether the products really have any positive effects to cure and reduce the severity of the disease, it is necessary to ensure scientific validation and periodic monitoring of the quality and efficacy (Himanshu and Phuja, 2012).

10.2 CONCEPT OF VALIDATION The concept of validation was first proposed by the Food and Drug Administration (FDA) in 1970. Validation is a concept that is fundamental to good manufacturing practices (GMP) and any quality assurance program. The USFDA defined validation as establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics. Assurance of product quality is derived from careful attention to a number of factors, including selection of quality materials, adequate product and process design, control of process, and in process and end product testing (Nash, 2003).

10.3 VALIDATION OF HERBAL DRUGS Validation of herbal drugs involves the following steps:

• Information about the herbal remedies could be gathered from traditional uses, practitioners, healers, farmers, nomads, authentic literature, and so on. • Such herbal remedies should be discussed in detail with a panel of experts for primary screening, involving herbalists, field veterinarians, pharmacologists, pharmacognosists, and so on, for their valuable uses for the purpose for which they are being reported by the users and only those remedies which seem worthy could be further processed for validation. • Identification of plant materials: Plants with therapeutic potentials for various ailments should be collected from suitable sources and botanically identified. • Properly identified plant material should be processed as per its traditional guidelines for the preparation of a remedy and then used in those proven clinical cases and similar experimental conditions for which the remedy has been prescribed. • Once these remedies have shown their efficacy in clinical and experimental conditions, they should be finally be selected and their ingredients again confirmed. • Extraction of active fractions and fractions of various plants should be subjected to respective biological activity and evaluation for identifying the fraction that possesses promising activity in in vitro/in vivo systems. • Purification of active fractions then chemical characterization of active principles should be undertaken. Validation of Herbal Drugs 87

• Pharmacodynamics studies of the active principles should be carried out. • Toxicological evaluation of the active principles should be undertaken in active collaboration with Veterinary Toxicologists and Pathologists. • Clinical evaluation of active principles should be carried out with the help of clinicians in multi-center clinical trials. • Active principles found to possess the promising activity could be subjected for the development of the drug in consultation with the pharmaceutical establishment.

10.4 PROCESS VALIDATION Validation is a concept that is fundamental to GMP and any quality assurance program. Validation of the individual steps of the process is called process validation. Process validation is defined as the collection and evaluation of data, from the process design stage through commercial production, which establishes scientific evidence that a process is capable of consistently delivering quality product. The process is developed in such way that the required parameters are achieved and it ensures that the output of the process will consistently meet the required parameters during routing production. This concept is applied in the pharmaceutical industry, but not that deeply or methodologically in the herbal industry. The activities relating to validation studies may be classified into three stages: process design, process qualification, and continued process verification (Alam, 2012).

10.5 VALIDATION ACCORDING TO WHO Qualification of critical equipment, process validation, and change control are particularly important in the production of herbal medicines with unknown therapeutically active constituents. In this case, the reproducibility of the production process is the main means for ensuring the consistency of quality, efficacy, and safety between batches. The written procedure should specify critical process steps and factors (such as extraction time, temperature, and solvent purity) and acceptance criteria, as well as the type of validation to be conducted (e.g. retrospective, prospective or concurrent) and the number of process runs.

10.6 VALUE ADDITION Value addition can be carried out based on knowledge for the purpose of exporting value-added material rather than raw material. This could be crude plant material, standardized plant extracts, partially purified extracts, and groups of phytochemicals or pure single phytochemicals. The extent of value addition carried out at the beginning could be the cleaning, drying, and sorting of natural products. Some degree of technological intervention is needed for value addition, depending on the type of extract required for the production of the herbal preparation at larger scales. Various dosage forms can also be formulated that may improve utility. The value addition of herbal products can further be done by purification of extracts and isolation of single bioactive molecules (Fabricant and Farnsworth, 2001). 88 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

10.7 CONCLUSION Validation of herbal drugs is beneficial in terms of developing economical, eco- friendly, easily accessible, effective, and safe drugs for enhancing health conditions and production. In addition to the development of new drugs with divergent therapeutic potentials, the process may often lead to unraveling a good pharmacological tool. The use of herbal medicine is the oldest form of health care. About 80% of the world’s population has faith in traditional medicine, particularly herbal drugs, for their primary health care. India has a rich tradition of herbal medicine as is evident from Ayurveda. As public interest in the use of herbal medicines grows, it is necessary to develop modern and objective standards for evaluating the quality of herbal medicines. Thus, there is a need for process validation in the manufacturing of herbal drugs in order to control the quality of herbal drugs. The reasons for doing process validation in the herbal manufacturing industry are manufacturers are required by law to confirm to GMP regulations, good business dictates that a manufacturer avoid the possibility of rejected or recalled batches, process validation helps to ensure product uniformity, reproducibility, and quality, and to make the process economical.

REFERENCES Alam S. Pharmaceutical Process Validation: An Overview. J Adv Pharm Education & Res. 2012, 2: 185–200. Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery and development. J Nat. Prod. 1997; 60: 52–60. Desai SR, Disouza JI, Shirwadkar BB. Process Validation: An approach for herbal tablet standardization. IJPPR. 2016; 8(2): 313–320. Fabricant DS, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect. 2001; 109: 69–75. Himanshu, Phuja, S. A review on pharmaceutical process validation. Int Res J Pharm. 2012; 3: 56–58. Nash R. Introduction. In: Nash RA, Wachter AH (ed.). Pharmaceutical Process Validation, Vol. 129, An International 3rd Edition, Revised and Expanded, Marcel Dekker, New York, 2003; 17–18. Patel V, Patel N. Review on quality safety and legislation of herbal medicine. Int J Res Ayurveda and Pharm. 2011; 2: 1486–1489. Phillipson JD. 50 years of medicinal plant research-every progress in methodology is a progress in science. Planta Med. 2003; 69: 491–495. Stability Study of 11 Plant Products

11.1 INTRODUCTION An herbal medicinal drug may be a single active constituent or an entire herb source and is considered to be a medicinal product. Most herbal drug products used are a group of constituents. Stable drug products maintain their identity, strength, and therapeutic effect within given specifications throughout the shelf life. Herbal medicinal products are of different natures, from thermo-labile to volatile. Stability testing is an obligatory requirement in the registration process for all medicinal products, including Herbal Medicinal Products (HMPs). Stability testing of herbal products is a complicated issue because the entire herb or herbal product is regarded as the active substance, regardless of whether the constituents with defined therapeutic activity are known (Kruse et al., 2011). The stability testing of herbal products checks the quality of herbal products, which varies with the time under the influence of environmental factors such as temperature, humidity, light, oxygen, moisture, other ingredients or excipients in the dosage form, particle size of drug, microbial contamination, trace metal contamination, leaching from the container, and so on, and also provides statistics for the determination of shelf-lives (Rangari, 2008). Therefore, evaluation of the parameters based upon chemical, physical, microbiological, therapeutic, and toxicological studies can serve as an important tool in stability studies. Based on the climatic conditions, only particular storage conditions can be determined. Stability studies should be performed on at least three production batches of the herbal products for the proposed shelf life, which is normally denoted as long term stability and is performed under natural atmospheric conditions. The tests are performed to define storage conditions and the product’s shelf life. For products on the market, the marketing authorization holder is legally obliged to undertake on-going stability studies to prove that the medicinal product can be used safely over the entire period of its shelf life (Roth-Ehrang, 2010). With the help of modern analytical techniques like spectrophotometry, HPLC, and HPTLC, and by employing proper guidelines, it is possible to generate sound stability data of herbal products and predict their shelf- life, which will help in improving the global acceptability of herbal products. In many aspects, stability testing of HMPs follows the same requirements as stability testing of chemically defined substances. However, some specific characteristics have to be taken into consideration:

• Herbal drugs and preparations (extracts) are the active pharmaceutical ingredient. • HMPs are complex in nature due to their high number of constituents.

89 90 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

• Constituents belong to different chemical classes with different analytical behaviors. • Constituents sometimes have very low concentrations in the finished product.

11.2 ROLE OF MARKERS Markers are chemically known compounds which may or may not have therapeutic effect, which are used to calculate the quantity of herbal medicinal ingredients in herbal medicinal products. The choice of the marker has to be justified. Finding the “right” analytical marker is a crucial need for stability testing of HMPs. Typical sources for finding markers are:

• Monographs and drafts • Experience, transfer from other plants/constituents • Literature research about known constituents • Scientific research

The search for suitable and new marker substances is an important interface between scientific research and the use of the results in the HMP industrýs routine quality control. The isolation and structure elucidation of chemically defined substances in a plant, drug and/or drug preparation not only helps to better understand the active principle of an HMP, it can also enhance analytical quality control (Sachan and Kumar, 2015). The requirement for getting suitable markers for analysis is shown in Figure 11.1.

11.3 ANALYTICAL METHODS FOR HERBAL PRODUCTS The analysis of herbal preparations is mostly done by running high performance liquid chromatography (HPLC) or gas chromatography (GC) and thin layer chromatography (TLC) methods, quantitative determinations by UV-Visible spectroscopy or combinations of these. HPLC and GC methods can be used for identification and purity testing, as well as the detection of single compounds for assay, and can be used during one analysis. LC and GC mass coupling are also tools for determination, but they are highly sophisticated and expensive methods. In consequence, analysis of HMPs should consider:

Characteristics for identity testing

Selective for assay

Robust method for stability and routine batch testing

FIGURE 11.1 Goal of an analytical marker for analysis of HMPs. Stability Study of Plant Products 91

• Different requirements for the different types of extracts • Use of markers for the active pharmaceutical ingredient (API) • Use of fingerprint chromatograms • Special hurdles to overcome for combination products

11.4 SHELF LIFE The determination of shelf life of herbal medicinal drug products is the same as that of chemically defined APIs, but the special nature of herbal products should be taken into consideration. It is recommended that in the case of a herbal medicinal product containing a natural product or a herbal drug preparation with constituents of known therapeutic activity, the variation in components during the proposed shelf life should not exceed ±5% of the initial assay value, unless it is justified to widen the range up to ±10% or even higher. The low marker concentration in the finished product could justify the wider range.

11.5 CHALLENGES IN STABILITY TESTING OF HERBAL MEDICINAL PRODUCTS An important part of quality control of herbal drug preparations is the evaluation of the chemical stability of a finished product during the storage period. Measuring the chemical stability is very challenging due to the complexity of a plant extract, which may contain thousands of different compounds. Many reports so far have focused on the stability of an isolated secondary metabolite and its decomposition products. However, results from these data do not always accurately reflect the chemical stability of the compound in an extract. Evaluating the stability of HMPs presents a number of challenges when compared to chemically defined substances. In particular:

• Active substances (herbal substances and/or herbal preparations) in HMPs consist of complex mixtures of constituents, and in most cases, the constituents responsible for the therapeutic effects are unknown. • The situation is further complicated when two or more herbal substances and/or herbal preparations are combined in an HMP. • In addition, many herbal substances/herbal preparations are known to be unstable.

As part of a total control strategy for herbal substances, herbal preparations, and HMPs, a set of test criteria including qualitative and quantitative parameters has been recognized as quality indicative. With regard to stability tests, chromatographic fingerprints as well as appropriate methods of assay via marker substances represent the fundamental part of this concept, laid down in shelf life specifications. Notwithstanding the appropriateness of this approach, its realization is often associated with analytical problems and high costs. In summary, HMPs have a number of characteristics that clearly differentiate them from chemically defined medicinal products, therefore, specific stability guidance needs to be established, 92 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines which covers particular aspects that existing specific herbal guidelines and general guidelines on stability do not address (Gafner and Bergeron, 2005).

11.6 PREDICTABLE CHANGES IN HERBAL MEDICINAL PRODUCTS The following predictable changes may occur in herbal medicinal products during storage and in shelf life determination: hydrolysis, oxidation, racemization, geometric isomerization, and temperature, moisture and light reactions. The other factors affecting the stability of herbal medicine are shown in Figure 11.2.

• Hydrolysis: Reaction with water takes place and results in degradation of the product. • Oxidation: The addition of an electro-negative atom (o), removal of an electro-positive atom, and radical formation results in decomposition of the natural products. • Racemization: Racemization is the process in which one enantiomer of a compound, such as an l-amino acid, converts to the other enantiomer. The compound then alternates between each form while the ratio between the (+) and (–) groups approaches 1:1, at which point it becomes optically inactive. • Geometric isomerization: Products can be change in trans or cis form. One form may be more therapeutically active. • Polymerization: There is combination of two or more identical molecules to form a much larger and more complex molecule. • Temperature: The rate of most chemical changes increases with an increase in temperature. Thus, the “tropical” area must be taken into consideration during preparation of the formula of the herbal substance.

Drug interaction Storage Insect condition attack

Moisture Packaging content interactions

Stability

Mold Content growth variation

Micro- Container organisms Environ- mental factors

FIGURE 11.2 Factors affecting the stability of herbal medicines. Stability Study of Plant Products 93

• Moisture: Moisture absorbed onto the surface of a solid drug will often increase the rate of decomposition if it is susceptible to hydrolysis. • Light: Many types of chemical reactions are induced by exposure to light of high energy. Autoxidation of volatile oil/fixed oil takes place and the substance becomes colored.

11.7 NOVEL APPROACHES FOR STABILITY IMPROVEMENTS IN NATURAL MEDICINES It has now been observed that many of the constituents present in the herbal formulation may react with each other and this raises serious concerns about the stability of such formulations, which is an important issue in the field of phytochemistry and natural medicines. Natural products are often prone to deterioration, especially during storage, leading to loss of the active component, production of metabolites with no activity, and, in extreme cases, production of toxic metabolites. Stability is defined as the capacity of a drug substance or drug product to remain within established specifications to maintain its density, strength, quality, and purity throughout the retest or expiration dating periods (Rockville, 1998). There are several tools employed to check natural product instability, including determination of the physical parameters and impurity profile, identification and quantification of all metabolites, and controlled storage conditions. In addition, various techniques are employed to make herbal products more stable such as nanoparticle coating for enhancing the shelf life of a natural product, semisolid preparations based on supercritical carbon dioxide, a novel approach for natural products, topical herbal formulations, liquid preparations coated with water-soluble cellulose derivative, polymeric plant-derived excipients in drug delivery, long chain fatty acid derivatives, chelating agents for stabilitzation of the aqueous plant extracts, suspension form of the herbal products, emulsion form of the herbal products, antioxidants and liquid formulations, linctuses for herbal preparations, transgenic plants and immunoprotective compounds, tablet formulation for volatile liquids, formulations without the use of a stabilizer, plant pigments, powders with oil composition containing an aqueous active substance, vitamin solutions, herbal compositions with high contents of herb medicine extracts, the use of colloidal silicon dioxide in the enhancement of the drying of herbal preparations, and so on (Thakur et al., 2011). According to WHO guidelines, the physical and chemical stability of a product in the container in which it is to be marketed should be tested under defined storage conditions and the shelf life should be established. The active and characteristic constituents should be specified and, if possible, content limits should be defined. Foreign matter, impurities, and microbial content should be defined or limited in the crude natural product (WHO, 2009).

11.8 CONCLUSION Stability testing of herbal products with known chemical constituents is the same as chemically defined APIs, but the majority of herbal medicinal products are complex in nature. Thus, one should take account of the particular requirements and conditions. 94 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Major studies are the same for both herbal medicinal and chemically defined products. Natural medicines are continuously gaining attention as the therapy for many of the ailments in the modern era. Hence, it becomes the prime responsibility of the herbal drug manufacturer to provide adequate stability for long-term storage and safety for consumption by the patients. As phytoformulations are a mixture of more than one active ingredient, care should be taken for the determination of the stability profile for natural medicines.

REFERENCES Gafner S, Bergeron C. The challenges of chemical stability testing of herbal extracts in finished products using state of the art analytical methodologies. Curr Pharm Anal. 2005; 1: 203–215. Kruse H, Kroll D, Steinfhoff T. On going stability testing of herbal medicinal products. Pharm Ind. 2011; 73(Nr. 8): 1401–1412. Rangari VD. Pharmacognosy and Phytochemistry, 2nd edition. Career Publications, Nashik, Maharashtra, 2008, pp. 78–100. Rockville MD. FDA draft guidance for industry, stability testing of drug substances and drug products. Glossary FDA. 1998: 1–10. Roth-Ehrang, A., Hubbert K., Lutz-Röder, PT. Wiedemann Steinhoff: Stability testing of herbal medicinal products. Pharm Ind. 2010; 72(Nr. &): 1166–1174. Sachan AK, Kumar A. Stability testing of herbal products. J Chem Pharm Res. 2015; 7(12): 511–514. Thakur L, Ghodasra U, Patel N, Dabhi M. Novel approaches for stability improvement in natural medicines. Pharmacogn Rev. 2011; 5(9): 48–54. WHO Technical Report Series, No. 953, Annex 2: Stability testing of active pharmaceutical ingredients and finished pharmaceutical products. 2009. Fingerprinting Techniques 12 for Herbal Drugs Standardization

12.1 INTRODUCTION Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world. But the quality control and quality assurance still remains a challenge because of the high variability of the chemical components involved. Herbal drugs, singularly and in combinations, contain a myriad of compounds in complex matrices in which no single active constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards for raw materials and standardization of finished herbal drugs. Traditionally, only a few markers of pharmacologically active constituents were employed to assess the quality and authenticity of complex herbal medicines. However, the therapeutic effects of herbal medicines are based on the complex interaction of numerous ingredients in combination, which is totally different from those of chemical drugs. The concept of fingerprinting has been increasingly used for the past few decades to determine the ancestry of plants, animals, and other microorganisms. Genotypic characterization of plant species and strains is useful as most plants, though belonging to the same genus and species, may show considerable variation between strains. Herbal medicines differ from those of the conventional drugs, thus some innovative methods are necessary for quality assessment of herbal drugs. Herbal drugs are consumed in most developed nations in the form of ethno-therapeutics of nutraceuticals or are used as the primary source of medicinal compounds or their intermediates. The fingerprint analysis approach is the most potent tool for quality control of herbal medicines because of its accuracy and reliability. Fingerprinting is a process that determines the concentrations of a set of characteristic chemical substances in an herb. Knowing the relative concentrations is a means of assuring the quality of herbal preparations. It can serve as a tool for identification, authentication, and quality control of herbal drugs. Based on the conception of phytoequivalence, the chromatographic fingerprinting and DNA fingerprints of herbal medicines could be utilized for addressing the problem of quality control of herbal medicines. In Figure 12.1, different fingerprinting techniques for herbal standardization have been shown.

12.2 PHYTOEQUIVALENCE AND CHROMATOGRAPHIC FINGERPRINTS OF HERBAL MEDICINES In general, one could use the chromatographic techniques to obtain a relatively complete picture of an herbal, which is in commonly called the chromatographic

95 96 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Chromatographic DNA fingerprinting fingerprinting

Hybridization- TLC, HPTLC, based methods: HPLC, GC RFLP and VNTR

chniques PCR-based

te method: RAPD, Fingerprinting AP-PCR, AFLP and SSR

Sequence-based methods: SNP and STR

FIGURE 12.1 Fingerprinting techniques in herbal drug standardization. TLC = Thin Layer Chromatography; HPTLC = High Performance Thin Layer Chromatography; HPLC = High Performance Liquid Chromatography; GC = Gas Chromatography; RFLP = Restriction Fragment Length Polymorphism; VNTR = Variable Number Tandem Repeats; RAPD = Randomly Amplified Polymorphic DNA; AP-PCR= Arbitrarily Primed Polymerase Chain Reaction; AFLP = Amplified Fragment Length Polymorphism; SSR= Simple Sequence Repeats; SNP = Single Nucleotide Polymorphism; STR = Simple Tandem Repeats. fingerprints of herbal medicines to represent the so-called phytoequivalence. Obtaining a good chromatographic fingerprint representing the phytoequivalence of herbs depends on several factors, such as the extracting methods, measurement instruments, measurement conditions, and so on. In fact, if we want to obtain an informative fingerprint of an herbal medicine, we need to have a good extracting method with which we could hopefully obtain almost all the pharmaceutically active compounds to represent the integrity of the herbal medicine (Cieśla, 2012; Gong et al., 2003). Furthermore, a chromatogram with good separation and a representative concentration profile of the bioactive components detected by a proper detector are also required. Thus, how to obtain a high quality chromatographic fingerprint for as much information of herbal medicines as possible is an important task for chemists and pharmacologists. In order to understand bioactivities and possible side effects of active compounds of the herbal medicines and to enhance product quality control, it seems that one needs to determine most of the phytochemical constituents of herbal products so as to ensure the reliability and repeatability of pharmacological and clinical research (Caoa et al., 2006; Gong et al., 2003).

12.2.1 evaluation of Chemical Fingerprints of Herbal Medicines It can be very difficult to select an optimal set integration parameters for chromatograms obtained from the analysis of complex samples which easily contain more than 100 peaks. Furthermore, the selection and extraction of peaks to include Fingerprinting Techniques for Herbal Drugs Standardization 97 in data analysis is difficult and partly subjective, and large amounts of the data in the chromatograms are discarded. The disadvantages of peak detection, integration, and introduction of a subjective peak selection can be avoided by using all collected data points in the chemometric analysis. Thus, the entire chromatographic profiles are utilized to perform direct chemometric analysis. Furthermore, another advantage of taking the entire chromatographic profile to perform direct chemometric analysis is that the peak shape can be included in data analysis, which will make the pretreatment of overlapping peaks much easier when one does an evaluation of the fingerprints (Tauler et al., 1992; Zhang et al., 2003).

12.2.1.1 Chromatographic Fingerprinting Chromatography is defined as the technique of isolation and identification of components of compounds or mixtures into their individual components by using the stationary phase and the mobile phase. Chromatography offers a very powerful separation ability, such that the complex chemical components in herbal extracts can be separated into many relatively simple subfractions (Liang et al., 2004). Chromatographic fingerprinting is the most powerful approach for the quality control of herbal medicines. The chromatographic fingerprints of herbal medicines are a chromatographic pattern produced from the extract of some common chemical components which may be pharmacologically active or have some chemical characteristics. Chromatographic fingerprinting can successfully demonstrate both the sameness and the differences between various samples and the authentication and identification of herbal medicines can be accurately conducted even if the number and/or concentration of chemically characteristic constituents are not very similar in different samples of herbal medicine (Xie, 2001). Thus, chromatographic fingerprinting should be considered to globally evaluate the quality of herbal medicines by considering the multiple constituents present in the herbal medicines. This technique can be employed for identification and authentication as well as for the determination of various adulterants and contaminants and for standardization purposes. In contrast to macroscopic, microscopic, and other molecular biological methods, this technique is not restricted to raw herbs, but can also be applied to pharmaceutical preparations (Liang et al., 2010). Chromatographic fingerprinting can be carried out using techniques such as thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC), gas chromatography (GC), and other hyphenated techniques. The construction of chromatographic fingerprints plays an important role in the quality control of complex herbal medicines. Chemical fingerprints obtained by chromatographic techniques are strongly recommended for the purpose of quality control of herbal medicines, since they might appropriately represent the chemical integrities of the herbal medicines, and therefore be used for authentication and identification of the herbal products. Thus, chromatographic fingerprinting should be considered to globally evaluate the quality of herbal medicines considering the multiple constituents present in the herbal medicines. Using chromatographic techniques like HPTLC and HPLC, a profile of their various chemical constituents is obtained. This is called chemoprofiling. The chemical fingerprints obtained 98 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines by chromatographic and electrophoretic techniques, especially by hyphenated chromatographies, are strongly recommended for the purpose of quality control of herbal medicines, since they might appropriately represent the chemical integrities of herbal medicines, and therefore be used for authentication and identification of the herbal products. Chemical constituents are isolated based on their affinities for particular organic solvents in an increasing order of polarity. They are resolved using suitable coloring reagents, resulting in characteristic patterns. The compound specific to that species (sterol, terpenoid, alkaloid, etc.) is characterized as a chemical marker. The medicinal utility of a particular plant species is related to the quantity of that marker compound present.

12.2.1.1.1 Thin Layer Chromatography This is one of the most popular and simple chromatographic techniques used for separation of compounds before instrumental chromatography methods such as GC and HPLC were established. TLC is used as an easier method of initial screening with a semi-quantitative evaluation together with other chromatographic techniques as there is relatively less change in the simple TLC separation of herbal medicines than with instrumental chromatography. Thin layer chromatography is a technique in which a solute undergoes distribution between two phases, a stationary phase acting through adsorption and a mobile phase in the form of a liquid (Wagner et al., 1996). The adsorbent is a relatively thin, uniform layer of dry, finely powdered material applied to a glass, plastic or metal sheet or plate. Glass plates are most commonly used (Figure 12.2). Separation may also be achieved on the basis of partition or a combination of partition and adsorption, depending on the particular type of support, its preparation, and its use with different solvents (Stahl, 1969).

Watch glass

in layer chromatography

Beaker Spot of mixture

Solvent

FIGURE 12.2 Thin layer chromatography. Fingerprinting Techniques for Herbal Drugs Standardization 99

In the phytochemical evaluation of herbal drugs, TLC is employed extensively for the following reasons:

• It enables rapid analysis of herbal extracts with minimum sample clean-up requirement. • It provides qualitative and semi-quantitative information of the resolved compounds.

TLC has the advantages of many fold possibilities of detection in analyzing herbal medicines. In addition, TLC is rather simple and can be employed for multiple sample analysis. For each plate, more than 30 spots of samples can be studied simultaneously. Thus, the use of TLC to analyze herbal medicines is still popular. In TLC fingerprinting, the data that can be recorded using a high-performance TLC (HPTLC) scanner includes information such as a chromatogram, retardation factor (Rf) values, the color of the separated bands, their absorption spectra, λ max, and shoulder inflection/s of all the resolved bands. All of these, together with the profiles on derivatization with different reagents, represent the TLC fingerprint profile of the sample. The information so generated has a potential application in the identification of an authentic drug, in excluding the adulterants, and in maintaining the quality and consistency of the drug (Svendsen, 1989). The advantages of using TLC/HPTLC to construct the fingerprints of herbal medicines are its simplicity, versatility, high velocity, specific sensitivity, and simple sample preparation. Thus, TLC is a convenient method of determining the quality and possible adulteration of herbal products. It is worth noting that the new techniques of TLC are also being updated, such as forced- flow planar chromatography (FFPC), rotation planar chromatography (RPC), over pressured-layer chromatography (OPLC), and electro planar chromatography (EPC). A simple but powerful preparative forced-flow technique was also reported; in this technique, hydrostatic pressure is used to increase mobile phase velocity. Parallel and serially-coupled layers open up new vistas for the analysis of a large number of samples (up to 216) for high throughput screening and for the analysis of very complex matrices (Funk and Droeschel, 1991; Gong et al., 2003).

12.2.1.1.2 High Performance Thin Layer Chromatography High Performance Thin Layer Chromatography (HPTLC) is the common fingerprinting method mainly used to analyze compounds with low or moderate polarities. The HPTLC technique is widely used in the pharmaceutical industry for quality control of herbs and health products, identification and detection of adulterants, substituents in the herbal products, and also helps in the identification of pesticide contents and Mycotoxins. The HPTLC method has several advantages, which are as follows:

• Several samples can be run simultaneously by the use of a smaller quantity of the mobile phase as compared to HPLC. • Mobile phases of pH 8 and above can be used for HPTLC. • Repeated detection (scanning) of the chromatogram with the same or different conditions is possible. 100 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

• HPTLC has been investigated for simultaneous assay of several components in a multi-component formulation. With this technique, authentication of various species of plant as well as the evaluation of stability and consistency of their preparations is possible.

HPTLC is one of the sophisticated instrumental techniques based on the full capabilities of TLC. It is a most flexible, reliable, and cost efficient separation technique. The advantage of automation, scanning, full optimization, selective detection principle, minimum sample preparation, hyphenation, and so on, enable it to be a powerful analytical tool for the chromatographic information of complex mixtures of pharmaceuticals, natural products, clinical samples, food stuffs, and so on (Attimarad et al., 2011).

12.2.1.1.3 Gas Chromatography Gas chromatography (GC), also known as gas liquid chromatography (GLC), is a technique for the separation of mixtures into components by a process which depends on the redistribution of the components between a stationary phase or support material in the form of a liquid, solid or combination of both and a gaseous mobile phase. GC analysis can often be used for authentication and quality control of herbal medicines having volatile bioactive constituents. It is well-known that many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by gas chromatography is very important in the analysis of herbal medicines. The high selectivity of capillary columns enables the simultaneous separation of many volatile compounds within comparatively short times. The GC analysis of volatile oils has a number of advantages (Figure 12.3). First, the GC of the volatile oil gives a reasonable “fingerprint” which can be used to identify the plant. The composition and relative concentration of the organic compounds in the volatile oil are characteristic of the particular plant and the presence of impurities in the volatile oil can be readily detected. Second, the extraction of the volatile oil is relatively straightforward and can be standardized and the components can be readily identified using GC-MS analysis. The advantage of GC clearly lies in its high sensitivity of detection for almost all the volatile chemical compounds.

Flow regulator Injection port Detector

Chart recorder

Column

Gas cylinder Oven

FIGURE 12.3 Gas chromatography. Fingerprinting Techniques for Herbal Drugs Standardization 101

However, the most serious disadvantage of GC is that this method is not convenient for the analysis of samples which are thermo-labile and non-volatile (Gong et al., 2003; Nyiredy, 2003; Yan, 2009).

