Authentication of Medicinal Plants Traded As Herbal Drugs by Using Systematics and Phytochemical Characterization

AUTHENTICATION OF MEDICINAL PLANTS TRADED AS HERBAL DRUGS BY USING SYSTEMATICS AND PHYTOCHEMICAL CHARACTERIZATION

SOFIA RASHID

Department of Plant Sciences Quaid-i-Azam University Islamabad Pakistan 2016 AUTHENTICATION OF MEDICINAL PLANTS TRADED AS HERBAL DRUGS BY USING SYSTEMATICS AND PHYTOCHEMICAL CHARACTERIZATION

A Thesis Submitted to the Quaid-i-Azam University in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY In Plant Sciences

(Plant Systematics and Biodiversity)

SOFIA RASHID

Department of Plant Sciences Quaid-i-Azam University Islamabad Pakistan 2016

This humble effort is dedicated to My Loving Family And Respected Teachers Whose prayers and affections are the source of strength and sign of success for my future CONTENTS

TITLES Page No

CHAPTER: 1 INTRODUCTION

1.1 Medicinal Plants as a Source of Herbal Medicine 01 1.2 Medicinal Plants Diversity in Pakistan 02 1.3 Medicinal Plants Trade 03 1.4 Standardization of Herbal Medicines 06 1.5 Risk Assessment Approaches of Herbal Drugs 09 1.6 Authentication of Crude Herbal Drugs 12 1.7 Taxonomic Evaluation 13 1.8 Pharmacognostic Evaluation 13 1.9 Phytochemical Evaluation 14 1.10 Chromatographic Evaluation 15 1.11 Background Justification of the Present Project 15 1.12 Objectives 17

CHAPTER: 2 MATERIALS AND METHODS

2.1 Collection and Preservation of Plant Material 18 2.2 Taxonomic Evaluation 18 2.2.1 Morphological Description 18 2.2.2 Palynological Studies 20 2.2.3 Foliar Epidermal Anatomy (LM) 20 2.3 Pharmacognostic Evaluation 21 2.3.1 Organoleptic Evaluation 22 2.3.2 Fluorescence and Solubility Analysis 22 2.3.3 Physicochemical Evaluation 22 2.3.3.1 Determination of Moisture Content 23 2.3.3.2 Determination of Ash Values 23 2.4 Phytochemical Evaluation 24 2.5 High Pressure Liquid Chromatography (HPLC) 26 2.6 Antioxidant Activity 28 2.7 Documentation of Indigenous Knowledge of Medicinal Plants 29 2.8 Assessment of Herbal Markets 30 2.9 Annexure 1: Key to Morphological Study 31 2.10 Annexure 2: Key to Pollen Study (LM & SEM) 32

2.11 Annexure 3: Key to Foliar Epidermal Anatomy (LM) 33 2.12 Annexure 4: Questionnaire for Ethnobotanical Data Collection 34 2.13 Annexure 5: Questionnaire for Market Assessment of Herbal Drugs 35

CHAPTER: 3 RESULTS AND DISCUSSION

3.1 Authentication of Herbal Drug Tukhm-e-Kalonji (Nigella sativa L.) 40 3.2 Authentication of Herbal Drug Tukhm-e-balango (Lallemantia royleana Benth.) 59 3.3 Authentication of Herbal Drug Belladona ( Atropa acuminata Royle.) 78 3.4 Authentication of Herbal Drug Dhatura ( Datura stramonium L.) 96 3.5 Authentication of Herbal Drug Chiraita ( Swertia cordata G. Don) 114 3.6 Authentication of Herbal Drug Zafran ( Crocus sativus L.) 132 3.7 Authentication of Herbal Drug Resha khatmi ( Althaea officinalis L.) 150 3.8 Authentication of Herbal Drug (Picrorhiza kurroa Royle ex Benth.) 167 3.9 Conclusion 184 3.10 Recommendations 185

CHAPTER: 4 REFERENCES 186

LIST OF PLATES Plate # Title Page # Plate 1 & 2 Collection sites of problematic medicinal plants 36 Plate 3 & 4 Collection of medicinal plant in the field 36 Plate 5 & 6 Collection of traditional knowledge of medicinal plants 36 Plate 7 & 8 Observation of herbal drugs during market surveys 37 Plate 9 & 10 Data collection from herbalist and hakims 37 Plate 11 & 12 Light and Scanning electron microscopy (LM & SEM) 37 Plate 13 & 14 Powder sample for analysis and Desicator for constant weight 38 Plate 15 & 16 Determination of Moisture content and Ash analysis 38 Plate 17 & 18 HPLC and Spectrophotometer for phytochemical analysis 38

