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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
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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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Contents
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
14.2.3Powder Studies......................................................... 127
14.2.4Histochemical 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 S
inusoidal 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