12.2.1.1.4 High Performance Liquid Chromatography High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of a small particle (3–50 µm) packing contained in a column with a small bore (2–5 mm), one end of which is attached to a source of pressurized liquid eluent (mobile phase). The three forms of high performance liquid chromatography most often used are ion exchange, partition, and adsorption. HPLC is a popular method for the analysis of herbal medicines because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. In general, HPLC can be used to analyze almost all the compounds in herbal medicines. Thus, over the past decades, HPLC has received the most extensive application in the analysis of herbal medicines (Figure 12.4). Reversed-phase (RP) columns may be the most popular columns used in the analytical separation of herbal medicines (Li et al., 2003; Tsai et al., 2002). It is necessary to notice that the optimal separation condition for the HPLC involves many factors, such as the different compositions of the mobile phases, their pH adjustment, pump pressures, and so on. Thus, a good experimental design for the optimal separation seems necessary. In order to obtain better separation, some new techniques have been recently developed in the research field of liquid chromatography. These are micellar electrokinetic capillary chromatography (MECC), high-speed counter-current chromatography (HSCCC), low-pressure size-exclusion chromatography (SEC), reversed-phase ion-pairing HPLC (RP IPC- HPLC), and strong anion-exchange HPLC (SAX-HPLC). They will provide new opportunities for good separation for some specific extracts of some herbal medicines (Li et al., 1999; Liu and Sheu, 1993). The advantages of HPLC lie in its versatility

Switching valve

Solvent degasser Gradient Mixing vessel High pressure valve for the delivery pump of mobile phase Sample injection loop Solvent reservoir Pre-column

Analytical column Data acquisition Waste Detector

FIGURE 12.4 HPLC chromatography. 102 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines for the analysis of the chemical compounds in herbal medicines, however, the commonly used detector in HPLC is a single wavelength UV detector; it seems to be unable to fulfill the task since many chemical compounds in herbal medicines are non-chromophoric compounds. Consequently, a marked increase in the use of HPLC analysis coupled with evaporative light scattering detection (ELSD) in a recent decade has demonstrated that ELSD is an excellent detection method for the analysis of non-chromophoric compounds (Revilla et al., 2001). This new detector provides the possibility for the direct HPLC analysis of many pharmacologically active components in herbal medicines, since the response of ELSD depends only on the size, shape, and number of eluate particles rather than the analysis structure and/ or chromophore of analytes as UV detectors require. This technique is especially suitable for the construction of the fingerprints of herbal medicines. Moreover, the qualitative analysis or structure elucidation of the chemical components in herbal drugs by simple HPLC is not possible, as they rely on the application of techniques using hyphenated HPLC, such as HPLC-IR, HPLC-MS, and HPLC-NMR for the analysis of herbal medicines (Lazarowych and Pekos, 1998; Zhang, 2004).

12.2.1.1.5 Electrophoretic Methods Capillary electrophoresis (CE) was introduced in the early 1980s as a powerful analytical and separation technique and has since been developed almost explosively. It allows an efficient way to document the purity/complexity of a sample and can handle virtually every kind of charged sample components ranging from simple inorganic ions to DNA. Thus, there has been an obvious increase of electrophoretic methods, especially capillary electrophoresis, used in the analysis of herbal medicines in the last decades. The more or less explosive development of capillary electrophoresis since its introduction has to a great extent paralleled that of liquid chromatography. The techniques most often used are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (CIEF). CE is promising for the separation and analysis of active ingredients in herbal medicines, since it needs only small amounts of standards and can analyze samples rapidly with very good separation ability. Also, it is a good tool for producing the chemical fingerprints of the herbal medicines, since it has similar technical characteristics as liquid chromatography. Recently, several studies dealing with herbal medicines have been reported and two kinds of medicinal compounds, that is, alkaloids and flavonoids, have been studied extensively (Yang and Smetena, 1995). In general, CE is a versatile and powerful separation tool with high separation efficiency and selectivity when analyzing mixtures of low-molecular-mass components. However, the fast development in capillary electrophoresis causes improvements of resolution and throughput rather than reproducibility and absolute precision. One successful approach to improve the reproducibility of both mobility and integral data has been based on internal standards. But many papers that were published unfortunately revealed a limited view of the real possibilities of CE in the field of fingerprinting herbal medicines (Liu and Sheu, 1993; Stuppner et al., 1992). CE and capillary electrochromatography approaches contribute to a better understanding of the solution behavior of herbal medicines, especially when additionally combined with the powerful spectrometric detectors (Liu and Sheu, 1992). Fingerprinting Techniques for Herbal Drugs Standardization 103

12.2.1.1.6 Hyphenated Techniques A chromatographic separation system online with a spectroscopic detector in order to obtain structural information on the analytes present in a sample has become the most important approach for the identification and/or confirmation of the identity of target and unknown chemical compounds. Chromatographic separation techniques can be coupled to various detection techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and so on. These hyphenated techniques provide information about the structure of the compounds present in the chromatogram and thus provide higher sensitivity in comparison to conventional approaches. Various hyphenated techniques used include liquid chromatography-mass spectrometry (LC-MS), liquid chromatography nuclear magnetic resonance (LC-NMR), gas chromatography- mass spectroscopy (GC-MS), and gas chromatography Fourier transform infrared spectrometry (GC-FTIR). The combination of column liquid chromatography or capillary gas chromatography with a UV-VIS (ultraviolet-visible) or a mass spectrometer (high-performance liquid chromatography-diode-array detector [HPLC-DAD], capillary electrophoresis-diode-array detector [CE-DAD], GC-MS and LC-MS, respectively) becomes the preferred approach for the analysis of herbal medicines. The data obtained from such hyphenated instruments are the so-called two-way data; one way for the chromatogram and the other way for the spectrum, which could provide much more information than the classic one-way chromatography (Patel et al., 2010).

12.2.1.1.6.1 Liquid Chromatography: Mass Spectrometry (LC-MS) Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. This technique has great importance because it can be used to characterize a wide variety of plant constituents ranging from small molecules to macromolecules such as peptides, proteins, carbohydrates, and nucleic acids. Recent advances in this instrument include electrospray, thermospray, and ion spray ionization techniques which offer the unique advantages of high detection, sensitivity, and specificity. Isotope pattern can also be detected with this technique (Pitt, 2009).

12.2.1.1.6.2 Liquid Chromatography: Nuclear Magnetic Resonance (LC-NMR) The combination of liquid chromatography (LC) and nuclear magnetic resonance (NMR) offers the potential of unparalleled chemical information from analytes separated from complex mixtures. This separation technique with NMR spectroscopy is one of the most powerful and time saving methods for the separation and structural elucidation of unknown compounds and mixtures, especially for the structural elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC-NMR improves the speed and sensitivity of the detection. The recent introduction of a pulsed field gradient technique in high resolution NMR as well as a three-dimensional technique improves the application for structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas of pharmacokinetics, toxicity studies, drug metabolism, and drug 104 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines discovery processes. Several other hyphenated NMR techniques have been developed to enhance the sensitivity of this technique. Liquid chromatography-solid-phase extraction- nuclear magnetic resonance (LC-SPE-NMR) increases the sensitivity of the instrument by utilizing a solid phase extraction device after the LC column. Capillary LC-NMR also practically lowers the detection limit to a nanogram range through the integration of capillary LC with NMR detection (Walker and O’Connell, 2008; Wang et al., 2001).

12.2.1.1.6.3 Gas Chromatography-Mass Spectrometry (GC-MS) Mass spectrometry is the most sensitive and selective method for molecular analysis and can yield information on the molecular weight as well as the structure of the molecule. Gas chromatography equipment can be directly interfaced with rapid scan mass spectrometers of various types. GC-MS is a unanimously accepted method for the analysis of volatile constituents of herbal medicines due to its sensitivity, stability, and high efficiency. The hyphenation with MS especially provides reliable information for the qualitative analysis of the complex constituents (Krone et al., 2010). The flow rate from the capillary column is generally low enough so that the column output can be fed directly into the ionization chamber of the MS. There are at least two significant advantages for GC-MS: 1) with the capillary column, GC-MS has, in general, very good separation ability, which can produce a chemical fingerprint of high quality, 2) with the coupled mass spectroscopy and the corresponding mass spectral database, the qualitative and relatively quantitative composition information of the herb investigated could be provided by GC-MS, which will be extremely useful for elucidating the relationship between the chemical constituents in herbal medicine and their pharmacology in further research. Thus, GC-MS should be the most preferable tool for the analysis of the volatile chemical compounds in herbal medicines (Gong et al., 2001 and Gong et al., 2003).

12.2.1.1.6.4 Gas Chromatography-Fourier Transform Infrared Spectrometry (GC-FTIR) Coupling capillary column gas chromatographs with a Fourier Transform Infrared Spectrometer provides a potent means for separating and identifying the components of different mixtures. Despite the widespread acceptance of chromatographic fingerprint techniques for quality control, the establishment of a characteristic fingerprint chromatogram for the quality control of herbal medicines remains a critical task. The ability to obtain a good chromatographic fingerprint depends on several factors such as the extraction method, measurement instrument, and measurement conditions (selection of mobile phase and stationary phase). However, in contrast to microscopic, macroscopic, and many molecular biology methods, chromatographic methods are not only restricted to raw herbs, but can also be applied to herbal preparations (Cai et al., 2005).

12.2.1.1.6.5 HPLC-DAD, HPLC-MS, and Others HPLC-DAD has become a common technique in most analytical laboratories in the world now. With the additional UV spectral information, the qualitative analysis of complex samples in herbal medicines turns out to be much easier than before. The HPLC technique is used for the analysis of the bioactive chemical compounds in plant and herbal medicines, especially the hyphenated HPLC techniques. Moreover, the combined HPLC- DAD-MS techniques take advantage of chromatography as a separation method and Fingerprinting Techniques for Herbal Drugs Standardization 105 both DAD and MS as identification methods. With the help of this hyphenation, in most cases, one could identify the chromatographic peaks directly online by comparison with literature data or with standard compounds, which has made the LC-DAD-MS become a powerful approach for the rapid identification of phytochemical constituents in botanical extracts. It can also be used to avoid the time-consuming isolation of all compounds to be identified (Rajani et al., 2001; Revilla et al., 2001).

12.2.1.1.6.6 Hyphenation of Capillary Electrophoresis (CE) This technique has also quickly been used for the analysis of samples from herbal medicines. The hyphenated CE instruments, such as CE-DAD, CE-MS, and CE-NMR have all appeared in the past decades. Coupling of capillary electrophoresis to mass spectrometry and other types of spectrometry allows both the efficient separation of CE and specific and sensitive detection to be achieved. In summary, as the hyphenated techniques in chromatographic and electrophoretic instruments develop, the ability of the analysis of herbal medicines, both in qualitative and quantitative respects, and the quality control of herbal medicines will become stronger. CE analysis can be driven by an electric field performed in narrow tubes which can result in the rapid separation of hundreds of compounds. It separates components by applying a voltage in between buffer-filled capillaries. The components are separated due to production of ions depending on their mass and charge. It is widely used in quantitative determination and analysis, particularly assay development and trace level determination. When MS is linked to CE, then it produces determination of the molecular weight of components, often termed as CE-MS. Separation is achieved from the etched surface of the capillaries that deliver samples to the electrospray ionization mass spectrometry (ESI MS). This technique runs in full automation and has higher sensitivity and selectivity. The new interface known as a coaxial sheath interface has been developed, which has potential for the alternative use of both CE-MS and LC-MS on the same mass spectrometer (Stockigt et al., 2002).

12.2.1.2 DNA Fingerprinting DNA analysis has been proven an important tool in herbal drug standardization and is useful for the identification of phytochemically indistinguishable genuine drugs from substituted or adulterated drugs. The DNA fingerprint genome remains the same irrespective of the plant part used, while the phytochemical constituents will vary with the part of plant used, the physiology, and the environment (Shikha and Mishra, 2009). This concept of fingerprinting has been increasingly applied in the past few decades to determine the ancestry of plants, animals, and other microorganisms. Genotypic characterization of plant species and strains is useful as most plants, though belonging to the same genus and species, may show considerable variation between strains. Additional motivation for using DNA fingerprinting on commercial herbal drugs is the availability of intact genomic DNA from plant samples after they are processed. Adulterants can be distinguished even in processed samples, enabling the authentication of the drug (Mihalov et al., 2000). The other useful application of DNA fingerprinting is the availability of intact genomic DNA specificity in commercial herbal drugs which helps in distinguishing adulterants even in processed samples (Carvalho, 2000). 106 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

12.2.1.2.1 Types of DNA Fingerprinting Techniques Used in Plant Genome Analysis Various types of DNA-based molecular techniques are utilized to evaluate DNA polymorphism, which includes hybridization-based methods, polymerase chain reaction (PCR)-based methods, and sequencing-based methods.

12.2.1.2.1.1 Hybridization-Based Methods In conventional DNA fingerprinting, hypervariable and repetitive sequences are detected with hybridization probes. Hybridization-based methods use cloned DNA elements or synthetic oligonucleotides as probes to hybridize the DNA of interest. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. The probes are labeled with radioisotopes or with conjugated enzymes which catalyze a color reaction to detect hybridization. Hybridization- based methods include:

a. Restriction Fragment Length Polymorphism (RFLP) b. Variable Number Tandem Repeats (VNTR)

(a) Restriction Fragment Length Polymorphism (RFLP): In this technique, plants may be differentiated by analysis of patterns derived from cleavage of their DNA. Restriction polymorphism is detected by using a hybridization probe. RFLPs involve fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure. PCR amplification of DNA is not required for this method. Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. RLFP markers have many advantages including reproducibility, codominant inheritance, no sequence information requirement, and easy scoring. But it also has a few limitations, such as low sensitivity, requirement of a large amount of high quality genomic DNA, and low stability and reproducibility (Smouse and Chakraborty, 1986).

(b) Variable Number Tandem Repeats (VNTR): Variable Number of Tandem Repeats (VNTR) loci are chromosomal regions in which a short DNA sequence motif (such as GC or AGCT) is repeated a variable number of times end-to-end at a single location (tandem repeat). This technique is similar to RFLP, but the probe used for Southern blotting exists as tandem repeats which occur as clusters among chromosomes. They show variations in length between individuals and each variant acts as an inherited allele (Li et al., 2012). Because of their variation between individuals, these DNA segments are useful for identifying individuals for such purposes as linking a suspect to a crime scene. These are the famed “DNA fingerprints.” Tandem repeat DNA sequences are also called satellite DNA. There are three main types:

• A satellite is a highly repetitive DNA sequence with each repeated sequence ranging from a thousand to several thousand base pairs. The entire satellite can be up to 100 million base pairs long, and tends to occur in regions Fingerprinting Techniques for Herbal Drugs Standardization 107

of heterochromatin. Satellites are abundant on the Y chromosome, which makes a handy tool for those studying paternal genetic transmission in mammals. • A minisatellite is an array of tandem repeats, with each repeat ranging from nine to 100 base pairs (but most commonly around 15 base pairs). The entire array is usually 500 to 30,000 base pairs long. These are most commonly found in euchromatin regions of the chromosome. • A microsatellite is an array of very short repeats (2–6 base pairs each), with the entire array ranging from 10,000 to 100,000 base pairs in length. They have so far been found in the euchromatin regions of vertebrate, insect, and plant chromosomes. The number of repeats varies among individuals in a population, making microsatellites particularly useful to the population geneticist.

12.2.1.2.1.2 Polymerase Chain Reaction-Based Methods Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA. PCR-based procedures are difficult to standardize due to the use of different DNA polymerase, buffer formulations, and equipment. Initially primers, the original DNA (extracted from the plant cell) which is to be amplified, a specific type of DNA polymerase, and the necessary chemicals for DNA synthesis are mixed. Then the following steps are carried out.

• Denaturation: DNA fragments are heated at high temperature (95°C for 30 seconds or 97°C for 15 seconds) which reduces the DNA double helix to a single helix to a single strand which becomes accessible to the primer. • Annealing: In this method, the temperature is lowered until the primers can hybridize or bind to complementary regions on the DNA. • Extension: The primers are used by DNA polymerase to initiate synthesis and new complementary strands of DNA are made.

PCR-based techniques include:

a. Randomly Amplified Polymorphic DNA (RAPD) b. Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) c. Amplified Fragment Length Polymorphism (AFLP) d. Simple Sequence Repeats (SSR)

(a) Randomly Amplified Polymorphic DNA (RAPD) and Arbitrarily Primed Polymerase Chain Reaction (AP-PCR): In these methods, a single arbitrarily chosen oligonucleotide is used as both the forward and reverse primer in the PCR reaction. This sequence consists of about 10 nucleotides in the case of RAPD and about 20 nucleotides in the case of AP-PCR. A product is produced when the primer binds on opposite strands, in the reverse orientation, and within an 108 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines amplifiable distance. PCR fragments are generated from different locations of the genome, because there are multiple sites within the genome for the primer to bind. Thus, multiple loci may be examined simultaneously. Use of a series of different primers shows the generation of a genetic fingerprint. The advantages of this technique include the high number of fragments, faster analysis, easy to operate, economical, and only a small quantity of target DNA is required. However, certain disadvantages are the low reproducibility and it is highly sensitive to laboratory changes.

(b) Amplified Fragment Length Polymorphism (AFLP): This fingerprinting technique is based on the detection of multiple DNA restriction fragments by means of PCR amplification and has the capacity to detect thousands of independent loci. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. Genomic DNA is digested by appropriate restriction enzymes, which cut the DNA at defined sequence sites. A subset of the resultant fragments is then ligated to synthetic double stranded adaptors (DNA segments) at each end and subsequently amplified using two specific adaptor homologous primers. The amplified and labeled restriction fragments are separated on denaturing gels or by capillary electrophoresis. The complexity of the AFLP profile depends on the primers and restriction enzymes chosen and on the level of sequence polymorphism between the tested DNA samples. The number of amplified bands of the preselected PCR is usually so high that a second round of PCR (selective PCR with fluorescent dyes) has to be performed to reduce the number of amplified products. This is done by using primers that possess 1–3 additional bases at the 3′ end. This technique has several advantages including higher reproducibility, resolution and sensitivity, reliability, robustness, and rapid amplification, but it is expensive and time consuming. Typically, 50–100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

(c) Simple Sequence Repeats (SSR): Simple sequence repeats are tandem repeats scattered throughout the genome. They can be amplified using primers that flank these regions. The technique has been successfully used to construct detailed genetic maps of several plant species and to study genetic variations within populations of the same species. It is a robust technique which requires a smaller amount of DNA, but separate SSR primers are required for each species.

12.2.1.2.1.3 Sequence-Based Methods In this technique, DNA sequences from the nuclear and chloroplast genomes are used for identification of plants at several taxonomic levels. DNA sequenced-based techniques have widely been used for authentication of herbs. The main two types of sequence-based methods include:

a. Single nucleotide polymorphism (SNP) b. Short tandem repeats (STR) Fingerprinting Techniques for Herbal Drugs Standardization 109

(a) Single Nucleotide Polymorphism (SNP): Single Nucleotide Polymorphisms or SNPs are variations in a DNA sequence that occur when a single nucleotide in the sequence is different from the norm in at least 1% of the population. When SNPs occur inside a gene, they create different variants or alleles of that gene. SNPs are common, occurring every 100–300 bases along the entire length of the human genome. Mutations in SNPs are very rare, so the sequences tend to be passed unchanged across generations. But because any given SNP is relatively common in the population, an analyst must examine dozens of SNPs to derive a true DNA fingerprint. For this reason, SNP analysis is rarely used in forensic cases.

(b) Short Tandem Repeats (STR): Short tandem repeats is a class of polymorphisms that occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 10 base pairs and is typically in the non-coding intron region, making it junk DNA. This technique is reliable, independent, and less affected by environmental changes. However, this is expensive and is affected by plant compounds or fungal contamination (Tautz 1989). The determination of common peaks in a set of chromatographic fingerprints provides useful qualitative and quantitative information on the characteristic components of the herbal medicines being investigated. Thus, chromatographic fingerprint analysis serves as a promising quality control tool for herbal medicines. DNA fingerprinting is another technique which is a promising tool for the authentication of medicinal plant species and for ensuring better quality herbs and nutraceuticals. DNA fingerprinting, apart from identifying alterations in the genotypes of plant species, can also use for the betterment of drug yield by tissue culturing. A DNA of interest can be stored as germplasm, which is then used for future cultivation as well as for the conservation of endangered plant species. Thus, the problem of quality assurance of herbal medicines has been solved to a great extent with the help of chromatographic and DNA fingerprint analysis.

12.3 SUMMARY In conventional drug analysis, fingerprinting is used to highlight the profiles of the sample matrix, which often is sufficient to give indications of the source and method of preparation. In herbal drugs, such a profile is dependent not only on the preparation processes, but also on the quality of the crude herb source material, which varies with different herb origins, sources, harvest times, and pretreatment processes. The consistency and stability of the chemical constituents observed in the profiles thus reflect more than just the conditions of the drug preparation process; they also reflect the source and quality of the raw herbs. The quality analysis (QA) and quality control (QC) of crude herbs by GAP guidelines as described earlier is, therefore, of prime importance to ensure the success of downstream GMP. In both GAP and GMP, fingerprinting analysis is used to appraise the quality of the herbal material of concern, and the key is to develop links between the marker compound-based chromatographic or spectroscopic profiles with the efficacy of herbal products. Chromatographic 110 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines profiles of major components are used to evaluate herbal growers and suppliers, to standardize raw materials, and to control formulation and tablet content uniformity. Ideally, bioactive compounds or components should be identified. When this is not possible, important chemical marker compounds are developed to allow fingerprinting analysis for the assessment of batch-to-batch consistency. Electrospray ionization was used in HPLC/MS for the detailed profiling of active components. In fingerprinting analysis, it is important to standardize all laboratory procedures to avoid artificial variations in results. The relative intensity of the peaks is important, and chromatographic fingerprints must be specific for the substance being analyzed. Hence, it is necessary to check fingerprints obtained from related botanical products and known adulterants to ensure that the method developed can distinguish true from false identifications.

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13.1 INTRODUCTION Spectroscopic techniques employ light to interact with matter and thus probe certain features of a sample to learn about its consistency or structure. Light is electromagnetic radiation, a phenomenon exhibiting different energies, and dependent on that energy, different molecular features can be probed. The spectroscopic techniques use instruments that share several common basic components, including a source of energy, a means for isolating a narrow range of wavelengths, a detector for measuring the signal, and a signal processor that displays the signal in a form convenient for the analyst (Orr et al., 2016). There are various spectroscopy techniques employed for herbal drug standardization. The important ones are explained below.

13.1.1 ultraviolet Spectroscopy (UV) Ultraviolet spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range. When a sample is exposed to light energy that matches the energy difference between a possible electronic transition within the molecule, a fraction of the light energy would be absorbed by the molecule and the electrons would be promoted to the higher energy state orbital. A spectrometer records the degree of absorption by a sample at different wavelengths and the resulting plot of absorbance (A) versus wavelength (λ) is known as a spectrum (Figure 13.1). UV-Vis Spectroscopy is beneficial in qualitative analysis as we can get spectra with specific solvent extractions and dissolution in a specific solvent. Spectra got can be used as a fingerprint of the sample extract if they are obtained using an authenticated standard raw drug sample. A library of spectra developed like this in the lab could be used to identify a given specimen by comparing the spectra with a developed library. Adulterants can be found out by UV spectral analysis. But initial spectra should be obtained using an authenticate specimen and tested multiple times with variations such as season of collection, place of collection, time of collection, and so on (Giridhar, 2015). UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and can be compared with that of the standard raw material. By also measuring the absorbance at a specific wavelength, the impurities can be detected. UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, and the presence of hetero atoms (Gupta et al., 2005). From the location of peaks and combinations of peaks, it can be concluded

113 114 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Reference

Deuterium/ Data Monochromator tungsten output Detector lamp

Sample

FIGURE 13.1 UV spectroscopy instrumentation. whether the compound is saturated or unsaturated, hetero atoms are present or not, and so on. UV absorption spectroscopy can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds. Many herbal drugs are either in the form of raw material or in the form of formulations. They can be assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at a specific wavelength (Agarwal and Paridhavi, 2012). Molecular weights of herbal compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.

13.1.2 infrared Spectroscopy (IR) Infrared spectroscopy is a technique based on the vibrations of the atoms of a molecule. Infrared spectroscopy involves the interaction of infrared radiation with matter. The energy at which any peak in an absorption spectrum appears corresponds to the frequency of a vibration of a part of a sample molecule (Jackson et al., 2002). Infrared spectroscopy exploits the fact that molecules absorb frequencies that are characteristic of their structure (Figure 13.2). Most of the analytical applications are confined to the middle IR region (2–15 µm) because absorption of organic molecules is high in this region (Shaw and Mantsch, 2002). Infrared spectroscopy has proven to be a powerful tool for the study of biological molecules and the application of this technique to biological problems is continually expanding, particularly with the advent of increasingly sophisticated sampling techniques such as infrared imaging

Sample

IR source Splitter Detector Processor Display

Reference

FIGURE 13.2 IR spectroscopy instrumentation. Spectroscopic Techniques 115

(Gremlich and Yan, 2000). This technique has been employed for a number of decades for the characterization of isolated biological molecules, particularly proteins and lipids (Shaw and Mantsch, 2000). However, the last decade has seen a rapid rise in the number of studies of more complex systems, such as diseased tissues. The efficiencies of herbal medicines depend on the amount of active components in them, which could vary significantly in content (Clark and Hester, 1996). Therefore, the quality control of herbal medicines is a very important issue. Identification and quality evaluation of herbal medicines is done by means of their component overlapping infrared spectra. True and false identification could be performed with specific peaks and their absorption ratios. IR spectroscopy has been widely applied in both qualitative and quantitative analysis of herbal drugs (Stuart, 2000). In addition, the application of IR spectroscopy to the detection of illegal additives and the rapid assessment of the quality of herbal medicines by fast inspection has also been proven.

13.1.3 Fourier Transform Infrared (FTIR) Spectroscopy Infrared spectroscopy is an analytical technique applied to the characterization of molecules. It has the potential to provide biochemical information without disturbing the biological sample. It is based on the fact that molecules absorb specific frequencies that are characteristic of their structure. The infrared region of the electromagnetic spectrum extends from the visible to the microwave. These absorptions are related to the strength of the bond. Consequently, the spectroscopic study of biological cells and tissue is an active area of research, with its primary goal being to elucidate how accurately infrared spectroscopy can determine whether cells or tissues are damaged (Pitt et al., 2005). FTIR stands for Fourier Transform Infrared, the preferred method of infrared spectroscopy. Fourier transforms infrared spectrometers, with their high signal-to-noise ratio and high precision in absorbance and wave number measurements, have caused a resurgence of interest in the use of infrared spectra for the identification of biomolecules (Wood et al., 1996).

13.1.3.1 Principle FTIR Spectroscopy is a technique based on the determination of the interaction between an IR radiation and a sample that can be solid, liquid or gaseous. When IR radiation is passed through a sample, some of the infrared radiation is absorbed by the sample and some of it is passed through (transmitted). In FTIR, the frequencies at which the sample absorbs and the intensities of these absorptions is measured. The frequencies are helpful for the identification of the sample’s chemical make-up due to the fact that chemical functional groups are responsible for the absorption of radiation at different frequencies. The concentration of a component can be determined based on the intensity of the absorption. A resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample just like a fingerprint, where no two unique molecular structures produce the same infrared spectrum (Figure 13.3) (Boyer et al. 2006). FTIR (Fourier Transform Infrared Spectroscopy) is one of the most widely used methods to identify the chemical constituents and elucidate a compound’s structures, and has been used as a requisite method to identify medicines in the pharmacopoeia 116 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Fixed mirror

Beam splitter Moving mirror

Source Collimator

Sample compartment

Detector

FIGURE 13.3 Block diagram of an FTIR spectrometer. of many countries. FTIR is an advanced technique which can further be utilized in the field of medicine for diagnosis as well as for treating ailments by discovering new drugs. Owing to the fingerprint characteristics and extensive applicability to the samples, FTIR has played an important role in pharmaceutical analysis in recent years. FTIR was developed in order to overcome the limitations encountered with dispersive instruments. The interferometer produces a unique type of signal which has all of the infrared frequencies “encoded” into it. The ease with which the FTIR works makes it more acceptable to work with not only in herbal analysis, but also in environmental studies, cancer detection, forensics, food analysis, and toxicology (Che Man et al., 2010).

13.1.3.1.1 Applications in Herbal Drug Analysis Use of FTIR in herbal analysis can be explained in quantitative and qualitative terms. A variety of natural drugs, medicinal plants, are available which has advanced the research in herbal drug systems. FTIR becomes a key instrument in estimating the herbal drugs, evaluating their components and therapeutic value, and proving their efficiency in treating ailments. FTIR techniques are also used in forensic science, paints, textiles, and cosmetics analysis as well as semiconductor analysis and pharmaceuticals. Physiological samples like malignant cells, bones, hairs, multilayer compounds like polymers, paintings, films, geological samples, and inclusions in stone can also be studied by FTIR (Crupi et al., 2002).