LIST OF TABLES Table # Title Page # Table 1 List of problematic medicinal plants of the study 19 Table 2 Authentication of herbal drug Tukhm-e-kalonji ( Nigella sativa L.) in 40 comparison with its adulterant Table 3 Comparative qualitative pollen morphology of Nigella sativa and Allium 43 cepa Table 4 Comparative quantitative pollen morphology of Nigella sativa and Allium 43 cepa Table 5 Comparative qualitative characters of foliar epidermal anatomy of Nigella 44 sativa and Allium cepa Table 6 Comparative quantitative characters of foliar epidermal anatomy of Nigella 44 sativa and Allium cepa Table 7 Fluorescence and solubility analysis of powdered drug of Nigella sativa 47 (Cold method) Table 8 Fluorescence and solubility analysis of powdered drug of Nigella sativa (Hot 48 method) Table 9 Fluorescence and solubility analysis of powdered drug of Allium cepa (Cold 49 method) Table 10 Fluorescence and solubility analysis of powdered drug of Allium cepa (Hot 50 method) Table 11 Physicochemical Parameters of Nigella sativa and Allium cepa 51 Table 12 Antioxidant activity of Ascorbic acid, Nigella sativa and Allium cepa 51 Table 13 Authentication of herbal drug Tukhm-e-balango ( Lallemantia royleana 59 Benth.) in comparison with its adulterant Table 14 Comparative qualitative pollen morphology of Lallemantia royleana and 62 Ocimum basilicum Table 15 Comparative quantitative pollen morphology of Lallemantia royleana and 62 Ocimum basilicum Table 16 Comparative qualitative characters of foliar epidermal anatomy of 63 Lallemantia royleana and Ocimum basilicum Table 17 Comparative quantitative characters of foliar epidermal anatomy of 63 Lallamentia royleana and Ocimum basilicum Table 18 Fluorescence and solubility analysis of powdered drug of Lallemantia 66 royleana (Cold method) Table 19 Fluorescence and solubility analysis of powdered drug of Lallemantia 67 royleana (Hot method) Table 20 Fluorescence and solubility analysis of powdered drug of Ocimum basilicum 68 (Cold method) Table 21 Fluorescence and solubility analysis of powdered drug of Ocimum basilicum 69 (Hot method) Table 22 Physicochemical Parameters of Lallemantia royleana and Ocimum basilicum 70 Table 23 Antioxidant activity of Ascorbic acid, Lallemantia royleana and Ocimum 70 basilicum Table 24 Authentication of herbal drug Belladona ( Atropa acuminata Royle.) in 78 comparison with its adulterant Table 25 Comparative qualitative pollen morphology of Atropa acuminata and 81 Solanum nigrum Table 26 Comparative quantitative pollen morphology of Atropa acuminata and 81 Solanum nigrum Table 27 Comparative qualitative characters of foliar epidermal anatomy of Atropa 82 acuminata and Solanum nigrum Table 28 Comparative quantitative characters of foliar epidermal anatomy of Atropa 82 acuminata and Solanum nigrum Table 29 Fluorescence and solubility analysis of powdered drug of Atropa acuminata 85 (Cold method) Table 30 Fluorescence and solubility analysis of powdered drug of Atropa acuminata 86 (Hot method) Table 31 Fluorescence and solubility analysis of powdered drug of Solanum nigrum 87 (Cold method) Table 32 Fluorescence and solubility analysis of powdered drug of Solanum nigrum 88 (Hot method) Table 33 Physicochemical Parameters of Atropa acuminata and Solanum nigrum 89 Table 34 Antioxidant activity of Ascorbic acid, Atropa acuminata and Solanum 89 nigrum Table 35 Authentication of herbal drug Dhatura ( Datura stramonium L.) in 96 comparison with its adulterant Table 36 Comparative qualitative pollen morphology of Datura stramonium and 99 Xanthium strumarium Table 37 Comparative quantitative pollen morphology of Datura stramonium and 99 Xanthium strumarium Table 38 Comparative qualitative characters of foliar epidermal anatomy of Datura 100 stramonium and Xanthium strumarium Table 39 Comparative quantitative characters of foliar epidermal anatomy of Datura 100 stramonium and Xanthium strumarium Table 40 Fluorescence and solubility analysis of powdered drug of Datura stramonium 103 (Cold method) Table 41 Fluorescence and solubility analysis of powdered drug of Datura stramonium 104 (Hot method) Table 42 Fluorescence and solubility analysis of powdered drug of Xanthium 105 strumarium (Cold method) Table 43 Fluorescence and solubility analysis of powdered drug of Xanthium 106 strumarium (Hot method) Table 44 Physicochemical Parameters of Datura stramonium and Xanthium 107 strumarium Table 45 Antioxidant activity of Ascorbic acid, Datura stramonium and Xanthium 107 