13.1.4 Mass Spectroscopy Mass Spectrometry is a powerful technique for identifying unknowns, studying molecular structure, and probing the fundamental principles of chemistry. It can provide us with useful structural information for drug discovery and has been recognized as a sensitive, rapid, and high-throughput technology for advancing drug discovery from herbal medicine in the post-genomic era. It is essential to develop an efficient, high-quality, high-throughput screening method integrated with Spectroscopic Techniques 117

Gas phase ions Ion sorting Ion detection Mass spectrum

Inlet Source Analyzer Ion Data detector system

Sample Vacuum pumps Data output introduction

FIGURE 13.4 Mass spectroscopy instrumentation. mass spectroscopy (Zhang et al., 2016). Mass spectroscopy is one of the primary spectroscopic methods for molecular analysis available to an organic chemist. It is a microanalytical technique requiring only a few nanomoles of the sample to obtain characteristic information pertaining to the structure and molecular weight of an analyte. It involves the production and separation of ionized molecules and their ionic decomposition products and finally the measurement of the relative abundance of the different ions produced (Yerlekar and Kshirsagar, 2014). It is, thus, a destructive technique in that the sample is consumed during analysis. In most cases, the nascent molecular ion of the analyte produces fragment ions by cleavage of the bond and the resulting fragmentation pattern constitutes the mass spectrum. Thus, the mass spectrum of each compound is unique and can be used as a “chemical fingerprint” to characterize the sample. Mass spectroscopy is the most accurate method for determining the molecular mass of a compound and its elemental composition. In this technique, molecules are bombarded with a beam of energetic electrons. The molecules are ionized and broken up into many fragments, some of which are positive ions. Each kind of ion has a particular ratio of mass to charge, that is, m/e ratio (value). For most ions, the charge is one, thus the m/e ratio is simply the molecular mass of the ion (Figure 13.4). Mass spectrometry (MS) has progressed to become a powerful analytical tool for both quantitative and qualitative applications. The ability of mass spectrometry to analyze proteins and other biological extracts is due to the advances gained through the development of soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) that can transform biomolecules into ions (Takats et al., 2004). MALDI, however, has the advantage of producing singly charged ions of peptides and proteins, minimizing spectral complexity. Regardless of the ionization source, the sensitivity of a mass spectrometer is related to the mass analyzer where ion separation occurs. Both quadrupole and time of flight (ToF) mass analyzers are commonly used.

13.1.5 nMR Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique used in quality control and research for determining the content and purity of a sample as well as its molecular structure (Ross et al., 2011). It is based on the fact that when a population of magnetic nuclei is placed in an external 118 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

Computer

Transmitter Gate and power Pulse programmer ampliﬁer Digital to analog converter Fourier transform

Accumulation Probe Spectrum

Analog to digital Receiver converter

FIGURE 13.5 NMR spectroscopy instrumentation. magnetic field, the nuclei become aligned in a predictable and finite number of orientations. The nuclei of many elemental isotopes have a characteristic spin (I). Some nuclei have integral spins (e.g., I = 1, 2, 3…), some have fractional spins (e.g., I = 1/2, 3/2, 5/2…), and a few have no spin, I = 0 (e.g., 12C, 16O, 32S). NMR can be used to determine molecular conformation in solution as well as studying physical properties at the molecular level such as conformational exchange, phase changes, solubility, and diffusion. NMR spectroscopy is a powerful tool for herbalists interested in the structure, dynamics, and interactions of herbal active constituents. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function (Darbeau, 2006). NMR identification of the main components in the essential oils or herbal extracts clarifies the possible chemotaxonomic differences between plant species. NMR evaluation and quality control of traditional and folk medicines, especially large scale analysis of herbal medicines, is complementary to common quality control methods (Saeidnia and Gohari, 2012). The instrumentation of NMR spectroscopy is shown in Figure 13.5.

13.2 CONCLUSION Spectroscopy techniques are the most commonly used methods in the standardization of herbal medicines, but the herbal system is not easy to analyze because of its complexity of chemical composition. Many cutting-edge analytical technologies have been introduced to evaluate the quality of medicinal plants and a significant amount of measurement data has been produced. Chemometric techniques provide a good opportunity for mining more useful chemical information from the original data. Comprehensive methods and hyphenated techniques associated with chemometrics used for extracting useful information and supplying various methods of data processing are now more and more widely used in medicinal plants. Spectroscopic Techniques 119

REFERENCES Agarwal SS, Paridhavi M. Herbal Drug Technology, 2nd Edition. University Press, Hyderabad, 2012, pp. 287–290. Boyer C, Bregere B, Crouchet S, Gaudin K, Dubost JP. Direct determination of niflumic acid in a pharmaceutical gel by ATR/FTIR spectroscopy and PLS calibration. J Pham Biomed Anal. 2006; 40: 433–437. Che Man YB, Syahariza ZA, Rohman A. Chapter 1. Fourier transform infrared (FTIR) spectroscopy: Development, techniques, and application in the analyses of fats and oils In Ress OJ. (Ed.), Fourier Transform Infrared Spectroscopy, Nova Science Publishers, New York: USA, 2010, pp. 1–26. Clark RJH, Hester RE. Biomedical Applications of Spectroscopy, Wiley, Chichester, UK, 1996. Crupi V, Majolino D, Mondello MR, Migliardo P, Venuti, V. FTIR spectroscopy: A powerful tool in pharmacology. J Pham Biomed Anal. 2002; 29: 1149–1152. Darbeau RW. Nuclear Magnetic Resonance (NMR) Spectroscopy: A Review and a Look at Its Use as a Probative Tool in Deamination Chemistry, 2006, pp. 401–425. Giridhar V. Quality control of herbal drugs through UV-Vis spectrophotometric analysis. Int J Ayurvedic Med. 2015; 6(1): 102–109. Gremlich HU, Yan B. Infrared and Raman Spectroscopy of Biological Materials, Marcel Dekker, New York, 2000. Gupta AK, Tandon N, Sharma M. Quality Standards of Indian Medicinal Plants, 1st Edition. ICMR, New Delhi, 2005, Volume 3. Jackson M, Moore DJ, Mantsch HH, Mendelsohn R. Vibrational spectroscopy of membranes. In Chalmers JM and Griffiths PR. (Eds.), Handbook of Vibrational Spectroscopy, Vol. 5. Wiley, Chichester, UK, 2002, pp. 3508–3518. Orr BJ, Haub JG, He Y, White RT. Spectroscopic applications of pulsed tunable optical parametric oscillators. In Duarte FJ. (Ed.), Tunable Laser Applications, 3rd Edition. CRC Press, Boca Raton, 2016, pp. 17–142. Pitt GD, Batchelder DN, Bennett R, Bormett RW, Hayward IP, Smith BJE, Williams KPJ, Yang YY, Baldwin KJ, Webster S. Engineering aspects and applications of the new Raman instrumentation. IEE Proc-Sci Meas Technol. 2005; 152(6): 241–318. Ross B, Tran T, Bhattacharya P, Watterson DM, Sailasuta N. Application of NMR spectroscopy in medicinal chemistry and drug discovery. Curr Top Med Chem. 2011; 11(1): 93–114. Saeidnia S, Gohari AR. Application of spectroscopy in herbal metabolomics. DARU J Pharma Sci. 2012; 20: 91. Shaw RA, Mantsch HH. Infrared spectroscopy in clinical and diagnostic analysis. In Meyers RA. (Ed.), Encyclopedia of Analytical Chemistry, Vol. 1. Wiley, Chichester, UK, 2000, pp. 83–102. Shaw RA, Mantsch HH. Vibrational spectroscopy applications in clinical chemistry. In Chalmers JM and Griffiths PR. (Eds.), Handbook of Vibrational Spectroscopy, Vol. 5. Wiley, Chichester, UK, 2002, pp. 3295–3307. Stuart BH. Infrared spectroscopy of biological applications. In Meyers RA. (Ed.), Encyclopedia of Analytical Chemistry, Vol. 1, Wiley, Chichester, UK, 2000, pp. 529–559. Takats Z, Wiseman JM, Gologan B, Cooks RG. Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science. 2004; 5695: 471–473. Wood BR, Quinn MA, Burden FR, McNaughton D. An investigation into FT-IR spectroscopy as a bio-diagnostic tool for cervical cancer. Biospectroscopy. 1996; 2: 143–153. Yerlekar A, Kshirsagar MM. A review on mass spectrometry: Technique and tools. Int J Eng Res Appl. 2014; 4(4): 17–23. Zhang A, Sun H, Wang X. Mass spectrometry-driven drug discovery for development of herbal medicine. Mass Spec Rev. 2016; 9999: 1–14.

Standardization 14 of Herbal Drugs

14.1 INTRODUCTION In recent years, there has been great demand for plant derived products in developed countries. These products are increasingly being sought out as medicinal products, nutraceuticals, and cosmetics (Sagar Bhanu et al., 2005). There are around 6000 herbal manufacturers in India. More than 4000 units produce Ayurveda medicines. Due to a lack of infrastructures, skilled manpower, reliable methods, and stringent regulatory laws, most of these manufacturers produce their product on a very tentative basis (Patel et al., 2006). In order to have a good coordination between the quality of raw materials, in-process materials, and the final products, it has become essential to develop reliable, specific, and sensitive quality control methods using a combination of classical and modern instrumental methods of analysis. Standardization is an essential measurement for ensuring the quality control of the herbal drugs (Shrikumar et al., 2006). Standardization expression is used to describe all measures which are taken during the manufacturing process and quality control leading to a reproducible quality. Standardization of a drug means confirmation of its identity, determination of its quality and purity, and detection of the nature of adulterants by various parameters, such as morphological, microscopical, physical, chemical, and biological observations (Figure 14.1). World Health Organization (WHO) provides guidelines for prevention, control, safety, and efficacy as well as evaluation and standardization of herbal materials (WHO, 1988, 1992, 1999, 2007). Standardization involves adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity by adding excipients or by mixing herbal drugs or herbal drug preparations. Botanical extracts made directly from crude plant material show substantial variation in composition, quality, and therapeutic effects (WHO, 1996). Standardized extracts are high-quality extracts containing consistent levels of specified compounds, and they are subjected to rigorous quality controls during all phases of the growing, harvesting, and manufacturing processes (Torey et al., 2010; Yadav and Dixit, 2008; Zhao et al., 2006). Standardization of herbal drugs is not an easy task as numerous factors influence the bio efficacy and reproducible therapeutic effect. In order to obtain quality oriented herbal products, care should be taken right from the proper identification of plants, season, and area of collection and their extraction and purification processes, thus rationalizing the combination in the case of polyherbal drugs (Bauer, 1998; Bisset, 1994; De Smet, 1999; Smillie and Khan, 2010). The herbal formulation in general can be standardized schematically in order to formulate the medicament using raw materials collected from different localities and a comparative chemical efficacy of different batches of formulation is to be observed.

121 122 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

• Qualitative • Shape • Quantitative • External marking • SEM studies • Power studies Macroscopic Microscopic • Color • Odor • Taste • Moisture • Texture BOTANICAL content • Fracture • Extractive values

PHYSICAL • Ash values ORGANOLEPTIC • STANDARDIZATION Fluorescence Microbial OF HERBAL DRUGS analysis contamination • Total viable BIOLOGICAL aerobic count • Determination of pathogens CHEMICAL • Aﬂatoxins Radioactive content contamination

Bioassay Antagonistic Qualitative Quantitative • Bacterial • HPTLC ﬁnger • HPTLC • Fungal • Analytical methods Toxicological printing GLC • • Determination of pesticide • Secondary HPLC residues metabolites • Determination of heavy metals • DNA ﬁnger printing

FIGURE 14.1 Standardization of herbal drugs.

The preparations with better clinical efficacy are to be selected. After all the routine physical, chemical and pharmacological parameters are to be checked for all the batches to select the final finished product and to validate the whole manufacturing process (EMEA, 1998, 2005; Mukherjee, 2002). The stability parameters for the herbal formulations which include physical, chemical, and microbiological parameters are as follows:

• Physical parameters include color, odor, appearance, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flow ability, flocculation, sedimentation, settling rate, and ash values. • Chemical parameters include limit tests, chemical tests, chemical assays, and so on. Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC, GC, UV, GC-MS fluorimetry, and so on. • Microbiological parameters include total viable content, total mold count, total enterobacterials, and their counts. Limiters can be utilized as a quantitative or semi-quantitative tool to ascertain and control the amounts of impurities, such as the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels and from the solvents, and so on. Standardization of Herbal Drugs 123

14.2 DIFFERENT TECHNIQUES INVOLVED IN STANDARDIZATION OF CRUDE DRUGS • Botanical methods • Physical methods • Chemical methods • Biological methods

14.2.1 botanical Methods 14.2.1.1 Macroscopic Methods The macroscopic identity of medicinal plants includes materials that are based on shape, size, color, surface characteristics, texture, and fracture. It is also known as organoleptic evaluation based on the study of the morphological and sensory profiles of whole drugs. Fractured surfaces in cinchona, quillia, and cascara barks and quassia wood are important characteristics. The aromatic odors of umbelliferous fruits and the sweet taste of liquorice are examples of this type of evaluation where the odor of a drug depends upon the type and quality of odorous principles (volatile oils) present (Dalal and Patel, 1995). The shape of drug may be cylindrical (sarpsilla), subcylindrical (podophyllum), conical (aconite), fusiform (jalap), and so on, while the size represents length, breadth, thickness, diameter, and so on. Color means the external color which varies from white to brownish-black and is an important diagnostic characteristic. The general appearance (external marking) of the weight of a crude drug often indicates whether it is likely to comply with prescribed standards, including furrows (alternate depressions or valleys), wrinkles (fine delicate furrows), annulations (transverse rings), fissures (splits), nodules (rounded outgrowths), and scars (spots left after fall of leaves, stems or roots). Taste is a specific type of sensation felt by the epithelial layer of the tongue (WHO, 2005). It may be acidic (sour), saline (salt like), saccharic (sweetish), bitter or tasteless (possessing no taste) (Table 14.1). Different macroscopic characteristics of herbal drugs are as follows.

14.2.1.1.1 Size A graduated ruler in millimeters is adequate for the measurement of the length, width, and thickness of the crude materials. Small seeds and fruits may be measured by aligning 10 of them on a sheet of calibrated paper, with 1 mm spacing between lines, and dividing the result by 10.

14.2.1.1.2 Color The color of the sample should be compared with that of a reference sample. Examine the untreated sample under diffuse daylight. An artificial light source with wavelengths similar to those of daylight may also be used.

14.2.1.1.3 Surface Characteristics, Texture, and Fracture Characteristics A magnifying lens (6×–10×) may be used. Wetting with water or reagents, as required, may be necessary to observe the characteristics of a cut surface. Touch the material to determine if it is soft or hard; bend and rupture it to obtain information on brittleness and the appearance of the fracture plane–whether it is fibrous, smooth, rough, granular, and so on. 124 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

TABLE 14.1 Macroscopic Characteristics of Herbal Drugs Test Observation Inference Color Light yellow Ginger, squill Light brown Fennel, dill Dark brown to violet Clove, ergot Red Cinchona, arjuna Orange Rhubarb Green Senna, digitalis Odor Characteristic Aromatic crude drugs Odorless Absence of aromatic crude drugs Taste Aromatic Dill, fennel Aromatic and pungent Ginger Sweet Liquorice Bitter Cinchona, nuxvomica Mucilagenous Isapghol Astringent Myrobalan

14.2.1.1.4 Odor First, determine the strength of the odor (none, weak, distinct, strong) and then the odor sensation (aromatic, fruity, musty, moldy, rancid, etc.). If the material is expected to be innocuous, place a small portion of the sample in the palm of the hand or in a beaker of suitable size and slowly and repeatedly inhale the air over the material. If no distinct odor is perceptible, crush the sample between the thumb and index finger or between the palms of the hands using gentle pressure. If the material is known to be dangerous, crush by mechanical means and then pour a small quantity of boiling water onto the crushed sample in a beaker.

14.2.2 Microscopic Methods This method is used for identification of drugs on a cellular level. By means of various microscopic techniques, the structural and cellular features of herbs are examined in order to determine their botanical origins and assess their qualities. This is used to determine the structure of organized drugs by their histological characters. It includes examination of the whole, certain parts or powdered crude drugs (Zhao et al., 2005). Quality control of herbal drugs has traditionally been based on appearance and today microscopic evaluation is indispensable in the initial identification of herbs, as well as in identifying small fragments of crude or powdered herbs and the detection of foreign matter and adulterants. This method is useful for identifying species from fragments or powders and for distinguishing species with similar morphological characters; it may also be useful for evaluating the pharmaceutical quality of herbs (Remington and Gennaroa, 1995). A primary visual evaluation, which seldom needs more than a simple magnifying lens, can be used to ensure that the plant is of the required species, and that the right part of the plant is being used (Ruzin, 1999). Standardization of Herbal Drugs 125

14.2.2.1 Microscopical Examination Microscopic analysis is needed to determine the correct species and/or that the correct part of the species is present. Using the microscope to determine the identity of herbal medicines, namely, microscopic authentication, refers to observing the cell structure and internal features using a microscope and its derivatives. Besides the ordinary light microscope, other microscopes have also been used to enhance the accuracy of authentication, such as the polarized light microscope and fluorescence microscope. Use of these microscopes expands the number of features available for use in identification (Liang et al., 2006, 2009; Zhao et al., 1997). Recent advancements in normal light microscopy have greatly enhanced its usefulness in the authentication of herbal medicines. Feature extraction and similarity measurement as well as the use of chord length distribution have been used effectively in the classification of starch grains (Tam et al., 2006). This provides greater accuracy and flexibility in capturing information about starch grains which are useful in authentication of herbal medicines (An et al., 2009; Chu et al., 2009; Li et al., 2008). The fluorescence microscope reveals the fluorescence emitted from herbal tissues under illumination. Many herbal tissues, by virtue of their chemical structures or secondary metabolites, have the ability to emit light of a specific wavelength following the absorption of light with a shorter wavelength and higher energy (Lau et al., 2004; Zhao, 2010). Details of the cell structure and arrangement of the cells are useful for differentiating similar species.

14.2.2.1.1 Equipment Use a microscope with an ocular micrometer to measure the size of small objects. The scales should be calibrated using a stage micrometer, consisting of a glass slide of usual size, upon which a scale is engraved or photographed, usually 1 or 2 mm long, in 0.1 and 0.01 mm divisions. The ocular micrometer consists of a small disc of glass, across the diameter of which is a 100-line scale that is engraved or photographed. The disc is placed into the eyepiece. A microscope is equipped with the following parts:

Lenses: Lenses providing a wide range of magnification and a substage condenser, a graduated mechanical stage, objectives with a magnification of 4×, 10× and 40×, and color filters of ground glass, for example, blue-green; high eyepoint eyepieces are preferred for wearers of spectacles. Lamp: A lamp, either separate or incorporated into the microscope. Micrometer: A stage micrometer and an ocular micrometer to be inserted into a 6x eyepiece and placed on the diaphragm or preferably, a micrometer eyepiece. Other parts: A set of polarizing filters; a set of drawing attachments for the microscope; a microburner (Bunsen type); slides and cover glasses of standard size and a set of botanical dissecting instruments.

14.2.2.1.2 Standard Procedure for Microscopic Identification Microscopic identification is used to examine transverse or longitudinal sections, powder, surfaces or disintegrated tissues of crude drugs and/or herbal proprietary medicines mounted on glass slides (Zhao et al., 1997). 126 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.2.2.1.3 Preliminary Treatment Dried parts of a plant may require softening before preparation for microscopy, preferably by being placed in a moist atmosphere or by soaking in water. For small quantities of material, place a wad of cotton wool moistened with water into the bottom of a test tube and cover with a piece of filter paper. Place the material being examined on the paper, stopper the tube, and allow it to stand overnight or until the material is soft and suitable for cutting. Use a desiccator for larger quantities of material, placing water into the lower part instead of the drying agent. The following steps should be adopted for different materials:

• Bark, wood, and other dense and hard materials usually need to be soaked in water or in equal parts of water, ethanol, and glycerol for a few hours or overnight until they are soft enough to be cut. Boiling in water for a few minutes may sometimes be necessary. • Any water-soluble contents can be removed from the cells by soaking in water. • Starch grains can be gelatinized by heating in water. In certain cases, material can be moistened with water for a few minutes to soften the surfaces and allow sections to be cut.

14.2.2.1.4 Preparation of Specimen A good quality specimen is essential for microscopic identification. The method of making slides should be chosen according to the nature of the material at hand and the purpose of the investigation.

14.2.2.1.5 Transverse Sections There are four main methods for mounting transverse sections.

• Free hand mounting: This is used for temporary slides. In this method, material is cut with the help of a blade and sliced smoothly from upper left toward lower right in a single motion. Avoid sawing back and forth; keep the specimen and blade lubricated with water. • Glide mounting: This method, using a gliding mounting machine, is suitable for lignum, ligneous roots, stems or other solid materials. It is also known as sliding microtome because of its composition of specimen feed, knife, holder, and specimen orientation. The sturdy construction gives it qualities that ensure excellent, reproducible sectioning results. The section thickness and knife inclination and declination can be adjusted. • Cryology mounting: This method is mainly used to make slides of animal tissue and fresh and young herbal tissue. Cut the sample into small pieces (about 1–2 cm in diameter) and embed them with cryomatrix on a crycasste; freeze them; slice using a machine; mount on glass slides and seal. • Paraffin mounting: This method entails embedding specimens in paraffin, then slicing the block. The steps include sampling, fixing, dehydration, vitrification, olefin immersion, olefin embedding, slicing, removing the paraffin, staining with, for example, safranin and fast green, vitrification Standardization of Herbal Drugs 127

after replacing the dyeing solution with a low to high gradient concentration of ethanol, and finally sealing the mounted specimen with gum arabic or neutral gum.

14.2.2.1.6 Clarification of Microscopic Particles The presence of certain cell contents, such as starch grains, aleurone grains, plastids, fats, and oils, may render sections non-translucent and obscure certain characteristics. Reagents that dissolve some of these contents can be used in order to make the remaining parts stand out clearly or produce a penetrating effect. This renders the section more transparent and reveals details of the structures. The most frequently used clarifying agents are chloral hydrate TS, Lactochloral TS, Sodium hypochlorite TS, Xylene R, and light petroleum R.

14.2.2.1.7 Sampling Sampling affects the accuracy of identification results; therefore, reliable, random procedures of sampling should be strictly followed. Sampling involves reference samples and test samples. Reference samples (RS) are essential for microscopic identification. This should be determined after strict botanical taxonomy identification of the original plant. For test samples (TS), origins, production place, specification, grade, and packaging style should be noted. Integrity of the package, hygienic level, water trace, extent of mildew, and contamination with foreign matter should also be checked and recorded in detail. The average quantity of samples for testing should be no less than three. One-third of the sample is used for experimental analysis, 1/3 is used for verification, while the remaining 1/3 is retained for at least a year.

14.2.2.1.8 Fragments In the case of pieces of tissue, place them on a slide, add wetting agent, and tease them apart with dissecting needles. Then add a cover skip.

14.2.2.1.9 Photography Photography with the digital method of storage makes it more and more convenient to save and share suitable pictures. Setting the functions of exposure time, contrast, crop selection, and microscopic measuring must be mastered.

14.2.3 Powder Studies Place 1 or 2 drops of water, glycerol/ethanol TS or chloral hydrate TS on a glass slide (other fluids may be used). Moisten the tip of a needle with water and dip into the powder. Transfer a small quantity of the material that adheres to the needle tip into the drop of fluid on the slide. Stir thoroughly, but carefully, and apply a cover glass. Press lightly on the cover glass with the handle of the needle, and remove excess fluid from the margin of the cover glass with a strip of filter paper. If the specimen is to be freed from air bubbles, boil carefully over a small flame of a microburner until the air is completely removed. Care should be taken to replace the fluid that evaporates so that the space beneath the cover glass is completely filled with fluid at the conclusion of the operation (WHO, 1998). The coarseness or fineness of a powder is classed 128 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

TABLE 14.2 Classification of Powders Descriptive Term Particle Size Coarse (2000/355) All the particles will pass through a No. 2000 sieve, and not more than 40% through a No. 355 sieve Moderately coarse (710/250) All the particles will pass through a No. 710 sieve, and not more than 40% through a No. 250 sieve Moderately fine (355/180) All the particles will pass through a No. 355 sieve, and not more than 40% through a No. 180 sieve Fine (180) All the particles will pass through a No. 180 sieve Very fine (125) All the particles will pass through a No. 125 sieve

TABLE 14.3 Sieve Numbers and Specifications Number of Nominal Size of Nominal Diameter Approximate Sieve (µm) Aperture (mm) of Wire (mm) Screening Area (%) 2000 2.00 0.90 48 710 0.710 0.450 37 500 0.500 0.315 38 355 0.355 0.224 38 250 0.250 0.160 37 250 0.250 0.160 37 212 0.212 0.140 36 180 0.180 0.125 35 150 0.150 0.100 36 125 0.125 0.090 34 90 0.090 0.063 35 75 0.075 0.050 36 45 0.045 0.032 34 according to the nominal aperture size expressed in micrometers of the mesh of the sieve through which the powder will pass, and is indicated in Table 14.2. The wire sieves used to sift powdered herbal materials are classified by numbers that indicate their nominal aperture size expressed in µm. The sieves are made of wire of uniform circular cross-section, and have the specifications indicated in Table 14.3.

14.2.4 Histochemical Detection This includes size, shape, and relative position of cells and tissues, and the chemical nature of cell wall, fragments of plant cells or tissues. This is necessary in initial identification of herbs, identification of small fragments of crude or powdered drugs, and detection of adulterants (insects, molds, fungi). Standardization of Herbal Drugs 129

• Starch grains • Aleurone grains • Fats, fatty oils, volatile oils and resins • Calcium oxalate/carbonate crystals • Lignified cell wall • Cellulose cell wall • Mucilage • Tannin

14.2.4.1 Cellulose Cell Walls Add 1–2 drops of iodinated zinc chloride TS and allow to stand for a few minutes; alternatively, add 1 drop of iodine (0.1 mol/L), allow to stand for 1 minute, remove excess reagent with a strip of filter paper and add 1 drop of sulfuric acid; cellulose cell walls are stained blue to blue-violet. On the addition of 1–2 drops of cuoxam, the cellulose cell walls will swell and gradually dissolve.

14.2.4.2 Lignified Cell Walls Moisten the powder or section on a slide with a small volume of phloroglucinol TS and allow to stand for about 2 minutes or until almost dry. Add 1 drop of hydrochloric acid and apply a cover glass; lignified cell walls are stained pink to cherry red.

14.2.4.3 Suberized or Cuticular Cell Walls Add 1–2 drops of Sudan red and allow to stand for a few minutes or warm gently; suberized or cuticular cell walls are stained orange-red or red.

14.2.4.4 Aleurone Grains Add a few drops of iodine/ethanol; the aleurone grains will turn yellowish-brown to brown. Then add a few drops of ethanolic trinitrophenol; the grains will turn yellow. Add about 1 mL of mercuric nitrate and allow to dissolve; the color of the solution turns brick red. If the specimen is oily, render it fat-free by immersing and washing it in an appropriate solvent before carrying out the test.

14.2.4.5 Calcium Carbonate Crystals or deposits of calcium carbonate dissolve slowly with effervescence when acetic acid (60 g/L) or hydrochloric acid (70 g/L) is added.

14.2.4.6 Calcium Oxalate Crystals of calcium oxalate are insoluble in acetic acid, but dissolve in hydrochloric acid without effervescence; they also dissolve in sulfuric acid, but needle-shaped crystals of calcium sulfate separate on standing after about 10 minutes. In polarized light, calcium oxalate crystals are birefringent. Calcium oxalate is best viewed after the sample has been clarified (e.g., with chloral hydrate).

14.2.4.7 Fats, Fatty Oils, Volatile Oils, and Resins Add 1–2 drops of Sudan red and allow to stand for a few minutes or heat gently, if necessary. The fatty substances are stained orange-red to red. Irrigate the preparation 130 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines with ethanol and heat gently; the volatile oils and resins dissolve in the solvent, while fats and fatty oils (except castor oil and croton oil) remain intact.

14.2.4.8 Hydroxyanthraquinones Add 1 drop of potassium hydroxide; cells containing 1,8-dihydroxyanthraquinones are stained red.

14.2.4.9 Inulin Add 1 drop each of 1-naphthol and sulfuric acid; spherical aggregations of crystals of inulin turn brownish-red and dissolve.

14.2.4.10 Mucilage Add 1 drop of Chinese ink to the dry sample; the mucilage shows up as transparent, spherically dilated fragments on a black background. Alternatively, add 1 drop of thionine to the dry sample, allow to stand for about 15 minutes, then wash with ethanol; the mucilage turns violet-red (cellulose and lignified cell walls are stained blue and bluish- violet, respectively).

14.2.4.11 Starch Add a small volume of iodine (0.02 mol/L); a blue or reddish blue color is produced. Alternatively, add a small volume of glycerol/ethanol and examine under a microscope with polarized light; birefringence is observed giving a Maltese cross effect with the arms of the cross intersecting at the hilum (Tong, 2007, 2008).

14.2.4.12 Tannin Add 1 drop of ferric chloride (50 g/L); it turns bluish-black or greenish-black.

14.2.4.13 Leaf Stomata In the mature leaf, four significantly different types of stoma are distinguished by their form and the arrangement of the surrounding cells, especially the subsidiary cells, as follows:

• Anomocytic or ranunculaceous (irregular-celled) type: The stoma is surrounded by a varying number of cells, generally not different from those of the epidermis. • Anisocytic or cruciferous (unequal-celled) type: The stoma is usually surrounded by three or four subsidiary cells, one of which is markedly smaller than the others. • Diacytic or caryophyllaceous (cross-celled) type: The stoma is accompanied by two subsidiary cells, the common wall of which is at right angles to the stoma. • Paracytic or rubiaceous (parallel-celled) type: The stoma has two subsidiary cells, of which the long axes are parallel to the axis of the stoma.

14.2.5 Measurement of Specimen • Stomata number • Stomatal index Standardization of Herbal Drugs 131

• Palisade ratio • Vein-islet number • Vein termination number • Lycopodium spore method

14.2.5.1 Determination of the Stomatal Index Place fragments of leaves, about 5 × 5 mm2 in size, in a test tube containing about 5 mL of chloral hydrate and heat on a water bath for about 15 minutes or until the fragments are transparent. Transfer a fragment to a slide and prepare it as described earlier, the lower epidermis uppermost, in chloral hydrate; place a small drop of glycerol/ethanol at one side of the cover glass to prevent the material from drying. Examine under a microscope with a 40X objective and a 6X eyepiece, equipped with a drawing apparatus. Mark on the drawing paper a cross (x) for each epidermal cell and a circle (o) for each stoma. Calculate the stomatal index as follows:

S×100 Stomatal index = ES+ where S = the number of stomata in a given area of leaf E = the number of epidermal cells (including trichrome) in the same area of leaf

14.3 PHYSICAL STANDARDIZATION OF HERBAL DRUGS Physical constants are sometimes taken into consideration to evaluate certain drugs. These include moisture content, specific gravity, optical rotation, refractive index, melting point, viscosity, and solubility in different solvents. All these physical properties are useful in identification and detection of constituents present in plant.

• Viscosity • Melting point • Solubility • Moisture content and volatile matter • Specific gravity • Density • Optical rotation • Refractive index • Bitterness value • Hemolytic activity • Swelling index • Foaming index • Ash value • Astringency 132 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.3.1 Foreign Organic Matter Herbal drugs should be made from the stated part of the plant and be devoid of other parts of the same plant or other plants. It should be entirely free from visible signs of contamination by molds or insects and other animal contamination, including animal excreta. No abnormal odor, discoloration, slime or signs of deterioration should be detected. Parts of the medicinal plant material or materials other than those named with the limits specified for the plant material concerned are termed as foreign organic matter. It may be any organism, part or product of an organism, other than that named in the specification and description of the plant material concerned. They should be entirely free from molds or insects, including excreta and visible contaminants such as sand and stones, poisonous and harmful foreign matter, and chemical residues. No poisonous, dangerous or otherwise harmful foreign matter or residue should be allowed. During storage, products should be kept in a clean and hygienic place so that no contamination occurs. Animal matter such as insects and “invisible” microbial contaminants, which can produce toxins, are also among the potential contaminants of herbal medicines. Macroscopic examination can easily be employed to determine the presence of foreign matter, although microscopy is indispensable in certain special cases (e.g., starch deliberately added to “dilute” the plant material). Macroscopic examination can conveniently be employed for determining the presence of foreign matter in whole or cut plant materials. Furthermore, when foreign matter consists, for example, of a chemical residue, TLC is often needed to detect the contaminants.

14.3.2 viscosity Viscosity of a liquid is constant at a given temperature and is an index of its composition. Hence, it can be used as a means of standardizing liquid drugs.

14.3.3 Melting Point In the case of pure photochemicals, melting points are very sharp and constant. The crude drugs from plant or animal origins, containing the mixed chemicals, are described with a certain range of melting point.

14.3.4 solubility The presence of an adulterant could be indicated by solubility studies, for example, pure asafoetida is soluble in carbon disulphide.

14.3.5 Moisture Content and Volatile Matter The moisture content of the drug should be minimized in order to prevent decomposition of the crude drug either due to chemical change or microbial contamination. The moisture content is determined by heating a drug at 105°C in an oven to a constant weight. For the drugs containing volatile constituents, the toulene distillation method is used. Standardization of Herbal Drugs 133

14.3.6 optical Rotation Optically active compounds have the property of rotating the plane of polarized light. This property is known as optical rotation. Normally, the optical rotation is determined at 25°C using a sodium lamp as the source of light. Castor oil has an optical rotation from +3.5° to +6°.

14.3.7 refractive Index When a ray of light passes from one medium to another of different density, the ratio of the velocity of light in vacuum to its velocity in the substance is termed as the refractive index of the second medium. It is constant for a pure drug and varies with the wavelength of the incident light, temperature, and pressure. The refractive index of castor oil is 1.4758–1.527.

14.3.8 ash Values and Extractives The residue remaining after incineration is the ash content of a drug. It involves non-volatile inorganic components. High ash value is indicative of contamination, substitution, adulteration or carelessness in preparing the crude drugs. To determine the ash content, the plant material is burned and the residual ash is measured as total and acid-insoluble ash. Total ash is the measure of the total amount of material left after burning and includes ash derived from the part of the plant itself and the acid- insoluble ash. The latter is the residue obtained after boiling the total ash with dilute hydrochloric acid and burning the remaining insoluble matter. The second procedure measures the amount of silica present, especially in the form of sand and siliceous earth. The ash remaining following ignition of herbal materials is determined by three different methods which measure total ash, acid-insoluble ash, and water-soluble ash.

14.3.9 total Ash Total ash is designed to measure the total amount of material produced after complete incineration of the drug material at as low a temperature as is possible (about 450°C) to remove all the carbons. This includes both “physiological ash,” which is derived from the plant tissue itself, and “non-physiological” ash, which is the residue of the extraneous matter (e.g., sand and soil) adhering to the plant surface. Total ash usually consists of carbonates, phosphates, silicates, and silica.

14.3.9.1 Determination of Total Ash Place about 2–4 g of the ground air-dried material in a previously ignited and tarred vessel (usually of platinum or silica). Spread the material in an even layer and ignite it by gradually increasing the heat to 500–600°C until it is white, indicating the absence of carbon. Cool in a desiccator and weigh. If carbon-free ash cannot be obtained in this manner, cool the crucible and moisten the residue with about 2 mL of water or a saturated solution of ammonium nitrate. Dry on a water bath, then on a hot plate, and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 134 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

30 minutes and then weigh without delay. Calculate the content of total ash in mg per g of air-dried material.

14.3.10 acid Insoluble Ash Acid insoluble ash is the residue obtained after extracting the total ash with dilute hydrochloride acid (HCl), and igniting the remaining insoluble matter. It gives an idea about the earthy matter, especially sand and siliceous earth.

14.3.10.1 Determination of Acid Insoluble Ash Add 25 mL of hydrochloric acid to the vessel containing the total ash, cover with a watch glass and boil gently for 5 minutes. Rinse the watch glass with 5 mL of hot water and add this liquid to the vessel. Collect the insoluble matter on an ashless filter paper and wash with hot water until the filtrate gets neutral. Transfer the filter paper containing the insoluble matter to the original vessel, dry on a hotplate, and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes and then weigh without delay. Calculate the content of acid insoluble ash in mg per g of air-dried material.

14.3.11 water Soluble Ash Total ash content which is soluble in water is called water soluble ash. It is good indicator of the presence of the previous extraction of water soluble salts in the drug or incorrect preparation or amount of inorganic matter. Water soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.

14.3.11.1 Determination of Water Soluble Ash Add 25 mL of water to the crucible containing the total ash and boil for 5 minutes. Collect the insoluble matter in a sintered glass crucible or on an ashless filter paper. Wash with hot water and ignite in a crucible for 15 minutes at a temperature not exceeding 450°C. Subtract the weight of this residue in mg from the weight of total ash. Calculate the content of water soluble ash in mg per g of air-dried material.

14.3.12 bitterness Value Medicinal plant materials that have a strong bitter taste are employed therapeutically, mostly as appetizing agents. Their bitterness stimulates secretions in the gastrointestinal tract, especially that of gastric juice. The bitter properties of plant material are determined by comparing the threshold bitter concentration of an extract of the materials with that of a dilute solution of quinine hydrochloride. The bitterness value is expressed in units equivalent to the bitterness of a solution containing 1 g of quinine hydrochloride in 2000 mL. The bitter sensation is not felt by the whole surface of the tongue, but is limited to the middle section of the upper surface of the tongue. Safe drinking water should be used as a vehicle for the extraction of herbal Standardization of Herbal Drugs 135 materials and for mouthwash after each tasting. Taste buds dull quickly if distilled water is used. 2000× c Bitterness value calculated in units per g using the following formula = ab× where a = the concentration of the stock test solution (ST) (mg/mL) b = the volume of test solution ST (in mL) in the tube with the threshold bitter concentration c = the volume of quinine hydrochloride R (in mg) in the tube with the threshold bitter concentration

14.3.13 Hemolytic Activity Many medicinal plant materials of the families Caryophyllaceae, Araliaceae, Sapindaceae, Primulaceae, and Dioscoreaceae contain saponins. The most characteristic property of saponins is their ability to cause hemolysis; when added to a suspension of blood, saponins produce changes in erythrocyte membranes causing hemoglobin to diffuse into the surrounding medium. The hemolytic activity of plant materials, or a preparation containing saponins, is determined by comparison with that of a reference material, saponin, which has a hemolytic activity of 1000 units/g.

a Hemolytic activity =×1000 b where 1000 = defined hemolytic activity of saponin standard a = quantity of saponin standard that produce total hemolysis (g) b = quantity of plant material that produce total hemolysis (g)

14.3.14 swelling Index Many herbal materials are of specific therapeutic or pharmaceutical utility because of their swelling properties—especially gums and those containing an appreciable amount of mucilage, pectin or hemicellulose. The swelling index is the volume in mL taken up by the swelling of 1 gm of plant material under specified conditions. Its determination is based on the addition of water or a swelling agent as specified in the test procedure for each individual plant material (either whole, cut or pulverized). Using a glass-stoppered measuring cylinder, the material is shaken repeatedly for 1 hour and then allowed to stand for a required period of time. The volume of the mixture (in mL) is then read. Note: The mixing of a whole herbal material with the swelling agent is easy to achieve, but cut or pulverized material requires vigorous shaking at specified intervals to ensure even distribution of the material in the swelling agent. 136 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.3.15 Foaming Index The foaming ability of an aqueous decoction of plant material and its extracts is measured in terms of foaming index. Many medicinal plant materials contain saponins that can cause persistent foam when an aqueous decoction is shaken.

14.3.15.1 Procedure Reduce the size of the herbal material to a coarse powder (sieve size no. 1250), weigh accurately, and transfer to a conical flask containing boiling water. Maintain at moderate boiling for 30 minutes. Cool and filter into a volumetric flask and add sufficient water through the filter to dilute to volume. Pour the decoction into test tubes and adjust the volume of the liquid in each tube with 10 mL water 10 mL. Close the mouth of the test tube and shake it in a lengthwise motion for 15 seconds, two shakes per second. Allow to stand for 15 minutes and measure the height of the foam. The results are assessed as follows:

• If the height of the foam in every tube is less than 1 cm, the foaming index is less than 100. • If the height of the foam is more than 1 cm in every tube, the foaming index is over 1000.

The foaming index can be calculated by using the following formula:

1000 Foaming index = a where a = the volume in mL of the decoction used for preparing the dilution in the tube where foaming to a height of 1 cm is observed.

14.3.16 extractive Value The amount of the active constituents present in a crude drug material when extracted with a specific solvent is called extractive value. It is employed for materials for which as yet no suitable chemical or biological assay exists. The following methods are used for determination of extractive value:

• Cold method • Hot method • Soxhlet method

14.3.17 total Solid Content The residue obtained when a prescribed amount of preparation is dried to constant weight under the specified conditions is called total solid content. For a powdered extract, the solid content is not less than 95% and for a semisolid extract, it is not less than 70%. Standardization of Herbal Drugs 137

14.3.18 water Content An excess of water in herbal materials may lead to microbial growth, the presence of fungi or insects, and deterioration following hydrolysis. Limits for water content should, therefore, be set for every given herbal material. This is especially important for materials that absorb moisture easily or deteriorate quickly in the presence of water. The azeotropic method is used to directly measure the water present in a material. It is determined by the titrimetric Karl Fisher method and gas chromatographic method. When the sample is distilled together with an immiscible solvent, such as toluene or xylene, the water present in the sample is absorbed by the solvent. The water and the solvent are distilled together and separated in the receiving tube on cooling. If the solvent is anhydrous, water may remain absorbed in it, leading to false results. It is, therefore, advisable to saturate the solvent with water before use. The test for loss on drying determines both water and volatile matter. It is determined by taking about 2–5 g of the prepared air-dried material or the quantity specified in the test procedure for the herbal material concerned, accurately weighed, in a previously dried and tared flat weighing bottle. Drying can be carried out either by heating to 100–105°C or in a desiccator over phosphorus pentoxide under atmospheric or reduced pressure at room temperature for a specified period of time. Dry until two consecutive weighings do not differ by more than 5 mg. Calculate the loss of weight in mg per g of air-dried material.

14.3.19 volatile Oil Content Volatile oils are the liquid components of the plant cells, immiscible with water, volatile at ordinary temperature, and can be steam distilled at ordinary pressure. Volatile oils are characterized by their odor, oil-like appearance, and ability to volatilize at room temperature. Chemically, they are usually composed of mixtures of monoterpenes, sesquiterpenes, and their oxygenated derivatives. Many herbal drugs contain volatile oil which is used as a flavoring agent, for example, clove volatile oil content not less than 15% v/w. In order to determine the volume of oil, the plant material is distilled with water and the distillate is collected in a graduated tube. The aqueous portion separates automatically and is returned to the distillation flask. If the volatile oils possess a mass density higher than or near to that of water or are difficult to separate from the aqueous phase owing to the formation of emulsions, a solvent with a low mass density and a suitable boiling point may be added to the measuring tube. The dissolved volatile oils will then float on top of the aqueous phase.

14.3.20 determination of Tannins Tannins are substances capable of turning animal hides into leather by binding proteins to form water-insoluble substances that are resistant to proteolytic enzymes. When this process is applied to the living tissue, it is known as an astringent action of tannins. Chemically, tannins are complex substances; they usually occur as mixtures of polyphenols that are difficult to separate and crystallize. They are easily oxidized 138 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines and polymerized in solution; if this happens, they lose much of their astringent effect and are, therefore, of little therapeutic value. Quantity of tannins as a percentage is determined by the following formula:

[(TT12−−T0)]× 500 Percentage quantity of tannins = w where w = the weight of the plant material in grams T1 = weight of material extracted in water T2 = weight of material not bound to hide powder T0 = weight of hide powder material soluble in water

14.3.21 loss on Drying (Volatile Matter) In order to measure volatile matter, a plant is diluted with water and the distillate is collected in a graduated tube. The aqueous portion separates and returns to the distillation flask. A solvent of low mass density with a suitable boiling point may be added to the measuring tube to easily separate the volatile oil.

14.4 CHEMICAL METHODS Most drugs have definite chemical constituents to which their biological or pharmacological activity is attributed. They can be separated by different chemical tests and assays. The isolation, purification, and identification of active constituents are chemical methods of evaluation. Qualitative chemical tests are used to identify certain drugs or to test their purity. The isolation, purification, and identification of active constituents are based on chemical methods of evaluation. Qualitative chemical tests are used in the detection of adulteration. The chemical evaluation also covers phytochemical screening carried out for establishing the chemical profile of a drug. Qualitative chemical tests include acid value, saponification value, and so on. Some of these are useful in the evaluation of resins (acid value, sulphated ash), balsams (acid value, saponification value, and bester values), volatile oils (acetyl and ester values), and gums (methoxy determination and volatile acidity).

14.4.1 analytical Methods In general, quality control is based on three important pharmacopoeia definitions- identity, purity, and content or assay. Pharmacopoeias are the best source to maintain quality control of herbal drugs (AOAC, 2005; WHO, 2000). Additional information, especially on chromatographic and/or spectroscopic methods, can be found in general scientific literature. The plant or plant extract can be evaluated by various biological methods to determine pharmacological activity, potency, and toxicity. A simple chromatographic technique such as TLC may provide valuable additional information to establish the identity of the plant material. Standardization of Herbal Drugs 139

This is especially important for those species that contain different active constituents. Qualitative and quantitative information can be gathered concerning the presence or absence of metabolites or breakdown of products (AOAC, 2005). TLC fingerprinting is of key importance for herbal drugs made up of essential oils, resins, and gums, which are complex mixtures of constituents that no longer have any organic structure. It is a powerful and relatively rapid solution to distinguish between chemical classes, where macroscopy and microscopy may fail. Instruments like ultraviolet and visible spectroscopy are easy to operate, and validation procedures are straightforward, but at the same time precise. Although measurements are made rapidly, sample preparation can be time consuming and works well only for less complex samples, and those compounds with absorbance in the UV-Visible region. HPLC is the preferred method for quantitative analysis of more complex mixtures. The separation of volatile components such as essential and fatty oils can be achieved with HPLC, but is best performed by GC or GC-MS. The quantitative determination of constituents has been made easy by recent developments in analytical instrumentation. Recent advances in the isolation, purification, and structure elucidation of naturally occurring metabolites have made it possible to establish appropriate strategies for the determination and analysis of quality and the process of standardization of herbal preparations. TLC, HPLC, GC, quantitative TLC (QTLC), and high performance TLC (HPTLC) can determine the homogeneity of a plant extract. Hyphenated chromatographic techniques are powerful tools, often used for standardization and to control the quality of both the raw material and the finished product. TLC and HPLC are the main analytical techniques commonly used. In cases when active ingredients are not known or are too complex, the quality of plant extracts can be assessed by a “fingerprint” chromatogram. Based on the concept of photo equivalence, the chromatographic fingerprints can be used for quality control of herbal medicines. Additionally, methods based on information theory, similarity estimation, chemical pattern recognition, spectral correlative chromatograms (SCC), multivariate resolution, and the combination of chromatographic fingerprints and chemometric evaluation for evaluating fingerprints are all powerful tools for quality control of herbal products.

14.4.2 thin Layer Chromatography (TLC) Thin layer chromatography is particularly valuable for the qualitative determination of small amounts of impurities. The principles of thin layer chromatography and application of this technique in pharmaceutical analysis are described in the International Pharmacopoeia (IP). As it is effective and easy to perform, and the equipment required is inexpensive, the technique is frequently used for evaluating herbal materials and their preparations. The following parameters should be determined while separating liquids: type of adsorbent and method of activation, method of preparation and concentration of the test and reference solutions; volume of the solutions to be applied on the plate; mobile phase and the distance of migration; drying conditions (including temperature) and method of detection; Rf values, fluorescence, and color. 140 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.4.2.1 Equipment The equipment consists of following parts:

• Glass plates of uniform thickness throughout their entire area, 15–20 cm long, and wide enough to accommodate the required number of test and reference solutions; • A device for spreading a uniform layer of coating material of desired thickness onto the glass plates; • A rack to hold the prepared plates (normally 10 plates with set spacings) during the drying period or for transportation and storage; the rack should be small enough to fit in a drying oven and desiccator; • A chromatographic chamber of transparent material, usually glass, with a tightly fitting lid, of suitable size to accommodate the test plates; • A suitable spraying implement with a fine spray nozzle, made of a material resistant to the reagents to be used; • An ultraviolet light source emitting short (254 nm) and long (365 nm) wavelengths.

14.4.2.2 Methodology Prepare slurry of the coating material and water or an aqueous solution and, using the spreading device, coat the cleaned plates with a layer about 0.25 mm thick. Dry the coated plates in air, heat to activate at 110°C for 30 minutes, and then allow to cool. Inspect the uniformity of the coating in transmitted light and the texture in reflected light. If the plates are not to be used immediately, store them in a desiccator containing silica gel. To form an edge, remove a narrow strip (2–5 mm wide) of the coating material from the sides of the plate. To achieve saturation, line at least half of the total area of the inside walls of the chamber with filter paper, pour into the chamber a sufficient quantity of the mobile phase to saturate the filter paper, and form a layer about 5 mm deep. Close the chamber and allow to stand for at least 1 hour at room temperature. All operations during which the plate is exposed to the air should preferably be carried out at a relative humidity of 50%–60%, and the plates should be handled with care. Then place spots of the test and reference solutions onto the starting line using a micropipette or a syringe graduated in µl. The spots should be at least 15 mm from the sides of the plate, and at least 15 mm apart. Mark the distance the mobile phase is intended to ascend as specified in the test procedure, usually 10–15 cm from the starting line. The results of separation can often be improved by applying the solutions to form a horizontal band 10–15 mm long and not more than 5 mm wide. Allow the spots to dry, then place the plate as nearly vertical as possible into the chamber, ensuring that the points of application are above the surface of the mobile phase. Close the chamber. Develop the chromatogram at room temperature unless otherwise specified in the test procedure, allowing the solvent to ascend the specified distance. Remove the plate, mark the position of the solvent front, and allow the solvent to evaporate at room temperature or as specified. Observe the spots produced in daylight, then under short-wave and long-wave ultraviolet light. Mark the center of each spot with a needle. Measure and record the distance from the center of each Standardization of Herbal Drugs 141 spot to the point of application, and indicate for each spot the wavelength under which it was observed. Then spray the spots with the specified reagent, and observe and compare the spots with those of a reference material.

14.4.2.3 Determination of Rf Value Calculate the ratio of the distance travelled on the adsorbent by a given compound to that travelled by the leading edge of the solvent (the Rf value) or the ratio of the distances moved by a compound and a stated reference substance (the Rr value) as follows:

a a Rf ==, Rf b c where a = the distance between the point of application and the center of the spot of the material being examined b = the distance between the point of application and the solvent front c = the distance between the point of application and the center of the spot of reference material

Note: Rf values may vary with each experiment depending on the saturation conditions in the chromatographic chamber, the activity of the adsorbent layer, and the composition of the mobile phase.

14.4.3 chemical Examination of Herbal Drugs Preliminary phytochemical screening is a part of chemical evaluation. These qualitative chemical tests are useful in identification of chemical constituents and detection of adulteration.

• Detection of alkaloids • Detection of carbohydrates and glycosides • Detection of phytosterols • Detection of fixed oils and fats • Detection of saponins • Detection of phenolic compounds and tannins • Detection of protein and free amino acids • Detection of gums and mucilage • Detection of volatile oils

14.4.3.1 Detection of Alkaloids The small portions of solvent free chloroform, alcoholic, and water extracts are stirred separately with a few drops of dilute hydrochloric acid and filtered. The filtrate may be tested carefully with various alkaloidal reagents, such as Mayer’s reagent (cream precipitate), Dragendrorff’s reagent (orange-brown precipitate), Hager’s reagent (yellow precipitate), and Wagner’s reagent (reddish-brown precipitate). 142 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.4.3.2 Detection of Carbohydrates and Glycosides Small quantities (200 mg) of alcoholic and aqueous extracts are dissolved separately in 5 mL of distilled water and filtered. The filtrate may be subjected to Molisch’s test to detect the presence of carbohydrates. Another small portion of extract is hydrolyzed with dilute hydrochloric acid for few hours in a water bath and is subjected to Liebermann-Burchard’s, Legal’s, and Borntranager’s tests to detect the presence of different glycosides. A small portion of extract is dissolved in water and treated with Fehling’s, Barfoed’s, and Benedict’s reagents to detect the presence of different sugars.

14.4.3.3 Detection of Phytosterols The petroleum ether, acetone, and alcoholic extracts are refluxed separately with a solution of alcoholic potassium hydroxide until complete saponification takes place. The saponification mixture is diluted with distilled water and extracted with ether. The ethereal extract is evaporated and the residue (unsaponifiable matter) is subjected to Liebermann’s and Burchard’s tests.

14.4.3.4 Detection of Fixed Oils and Fats A few drops of 0.5 N alcoholic potassium hydroxide is added to a small quantity of petroleum ether or benzene extract along with a drop of phenolphthalein. The mixture is heated on a water bath for 1–2 hours. Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

14.4.3.5 Detection of Saponins About 1 mL of alcoholic and aqueous extracts are diluted separately with distilled water to 20 mL and shaken in a graduated cylinder for 15 minutes. A 1-cm layer of foam indicates the presence of saponins. The test solution may be subjected to a test for hemolysis.

14.4.3.6 Detection of Phenolic Compounds and Tannins Small quantities of alcoholic and aqueous extracts in water are tested for the presence of phenolic compounds and tannins with dilute ferric chloride solution (5%), 1% solution of gelatin containing 10% sodium chloride, and 10% lead acetate and aqueous bromine solutions.

14.4.3.7 Detection of Proteins and Free Amino Acids Small quantities of alcoholic and aqueous extracts are dissolved in a few mL of water and subjected to Millon’s Biuret and Ninhydrin tests.

14.4.3.8 Detection of Gums and Mucilages About 10 mL of aqueous extract is added to 25 mL of absolute alcohol with constant stirring. The precipitate is dried in air. The precipitate is examined for its swelling properties and for the presence of carbohydrates.

14.4.3.9 Detection of Volatile Oil About 50 g of powdered material is taken in a volatile oil estimation apparatus and subjected to hydro distillation for the detection of volatile oil. The distillate Standardization of Herbal Drugs 143 is collected in the graduated tube of the assembly in which the aqueous portion is automatically separated from the volatile oil if it is present in the drug, and returned back to the distillation flask.

14.4.4 radioactive Contamination This exposure cannot be avoided because of many naturally occurring sources, including radionucleotides, in the ground and atmosphere. The range of radionuclides that may be released into the environment as the result of a nuclear accident might include long-lived and short-lived fission products, actinides, and activation products. Microbial growth in herbals is not usually irradiated. This process may sterilize the plant material, but the radioactivity hazard should be taken into account. The nature and the intensity of radionuclides released may differ markedly and depend on the source (reactor, reprocessing plant, fuel fabrication plant, isotope production unit, etc.). Dangerous contamination, however, may be the consequence of a nuclear accident. The WHO has developed guidelines in the event of a widespread contamination by radionuclides resulting from major nuclear accidents. The quantity of radioactivally contaminated herbal medicine normally consumed by an individual is unlikely to be a health risk. Therefore, at present, no limits are proposed for radioactive contamination (De Smet, 1992; WHO, 2000).

14.5 BIOLOGICAL METHODS Some drugs have specific biological and pharmacological activities which are utilized for their evaluation. Actually, this activity is due to a specific type of constituents present in the plant extract. For evaluation, the experiments were carried out on both intact and isolated organs of living animals. With the help of bioassays (testing the drugs on living animals), the strength of a drug in its preparation can also be evaluated (Ansari, 2011; Kokate et al., 2005; Williamson et al., 1996). Drugs which cannot be assayed by chemical or physical means are evaluated by biological methods.

14.5.1 bioassay It is well established that the biological potency of the herbal constituents is due to not one, but a mixture of bioactive plant constituents and the relative properties of a single bioactive compound can vary from batch to batch while the biological activity remains within the desirable limits (1). Need of Bioassay

1. Bioassay helps to determine the concentration of the unknown compound in addition to the potency. 2. Substances which are used in biological systems like drugs, vaccines, toxins, disinfectants, and antiseptics, and so on, can be standardized through bioassay. 3. Specificity of the compound can also be determining using bioassay, for example, identification of the type of bacteria for which a suitable drug can be selected. 4. Estimation of Vitamin B-12 can be done by bioassay. 5. Bioassay is a reliable option in cases where no assay is available. 144 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

6. Sometimes the chemical composition of samples is different, but they have the same biological activity, for example, cardiac glycosides isolated from different sources, catecholamines, and so on. 7. For samples where no other methods of assays are available, bioassay is the reliable option. 8. When a chemical method is not available, or is too complex or insensitive to low doses, bioassay can be done. 9. Determination of the side effect profile, including the degree of drug toxicity.

14.5.1.1 Types of Bioassays Basically, there are two types of bioassays as per the technique used in determination of the sample under test.

1. End point or quantal assay 2. Graded response assay

14.5.1.1.1 End Point or Quantal Assay This is the simplest bioassay, which produces an “All or None” response in different animals. In this bioassay, the pharmacological effect produced by the threshold dose of the sample is determined and compared with the standard drug or solution. Determination of LD50 (LD = Lethal dose) or ED50 (ED = effective dose) is done by this method, for example, cardiac arrest produced by digitalis in cats, hypoglycemic convulsions in mice, and so on.

14.5.1.1.2 Graded Response Assay The response produced in this bioassay is based on the dose of the sample. As the dose of the sample increases, there is a rise in the response of the tissue. However, after certain doses, the response of the tissue dose not rise any further; this condition is known as the ceiling effect. The curve is obtained by plotting a graph between the dose and response on the X and Y axes, respectively (Figure 14.2). The curve is sigmoid in shape, however, a straight line curve is obtained from a log dose.

Concentration of unknown compound Threshold dose of standarrd = ×Concentration of standard Threshold dose of test

Based on the method used during the grade point assay procedure for determination of type of activity and potency of the sample, four methods of assays are classified as:

1. Matching point or bracketing method 2. Interpolation assay 3. Three point (2 + 1) assay 4. Four point (2 + 2) assay

1. Matching point or bracketing method: In this method, various doses of the test sample are administered and compared with the constant dose of the Standardization of Herbal Drugs 145

Maximal response

0.2 0.4 0.6 0.8 1.6 3.2

FIGURE 14.2 Graded response.

standard in the same manner as bracketing by increased and decreased doses of the test sample. Disadvantages of this method are that it is applicable only when the sample of the test drug is too small and it is difficult to estimate the margin of error (Figure 14.3), for example, histamine on guinea pig ileum, posterior pituitary on rat uterus, and so on. 2. Interpolation assay: This bioassay is performed to determine the quantity of preparation of unknown potency which produces a significant effect on test animals or isolated organs or tissues under standard conditions. The response produced by the unknown is expressed as a percentage response of the standard, and the amount of the test compound required to produce the same pharmacological response as the standard is compared. 3. Multi point bioassay: This bioassay involves both interpolation and bracketing methods. It can further be divided as 3 point (2 + 1), 4 point (2 + 2) and 6 point (3 + 3) bioassay. This procedure of 2 + 1 or 2 + 2 is repeated 3 times or 4 times based on the method of crossing over of all the samples. a. Three point assay (2 + 1 dose assay): It is fast and convenient and indicates two response of Standard (S) and one response of Test (T).

Bracketing

Test (T)

S1 S2 S1 T S2

FIGURE 14.3 Matching method. 146 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

• Methodology: A log dose response (LDR) curve is plotted with different concentrations of standard drug solutions and a given test

solution. Two doses of the standard, Q1 and Q2, are selected in the ratio of 2:3 from the linear part of the LDR and responses S1 and S2 are obtained, respectively. One test dose R is selected and gets the response T between S1 and S2. Data is recorded as:

Q1 Q2 R

R Q1 Q2

Q2 R Q1

Q1 Q2 R (T −S1) • Calculation: Log potency ratio [M] = × log dose ratio. (S2S− 1) b. 4 point assay (2 + 2 dose assay): This indicates two responses of the standard (S) and two responses of the test (T) sample, for example, Ach bioassay. • Methodology: A log dose response (LDR) curve is plotted with different concentrations of the standard solution and the given test

solution. Two doses of the standard, that is, Q1 and Q2, are selected from the linear part of the dose response curve (DRC) and the

responses S1 and S2 are recorded (Figure 14.4). Two test doses, R1 and R2, are selected and responses T1 and T2 between S1 and S2 are obtained. Here, Q2/Q1 = R2/R1. Data is recorded as:

Q1 Q2 R1 R2

Q2 R1 R2 Q1

R1 R2 Q1 Q2

R2 Q1 Q2 R1

14.5.2 Microbial Contamination 14.5.2.1 Total Viable Aerobic Count The total viable aerobic count (TVC) of the herbal material being examined is determined by methods such as membrane-filtration, plate count or serial dilution.

S1 S2 T2 T1 S2 T2 T1 S1 T2 T1 S1 S2 T1 S1 S2 T2

FIGURE 14.4 Four point assay. Standardization of Herbal Drugs 147

Aerobic bacteria and fungi (molds and yeasts) are determined by the TVC. Usually, a maximum permitted level is set for certain products, but when the TVC exceeds this level, then it is unnecessary to proceed with the determination of specific organisms; the material should be rejected without being subjected to further testing.

14.5.2.1.1 Test Procedure 14.5.2.1.1.1 Plate Count Petri dishes of 9–10 cm in diameter are used for bacteria. To one dish, add a mixture of 1 mL of the pre-treated herbal material and about 15 mL of liquefied casein-soybean digest agar at a temperature not exceeding 45°C. Alternatively, spread the material on the surface of the solidified medium in a Petri dish. If necessary, dilute the material to obtain an expected colony count of not more than 300. Prepare at least two dishes using the same dilution, invert them, and incubate them at 30–35°C for 48–72 hours, unless a more reliable count is obtained in a shorter period of time. Count the number of colonies formed and calculate the results using the plate with the largest number of colonies, up to a maximum of 300. However, for determination of fungi, the casein-soybean digest agar is replaced with liquefied Sabouraud glucose agar and colonies should not number more than 100. Incubation should be performed at 20–25°C for 5 days, unless a more reliable count is obtained in a shorter period of time.

14.5.2.1.1.2 Membrane Filtration Use membrane filters with a nominal pore size of not greater than 0.45 µm, and with a proven effectiveness at retaining bacteria. For example, cellulose nitrate filters are used for aqueous, oily, and weakly alcoholic solutions, whereas cellulose acetate filters are better for strongly alcoholic solutions. Generally, filter discs of about 50 mm in diameter are used. However, filters of a different diameter are also used to adjust the volumes of the dilutions and washings accordingly.

14.5.2.2 Aflatoxins Aflatoxins are the poisonous substances in the spores of the fungiAspergillus flavus and Aspergillus parasiticus. The toxin is known to produce cancer in human beings living in warm and humid regions of the world. Whenever testing for aflatoxins is required, this should be done after using a suitable clean-up procedure during which great care should be taken not to expose any personnel or the working or general environment to these dangerous and toxic substances. Stored nuts and cereals are contaminated by the fungus. The presence of aflatoxins can be determined by chromatographic methods using standard aflatoxins B1, B2, G1, and G2 mixtures. The recommended quantity of aflatoxins are; the IP method—not more than 2 µg/kg of aflatoxins B1 and total aflatoxins 4 µg/kg, and the United States of Pharmacopoeia (USP) method—not more than 5 ppb of aflatoxins B1 and total aflatoxins 20 ppb.

14.5.3 toxicological Standardization • Determination of pesticides • Determination of arsenic and heavy metals 148 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

14.5.3.1 Pesticides Herbs and herbal products must be free from toxic chemicals or at least controlled for the absence of unsafe levels (WHO, 2000). Herbal drugs are liable to contain pesticide residues, which accumulate from agricultural practices, such as spraying, treatment of soils during cultivation, and administering of fumigants during storage. Many pesticides contain chlorine in the molecule, which, for example, can be measured by analysis of total organic chlorine. In an analogous way, insecticides containing phosphate can be detected by measuring total organic phosphorus. Different types of pesticides are fungicides, herbicides, insecticides, acarcicides, nematocides, rodenticides, and bactericides. Chromatography (mostly column and gas) is recommended as the principal method for the determination of pesticide residues; column and gas chromatography (GC) are most frequently used. These methods may be coupled with mass spectrometry (MS). Impurities present in herbal drugs are removed by partition and/or adsorption chromatography, and individual pesticides are measured by GC, MS or GC-MS. WHO has laid down general limits for pesticide residues in medicine (De Smet, 1997; WHO, 1996, 1998). It is desirable to test unknown herbal materials with broad groups of compounds rather than for individual pesticides. Various methods are available, that is, pesticides containing chlorine in the molecule can be detected by the measurement of total organic chlorine; insecticides containing phosphate can be measured by analysis for total organic phosphorus; whereas pesticides containing arsenic and lead can be detected by measurement of total arsenic or total lead, respectively. Similarly, the measurement of total bound carbon disulfide in a sample will provide information on whether residues of the dithiocarbamate family of fungicides are present. Other pesticides of plant origin are tobacco leaf and nicotine; pyrethrum flower, pyrethrum extract, and pyrethroids; derris root and rotenoids.

14.5.3.1.1 Determination of Pesticide Residues An acceptable residual limit (ARL) of pesticide in mg of plant material per kg can be calculated on the basis of the maximum acceptable daily intake of the pesticide for humans (ADI) as recommended by WHO and the mean daily intake (MDI) of the medicinal plant material. Pesticide content should not be more than 1%.

ADIE××60 ARL = MDI×100 where ADI = Maximum acceptable daily intake of pesticides (mg/kg of body weight) E = Extraction factor, which determines the transition rate of the pesticides from the plant material into the dosage form MDI = Mean daily intake of medicinal plant 60 in numerator = Adult body weight 100 in denominator = Consumption factor 14.5.3.2 Determination of Arsenic and Heavy Metals Contamination by toxic metals can either be accidental or intentional. Contamination by heavy metals such as mercury, lead, copper, cadmium, and arsenic in herbal Standardization of Herbal Drugs 149 remedies can be attributed to many causes, including environmental pollution, and can pose clinically relevant dangers for the health of the user and should, therefore, be limited. Arsenic and heavy metals are dangerous even in trace amounts and must be removed from herbal drugs. Arsenic is abundant in nature and its presence in herbal materials should be no different from its wide occurrence in foods. Atomic absorption spectrometry (AAS) is used for the determination of the amount or concentration of specific heavy metals. AAS uses the phenomenon that atoms in the ground state absorb light of a specific wavelength, characteristic of the particular atom, when the light passes through an atomic vapor layer of the element to be determined. The contamination of medicinal plant materials with arsenic and heavy metals can be attributed to many causes, including environmental pollution and traces of pesticides. The contents of lead and cadmium may be determined by inverse voltametry or by atomic emission spectrophotometry. The following maximum amounts in dried plant materials, which are based on the ADI values, are proposed for lead (10 mg/kg) and cadmium (0.3 mg/kg).

14.6 VALIDATION Validation of herbal products is an important step toward the standardization of herbs where fakers selling adulterated herbal medicines are common. It is necessary to ensure scientific validation and periodic monitoring of the quality and efficacy of herbal products by drug control administrators where herbal products are marketed as therapeutic agents, and irrespective of whether the products really have any positive effects to cure and reduce the severity of the disease. Validation is the process of proving that an analytical method is acceptable for its intended purpose for pharmaceutical methods. It includes studies on specificity, linearity, accuracy, precision, range, detection, and quantitative limits, depending on whether the analytical method used is qualitative or quantitative (De Smet, 1997). It is feasible that the introduction of scientific validation would control the production of impure or adulterated herbal products and would eventually ensure their rational use. This leads to the regulation of the industry so that only qualified personnel and health providers are allowed to prescribe the medication. It is advisable to use official monographs published in a pharmacopoeia so that standards are defined and available, and that the analytical procedures used are fully validated. This is of major importance, since validation can be a rather time-consuming process.

14.7 DETERMINATION OF ARSENIC AND HEAVY METALS Labelling of herbal products should be appropriate so as to reduce the risk of inappropriate uses and adverse reactions. Information about the quality of herbal drugs to the consumer is an important phenomenon regarding the safe use of herbal drugs. The label is the primary source of providing herbal drug information. Unfortunately, no any organization or government body exists that certifies herbs or a supplement as being labelled correctly. It is equally truth that herbal remedy labels often cannot be trusted to reveal what is in the container. It cannot be assumed to be “standardized” as written on the label because there is no legal definition of the word 150 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

“standardized.” The product has been manufactured according to pharmacopoeia standards, listing of active ingredients and amounts, directions regarding dosage and frequency of intake of the drug, must be in the label.

14.8 CONCLUSION Herbal drugs are usually mixtures of many constituents. The active principle(s) is (are), in most cases, unknown. Selective analytical methods or reference compounds may not be available commercially. The need for standardization of herbals is now very essential given the global acceptance of herbal products as remedies for various diseases and ailments. Safety and efficacy assurance of herbal drugs require monitoring of the quality of the product from collection through processing to the finished packaged product. Strict guidelines have to be followed for the successful production of a quality herbal drug. Among them are proper botanical identification, phytochemical screening, and standardization. The WHO guideline is a recommended universal approach that should be followed by various government agencies for herbal quality control and herbal monography. It should be prepared using the various quality parameters. This will strengthen the regulatory process and minimize quality breaches. Quality control and the standardization of herbal medicines involve several steps. The source and quality of raw materials and good agricultural practices and manufacturing processes are certainly essential steps for the quality control of herbal medicines and play a pivotal role in guaranteeing the quality and stability of herbal preparations.

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EMEA. Guidelines on Quality of Herbal Medicinal Products/Traditional Medicinal Products, EMEA/CVMP/814OO Review. European Agency for the Evaluation of Medicinal Products (EMEA), London, 2005. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, 31st edition. Nirali Prakshan, India, 2005, pp. 97–131. Lau PE, Peng Y, Zhao ZZ. Microscopic identification of Chinese patent medicine [1]: Wu Zi Yan Zong Wan. Nat Med. 2004; 6: 258–265. Li J, Yi T, Lai HS, Xue D, Jiang H, Peng HC, Zhang H. Application of microscopy in authentication of traditional Tibetan medicinal plant Halenia elliptica. Microsc Res Tech. 2008; 71: 11–19. Liang ZT, Chen HB, Zhao ZZ. An experimental study on four kinds of Chinese herbal medicines containing alkaloids using fluorescence microscope and microspectrometer. J Microsc. 2009; 233: 24–34. Liang ZT, Jiang ZH, Leung KSY, Peng Y, Zhao ZZ. Distinguishing the medicinal herb Oldenlandia diffusa from similar species of the same genus using fluorescence microscopy. Microsc Res Tech. 2006; 69: 277–282. Mukherjee PW. Quality Control of Herbal Drugs: An Approach to Evaluation of Botanicals, Business Horizons Publishers, New Delhi, India, 2002. Patel PM, Patel NM, Goyal RK. Evaluation of marketed polyherbal antidiabetic formulations uses biomarker charantin. Pharma Rev. 2006; 4(22): 113. Remington JP, Gennaro AR (Eds.) Quality control methods. In Remington: The Science and Practice of Pharmacy, 19th Edition. MACK, Easton PA, 1995, pp. 118–119. Ruzin SE. Plant Microtechnique and Microscopy, Oxford Univ. Press, New York, 1999, p. 2. Sagar Bhanu PS, Zafar R, Panwar R. Herbal drug standardization. Indian Pharmacist. 2005; 4(35): 19–22. Shrikumar S, Maheshwari U, Sughanti A, Ravi TK. WHO guidelines for herbal drug standardization, 2006. Smillie TJ, Khan IA. A comprehensive approach to identifying and authenticating botanical products. Clin Pharmacol Ther. 2010; 87: 175–186. Tam CH, Peng Y, Liang ZT, He ZD, Zhao ZZ. Application of Microscopic Techniques in Authentication of Herbal Tea—Ku-Ding-Cha. Microsc Res Tech. 2006; 69: 927–932. Tong CS, Choy SK, Chiu SN, Zhao ZZ, Liang ZT. Characterization of shapes for use in classification of starch grains images. Microsc Res Tech. 2008; 71(9): 651–658. Tong CS, Choy SK, Zhao ZZ, Liang ZT, Chen HB. Identification of starch grains in microscopic images based on granulometric operations. Microsc Res Tech. 2007; 70(8): 724–732. Torey A, Sasidharan S, Yeng C, Latha LY. Standardization of cassia spectabilis with respect to authenticity, assay and chemical constituent analysis. Molecules. 2010; 15(5): 3411–3420. WHO. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1988. WHO. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1992. WHO. Quality Assurance of Pharmaceuticals: A Compendium of Guidelines and Related Materials, Good Manufacturing Practices and Inspection, World Health Organization, Geneva, 1996. WHO. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1998. WHO. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1999. WHO. The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 2000–2002 (WHO/PCS/01.5), International Programme on Chemical Safety, World Health Organization, Geneva, 2000. 152 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

WHO. Guidelines for sampling of pharmaceutical products and related materials. In Addae- Mensah I, Beltramini H, Haggag AA, Hoogmartens J, Shaohong J, Molzon JA, Paál TL, van Zyl AJ (Eds.), WHO Expert Committee on Specifications for Pharmaceutical Preparations, Thirty-ninth report. World Health Organization, Geneva, 2005, pp. 101–106. WHO. Guidelines for Good Manufacturing Practices (GMP) for Herbal Medicines, World Health Organization, Geneva, 2007. Williamson E, Okpako DT, Evans FJ. Pharmacological Methods in Phytotherapy Research, Volume 1. Selection, Preparation and Pharmacological Evaluation of Plant Material. John Wiley and Sons, Chichester, 1996. Yadav NP, Dixit VK. Recent approaches in herbal drug standardization. Int J Integr Biol. 2008; 2: 195–203. Zhao Z. Application of microscopic techniques for the authentication of herbal medicines. In: Méndez-Vilas A and Díaz J (Eds.). Microscopy: Science, Technology, Applications and Education. Formatex, Badajoz, 2010, pp. 803–12. Zhao ZZ, Hu YN, Liang ZT, Yuen JPS, Jiang ZH, Leung KSY. Authentication is fundamental for standardization of Chinese medicines. Planta Med. 2006; 72(10): 865–874. Zhao ZZ, Hu YN, Wong YW, Wong WCG, Wu K, Jiang ZH, Kang, T. Application of Microscopy in Authentication of Chinese Patent Medicine-Bo Ying Compound. Microsc Res Tech. 2005; 67: 305–311. Zhao ZZ, Shimomura H, Sashida Y, Ishikawa R, Ohamoto T, Kazami T. Identification of crude drugs in traditional Chinese patent medicines by means of microscope and polariscope (2): Polariscopic characteristics of stone cells, vessels and fibres. Nat Med. 1997; 51: 504–511. 15 Omics Techniques

15.1 INTRODUCTION The increasing use of herbal medicine around the globe requires new scientific approaches for their standardization. Evaluation of physical and chemical parameters is the most common method for standardization. Standardization of herbal plants is a critical issue to ensure the quality of the research process for safety and efficacy of the research products, which are critical to scientists and regulators for ensuring the quality and interoperability of herbal products (Shukla et al., 2009). The introduction of omic techniques such as genomics, proteomics, and transcriptomics, as well as various profiling approaches, including metabolomics and metabomics, leads to examining the molecular effects of mixtures of chemical agents. By using this technique, one can easily have in-depth knowledge of pharmacodyanamics, pharmacokinetics, and toxicological characterization of the active constituents of an herbal plant. Omic technique is an important tool for the fingerprinting and quality control of herbal medicine (Holmes et al., 2010).

15.2 OMICS TECHNIQUES Different types of omic techniques are known, such as genomics, proteomics, transcriptomics, metabolomics, and metabomics. These techniques are summarized below.

15.2.1 genomics and its Modified Techniques The study of the human genome along with detection of the variability of the DNA is called genomics. DNA chip technology has proven to be a powerful tool that could be used for analyzing mixed herbal preparations (Lai et al., 2010). It is a rapid, high-throughput, and information-rich tool for genotyping, quality assurance, and species confirmation. Likewise, DNA barcoding is a method for herbal preparation identification. This method is used for recognition and identification of unknown species of plants by comparing the DNA barcode sequences to the library sequences of known species (Lam et al., 2010). Microarray is referred to as transcriptomic technologies, one of the most powerful tools for explicating the mechanisms which are involved at the molecular level and various processes underlying the complex pharmacological action of herbal formulations (Evans et al., 1960).

15.2.2 Proteomics Proteomics is the research area enlightening the temporal dynamics of proteins articulated in a given biological compartment at a given time (Epstein et al., 2010).

153 154 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

This technique is a post-translational modification of gene products. Two- dimensional gel electrophoresis is the most proficient analytical separation technique for proteins. The large size of the globular structures of proteins is a major hurdle for chromatographic high-resolution separations due to the unfavorable diffusion coefficients in the separation mechanism (Calvel et al., 2010). The two approaches based on mass spectrometry are the most frequently used for global quantitative protein profiling: (1) two-dimensional electrophoresis (2DE) followed by staining, selection, and identification by mass spectrometry and (2) isotope tags to label proteins, separation by multidimensional liquid chromatography, and mass spectrometry analysis. These approaches are supplemented with useful information provided by molecular imaging (Mateos et al., 2010). Proteomics can prove a valuable tool for quality control, toxicity studies, and standardization of preparations and decoctions. In comparison to genomic and transcriptomic approaches, proteomic assays have been successfully used for describing the mechanisms of action of many different herbal preparations (Li et al., 2011). Today, proteomics is becoming a valuable tool for elucidating the multi-target effects of complex herbal preparations, discovery of single bioactive compounds, development of active fractions, characterization of secure herbal prescriptions, and eventually modified molecular diagnosis (Cho, 2007). The major limitation of proteomics is that they are specific to a tissue. Unlike blood-based targets or respective tumor tissues, from which the pertinent biologic matrix is practical to obtain, tissue samples from organs such as lung, kidney, heart or brain are not easily obtained for proteomic screens (Andrew et al., 2012).

15.2.3 transcriptomics This is a method that analyzes the expression level of genes by measuring the transcriptome. Transcriptomics make use of high density or high-throughput methods for assessing messenger ribonucleic acid (mRNA) expression (Powell and Kroon, 1994). Transcriptomics are actually used as a platform for translational medicine, and DNA microarray technology is used to analyze the biological events induced in different herbal formulas to conclude their therapeutic potential, as well as their safety. The fundamental limitation of using transcriptomics assays is that mRNAs are not the main products, but are the intermediate products of disease, which fail to adequately predict the clinical effect (Mendrick, 2011).

15.2.4 Metabolomics The systematic study of inimitable chemical fingerprints with definite cellular processs is called metabolomics. It includes the study of small-molecule metabolite profiles. The term metabolomics is mainly framed for exhaustive, nonbiased, high-throughput analyses of complex metabolite mixtures mainly of plant extracts (Figure 15.1). The metabolome represents all metabolites collections in a biological organism, which are mainly the products of its gene expression (Johnson et al., 2012). Metabolomic profilings are used for analysis of extracts by using Fourier transform ion cyclotron mass spectrometry (FTMS). Omics Techniques 155

Sample collection Chemical analysis Sample preparation urine, blood, etc. NMR, HPLC, etc.

Identify altered metabolite in Data processing Data analysis response to disease state

Information potential model biological marker

FIGURE 15.1 Workflow for a metabolomic experiment.

15.2.5 application of Omics Techniques in the Context of Herbal Medicine Omic technique is mainly used for identification of biomedical resources such as genomic technique in DNA sequencing and fingerprinting or DNA microarrays. The newer trends are established for the use of omic techniques in the field of herbal plants related to pharmacological experiments. The proteomic technique has been applied for the treatment of cardiovascular diseases, epilepsy, cancer, and so on, with herbal medicines (Gowda et al., 2008). Proteomics is one of the most useful techniques with which to identify different species. These applications would be very valuable tools for the quality control, toxicity studies, and standardization of herbal preparations (Wang et al., 2011). A number of DNA polymorphism-based assays have been developed for the identification of herbal products. DNA chips with DNA sequences proved to be more powerful tools that could be also used for analyzing mixed herbal preparations (Burian et al., 2012). This technique can be properly coupled with bioinformatics and statistical information and can be used for pharmacodyanamics, pharmacokinetics, and toxicological characterization of herbal drugs (Trevino et al., 2007).

15.3 CONCLUSION The different omic techniques are used all over the world today for standardization and quality control of herbal formulas, mechanisms of action, characterization, identification of molecular mechanisms for prediction of side effects, and interactions with other drugs. All the omic processes are gradually but firmly approaching the area of herbal standardization. 156 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

REFERENCES Andrew AM, Vasilis V, Kennon JH. Omics screening for pharmaceutical efficacy and safety in clinical practice. J Pharmacogenomics Pharmacoproteomics. 2012; S5: 1–19. Burian A. Omic techniques in systems biology approaches to traditional Chinese medicine research: Present and future. J Ethn. 2012; 140(3): 535–544. Calvel P, Antoine DR, Bernard J, Charles P. Testicular postgenomics: Targeting the regulation of spermatogenesis. Phil Trans R Soc B. 2010; 365(1546): 1481–500. Cho WCS. Application of proteonomics in Chinese medicine research. Am J Chinese Med. 2007; 35(6): 911–922. Epstein RS, Moyer TP, Aubert RE, Kane DJ, Xia F. Warfarin genotyping reduces hospitalization rates results from the MM-WES (Medco-Mayo Warfarin Effectiveness study). J Am Coll Cardiol. 2010; 55(25): 2804–2812. Evans DA, Manley KA, McKusick VA. Genetic control of isoniazid metabolism in man. Br Med J. 1960; 2: 485–491. Gowda N, Zhang S, Haiwei G, Asiago V, Shanaiah N, Raftery D. Metabolomics-based method for early disease diagnostics: A review. Expert Rev Mol Diagn. 2008; 8(5): 617–633. Holmes C, McDonald F, Jones M, Ozdemir V, Janice E. Graham. Standardization and omics science: Technical and social dimensions are inseparable and demand symmetrical study. OMICS A J Inte Bio. 2010; 14(3): 3–10. Johnson CH, Patterson AD, Idle JR, Gonzalez FJ. Xenobiotic metabolomics: Major impact on the metabolome. Annu Rev Pharmacol Toxicol. 2012; 52: 37–56. Lai J, Li R, Xu X, Jin W, Xu M, Zhao H. Genome-wide patterns of genetic variation among elite maize inbred lines. Nat Genet. 2010; 42(11): 1027–1030. Lam HM, Xu X, Liu X, Chen W, Yang G, Wong FL. Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nat Genet. 2010; 42(12): 1053–1059. Li ZH, Alex D, Siu SO, Chu IK, Renn J, Winkler C. Combined in vivo imaging, and omics approaches reveal metabolism of icaritin and its glycosides in zebrafish larvae. Mole Biosyste. 2011; 7(7): 2128–2138. Mateos CPJ, Macaya C, Azcona L, Modrego J, Mahillo E. Different expression of proteins in platelets from aspirin-resistant and aspirin-sensitive patients. Thromb Haemost. 2010; 103(1): 160–170. Mendrick DL. Transcriptional profiling to identify biomarkers of disease and drug response. Pharmacogenomics. 2011; 12(12): 235–249. Powell EE, Kroon PA. Low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme, A reductase gene expression in human mononuclear leukocytesis regulated coordinately and parallels gene expression in human liver. J Clin Invest. 1994; 93(5): 2168–2174. Shukla SS, Saraf S, Saraf S. Approaches towards standardization and quality assessment of herbals. J Res Educ Indian Med. 2009; 15(1): 25–32. Trevino V, Falciani F, Hugo ABS. DNA microarrays: A powerful genomic tool for bio medical and clinical research. Mol Med. 2007; 13(9): 527–541. Wang L, McLeod HL, Weinshilboum RM. Genomics and drug response. N Engl J Med. 2011; 364(12): 1144–1153. Toxicity Study of 16 Plant Materials

16.1 INTRODUCTION Herbal medicines are the synthesis of therapeutic experiences of generations of practicing physicians of indigenous systems of medicine for over hundreds of year. Medicinal plants are a source of raw materials for both traditional systems of medicine and modern medicine. These medicines are also in great demand in the developed world for primary health care because of their efficacy, safety, and lesser side effects. They also offer therapeutics for age-related disorders like memory loss, osteoporosis, immune disorders, and so on. The herbal drug preparation in its entirety is regarded as the active substance and the constituents are either of known therapeutic activity or are chemically defined substances or group of substances generally accepted to contribute substantially to the therapeutic activity of the drug. Indian materia medica includes about 2000 drugs of natural origin which are derived from different traditional systems and traditional practices (Mukherjee et al., 2016). The therapeutic potential of herbal drugs depends on their form: whether parts of a plant or simple extracts or isolated active constituents. Herbal medicine is still the mainstay of about 75%–80% of the world’s population, mainly in the developing countries, for primary health care because of better cultural acceptability and better compatibility with the human body. Traditionally, herbs and herbal products have been considered to be nontoxic and have been used by the general public and traditional medicinal doctors worldwide to treat a range of ailments. The fact that something is natural does not necessarily make it safe or effective, however. The active ingredients of plant extracts are chemicals that are similar to those in purified medications, and they have the same potential to cause serious adverse effects. While the literature documents severe toxicity resulting from the use of herbs, on many occasions the potential toxicity of herbs and herbal products has not been recognized (WHO, 2004). Most herbal remedies when used as directed and under the supervision of knowledgable individuals are safe, but the potential for adverse effects certainly exists. Preclinical studies of herbal drugs provide scientific justification for their traditional use and prove that they are safe and efficacious (WHO, 2000).

16.2 NEED OF HERBAL TOXICITY TESTING The principal need for toxicological assessment of any herbal medicine is to identify adverse effects and to determine limits of exposure levels at which such effects occur (Gamaniel, 2000). Because herbs are classified as a dietary supplements and not food or drugs, they do not have to go through the pre-market testing that drugs and food additives do. Acute toxicity testing is conducted to get information regarding the

157 158 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines safety and further evaluate the biological activity and mechanism of action of the drug. The information generated by the test is used in hazard identification and risk management of the drugs (Akila and Manickavasakam, 2012). In medicinal plants, one or more than one biological activities and plants have been used traditionally in the various herbal formulations. Table 16.1 lists various plants, plant parts, type of extract, and medicinal uses whose toxicity profiles have been reported and found to be safe. These plants have been used in different pharmaceutical and commercial

TABLE 16.1 Toxicity Profile of Traditional Herbal Plants S.N. Biological Source Plant Parts Use References 1. Solanum nigrum Whole Pain, fever, inflammation Marwa et al. (2013) plant 2. Terminalia chebula Fruit Laxative, carminative Panunto et al. (2011) 3. Curcuma amada Rhizome Skin disease Karchuli and Pradhan (2011) 4. Phyllanthus niruri Leaf Skin diseases Asare et al. (2011) 5. Tamarindus indica Stem bark Dysentery, jaundice Nwodo et al. (2011) 6. Gloriosa superba Root Blood pressure Malpani Arati (2011) 7. Pistacia vera Leaf Analgesic, carminative Hosseinzadeha et al. (2011) 8. Abutilon indicum Leaf Laxative, diuretic Pingale and Virkar (2011) 9. Clerodendron Leaf Asthma, fever, burning Das Sudipta et al. (2011) infortunatum sensation 10. Phyllanthus amarus Aerial Anti- Pingale and Shewale bacterial, anti-fungal (2011) 11. Cassia tora Leaf Skin disease Singhal and Kansara (2012) 12. Curcuma caesia Rhizome Leprosy, asthma Das et al. (2012) 13. Ziziphus jujuba Leaf Anti-diarrheal Rao and Lakshami (2012) 14. Rauwolfia serpentina Root Blood pressure Azmi and Qureshi (2012) 15. Cassia auriculata Leaf Heptoprotective, Kainsa et al. (2013) antioxidant 16. Cyperus rotundus Fruit Anti-diarrheal, Shamkuwar et al. (2012) antispasmodic 17. Combretum Molle Leaf Anti-bacterial, anti-fungal Yeo et al. (2012) 18. Lygodium flexuosum Whole Hepatoprotective Pallara et al. (2012) plant 19. Calotropis procera Root barks Hydrophobia Ouedraogo et al. (2013) 20. Cosmos Caudatus Leaf Anti-aging agent Amna et al. (2013) 21. Alstonia scholaris Bark Fevers, abdominal Bandwane et al. (2011) disorders 22. Albizzia odoratissima Bark Skin disease, rheumatism Kumar et al. (2011) 23. Cassia fistula Seed Skin diseases, fever Subramanion et al. (2011)

(Continued) Toxicity Study of Plant Materials 159

TABLE 16.1 (Continued) Toxicity Profile of Traditional Herbal Plants S.N. Biological Source Plant Parts Use References 24. Calotropis gigantea Root Immunomodulatory, Bulani et al. (2011) hepatoprotective 25. Mallotus philippensis Leaf Anti-fungal, antimicrobial Ramakrishna et al. (2011) 26. Ageratum conyzoides Leaf Diuretic, antipyretic Dash and Murthy (2011) linn 27. Bouvardia ternifolia Aerial Hepatoprotective, Garrido et al. (2012) anti-inflammatory 28. Leucas aspera Aerial Anti-fungal, anti-microbial Kripa et al. (2011) 29. Adina cordifolia Leaf Antifertility, Sharma et al. (2012) anti-inflammatory 30. Citrullus colocynthis Root Antimicrobial, antimalarial Agarwal et al. (2012) 31. Shorea robusta Leaf Analgesic, Supriya et al. (2012) Anti-inflammatory 32. Crinum defixum Bulbs Pimples, Shilpa et al. (2012) body swelling, dropsy, carbuncles 33. Moringa oleifera Leaves Anti-inflammatory Kasolo et al. (2012) 34. Pterospermum Leaf Analgesic, antioxidant, Nandy and Datta (2012) acerifolium antiulcer 35. Spathodea campanulata Leaf Hypoglycaemic, Coolborn et al. (2012) antioxidant 36. Tamarindus indica Leaf Anti-inflammatory, Goyal et al. (2013) antimicrobial 37. Dalbergia latifolia Root Muscle relaxant, for Prasad et al. (2013) diabetes 38. Pisonia Aculeata Leaves Hepatoprotective and Ghode et al. (2013) antioxidant 39. Averrhoa carambola Leaf Analgesic Pessoa et al. (2013) 40. Aristolichia indica Aerial Antibacterial, antioxidant Mall et al. (2011) 41. Cuscuta reflexa Whole Antitumor Chatterjee et al. (2011) 42. Alangium lamarckii Root Anti-inflammatory Ahad et al. (2011) 43. Semecarpus anacardium Nut Anti-arthritis, antioxidant Chakraborty and Asdaq (2011) 44. Bauhinia Variegata Linn Root Antimicrobial, anti- Sharma et al. (2011) inflammatory, hepatoprotective 45. Piliostigma thonningii Leaf Skin disease Daniyan et al. (2011) 46. Bauhinia vahlii Whole Antidiabetic Das et al. (2012) 47. Pistacia integerrima Bark Antioxidant, Ismail et al. (2012) antidepressant 48. Murraya Paniculata Leaves Stimulant astringent Gautam et al. (2012)

(Continued) 160 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

TABLE 16.1 (Continued) Toxicity Profile of Traditional Herbal Plants S.N. Biological Source Plant Parts Use References 49. Fagara heitzii Stem and Analgesic Lembe et al. (2012) barks 50. Chrozophora plicata Leaf Tantircuse and jaundice Kadiri and Rao (2013) 51. Nigella damascena Seed Amenorrhea diuretic Yacine et al. (2013) 52. Cocos nucifera Leaf Nourishing hair Paul et al. (2011) 53. Hemidesmus indicus Leaf Leucorrhoea, bronchitis Moideen et al. (2011) 54. Madhuca latifolia Seed Leprosy, peptic ulcer Bindu et al. (2011) 55. Eugenia jambolana Fruit Antioxidant,anti- Baliga (2011) inflammatory 56. Capparis zeylanica Root Haque and Haque (2011) 57. Momordica dioica Seed Asthma, leprosy Rakh et al. (2012) 58. Anacyclus pyrethrum Root Antibacterial, Kuttan et al. (2012) antidepressant 59. Albizia amara Bark Inflammations Khuddus et al. (2013) 60. Astercantha longifolia Seed Gout and rheumatoid Rajina and Dominic arthritis (2013) 61. Tinospora cordifolia whole Antidiabetic, anti-athritic Pingale (2011) 62. Clitoria ternatia Root Anti-diarrheal, Deka and Kalita (2011) antihistaminic 63. Rubia cordifolia linn. Root Gastrointestinal, Deoda et al. (2011) cardiovascular 64. Mucuna pruriens Whole Dysmenorrhea, Parekar and Somkuwar plant amenorrhea (2011) 65. Coccinia indica Root Skin diseases, ulcer Baghel et al. (2011) 66. Plectranthus Leaf Antibacterial, antimalarial Pillai et al. (2011) amboinicus 67. Pongamia pinnata Seeds & Ulcers, liver pain Aneela et al. (2011) sprouts 68. Anogeissus acuminata Leaf Antidiabetic Hemamalini and Vijusha (2012) 69. Eichhornia crassipes Leaves and Antimicrobial Lalitha et al. (2012) shoot 70. Albizia lebbeck Leaf For snake poison Sivakumar et al. (2013) 71. Boerharia diffusa Whole Headache, anxiety Venkateswarlu and Rao (2013) 72. Momordica dioica Fruit Hepatoprotective, Yadav et al. (2013) antibacterial 73. Ziziphus xylopyrus Stem bark Antidepressant, Mishra et al. (2012) antimicrobial 74. Bauhinia variegata Leaf/bark/ Antioxidant Singh et al. (2012) seed 75. Annona squamosa Leaf Antitumor, antidiabetic Madhu et al. (2012) and antilipidemic Toxicity Study of Plant Materials 161

Bioactivity guided isolation, chemical characterization Herbal medicinal Pure chemical product molecule

Metabolic studies with CYtP450

QSAR, ADMET modeling and simulation

Comparison of compound profile with xenobiotic data libraries High throughput toxicogenomic studies, microarrays, proteomics, metabonomics

Animal studies: Phase-I Phase-II Phase-III Cytotoxic assays acute, subacute and clinical clinical clinical chronic toxicity trials trials trials

Phase-IV clinical trials

FIGURE 16.1 Toxicity evaluation of herbal drugs. formulations. The processes involved in the toxicological evaluation of complex herbal extracts and chemically characterized isolated compounds are schematically represented through Figure 16.1.

16.3 TOXICITY OF HERBS Despite the growing market demand for herbal medicines, there are several issues associated with their safety. Very few (<10%) marketed herbal products are standardized and strictly follow quality control measures (Winston and Maimes, 2007). There is a perception that “natural” drugs are safe and have no side effects. Unfortunately, even “natural” drugs may have significant toxicity (De Smet, 1997). For the majority of these products in use, very little is known about their active or 162 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines toxic constituents. In fact, a good proportion of commonly used pharmaceuticals are derived from natural substances. The safety of combining natural remedies with conventional medical therapy has not been well studied. Many plants produce toxic secondary metabolites as a natural defense from adverse conditions. Some of these are Aconitum columbianum, Blighia sapida, Trifolium hybridum, Digitalis purpurea, Gymnocladius dioica, Hyoscyamus niger, Solanum nigrum, Sanguinaria canadensis, Atropa belladonna, Physostigma venenosum, Pteridium aquilinum, and Podophyllum peltatum. These toxic substances are not distinguished from therapeutically active ingredients. Allergic reactions to chamomile tea used to treat colic have been reported. Infant death has been reported following maternal ingestion of teas containing pyrolizidine alkaloids found in herbs such as comfrey (Huxtable, 1992). There certain phytochemicals alkaloids, flavonoids, terpenoids, and saponins which can mimic or antagonize the functions of signaling molecules, neuropeptides, hormones, and neuropeptides in humans (Daniels et al., 2008; Ismail et al., 2008; Klowden, 2007; Nassel and Winther, 2010). Some lipid soluble terpenes have shown inhibitory properties against mammalian cholinesterase (Savelev et al., 2004), while some interact with the gamma-aminobutyric acid-ergic (GABAergic) system in vertebrates (Rattan, 2010). Saponins contain phytochemicals having potent antimicrobial properties and these are potent surfactants that can disrupt the lipid rich cellular membranes of human erythrocytes (Francis et al., 2002). The presence of toxic minerals and heavy metals like cadmium, lead, mercury, and arsenic also produce implications in the toxicity of herbs. The investigation into herbal toxicity is limited by the following: the lack of a good animal model, a passive reporting system, analytical methodology that is not well characterized, limited knowledge of active ingredients and chemical interactions, limited knowledge of the mechanism of action, variability in the preparation method, and interpatient variability.

16.4 SAFETY AND EFFICACY OF HERBALS It is one of the most essential and demanding tasks for scientists working in herbal drug development to investigate the efficacy of herbal medicine, to scrutinize adverse effects, to identify marker compounds or therapeutic agents in medicinal or botanical preparations, and to separate contaminants from herbal mixtures. Most important reasons or causes of botanical or herbal drug toxicity are improper identification or authentication of botanical or herbals, improper or mislabeling of plant material, contamination of herbals with microorganisms, contamination of herbals with fungal toxins such as aflatoxin, contamination of herbals with pesticides and heavy metals, interaction with conventional drugs upon concomitant intake, improper or unprofessional processing, and inadequate standardization. The demand for herbal medicines increases daily in India as well as the international market, which is why the main concern of herbs is not only their use, but their safety, too. Surveys say only 10% of herbals in the global market are standardized with special reference to markers or active principles and their quality control parameters. The majority of the herbals or plant derived natural products which are used by the majority of the world population needs strict development of standardization and quality control parameters. For the majority of these products in use, very little is known about their Toxicity Study of Plant Materials 163 active or toxic constituents (Winston and Maimes, 2007). This issue raises concerns about the safety and efficacy of herbal drugs. For avoiding potential harmful effects, toxicity testing can reveal some hazards that may be associated with the safer use of herbs or herbal drugs.

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Biological Markers 17 for Quality Control of Herbal Medicines

17.1 INTRODUCTION The term marker compounds can be defined as standard reference compounds used for the purpose of comparison and quality control purposes. Development of a marker provides a suitable and an important parameter for quality control of plants and herbal formulations. Selection of biological markers is crucial for the quality control of herbal medicines, including authentication of genuine species, harvesting the best quality raw materials, evaluation of post-harvesting handling, assessment of intermediates and finished products, and detection of harmful or toxic ingredients. Marker assisted selection of desirable chemotypes along with authentication of species identity for the prediction of the concentration of active phytochemicals may be required for quality control in the use of plant materials for pharmaceutical purposes. Identification of DNA markers that can correlate DNA fingerprinting data with the quantity of selected phytochemical markers associated with that particular plant would have wide applications in quality control of raw materials. Ideal chemical markers should be the therapeutic components of herbal medicines. However, for most herbal medicines, the therapeutic components have not been fully elucidated or easily monitored. Bioactive, characteristic, main, synergistic, correlative, toxic, and general components may be selected. Quality control of herbal medicines aims to ensure their consistency, safety, and efficacy. Chemical fingerprinting has been demonstrated to be a powerful technique for the quality control of herbal medicines. A chemical fingerprint is a unique pattern that indicates the presence of multiple chemical markers within a sample.

17.2 MARKERS ARE CATEGORIZED INTO TWO CLASSES a. DNA markers are reliable for informative polymorphisms as the genetic composition is unique for each species and is not affected by age, physiological conditions, and environmental factors. DNA can be extracted from fresh or dried organic tissue of the botanical material; hence, the physical form of the sample for assessment does not restrict detection (Butterweck, 2003). b. Chemical markers generally refer to biochemical constituents, including primary and secondary metabolites and other macromolecules such as nucleic acids (Butterweck and Schmidt, 2007).

169 170 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

17.2.1 dna Markers DNA markers as a new pharmacognostic tool. Various types of DNA-based molecular techniques are utilized to evaluate DNA polymorphism. These are hybridization- based methods, Polymerase Chain Reaction (PCR)-based methods, and sequencing- based methods. These markers have shown remarkable utility in the quality control of commercially important botanicals like Ginseng, Echinacea, and Atractylodes. Although DNA analysis is currently considered to be cutting-edge technology, it has certain limitations due to which its use has been limited to academia. Another important issue is that DNA fingerprints will remain the same irrespective of the plant part used, while the phytochemical content will vary with the plant part used, its physiology, and the environment.

17.2.1.1 Applications of Biological Markers DNA-based molecular markers have proven their utility in fields like taxonomy, physiology, embryology, genetics, and so on. The applications of biological markers are as follows:

a. Genetic variation/genotyping: RAPD-based molecular markers have been found to be useful in differentiating different accessions of herbal drugs collected from different geographical regions. Interspecies variation has been studied using RFLP and RAPD in different genera such as Glycerrhiza, Echinacea, Curcuma, and Arabidopsis. RAPD has served as a tool for the detection of variability in Jojoba (Simmondsia chinensis L. Schneider), Vitis vinifera L., and tea (Camellia sinesis). b. Authentication of medicinal plants: Sequence Characterized Amplified Region (SCAR), AP–PCR, RAPD, and RFLP have been successfully applied for differentiation of these plants and to detect substitution by other closely related species. Certain rare and expensive medicinal plant species are often adulterated or substituted by morphologically similar, easily available or less expensive species. For example, Swertia chirata is frequently adulterated or substituted by the cheaper Andrographis paniculata. c. Marker assisted selection of desirable chemo types: AFLP analysis has been found to be useful in predicting phytochemical markers in cultivated Echinacea purpurea germplasm and some related wild species. DNA profiling has been used to detect the phylogenetic relationship among Acorus calamus chemotypes differing in their essential oil composition. d. Medicinal plant breeding: Molecular markers have been used as a tool to verify sexual and apomictic offspring of intraspecific crosses in Hypericum perforatum, a well-known antihelminthic and diuretic. e. Applications in foods and nutraceuticals: Roundup ready soybeans, maize, cecropin, and capsicum have been successfully discriminated from non-GM products using primers specific for inserted genes and crop endogenous genes. Biological Markers for Quality Control of Herbal Medicines 171

17.2.2 chemical Markers Selection of chemical markers is crucial for the quality control of herbal medicines, including authentication of genuine species, harvesting the best quality raw materials, evaluation of post harvesting handling, assessment of intermediates and finished products, and detection of harmful or toxic ingredients. Chemical markers as chemically defined constituents or groups of constituents of an herbal medicinal product are of interest for quality control purposes regardless of whether they possess any therapeutic activity as defined by European Medicines Agency (EMEA). Chemical markers can be further categorized as therapeutic components, bioactive components, synergistic components, characteristic components, main components, correlative components, toxic components, and general components used with fingerprint spectra. All markers may contribute to the evaluation, standardization, and safety assessment of herbal medicines.

17.2.2.1 Therapeutic Components Therapeutic components possess the direct therapeutic effects of an herbal medicine. They may be used as chemical markers for both qualitative and quantitative assessments. A therapeutic component originated from the bulbs of Bulbus fritillariae is commonly prescribed as an antitussive and expectorant. Verticine, verticinone, and imperialine were identified as the major therapeutic components that account for the antitussive effect (Li et al., 2006; Lin et al., 2006a,b). Therefore, isosteroidal alkaloids were selected as the chemical markers for the quality assessment of Bulbus Fritillariae using a series of chromatographic techniques such as pre-column derivatizing gas chromatographyflame ionization detection (GC-FID), direct GC-FID, gas chromatography—mass spectrometry (GC-MS), pre-column derivatizing high-performance liquid chromatography—ultraviolet detection (HPLC-UV), high-performance liquid chromatography—evaporative light scattering detection (HPLC-ELSD), and high-performance liquid chromatography—mass spectrometry (HPLC-MS) methods (Lin et al., 2001).

17.2.2.2 Bioactive Components Bioactive components are structurally different chemicals within an herbal medicine. Although the individual components may not have direct therapeutic effects, the combination of their bioactivities does contribute to the therapeutic effects. Bioactive components may be used as chemical markers for qualitative and quantitative assessment. Bioactive components, including isoflavonoids and saponins, were used simultaneously in the evaluation of the quality of Radix Astragali (Song et al., 2007; Yu et al., 2007; Zhang et al., 2007).

17.2.2.3 Synergistic Components Synergistic components do not directly contribute to the therapeutic effects or related bioactivities. However, they act synergistically to reinforce the bioactivities of other components, thereby modulating the therapeutic effects of the herbal medicine. Synergistic components may be used as chemical markers for qualitative and quantitative assessment. Naphthodianthrone, hypericin, and hyperforin 172 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

HO CH3 HH

H H H N N H H H OH O H CH3 H H CH3 H H

HH HO HO Imperialine H Verticinone O OH

OH OHO HO O OH OH HO O OH HO OH O CH3 HO O O OH

HO OHO HO O Naphthodianthrone Rutin OH

FIGURE 17.1 Synergistic components as chemical markers.

(a phloroglucinol derivative) were identified as the major components that contribute to the pharmacological activities of St John’s wort (Butterweck, 2003; Butterweck and Schmidt, 2007). Rutin is a ubiquitous flavonoid that demonstrated synergistic antidepressant actions in St John’s wort (Noldner and Schota, 2002). Figure 17.1 shows the structure of chemical markers.

17.2.2.4 Characteristic Components While characteristic components may contribute to the therapeutic effects, they must be specific and unique ingredients of an herbal medicine. Valerenic acids, the characteristic components of valerian derived from the roots of Valeriana officinalis L., have sedative effects and improve sleep quality (Bent et al., 2006; Taibi et al., 2007). Valerenic acids are used as chemical markers to evaluate the quality of valerian preparations although their sedative effects have not been fully elucidated.

17.2.2.5 Main Components The main components are the most abundant in an herbal medicine (or significantly more abundant than other components). They are not characteristic components and their bioactivities may not be known. Main components may be used for both qualitative and quantitative analysis of herbal medicines, especially for differentiation and stability evaluation. Flavonoids including epimedin A, B, C and icariin are Biological Markers for Quality Control of Herbal Medicines 173 the main components of Herba Epimedii. Total flavonoids and icariin are used as chemical markers for Herba Epimedii (Pei and Guo, 2007).

17.2.2.6 Correlative Components Correlative components in herbal medicines have a close relationship with one another. Correlative components can be used as chemical markers to evaluate the quality of herbal medicines originated from different geographical regions and stored for different periods of time. Psoralen and isopsoralen are used as chemical markers for assessing the quality of Fructus Psoraleae (Qiao et al., 2006). Figure 17.2 shows the structure of chemical markers.

17.2.2.7 Toxic Components Traditional Chinese medicine literature and modern toxicological studies have documented some toxic components of medicinal herbs. For instance, aristolochic acids (AAs) and pyrrolizidine alkaloids (PAs) may cause nephrotoxicity and hepatotoxicity, respectively (Cosyns, 2003; Fu et al., 2004).

17.2.2.8 Applications of Chemical Markers • Identification of adulterants. • Differentiation of herbal medicines with multiple sources. • Determination of the best harvesting time. • Confirmation of collection sites. • Assessment of processing methods. • Quality evaluation of herbal parts. • Identification and quantitative determination of proprietary products.

CO H O 2

NO O O 2

OO Isopsoralen OCH3 Aristolochic acids O HO O O O

HO OH O O O O OH OH O OH O Psoralen Icarrin OH OH

FIGURE 17.2 Correlative components as chemical markers. 174 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines

• Stability test of proprietary products. • Diagnosis of herbal intoxication. Toxic components may be used as chemical markers in screening methods, for example, rapid diagnosis of acute hidden aconite poisoning in urine samples by HPLC-MS. • Lead compounds for new drug discovery.

17.3 CONCLUSION Quality control of herbal medicines aims to ensure their quality, safety, and efficacy. The use of chromatographic techniques and marker compounds to standardize botanical preparations has limitations because of their variable sources and chemical complexity. Markers can have a vital role in various applications such as applications of molecular markers in herbal drug technology for authentication, detection of adulteration/substitution of medicinal plants, marker assisted selection of desirable chemotypes, DNA markers as new pharmacognostic tools, and marker applications in foods and nutraceuticals for the purpose of safety and efficacy of the drugs. DNA-based molecular markers have utility in the fields like taxonomy, physiology, embryology, genetics, and so on. Chemical markers are pivotal in the current practice of quality control. Chemical markers should be used at various stages of the development and manufacturing of an herbal medicine, such as authentication and differentiation of species, collecting and harvesting, quality evaluation and stability assessment, diagnosis of intoxication, and discovery of lead compounds. Lack of chemical markers remains a major problem for the quality control of herbal medicines. Furthermore, there are many technical challenges in the production of chemical markers. For example, temperature, light, and solvents often cause degradation and/or transformation of purified components; isomers and conformations may also cause confusion of chemical markers. The fingerprinting profile of the marker compounds in plant drugs, which shows the presence/percentage of the active principle along with the closely related bioactive principles, is necessary for all herbal formulations.

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Lin BQ, Ji H, Li P, Jiang Y, Fang W. Selective antagonism activity of alkaloids from Bulbs Fritillariae at muscarinic receptors: Functional studies. Eur J Pharmacol. 2006b; 551: 125–130. Lin G, Li P, Li SL, Chan SW. Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb. J Chromatogr A. 2001; 935: 321–338. Noldner M, Schota K. Rutin is essential for the antidepressant activity of Hypericum perforatum extracts in the forced swimming test. Planta Med. 2002; 68: 577–580. Pei LK, Guo BL. A review on research of raw material and cut crude drug of Herba Epimedii in last ten years (Chinese). Zhongguo Zhongyao Zazhi 2007; 32: 466–471. Qiao CF, Han QB, Song JZ, Mo SF, Kong LD, Kung HF, Xu HX. Quality assessment of Fructus Psoraleae. Chem Pharm Bull. 2006; 54: 887–890. Song JZ, Mo SF, Yip YK, Qiao CF, Han QB, Xu HX. Development of microwave assisted extraction for the simultaneous determination of isoflavonoids and saponins in Radix Astragali by high performance liquid chromatography. J Sep Sci. 2007; 30: 819–824. Taibi DM, Landis CA, Petry H, Vitiello MV. A systematic review of valerian as a sleep aid: Safe but not effective. Sleep Med Rev. 2007; 11: 209–230. Yu QT, Qi LW, Li P, Yi L, Zhao J, Bi Z. Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi (Radix Astragali) by HPLC-DAD-ELSD. J Sep Sci. 2007; 30: 1292–1299. Zhang X, Xiao HB, Xue XY, Sun YG, Liang XM. Simultaneous characterization of isoflavonoids and astragalosides in two Astragalus species by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry. J Sep Sci. 2007; 30: 2059–2069.

Pollutants for 18 Herbal Drugs

18.1 INTRODUCTION Herbal medicines have been used for the treatment of diseases for many thousands of years and are recognized as valuable, readily available resources for health care. The use of medicinal plants is increasing day by day due to their effectiveness and safety profile. Growing tendencies and an increase in demand for food safety has drawn the attention of researchers to the risk factors associated with the consumption of contaminated foodstuffs, that is, pesticides, heavy metals, and/or toxins, and so on. (Abdollatif et al., 2009). To have the desired therapeutic outcomes, the quality of finished products and plant raw materials must be ensured. It has been reported that high heavy metals, contaminants, and residues are associated with the extensive pollution of herbal medicine (Gasser et al., 2009). Pollution of the atmosphere and soil, followed by plants and animals with Ni originate from sources such as stainless steel, glass and ceramic production industries, catalytic converters, cigarette smoke, and medical prostheses and utensils. Polluted irrigation water, automobile and industrial exhausts, pesticides, and fertilizers play important roles in contamination of medicinal plants with copper and cobalt (Girisha and Ragavendra, 2009).

18.2 WHO GUIDELINES FOR CONTAMINANTS AND RESIDUES According to the World Health Organization (WHO), over 70%–80% of the world’s population living in rural areas relies on non-conventional medicine for the treatment of their ailments. The World Health Organization (WHO) estimated that within the last 15 years, the demand for medicinal plants has reached around $14 billion annually and could reach $5 trillion by 2050 (Akerele, 1992). However, owing to the nature and sources of these medicinal plants, they are sometimes contaminated with toxic metals such as lead, arsenic, mercury, and cadmium, which pose serious health risks to consumers. It is critical to examine the source materials and extracts for toxic metals in order to ensure the safety, efficacy, and quality of medicinal plants. The WHO has given certain guidelines for controlling the quantity of these toxic substances. The objectives of these guidelines are to provide:

• Guiding principles for assessing the quality in relation to the safety of herbal medicines, with specific reference to contaminants and residues • Model criteria for use in identifying possible contaminants and residues • Technical procedures for controlling the quality of finished herbal products

However, economic constraints should not prevent the implementation of testing for contaminants. If resources are insufficient, the establishment of a national or

177 178 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines regional pesticide control laboratory could be a suitable solution and cooperation with laboratories from academia or research centers or with specialized laboratories working in food-related areas should be considered (Anonymous, 1998).

18.2.1 Pesticides Pesticides are defined as any substance intended for preventing, destroying, attracting, repelling or controlling any pest including unwanted species of plants or animals during production, storage, transport, distribution, and processing. The term includes substances intended for use as a plant-growth regulator, defoliant, desiccant, fruit- thinning agents or sprouting inhibitors, and substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport. The term normally excludes fertilizers and plant nutrients (Anonymous, 2000).

18.2.2 Pesticide Residue Pesticide residues are any specified substance in food, agricultural commodities or animal feed resulting from the use of a pesticide. The term includes any derivatives of a pesticide, such as conversion products, metabolites, reaction products, and impurities which are considered to be of toxicological significance (WHO, 2002).

18.2.3 Pesticidal Toxicity Pesticides are also the major concern related to the quality of herbal drugs. Pesticides used for protection from certain insects are finally transported to the human body by different routes. Aspergillus species are the major contaminants in herbals, responsible for poor performance, liver lesions, and immunosuppression. The contamination of pollutants in herbals remains and continues during their transportation and storage. Such contaminated herbs are one of the major potential sources of pollutants in the human organs and systems. Quality of the finished products and plant raw materials must be ensured to get the desired therapeutic outcome. Due to undesired toxicity in plants, WHO has made it mandatory to estimate the pollutants in every batch.

18.3 HEAVY METAL TOXICITY TO PLANTS Medicinal plants can accumulate heavy metals when grown on polluted environmental media, such as roadsides and metal mining and smelting operations. Furthermore, heavy metal contamination in medicinal herbs can also be due to anthropogenic processes involving the application of synthetic fertilizers, organic manures, lime and industrial effluents that contaminate the agro ecosystem or during transportation and unhygienic storage conditions (Okatch et al., 2012). Pollution present in the environment such as heavy metals, pesticides, radioactive particles, mycotoxins, and microbes including pathogens, can cause serious problems for the quality of herbal and herbal drugs. Due to environmental pollution, several toxic compounds have been accumulating at alarming level on plants. Heavy metals such as Cu, Zn, Cr, Fe, and Co are required in very trace quantities for the proper functioning of enzyme Pollutants for Herbal Drugs 179 systems, hemoglobin formation, and vitamin synthesis in humans and for growth and photosynthesis in plants (Annan et al., 2010). The use of fertilizers containing cadmium (Cd), organic mercury or lead-based pesticides could also contaminate medicinal plant materials with toxic metals. An imbalance of these essential metals may leads to metabolic disturbances. However, Pb, Cd, As, and Hg are toxic metals that are not required by the body, and they produce deleterious effects upon exposure even at very low concentrations (Baye and Hymete, 2013).

18.4 POTENTIALLY HAZARDOUS CONTAMINANTS AND RESIDUES IN HERBAL MEDICINES Contamination can be avoided and controlled through quality assurance measures such as good agricultural and collection practices (GACP) for medicinal plants and good manufacturing practices (GMP) for herbal medicines. Chemical and microbiological contaminants can result from the use of human excreta, animal manures, and sewage as fertilizers. As noted in the WHO guidelines on GACP for medicinal plants, human excreta must not be used as a fertilizer, and animal manure should be thoroughly composted. Toxic elements and other chemical contaminants, including solvents originating from products intended for use in households and industrial chemicals, can be concentrated in composted sewage. Table 18.1 shows examples of potentially hazardous contaminants and residues that may occur in herbal medicines.

18.5 CHEMICAL CONTAMINANTS

18.5.1 toxic Metals and Non-Metals Contamination of herbal materials with toxic substances such as arsenic can be attributed to many causes. These include environmental pollution such as:

• Contaminated emissions from factories and leaded petrol • Contaminated water including runoff water • Soil composition and fertilizers

Pesticides containing arsenic and mercury were widely used until a few years ago. As toxic substances are likely to be present in many foods due to their abundance in nature, it is important that concomitant ingestion of herbal products not add to the total concentration of toxic metals consumed by people according to guidelines given by WHO.

18.5.2 Persistent Organic Pollutants (POPs) Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bio-accumulate through the food web, and pose a risk of causing adverse effects to human health and the environment. With the evidence of long- range transport of these substances to regions where they have never been used or produced and the consequent threats they pose to the environment of the whole globe, 180 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines )

Continued ( Possible Sources Possible growth, manufacturing process manufacturing growth, cultivation/growth storage storage and storage storage Polluted soil and water during cultivation/ Polluted soil and water during soil and water, Polluted air, during cultivation/growth soil, water Air, processing, transportation, and Post-harvest processing, transportation and Post-harvest Soil, post-harvest processing, transportation, Soil, post-harvest processing, transportation, and Post-harvest Soil, excreta processing Post-harvest processing Post-harvest Soil and water Specific Examples dieldrin, endrin, heptachlor aeruginosa, salmonella species, shigella species, escherichia coli Lead, cadmium, mercury, chromium Lead, cadmium, mercury, Dioxin aldrin, chlordane, DDT, Cs-134, Cs-137 Mycotoxins Bacterial endotoxins Staphylococcus aureus, pseudomonas Staphylococcus molds Yeast, Amebae, nematoda Cockroach earthworms Mouse excreta, Acetone, methanol, ethanol Contaminants Subgroup Toxic metals and non-metals Toxic Persistent organic pollutants Persistent organic Radionuclide Biological toxins Bacteria Fungi Parasites Insects Others Organic solvents Organic Group materials Toxic and hazardous Toxic Micro-organisms Animals Chemical contaminants TABLE 18.1 Classificationof Contaminants Major Residuesand Herbalin Medicines Classification General Biological contaminants Solvents Pollutants for Herbal Drugs 181 Possible Sources Possible Air, soil, water, during cultivation soil, water, Air, during cultivation soil, water, Air, during cultivation soil, water, Air, Post-harvest processing Post-harvest During cultivation Manufacturing process Manufacturing Specific Examples hydrocarbons, organophosphorus hydrocarbons, bromide, sulfur dioxide Carbamate, chlorinated 2,4-D, 2,4,5-T Dithiocarbamate Ethylene oxide, phosphine, methyl oxide, phosphine, methyl Ethylene Thiamethoxam Acetone, methanol, ethanol, butanol Residues Subgroup Insecticides Herbicides Fungicides Chemical agents Antiviral agents Antiviral Organic solvents Organic Group ) agents Pesticides Fumigants Disease control Continued Dichlorodiphenyltrichloroethane.

DDT

Agrochemical residues Classificationof Contaminants Major Residuesand Herbalin Medicines TABLE 18.1 ( 18.1 TABLE Classification General Residual solvents Note: 182 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines the international community has on several occasions called for urgent global action to reduce and eliminate releases of these chemicals. The use of persistent pesticides, such as DDT and benzene hexachloride (BHC), in agriculture has been banned for many years in many countries. However, they are still found in the areas where they were previously used and often contaminate medicinal plants growing nearby.

18.5.3 residual Solvents A range of organic solvents is used for manufacturing herbal medicines, and can be detected as residues of such processing in herbal preparations and finished herbal products. Solvents are classified by International Conference on Harmonisation (ICH), according to their potential risk, into: • Class 1 (solvents to be avoided such as benzene) • Class 2 (limited toxic potential such as methanol or hexane) • Class 3 (low toxic potential such as ethanol)

18.6 RADIOACTIVE CONTAMINATION A certain amount of exposure to ionizing radiation is unavoidable because many sources, including radionuclides, occur naturally in the ground and the atmosphere. Dangerous contamination may be the consequence of a nuclear accident or may arise from other sources. WHO has given guidelines for use in the event of widespread contamination by radionuclides resulting from a major nuclear accident. Examples of such radionuclides include long-lived and short-lived fission products, actinides, and activation products.

18.7 MYCOTOXINS AND ENDOTOXINS The presence of mycotoxins in plant material can pose both acute and chronic risks to health. Mycotoxins are usually secondary metabolic products, which are nonvolatile, have a relatively low molecular weight, and may be secreted onto or into the medicinal plant material. They are thought to play a dual role, first in eliminating other microorganisms competing in the same environment, and second in helping parasitic fungi to invade host tissues. Mycotoxins produced by species of fungi including aspergillus, fusarium, and penicillium are the most commonly reported. Mycotoxins comprise four main groups, namely, afatoxins, ochratoxins, fumonisins, and tricothecenes, all of which have toxic effects. Endotoxins are found mainly in the outer membranes of certain gram-negative bacteria and are released only when the cells are disrupted or destroyed. They are complex lipopolysaccharide molecules that elicit an antigenic response, cause altered resistance to bacterial infections, and have other serious effects.

18.8 BIOLOGICAL CONTAMINANTS

18.8.1 Microbiological Contaminants Herbs and herbal materials normally carry a large number of bacteria and molds, often originating in soil or derived from manure. While a large range of bacteria and fungi form Pollutants for Herbal Drugs 183 the naturally occurring microflora of medicinal plants, aerobic spore-forming bacteria frequently predominate. Current practices of harvesting, production, transportation, and storage may cause additional contamination and microbial growth. Proliferation of microorganisms may result from failure to control the moisture levels of herbal medicines during transportation and storage, as well as from failure to control the temperatures of liquid forms and finished herbal products. The presence of escherichia coli, Salmonella spp., and molds may indicate poor quality of production and harvesting practices.

18.8.2 Parasitic Contamination Parasites such as protozoa and nematoda and their ova may be introduced during cultivation and may cause zoonosis, especially if uncomposted animal excreta are used. Contamination with parasites may also arise during processing and manufacturing if the personnel carrying out these processes have not taken appropriate personal hygiene measures.

18.8.3 agrochemical Residues The main agrochemical residues in herbal medicines are derived from pesticides and fumigants. Pesticides may be classified on the basis of their intended use, for example, as follows:

• Insecticides • Fungicides and Nematocides • Herbicides • Other Pesticides (e.g., ascaricides, molluscicides, and rodenticides)

Examples of fumigants include ethylene oxide, ethylene chlorohydrin, methyl bromide, and sulfur dioxide.

18.9 PESTICIDE RESIDUES Medicinal plant materials may contain pesticide residues, which accumulate as a result of agricultural practices, such as spraying, treatment of soils during cultivation, and administration of fumigants during storage. Examples of pesticides include benzene hexachloride (BHC), lindane, methoxychlor, organophosphorus pesticides: carbophenothion, chlorpyrifos, methylchlorpyrifos, coumaphos, demeton, dichlorvos, dimethoate, ethion, fenchlorphos, malathion, methyl parathion, pesticides of plant origin: tobacco leaf extract, pyrethrum flower, and pyrethrum extract; derris and Lonchocarpus root and rotenoids. Only the chlorinated hydrocarbons and organophosphorus pesticides (e.g., carbophenothion) have a long residual action.

18.10 BIOCHEMICAL CHANGES IN MEDICINAL PLANT LEAVES AS A BIOMARKER OF POLLUTION Pollution is a serious issue with the major sources being fuel wood and biomass burning, fuel adulteration, vehicle emission, and traffic congestion. Air pollution 184 Fingerprinting Analysis and Quality Control Methods of Herbal Medicines is one of the severe problems the world is facing today. It deteriorates ecological conditions and can be defined as a fluctuation in any atmospheric constituent from the value that would have existed without human activity (Tripathi and Gautam, 2007). Over the years, there has been a continuous growth in human population, road transportation, vehicular traffic, and industries which increases the concentration of gaseous and particulate pollutants (Joshi et al., 2009). To develop the importance of herbal plants as bio-indicators of air pollution requires appropriate selection of herbal tree and plant species. Air pollution tolerance index (APTI) is performed by studying the leaf pH, ascorbic acid, total chlorophyll, and relative water content. It is known that Bambusa Bambos, Washingtonia robusta, Mangifera indica, Acacia Arabica, and Albizia amara were found to have high air pollution tolerance index (APTI) values and Spathodea campanulata and Magifera caesia have low APTI values as sensitive plants (Krishnaveni et al., 2013). This is an attempt to bio-monitor the environment by analyzing the APTI of plant species present. The efficiency of plants in absorbing pollutants is such that they can produce clean air. Bernatsky A has suggested that green belts might help to reduce air pollution. Plants growing in an air polluted environment respond and show significant changes in their morphology and biochemistry (Bernatsky, 1969).

18.10.1 aPTI Factors It has been observed that acidic pollutants reduce the leaf pH and the decline is more pronounced in sensitive species. This shift of cell sap pH toward the acidic side could decrease the efficiency of conversion of hexose sugar to ascorbic acid and is pH dependent, that is, the activity is greater at higher pHs and lower at lower pHs. Hence, pH on the higher side could provide tolerance in plants against pollutants as reported by some authors (Rabe and Kreeb, 1980). The chlorophyll content of plants signifies their photosynthetic activity as well as the growth and development of the biomass. It is evident that the chlorophyll contents of plants vary from species to species and with the age of the leaf, as well as with the pollution level and other biotic and abiotic conditions (Lakshmi et al., 2009). The decrease in foliar chlorophyll concentration in plants might be due to the destruction of chlorophyll, reversible swelling of thylakoids, and inhibition of RuBp carboxylase. Low chlorophyll content in the winter season might be due to the high pollution levels, temperature stress, low sunlight intensity, and a short photoperiod (Speeding and Thomas, 1973; Wellburn et al., 1972).

18.11 CONCLUSION The role of plants in developing a healthy atmosphere is very desirable in the context of the deteriorating environment resulting from increased urbanization, industrialization, and improper environmental management. It is necessary that the plants used must be tolerant to air pollution. Vegetation naturally cleanses the atmosphere by absorbing gases and some particulate matter through leaves. Plants have a very large surface area and their leaves function as an efficient pollutant-trapping device. Some plants have been classified according to their degree of sensitivity and tolerance towards Pollutants for Herbal Drugs 185 various air pollutants. The importance of trees in urban environments is now widely recognized, because they, too, cleanse particular air pollution and help to make cities and towns more agreeable places to live in.

REFERENCES Abdollatif GA, Ardalan M, Mohammadi MT, Hosseini HM, Karimian N. Solubility test in some phosphate rocks and their potential for direct application in soil. World App Sci J. 2009; 6: 182–190. Akerele O. WHO guidelines for the assessment of herbal medicines. Fitoterapi. 1992; 63: 99–104. Annan K, Kojo AI, Cindy A, Samuel A, Tunkumgnen BM. Profile of heavy metals in some medicinal plants from Ghana commonly used as components of herbal formulations. Pharmacognosy Res. 2010; 2(1): 41–44. Anonymous. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva, 1998. Anonymous. Pesticide Residues in Food—Methods of Analysis and Sampling. Codex Alimentarius. Vol. 2A, Part 1, 2nd Edition. Joint FAO/WHO Food Standards Programme, Rome, 2000. Baye H, Hymete A. Levels of heavy metals in common medicinal plants collected from environmentally different sites. Middle-East J Sci Res. 2013; 13(7): 938–943. Bernatsky A. On the influence of air pollution on plants and animals, Proc. First European Cong., Wageningen, Netherlands, 1969; 385–95. Gasser UB, Klier AV, Kuhn B. Irrespective of their site of collection. Current findings on the heavy metal content in herbal On the other hand, some plants tend to accumulate drugs. Pharmeur Sci Notes. 2009; 1: 37–50. Girisha ST, Ragavendra VB. Accumulation of heavy metals in leafy vegetables grown in urban areas by using sewage water its effect. Arch Phytopathol Plant Protection. 2009; 42: 956–959. Joshi N, Chauhan A, Joshi PC. Impact of industrial air pollutants on some biochemical parameters and yield in wheat and mustard plants. Environmentalist 2009; 29: 398–404. Krishnaveni M, Amsavalli L, Chandrasekar R, Durairaj S, Madhiyan P. Biochemical changes in medicinal plant leaves as a biomarker of pollution. Res J Pharm Tech. 2013; 6(5): 537–543. Lakshmi PS et al. Air pollution tolerance index of various plant species growing in industrial areas. The Ecoscan. 2009; 2: 203–206. Okatch H, Ngwenya B, Raletamo KM, Andrae-Marobela K. Determination of potentially toxic heavy metals in traditionally used medicinal plants for HIV/AIDS opportunistic infections in Ngamiland District in Northern Botswana. Anal Chim Acta. 2012; 730: 42–48. Rabe P, Kreeb KH. Bioindication of air pollution by chlorophyll destruction in plant leaves. Oikos. 1980; 34: 163–167. Speeding DJ, Thomas WJ. Effects of sulphur oxide on the metabolism of glycolic acid Barley (Hordeum vulgare) leaves. Australian J Biol Sci. 1973; 26: 281–286. Tripathi AK, Gautam M. Biochemical parameters of plants as indicators of air pollution. J Environ Biol 2007; 28: 127–132. Wellburn AR et al. Effects of SO2 and NO2 polluted air upon the ultra structure of chloroplasts. Environ Pollut. 1972; 3: 37–49. WHO. Traditional Medicine Strategy: 2002–2005, World Health Organization, Geneva, 2002.

Index

A AP–PCR, see Arbitrarily primed polymerase chain reaction AAs, see Aristolochic acids APTI, see Air pollution tolerance index AAS, see Atomic absorption spectrometry Aqueous alcoholic extraction by fermentation, ABC transporters, see ATP-binding cassette 26–27 transporters Arabidopsis, 68 Acacia Arabica (A. Arabica), 184 Arbitrarily primed polymerase chain reaction Acceptable residual limit (ARL), 148 (AP–PCR), 68, 107–108, 170 Acceptance criteria, 10 Arista, 79 Acidic pollutants, 184 Aristolochic acids (AAs), 70, 71, 173 Acid insoluble ash, 134 ARL, see Acceptable residual limit Acorus calamus (A. calamus), 170 Arsenic (As), 162, 177, 179 chemotypes, 68 determination, 148–150 Active pharmaceutical ingredient (API), 64 “Asava and arista” Ayurvedic preparations, 26 Acute toxicity testing, 157–158 Asavas, 79 Aflatoxins, 147, 182 Ascaricides, 183 AFLP, see Amplified fragment length “Ashtasthana pareeksha”, 4 polymorphism Ash values and extractives, 133 Age-related disorders, 157 Aspergillus flavus (A. flavus), 147 Agricultural practices, 148, 183 Aspergillus parasiticus (A. parasiticus), 147 Agrochemical residues, 183 Aspergillus species, 178 Air pollution, 183–184 Astringent action of tannins, 137 Air pollution tolerance index (APTI), 184 ASU medicine, see Ayurveda, Siddha, and Unani Ajmalicine, 13 medicine Albizia amara (A. amara), 184 Atomic absorption spectrometry (AAS), 149 Alcohol, 14 ATP-binding cassette transporters (ABC Aleurone grains, 129 transporters), 76 Alkaloids, 32–33, 102, 162 Atractylodes, 68 phytochemical analysis tests, 39–40 Australia, regulatory aspects and approval of reagents, 141 herbal drugs in, 53 Allium sativum, see Garlic Authentication of medicinal plants, 68, 170 Amarogentin, 32 Automobile, 177 Amino acids, 33 Ayurveda, Siddha, and Unani medicine (ASU Amplified fragment length polymorphism medicine), 50 (AFLP), 68, 107, 108, 170 Ayurvedic/Ayurveda, 4 Amplitude, 20 drugs, 5 Anabolic steroids, 36 medicines, 121 Analytical methods, 7, 63, 138–139 pharmacopoeia of India, 5 for herbal products, 64, 90–91 preparations, 26 Andrographis paniculata (A. paniculata), Azeotropic 170 method, 137 Andrographolide, 32 mixtures, 14 Anisocytic cell type, 130 Annealing, 107 B Anomocytic cell type, 130 Anthocyanin, phytochemical screening of, 40 Baljel’s tests, 43 Anthraquinone, phytochemical analysis tests Balsams, 138 for, 40 Bambusa Bambos (B. Bambos), 184 Anthropogenic processes, 178 Benzene, 182 Anupaan, 78 Benzene hexachloride (BHC), 182, 183 API, see Active pharmaceutical ingredient β-himachalene, 35

187 188 Index

Betulic acid, 35 Capillary isoelectric focusing (CIEF), 102 BHC, see Benzene hexachloride Capillary LC-NMR, 104 Biliary clearance, 77 Capillary zone electrophoresis (CZE), 102 Bioactive components, 69, 171 Carbohydrates detection, 142

Bioassay, 143–144 Carbon dioxide (CO2), 27, 28 end point or quantal assay, 144 Carbophenothion, 183 graded response assay, 144–146 Cardiac glycosides, see Steroidal glycosides types, 144 Cardiac glycosides, phytochemical analysis tests Bioavailability enhancers, need for, 75–76 for, 40 Bioavailability of herbal drugs, 75 Carvacrol, 35 drug absorption barriers, 76 Caryophyllaceous cell type, see Diacytic cell mechanism of action of bioenhancers, 76–77 type medicinal plants and compounds as drug Castor oil, 133 bioavailability enhancers, 77–78 Catechin test, see Matchstick test need for bioavailability enhancers, 75–76 CCE, see Counter current extraction Bioenhancers, 75, 76 CE-DAD, see Capillary electrophoresis-diode- mechanism of action, 76–77 array detector Biological contaminants, 182–183 CE, see Capillary electrophoresis Biological markers, 67; see also Stability studies Ceiling effect, 144 of herbal medicines Cellulose cell walls, 129 applications, 170 CGE, see Capillary gel electrophoresis categorized into two classes, 169 Chamomile tea, 162 chemical markers, 69–71, 171–174 Characteristic components, 69–70, 172 DNA markers, 67–68, 170 Characterization of herbal substance/preparation, for quality control of herbal medicines, 169 9–10 Biological methods for herbal drugs, 143 Character of drug, 15 bioassay, 143–146 Chemical contaminants, 179 microbial contamination, 146–147 POPs, 179–182 toxicological standardization, 147–149 residual solvents, 182 Biomedical resources, 155 toxic metals and non-metals, 179 Bitterness value, 134–135 Chemical fingerprint(ing), 67, 117, 169 Bitter principles, 32 chromatograms, 96–97 Black cumin (Cuminum cyminum), 77 chromatographic fingerprinting,97 –105 Borntragor’s test, 40 DNA fingerprinting, 105–109 Botanical Drug Products, 62 evaluation of herbal medicines, 96 Botanical Drug Substances, 62 Chemical markers, 69, 169, 171 Botanical methods, 123–124 applications, 71, 173–174 BP, see British Pharmacopoeia bioactive components, 69, 171 Bracketing method, see Matching point method characteristic components, 69–70, 172 British Pharmacopoeia (BP), 23 correlative components, 71, 173 Bulbus fritillariae (B. fritillariae), 69, 171 main components, 71, 172–173 synergistic components, 69, 171–172 C therapeutic components, 69, 171 toxic components, 71, 173 Cadmium (Cd), 149, 162, 177, 178 Chemical methods for herbal drugs, 138 Calcium carbonate, 129 alkaloids detection, 141 Calcium oxalate, 129 analytical methods, 138–139 CAM, see Complementary/Alternative Medicines chemical examination of herbal drugs, 141 Camellia sinesis, see Tea detection of carbohydrates and glycosides, 142 Camphor, 34 detection of fixed oils and fats, 142 Canadian regulatory system, 51 detection of gums and mucilages, 142 Canthin-6-one, 33 detection of phenolic compounds and Capillary electrophoresis-diode-array detector tannins, 142 (CE-DAD), 103 detection of proteins and free amino Capillary electrophoresis (CE), 102, 108 acids, 142 hyphenation, 105 phytosterols detection, 142 Capillary gel electrophoresis (CGE), 102 radioactive contamination, 143 Index 189

saponins detection, 142 Contaminated herbs, 178 TLC, 139–141 Content or assay, 7, 63 volatile oil detection, 142–143 Continued process verification, 87 Chemical parameters for herbal formulation, 122 Continuous apparatus, 23, 24 Chemical stability, 91 Continuous countercurrent extraction, 21 Chemistry, Manufacture, and Control (CMC), 62 Copper (Cu), 178 Chemoprofiling, 97 Copper acetate test, 43 Chloral hydrate TS, 127 Correlative components, 71, 173 Chlorinated hydrocarbons, 183 Co-solvents, 27 Chloroform, 14 Cost of drug, 16 Chlorogenic acid test, 42 Coumarins, phytochemical analysis tests for, Chlorophyll, 184 40–41 Chromanone, 33 Counter current extraction (CCE), 25 Chromatogram, 99 Cover and run down method, 22 Chromatographic fingerprinting,9 , 95, 97 Cross-celled type, see Diacytic cell type analysis, 61–62 Cruciferous cell type, see Anisocytic cell type chemical fingerprints evaluation of herbal Crude plant materials, 2 medicines, 96 Cryology mounting, 126 chromatographic techniques, 95–96 Crystallization, 14–15 construction of, 97–98 Cuminum cyminum, see Black cumin electrophoretic methods, 102 Cuprocyanate test, 41 GC, 100–101 Curcuma, 68 of herbal medicines, 95 Cuticular cell walls, 129 HPLC, 101–102 Cutting-edge technology, 170 HPTLC, 99–100 Cyanogenic glycosides, 32 hyphenated techniques, 103–105 phytochemical analysis tests for, 41 TLC, 98–99 CYP1A1, 76 Chromatographic separation system, 103 CYP1B2, 76 Chromatographic technique, 138, 171 CYP2E1, 76 Chromatography, 97, 148 CYP3A4, 76 Chromium (Cr), 178 CZE, see Capillary zone electrophoresis CIEF, see Capillary isoelectric focusing 1,8-Cineole, 35 D CMC, see Chemistry, Manufacture, and Control Coaxial sheath interface, 105 Daidezein, 36 Cobalt (Co), 178 Dasmularista, 27 Column chromatography, 148 DDT, see Dichlorodiphenyltrichloroethane Commercial scale percolator, 21, 22 Decoctions, 13, 26 Committee for Herbal Medicinal Products Denaturation, 107 (HMPC), 52 Department of Indian Systems of Medicine and Community monographs, 52 Homeopathy (ISMH), 5, 50 Complementary/Alternative Medicines (CAM), Derivative thermogravimetry (DTG), 79 47, 49, 52, 55 Detection techniques, 103 Complementary medicine, 52 Diacytic cell type, 130 Complex modulus, 81 Dichlorodiphenyltrichloroethane (DDT), 182 Compound-oriented approach, 62 Differential scanning calorimetry (DSC), 79, 80 Concentrated infusions, 26 Differential thermal analysis (DTA), 79 Concentrated preparations, 16 applications, 81 Concentration of product, 16 characteristics, 80–81 Condensed tannins, 36 curve, 80 Contaminants Dihydroquercetin, 26 biological, 182–183 Dilute products, 16 chemical, 179–182 Diosgenin, 36 potentially hazardous contaminants and Directive 2001/EC, 52

residues in herbal medicines, 179, Diterpenes (C20), 34 180–181 phytochemical analysis tests for, 43 WHO guidelines for and, 177–178 DMA, see Dynamic mechanical analysis 190 Index

DMTA, see Dynamic Mechanical Thermal Efficacy of herbals, 162–163 Analysis Egallitannins, 36 DNA Electrical energy, extractions by, 20 analysis, 68, 105, 170 Electrolytes, 15 barcoding, 153 Electrophoretic methods, 102 chip technology, 153 Electro planar chromatography (EPC), 99 DNA-based molecular techniques, 170 Electrospray ionization mass spectrometry (ESI microarray technology, 154 MS), 105 polymorphism-based assays, 155 Electrospray ionization (ESI), 117 segments, 108 ELSD, see Evaporative light scattering detection DNA fingerprinting, 95, 105, 169, 170; see also EMA, see European Medicines Agency Chromatographic fingerprinting EMEA, see European Medicines Agency (EMA) hybridization-based methods, 106–107 Endotoxins, 182 PCR-based methods, 107–108 End point assay, 144 sequence-based methods, 108–109 Environmental pollution, 178, 179 types, 106 EPC, see Electro planar chromatography DNA markers, 67, 68, 169, 170; see also Epimedin A, 172 Chemical markers Epimedin B, 172 Dose response curve (DRC), 146 Epimedin C, 172 Double maceration, 18 ESI, see Electrospray ionization Douglas fir bark extracts, 26 ESI MS, see Electrospray ionization mass Dragendorff’s test, 39 spectrometry Dragendroff’s reagent, 141 Ethanol, 14, 27, 76, 182 DRC, see Dose response curve Ethnobotany, 55 Drug Ethnopharmacological information, 56 absorption barriers, 76 Ethnopharmacology of medicinal plants, 55 adulteration, 11 of Chhattisgarh, India, 57–59 character, 15 need of documentation, 57 cost, 16 phytotherapy, 55–56 discovery process, 77 practicing herbal medicine, 56–57 leads, 56 Ethylene chlorohydrin, 183 metabolizing enzymes, 76 Ethylene oxide, 183 stability, 16 Eugenol, 34 therapeutic value, 15 European Communities (EC), 52 Drugs and Cosmetics Act, 50 European herbal guidelines, 52 DSC, see Differential scanning calorimetry European Medicines Agency (EMA), 52, DTA, see Differential thermal analysis 69, 171 DTG, see Derivative thermogravimetry Evaporative light scattering detection (ELSD), Dynamic mechanical analysis (DMA), 79, 81, 83; 102 see also Thermo-mechanical analysis Exhaustive extraction, 20 (TMA) Extension, 107 instrument and working, 81–82 Extensive characterization, 10 principles, 81 Extraction method, 13 sinusoidal oscillation and response of linear- character of drug, 15 viscoelastic material, 82 concentration of product, 16 Dynamic Mechanical Thermal Analysis cost of drug, 16 (DMTA), 81 experimental conditions for methods, 17 factors affecting choice, 15–16 E procedures for herbal drugs, 16–28 recovery of solvent from the marc, 16 EC, see European Communities regeneration of solvent, 14–15 Echinacea, 68 selection of solvents, 14 Echinacea purpurea (E. purpurea), 170 solutions, 15 germplasm, 68 solvent, 13–14, 16 Economic constraints, 177–178 stability of drug, 16 ED, see Effective dose therapeutic value of drug, 15 Effective dose (ED), 144 Extractive value, 136 Index 191

F G Fats, 129–130 GABAergic, see Gamma-aminobutyric detection, 142 acid-ergic phytochemical analysis tests for, 43 GACP, see Good agricultural and collection Fatty oils, 129–130 practices FDA, see Food and Drug Administration Gallotannins, 36 Feigel’s test, 43 Gamma-aminobutyric acid-ergic (GABAergic), 162 Fermentation, aqueous alcoholic extraction by, Gamma glutamyl transpeptidase (GGT), 77 26–27 Garlic (Allium sativum), 77

Ferric chloride test (FeCl3 test), 40–42 Gas chromatography (GC), 7, 63, 64, 90, 97, Ferriferrocyanide test, 41 100–101, 137, 148 Ferruginol, 35 Gas chromatography–flame ionization detection Fertilizers, 177, 179 (GC-FID), 69, 171 FFPC, see Forced-flow planar chromatography Gas chromatography-Fourier transform infrared Fingerprint spectrometry (GC-FTIR), 103, 104 analysis, 62 Gas chromatography–mass spectrometry (GC- chromatogram, 139 MS), 66, 69, 103, 104, 171 Fingerprinting techniques, 95; see also Gas liquid chromatography (GLC), 100 Spectroscopic techniques Gas–liquid extraction, 13 in herbal drug standardization, 96 Gastro-intestinal absorption (GI absorption), 75 phytoequivalence and chromatographic Gastrointestinal tract (GIT), 77 fingerprints of herbal medicines, GC-FID, see Gas chromatography–flame 95–109 ionization detection Finished herbal products, 2, 3 GC-FTIR, see Gas chromatography-Fourier Fixed oils transform infrared spectrometry detection, 142 GC-MS, see Gas chromatography–mass phytochemical analysis tests for, 43 spectrometry Flavans, 34 GC, see Gas chromatography Flavonoids, 33–34, 71, 102, 162, 172 Gelatin test, 42 phytochemical analysis tests for, 41 Genetically modified products (GM products), 68 Fluid extracts, 13 Genetic variation/genotyping, 68, 170 Fluorescence microscope, 124 Genistein, 36 Fluorescence test, 41 Genomic Foaming index, 50, 136 DNA, 108 Food and modified techniques, 153 biological markers applications in, 170 technique, 153, 155 DNA markers applications in, 68 Genotypic characterization of plant, 105 safety, 177 Gentiopicrin, 32 Food and Drug Administration (FDA), Geometric isomerization, 92 52, 62, 86 German Federal Health Agency, 51 Forced-flow planar chromatography Germany’s Commission E for phytotherapy and (FFPC), 99 herbal substances, 51 Force motor, 81 Germplasm, 109 Foreign organic matter, 132 GGT, see Gamma glutamyl transpeptidase Fourier Transform Infrared spectroscopy (FTIR GI absorption, see Gastro-intestinal absorption spectroscopy), 115–116 Ginger (Zingiber officinale), 77 4 point assay (2 + 2 dose assay), 146 Ginseng, 68, 170 Fragments, 127 GIT, see Gastrointestinal tract Free amino acids detection, 142 GLC, see Gas liquid chromatography Free hand mounting, 126 Glide mounting, 126 Fresh infusion, general method for preparing, 26 Global quantitative protein profiling, 154 Fructus Psoraleae (F. Psoraleae), 71, 173 Glycerrhiza, 68 FTIR spectroscopy, see Fourier Transform Glycitein, 36 Infrared spectroscopy Glycosides, 14, 31–32 Fumigants, 183 detection, 142 Fumonisins, 182 phytochemical analysis test for cardiac, 40 192 Index

Glycyrrhiza glabra, see Liquorice terms relating to, 2 GM products, see Genetically modified usage, 153 products WHO guidelines on safety monitoring, 48 GMPs, see Good manufacturing practices Herbal(s) Goldbeater’s skin test, 42 acute toxicity testing, 157–158 Good agricultural and collection practices analytical methods for herbal products, 64, (GACP), 48, 179 90–91 WHO guidelines on, 48–49 materials, 3, 182 Good manufacturing practices (GMPs), 5, 7, 50, medicinal drugs, 63 86, 179 plants, 57 Graded response assay, 144–146 preparations, 3 Gums, 138 regulation in India, 5 detection, 142 remedies, 157 phytochemical analysis test for, 43 safety and efficacy of, 162–163 Gustatory nerves, 32 specifications for herbal substances, 9 toxicity evaluation of herbal drugs, 161 H toxicity profile of traditional herbal plants, 158–160 Hager’s reagent, 141 toxicity testing, 157 Hager’s test, 40 Herbs, 2, 182 Hazard, 6 toxicity of, 161–162 HCl, see Hydrochloride acid Hesperidin, 32 Heavy metal(s), 148, 162, 178 Hexane, 182 determination, 148–150 High-performance liquid chromatography-diode- toxicity to plants, 178–179 array detector (HPLC-DAD), 103, Hecogenin, 36 104–105 Hemolytic activity, 135 High-speed counter-current chromatography Herba Epimedii (H. Epimedii), 71, 173 (HSCCC), 101 Herbal drugs, 1, 2, 85 High performance liquid chromatography bioavailability, 75–78 (HPLC), 7, 63, 64, 66, 90, 97, chemical examination, 141–143 101–102, 139 FTIR applications in herbal drug analysis, High performance liquid chromatography– 116 evaporative light scattering detection India’s strength in herbal technology, 2 (HPLC-ELSD), 69, 171 quality control, 6–8 High performance liquid chromatography–mass regulations in India, 50–51 spectrometry (HPLC-MS), 69, 102, technology, 45 104–105, 171, 174 WHO guidelines for herbal drug High performance liquid chromatography– standardization, 49–50 ultraviolet detection (HPLC-UV), Herbal formulation, 2, 121–122 69, 171 WHO guidelines for quality control of, 49 High performance thin layer chromatography Herbal medicinal products (HMP), 63, 89 (HPTLC), 97, 99–100, 139 challenges in stability testing, 65, 91–92 High Pressure Extraction (HPE), 28 goal of analytical marker for analysis, 90 High pressure liquid chromatography, see High predictable changes in, 92–93 performance liquid chromatography specific characteristics, 64 (HPLC) Herbal medicines (HMs), 1, 12, 45, 61, 79, 95, Hirsutine, 33 157, 177 Histochemical detection, 128 finished herbal products or herbal medicinal aleurone grains, 129 products, 3 calcium carbonate, 129 guidelines for regulation in Southeast Asia calcium oxalate, 129 region, 48 cellulose cell walls, 129 herbal materials, 3 cuticular cell walls, 129 herbal preparations, 3 fats, fatty oils, volatile oils, and resins, practicing, 56–57 129–130 registration, 46 hydroxyanthraquinones, 130 regulation, 46, 49 inulin, 130 Index 193

leaf stomata, 130 I lignified cell walls, 129 mucilage, 130 Icarrin, 70, 71, 172, 173 starch, 130 ICH, see International Conference on suberized cell walls, 129 Harmonization tannin, 130 Identity, 6, 62 HMP, see Herbal medicinal products IDMA, see Indian Drug Manufacturers Association HMPC, see Committee for Herbal Medicinal Immiscible solvent, 137 Products Imperialine, 69, 70, 171, 172 HMs, see Herbal medicines In-process tests, 10 Homeopathy, 4 Inclusion complexes, 27 Hot continuous extractions, 23 IND, see Investigations of New Drugs HPE, see High Pressure Extraction India HPLC-DAD-MS techniques, 104–105 herbal drug regulations in, 50–51 HPLC-DAD, see High-performance herbal regulation in, 5 liquid chromatography-diode- ethnopharmacognostical studies of medicinal array detector plants, 57–59 HPLC-ELSD, see High performance liquid Indian Drug Manufacturers Association chromatography–evaporative light (IDMA), 50 scattering detection Indian Herbal Pharmacopoeia, 50 HPLC-IR, 102 Indian Pharmacopoeia (IP), 23, 50 HPLC-MS, see High performance liquid Indian system of medicine (ISM), 3–5, 85 chromatography–mass spectrometry Industrial exhausts, 177 HPLC-NMR, 102 Infrared spectroscopy (IR spectroscopy), 103, HPLC-UV, see High performance liquid 114–115 chromatography–ultraviolet detection Infusions, 13, 26 HPLC, see High performance liquid In situ parameters, 21 chromatography Interferometer, 116 HPTLC, see High performance thin layer International Conference on Harmonization chromatography (ICH), 52, 182 HSCCC, see High-speed counter-current International Pharmacopoeia (IP), 139 chromatography Interpolation assay, 145 Hybridization-based methods, 106–107 Interspecies variation, 68 Hydrochloride acid (HCl), 134 Inulin, 130 Hydrocotyline, 33 Investigations of New Drugs (IND), 62 Hydrofluorocarbon-134a, 28 IP, see Indian Pharmacopoeia; International Hydrolysable tannins, 36 Pharmacopoeia Hydrolysis, 92, 137 Iron (Fe), 178 Hydrophilic nature of aqueous stagnant layer, 76 Irregular-celled type, see Anomocytic cell type Hydroxyanthraquinones, 130 IR spectroscopy, see Infrared spectroscopy Hyoscine, 13 ISM, see Indian system of medicine Hyperforin, 69, 171 ISMH, see Department of Indian Systems of Hypericin, 69, 171 Medicine and Homeopathy Hypericum perforatum (H. perforatum), Isobaric process, 28 68, 170 Isopsoralen, 70, 71, 173 Hyphenated chromatographic techniques, 139 Isosteroidal alkaloids, 69 Hyphenated HPLC, 102 Hyphenated techniques, 97, 103; see also J Spectroscopic techniques GC-FTIR, 104 Jashpur, Chhattisgarh, 57 GC-MS, 104 Jojoba (Simmondsia chinensis L. Schneider), HPLC-DAD, 104–105 68, 170 HPLC-MS, 104–105 hyphenation of CE, 105 K LC-MS, 103 LC-NMR, 103–104 Kaempferol, 34 others HPLC-DAD, 104–105 Kanakasava, 27 194 Index

Karpurasava, 27 modification of general processes, 18 Keller–Killani test, 40 process, 18 Kinetic maceration, 19 Macromolecules, 103, 169 Macroscopic examination, 131 L Macroscopic methods of herbal drugs, 123–124; see also Microscopic methods Labelling of herbal products, 149 Magifera caesia (M. caesia), 184 Lactochloral TS, 127 Main components, 71 Lactones, phytochemical analysis tests for, 43 MALDI, see Matrix-assisted laser desorption Large scale extractor, 25 ionization LC-MS, see Liquid chromatography-mass Mangifera indica (M. indica), 184 spectrometry Mangiferin, 34 LC-NMR, see Liquid chromatography nuclear Marc, recovery of solvent from, 16 magnetic resonance Markers, 90 LC-SPE-NMR, see Liquid chromatography- assisted selection of chemo types, 68 solid-phase extraction-nuclear compounds, 67, 169 magnetic resonance Mass spectrometry (MS), 7, 103, 116–117, 148 LD, see Lethal dose analysis, 154 LDR, see Log dose response Matching point method, 144–145 Lead (Pb), 149, 162, 177, 179 Matchstick test, 42 acetate tests, 41 Matrix-assisted laser desorption ionization compounds, 174 (MALDI), 117 Leaf stomata, 130 Mayer’s reagent, 141 Legal’s test, 43 Mayer’s test, 40 Lenses, 125 MDI, see Mean daily intake Lethal dose (LD), 144 Mean daily intake (MDI), 148 Leucocyanidin, 34 MECC, see Micellar electrokinetic capillary Lieberman–Burchard test, 42 chromatography Liebermann’s and Burchard’s tests, 142 Medical systems, 4 Light, 93, 113 Medicinal compounds, 102 petroleum R, 127 Medicinal plants, 1, 31, 56, 157–158, 177–178 Lignified cell walls, 129 biochemical changes in medicinal plant Limonene, 35 leaves as pollution biomarker, Lindane, 183 183–184 Linear Variable Differential Transformer breeding, 68, 170 (LVDT), 82 materials, 134, 183 Lipid solubility, 75 Medicines and Healthcare products Regulatory Lipid soluble terpenes, 162 Agency (MHRA), 52 Liposomes, 27 Melting point, 132 Liquid chromatography-mass spectrometry (LC- Membrane-filtration method, 146, 147 MS), 103 Menstruum, 14 Liquid chromatography-solid-phase extraction- Menthol, 34 nuclear magnetic resonance (LC-SPE- Mercury (Hg), 162, 177, 179 NMR), 104 Metabolomics, 153–154 Liquid chromatography nuclear magnetic Metabomics, 153 resonance (LC-NMR), 103–104 Metals, 178 Liquid–liquid extraction, 13 Methanol, 14, 27, 182 Liquorice (Glycyrrhiza glabra), 77 Methoxychlor, 183 Log dose response (LDR), 146 Methyl bromide, 183 Low-pressure size-exclusion chromatography Methylene chloride, 14 (SEC), 101 MHRA, see Medicines and Healthcare products Luteolin, 34 Regulatory Agency LVDT, see Linear Variable Differential Transformer Micellar electrokinetic capillary chromatography M (MECC), 101 Microarray technology, 153 Maceration, 17 Microbes, 178 circulatory extractions, 19 Microbial contamination, 146–147 Index 195

Microbiological contaminants, 182–183 Nibmin, 35 and foreign materials, 66 Nichghat, 57 Microbiological parameters for herbal Nickel (Ni), 177 formulation, 122 NMR, see Nuclear magnetic resonance Microorganisms, 183 Non-electrolytes, 15 Micro particles, 27 Non-metals, 179 Microsatellite DNA, 107 “Non-physiological” ash, 133 Microscopic authentication, 124 Nootkatone, 35 Microscopic methods, 124 Nuclear magnetic resonance (NMR), 103 clarification of microscopic particles, 127 spectroscopy, 117–118 equipment, 125 Nucleic acids, 169 fragments, 127 Nutraceuticals, applications in, 68 microscopical examination, 125 photography, 127 O preliminary treatment, 126 preparation of specimen, 126 Ochratoxins, 182 sampling, 127 Ocular micrometer, 125 standard procedure for microscopic Odor, 124 identification, 125 aromatic, 123 transverse sections, 126–127 sensation, 124 Minisatellite DNA, 107 Office of Dietary Supplements, 52–53 Miscella, residual insoluble drug plant Official extractor, 23, 24 material, 14 Oil, 27 Modern medicine, 78 castor, 130 Modified Borntragor’s test, 40 croton, 130 Moisture, 93 volatile, 50 content and volatile matter, 132 Oleanolic acid, 35 Molecular markers, 68, 170 Omics techniques, 153; see also Fingerprinting Molluscicides, 183 techniques; Spectroscopic techniques Monographs, 5, 23, 26, 50 application in herbal medicine context, 155 community, 53 genomics and modified techniques, 153 Monoterpenes, 34 metabolomics, 154–155 MS, see Mass spectrometry proteomics, 153–154 Mucilage(s), 130 transcriptomics, 154 detection, 142 Opium Tincture, 18 phytochemical analysis test for, 43 OPLC, see Over pressured-layer chromatography Multidimensional liquid chromatography, 154 Optical rotation, 133 Multi point bioassay, 145–146 Organic liquids, 14 Mycotoxins, 99, 178, 182 Organic solvents, 28 Myricetin, 34 mixtures, 14 non-polar, 36 N Organoleptic evaluation, 123 Organophosphorus pesticides, 183 Nanocoating of active components of herbal Organs, 154 formulation, 66–67 Over pressured-layer chromatography Nanoparticles, 27 (OPLC), 99 Naphthodianthrone, 69–70, 171, 172 Oxidation, 92 Naringenin, 34 Oximatrine, 33 Naringin, 33 National policy on TM, 49 P Natural drugs, 161 Natural medicines, novel approaches for stability PAL, see Phenylalanine ammonia lyase improvements in, 93 Paracytic cell type, 130 Natural products, 31, 93 Paraffin mounting, 126–127 Naturopathy, 4 Parallel-celled type, see Paracytic cell type Nematoda, 183 Parasites, 183 Niaziridin, 77 Parasitic contamination, 183 196 Index

Particulate drug delivery systems, 27 standardization, 10 PAs, see Pyrrolizidine alkaloids test for cardiac glycosides, 40 Pattern-oriented approach, 62 test for diterpenes, 43 PCR-based methods, see Polymerase chain test for gums and mucilages, 43 reaction-based methods test for saponins, 41 Percentage quantity of tannins, 138 tests for alkaloids, 39–40 Percolation, 20 tests for anthraquinone, 40 modification of general process, 21–22 tests for coumarins, 40–41 procedure, 21 tests for cynogenetic glycosides, 41 reserved, 22 tests for fixed oils and fats, 43 Persistent organic pollutants (POPs), 179–182 tests for lactones, 43 Persistent pesticides, 182 tests for phenolics and flavonoids, 41 Pesticidal toxicity, 178 tests for steroids, 42 Pesticide residue, 178 tests for tannins, 42 determination, 148 tests for triterpenes, 42 Pesticides, 177–179, 183 Phytochemistry, 39 P-glycoprotein (P-gp), 76, 77 Phyto-constituent detection methods, 39 Pharmacopoeial tests, 10 phytochemical analysis, 39–43 Pharmacopoeias, 138 Phyto-constituents, 31 Pharmacopoeia standards, 150 alkaloids, 32–33 Pharmacovigilance systems, 48 classes, 31 Phenazone test, 42 flavonoids, 33–34 Phenolic(s), 35 glycosides, 31–32 compounds detection, 142 phenolics, 35 phytochemical analysis tests for, 41 saponins, 35–36 Phenols, 35 steroids, 36 Phenylalanine ammonia lyase (PAL), 35 tannins, 36 Phloroglucinol derivative, 172 terpenes, 34–35 Photography, 127 Phytoequivalence, 95 Photometric analysis, 63 chemical fingerprints evaluation of herbal Physical instability, 65 medicines, 96–109 Physical parameters for herbal formulation, 122 chromatographic fingerprinting,97 –105 Physical standardization of herbal drugs, 131 chromatographic techniques, 95–96 acid insoluble ash, 134 DNA fingerprinting, 105–109 ash values and extractives, 133 of herbal medicines, 95 bitterness value, 134–135 Phytonics process, 28 extractive value, 136 Phytopharmaceuticals, 14 foaming index, 136 Phytotherapy, 55–56 foreign organic matter, 132 Piezoelectric ultrasound transmitters, 20 hemolytic activity, 135 Pilular extracts, 13 melting point, 132 Piperine, 77 moisture content and volatile matter, 132 Piper nigrum Linn, 77 optical rotation, 133 1-Piperoyl piperidine, see Piperine refractive index, 133 Plant constituents solubility, 132 classes of phyto-constituents, 31–36 swelling index, 135 separation and isolation, 31 tannins determination, 137–138 Plants, 55, 56 total ash, 133–134 chemistry, 39 total solid content, 136 extract, 91 viscosity, 132 genome analysis, 106 volatile oil content, 137 materials, 8 water content, 137 Plate count method, 146, 147 water soluble ash, 134 Pollutants for herbal drugs “Physiological ash”, 133 biochemical changes in medicinal plant Phytochemicals, 13, 31, 39 leaves, 183–184 analysis, 39 biological contaminants, 182–183 screening of anthocyanin, 40 chemical contaminants, 179–182 Index 197

heavy metal toxicity to plants, 178–179 Quantitative TLC (QTLC), 139 mycotoxins and endotoxins, 182 Quercetin, 34 pesticide residues, 183 Quercitrin, 34 potentially hazardous contaminants and Quillaja saponaria, see Saponaria vaccaria residues, 179, 180–181 radioactive contamination, 182 R WHO guidelines for contaminants and residues, 177–178 Racemization, 92 Polluted irrigation water, 177 Radioactive contamination, 143, 182 Pollution, 183 Radioactive particles, 178 air, 183–184 Radionuclides, 182 biomarker, 183–184 Radix Astragali (R. Astragali), 171 environmental, 178, 179 Randomly amplified polymorphic DNA (RAPD), Polymerase chain reaction-based methods (PCR- 107–108, 170 based methods), 67, 106, 107–108 RAPD-based molecular markers, 68, 170 Polymerization, 92 Ranunculaceous cell type, see Anomocytic cell type Polyphenolics, 35 RAPD, see Randomly amplified polymorphic DNA POPs, see Persistent organic pollutants Reciprocal piezoelectric effect, 20 Powdered extracts, 13 Rectification, 13, 14 Powder studies, 127–128 Re-extraction, 13 Precipitation of Hg from HgNO, 41 Reference samples (RS), 127 Preclinical studies of herbal drugs, 157 Reference standard, 10 Predictable changes Reflux condenser, 23 in herbal drug material, 65–66 Refractive index, 133 in herbal medicinal products, 92–93 Regeneration of solvent, 14–15 Preliminary phytochemical screening, 141 Registered medicines, 53 Process design, 87 Regulation of herbal medicine, 49 Process qualification, 87 Regulatory aspects for herbal drugs, 45; see Process validation, 87 also Quality control of herbal drugs/ Profiling approaches, 153 medicine Proteins, 154 aim of regulatory guidelines for herbal detection, 142 medicines, 46 Proteomics, 153–154 Australia, 53 technique, 155 European herbal guidelines, 52 Protozoa, 183 guidelines for regulation of herbal medicines Psoralen, 70, 71, 173 in Southeast Asia region, 48 Purity, 6–7, 63 herbal drug regulations in India, 50–51 and quality of herbs, 47 National policy on TM, 49 Pyrrolizidine alkaloids (PAs), 71, 173 objectives, 48 regulation, 46 Q regulation and registration of herbal medicines, 46 QTLC, see Quantitative TLC regulation of herbal medicine, 49 Quadrupole analyzers, 117 regulatory aspects and approval of herbal Qualitative chemical tests, 138 drugs in different countries, 51–53 Quality control, 138 regulatory requirements, 47–50 Quality control of herbal drugs/medicine, 6, 61, United States of America, 52–53 62–63, 115, 124, 169; see also Herbal WHO guidelines for herbal drug medicines (HMs); Regulatory aspects standardization, 49–50 for herbal drugs WHO guidelines for herbals, 47 content or assay, 7, 63 WHO guidelines for quality control of herbal identity, 6, 62 formulation, 49 problems influencing, 8 WHO guidelines on GACP for medicinal purity, 6–7, 63 plants, 48–49 biological markers for herbal medicines, 67–71 WHO guidelines on safety monitoring stability studies of herbal medicines, 63–67 of herbal medicines in Quantal assay, 144 pharmacovigilance systems, 48 198 Index

Repeated maceration, 18 SEC, see Low-pressure size-exclusion Re-percolation, 20–22 chromatography Reserpine, 33 Sehpaan, 78 Reserved percolation, 22 Semi-concentrated preparations, 16 Residual solvents, 182 Semisolid extracts, see Pilular extracts Residues Senecionine, 33 agrochemical, 183 Separate Drugs Consultative Committee (SDCC), pesticide, 183 50 potentially hazardous contaminants and residues Separate Drug Technical Advisory Board in herbal medicines, 179, 180–181 (SDTAB), 50 WHO guidelines for and, 177–178 Sequence Characterized Amplified Region Resins, 34, 129–130, 138 (SCAR), 68, 170 Restriction fragment length polymorphism Sequencing-based methods, 106, 108–109 (RFLP), 106, 170 Serial dilution method, 146

Retardation factor values (Rf values), 99 Sesquiterpenes (C15), 34 determination, 141 SFE, see Supercritical fluid extraction Reversed-phase columns (RP columns), 101 Shaking, 19 Reversed-phase ion-pairing HPLC (RP Shelf life, 63, 65, 89, 91 IPCHPLC), 101 Shinoda’s test for flavonoids, 41 RFLP, see Restriction fragment length Short tandem repeats (STR), 108, 109 polymorphism Siddha, 4 Rf values, see Retardation factor values Simmondsia chinensis L. Schneider, see Jojoba Risk, 6 Simple maceration, 19 Risk analysis, 6 Simple sequence repeats (SSR), 108 Risk assessment, 6 Single nucleotide polymorphism (SNP), 108, 109 Risk communication, 6 Sinusoidal force, 82 Risk management, 6 Sliding microtome, 126 Rodenticides, 183 Small scale or laboratory scale extraction, 23 Rotation planar chromatography (RPC), 99 continuous apparatus, 23, 24 RPC, see Rotation planar chromatography hot continuous extractions, 23 RP columns, see Reversed-phase columns SNP, see Single nucleotide polymorphism RP IPCHPLC, see Reversed-phase ion-pairing Sodium hypochlorite TS, 127 HPLC Solid–liquid extraction, 13 RS, see Reference samples Solubility, 132 Rubiaceous cell type, see Paracytic cell type Solute, 15 Rutin, 69, 70, 172 Solutions, 15 Solvent(s), 15, 16, 182 S for extraction, 13–14 polarity, 28 Safety regeneration, 14–15 of herbal medicine, 48 selection, 14 of herbals, 162–163 Southeast Asia region, guidelines for regulation Salkowski test, 42 of herbal medicines in, 48 Sapogenins, 35 Soxhlet extractor, 23, 24 Saponaria vaccaria (Quillaja saponaria), 35 Soxhletion, 23 Saponins, 35–36, 135, 162 Spathodea campanulata (S. campanulata), 184 detection, 142 Specifications for herbal substances, 9 phytochemical analysis tests for, 41 Specimen measurement, 130–131 Satellite DNA, 106–107 Spectral correlative chromatograms (SCC), 139 SAX-HPLC, see Strong anion-exchange HPLC Spectroscopic techniques, 113; see also SCAR, see Sequence Characterized Amplified Fingerprinting techniques; Omics Region techniques SCC, see Spectral correlative chromatograms FTIR spectroscopy, 115–116 SDCC, see Separate Drugs Consultative IR spectroscopy, 114–115 Committee mass spectrometry, 116–117 SDTAB, see Separate Drug Technical Advisory NMR spectroscopy, 117–118 Board UV spectroscopy, 113–114 Index 199

Spectrum, 113 pharmacopoeial tests and acceptance absorption, 114 criteria, 10 electromagnetic, 115 reference standard, 10 mass, 117 specification, 9 SSR, see Simple sequence repeats Starch, 130 Stability of drug, 16 Steam distillation, 27 Stability studies of herbal medicines, 63–65; Steroidal/steroids, 36 see also Biological markers glycosides, 36 analytical methods for herbal products, 64 phytochemical analysis tests for, 42 challenges in stability testing of herbal sapogenin, 35 medicinal products, 65 saponins, 35, 41 factors affecting stability of natural Stirring, 19 medicines, 66 Stomatal index determination, 131 importance of stability testing, 66–67 STR, see Short tandem repeats predictable changes in herbal drug material, Strong anion-exchange HPLC (SAX-HPLC), 101 65–66 Suberized cell walls, 129 shelf life, 65 Sulfur dioxide, 183 specific characteristics of herbal medicinal Supercritical carbon dioxide technique, 67 products, 64 Supercritical fluid extraction (SFE), 27–28 Stability study of plant products, 89 Swelling index, 135 analytical methods for herbal Swertia chirata (S. chirata), 170 products, 90–91 Synergistic components, 69, 171–172 challenges in stability testing of herbal Synthetic drugs, 85 medicinal products, 91–92 factors affecting stability of herbal T medicines, 92 markers, 90 Tannic acid test, 40 novel approaches for stability improvements Tannin(s), 36, 130 in natural medicines, 93 detection, 142 predictable changes in herbal medicinal determination, 137–138 products, 92–93 phytochemical analysis tests for, 42 shelf life, 91 Taspine, 33 Stability testing, 89 Taxifolin, 26, 34 challenges in, 65 “Taxol” drug, 34, 77 of herbal products, 63, 89 Tea (Camellia sinesis), 68, 170 importance, 66–67 Temperature, 79, 80, 92 Stable drug products, 89 Terpenes, 34–35 Stage micrometer, 125 Terpenoids, 34–35, 162 Standardization of herbal drugs, 121, 122, 153 Terpinen-4-ol, 34 biological methods, 143–149 Test samples (TS), 127 botanical methods, 123–124 TG, see Thermal gravimetry chemical methods, 138–143 TGA, see Thermogravimetric analysis determination of arsenic and heavy metals, TG curve, see Thermogravimetric curve 149–150 Theaflavins, 36 different techniques in standardization of Therapeutic components, 69, 171 crude drugs, 123 Therapeutic value of drug, 15 herbal formulation, 121–122 Thermal analysis of herbal drugs, 79 histochemical detection, 128–130 DMA, 81–82 measurement of specimen, 130–131 DTA, 80–81 microscopic methods, 124–127 TG, 79–80 physical standardization of herbal drugs, TMA, 82–84 131–138 Thermal gravimetry (TG), 79 powder studies, 127–128 applications of thermogravimetric analysis, 80 validation, 149 characteristics, 80 Standardization of herbal formulation, 8 Thermo-mechanical analysis (TMA), 79, 82, 83; characterization, 9–10 see also Dynamic mechanical analysis in-process tests, 10 (DMA) 200 Index

Thermo-mechanical analysis (TMA) (Continued) Triterpenoids, 41 applications, 83–84 TS, see Test samples instrumentation, 82 Tschugajen test, 42 Thermogravimetric analysis (TGA), 79 Turbo extraction, see Vortical extraction Thermogravimetric curve (TG curve), 79–80 TVC, see Total viable aerobic count Thin layer chromatography (TLC), 7, 63, 64, 66, Two-dimensional electrophoresis (2DE), 154 90, 97, 98–99, 138, 139 Two-dimensional gel electrophoresis, 154 equipment, 140 fingerprinting, 139 U methodology, 140–141 Rf value determination, 141 UDP, see Uridine diphosphate Three point assay (2 + 1 dose assay), 145 Ultrasound extraction, 19–20 Thujone, 34 Ultraviolet (UV), 113 Time of flight mass analyzers (ToF mass absorption spectroscopy, 113–114 analyzers), 117 spectroscopy, 113–114, 139 Tinctures, 13 Ultraviolet/visible spectroscopy (UV/VIS), 7, Titrimetric Karl Fisher method, 137 103, 113 TLC, see Thin layer chromatography Unani, 4–5 TM, see Traditional Medicines Unequal-celled type, see Anisocytic cell type TMA, see Thermo-mechanical analysis United States of America, regulatory aspects and ToF mass analyzers, see Time of flight mass approval of herbal drugs in, 52–53 analyzers United States of Pharmacopoeia (USP), 147 Toluene, 137 Upper Ghat, 57 Total ash, determination of, 133–134 Uridine diphosphate (UDP), 77 Total flavonoids, 71 USP, see United States of Pharmacopoeia Total solid content, 136 UV, see Ultraviolet Total viable aerobic count (TVC), 146–147 UV/VIS, see Ultraviolet/visible spectroscopy Toxic components, 71, 173, 174 Toxicity of herbs, 161–162 V Toxicity study of plant materials herbal toxicity testing, 157–161 Valerenic acids, 69–71, 172 safety and efficacy of herbals, 162–163 Valeriana officinalis (V. officinalis), 172 toxicity of herbs, 161–162 Valeriana officinalis L., 71 Toxic metals, 148, 177, 179 Validation, 85, 86, 87 Toxicological standardization for herbal drugs, of herbal drugs, 86–87 147; see also Physical standardization of herbal products, 149 of herbal drugs process, 87 determination of arsenic and heavy metals, value addition, 87 148–149 Value addition, 87 pesticides, 148 Vanillin-hydrochloric acid test, 42 Toxic substances, 162 Variable number tandem repeats (VNTR), Traditional Chinese medicine, 173 106–107 Traditional herbal plants, toxicity profile of, Verticine, 69, 171 158–160 Verticinone, 69, 70, 171, 172 Traditional Medicines (TM), 47, 48, 55 Vincristine, 13 National policy on, 49 Viscosity, 132 program, 55 Visible contaminants, 131 Traditional use section, 52 Visible spectroscopy, 139 Transcriptomics, 153, 154 Vitis vinifera L. (V. vinifera L.), 68, 170 Transverse sections, 126–127 VNTR, see Variable number tandem repeats Tricothecenes, 182 Volatile matter, 138 “Trikatu”, 77–78 Volatile oil(s), 129–130, 138

Triterpenes (C30), 34 content, 137 phytochemical analysis tests for, 42 detection, 142–143 saponins, 35 Volatility, 66 Triterpenoidal sapogenin, 35 Vortical extraction, 19 Index 201

W guidelines for quality control of herbal formulation, 49 Wagner’s reagent, 141 guidelines on GACP for medicinal plants, 48–49 Wagner’s test, 40 guidelines on safety monitoring of herbal Washingtonia robusta (W. robusta), 184 medicines, 48 Water, 14 content, 137 X soluble ash, 134 water-soluble plant constituents, 26 Xylene, 27, 137 Well-established use section, 52 Xylene R, 127 WHO, see World Health Organization Wogonin, 34 Y World Health Organization (WHO), 1, 31, 47, 55, 61, 79, 86, 121, 177 Yoga, 4 guidelines for contaminants and residues, 177–178 Z guidelines for herbal drug standardization, 49–50 Zingiber officinale, see Ginger guidelines for herbals, 47 Zoonosis, 183