strumarium Table 46 Authentication of herbal drug Chiraita ( Swertia cordata G. Don) and its 114 adulterant Table 47 Comparative qualitative pollen morphology of Swertia cordata and Swertia 117 paniculata Table 48 Comparative quantitative pollen morphology of Swertia cordata and Swertia 117 paniculata Table 49 Quantitative characters of foliar epidermal anatomy of Swertia cordata and 118 Swertia paniculata Table 50 Quantitative characters of foliar epidermal anatomy of Swertia cordata and 118 Swertia paniculata Table 51 Fluorescence and solubility analysis of powdered drug of Swertia cordata 121 (Cold method) Table 52 Fluorescence and solubility analysis of powdered drug of Swertia cordata 122 (Hot method) Table 53 Fluorescence and solubility analysis of powdered drug of Swertia paniculata 123 (Cold method) Table 54 Fluorescence and solubility analysis of powdered drug of Swertia paniculata 124 (Hot method) Table 55 Physicochemical Parameters of Swertia cordata and Swertia paniculata 125 Table 56 Antioxidant activity of Ascorbic acid, Swertia cordata and Swertia paniculata 125 Table 57 Authentication of herbal drug Zafran ( Crocus sativus L.) in comparison with 132 its adulterant Table 58 Comparative qualitative pollen morphology of Crocus sativus and 135 Carthamus tinctorius Table 59 Comparative quantitative pollen morphology of Crocus sativus and 135 Carthamus tinctorius Table 60 Comparative qualitative characters of foliar epidermal anatomy of Crocus 136 sativus and Carthamus tinctorius Table 61 Comparative quantitative characters of foliar epidermal anatomy of Crocus 136 sativus and Carthamus tinctorius Table 62 Fluorescence and solubility analysis of powdered drug of Crocus sativus 139 (Cold method) Table 63 Fluorescence and solubility analysis of powdered drug of Crocus sativus (Hot 140 method) Table 64 Fluorescence and solubility analysis of powdered drug of Carthamus 141 tinctorius (Cold method) Table 65 Fluorescence and solubility analysis of powdered drug of Carthamus 142 tinctorius (Hot method) Table 66 Physicochemical Parameters of Crocus sativus and Carthamus tinctorius 143 Table 67 Antioxidant activity of Ascorbic acid, Crocus sativus and Carthamus 143 tinctorius Table 68 Authentication of herbal drug Resha khatmi ( Althaea officinalis L.) in 150 comparison with its adulterant Table 69 Comparative qualitative pollen morphology of Althaea officinalis and 152 Hibiscus rosa-sinensis Table 70 Comparative quantitative pollen morphology of Althaea officinalis and 152 Hibiscus rosa-sinensis Table 71 Comparative qualitative characters of foliar epidermal anatomy of Althaea 153 officinalis and Hibiscus rosa-sinensis Table 72 Comparative quantitative characters of foliar epidermal anatomy of Althaea 153 officinalis and Hibiscus rosa-sinensis Table 73 Fluorescence and solubility analysis of powdered drug of Althaea officinalis 156 (Cold method) Table 74 Fluorescence and solubility analysis of powdered drug of Althaea officinalis 157 (Hot method) Table 75 Fluorescence and solubility analysis of powdered drug of Hibiscus rosa- 158 sinensis (Cold method) Table 76 Fluorescence and solubility analysis of powdered drug of Hibiscus rosa- 159 sinensis (Hot method) Table 77 Physicochemical Parameters of Althaea officinalis and Hibiscus rosa-sinensis 160 Table 78 Antioxidant activity of Ascorbic acid, Althaea officinalis and Hibiscus rosa- 160 sinensis Table 79 Authentication of herbal drug Kaur (Picrorhiza kurroa Royle ex Benth.) in 167 comparison with its adulterant Table 80 Comparative qualitative pollen morphology of Picrorhiza kurroa and Lagotis 169 cashmeriana Table 81 Comparative quantitative pollen morphology of Picrorhiza kurroa and 169 Lagotis cashmeriana Table 82 Comparative qualitative characters of foliar epidermal anatomy of 170 Picrorhiza kurroa and Lagotis cashmeriana Table 83 Comparative quantitative characters of foliar epidermal anatomy of 170 Picrorhiza kurroa and Lagotis cashmeriana Table 84 Fluorescence and solubility analysis of powdered drug of Picrorhiza kurroa 173 (Cold method) Table 85 Fluorescence and solubility analysis of powdered drug of Picrorhiza kurroa 174 (Hot method) Table 86 Fluorescence and solubility analysis of powdered drug of Lagotis 175 cashmeriana (Cold method) Table 87 Fluorescence and solubility analysis of powdered drug of Lagotis 176 cashmeriana (Hot method) Table 88 Physicochemical Parameters of Picrorhiza kurroa and Lagotis cashmeriana 177 Table 89 Antioxidant activity of Ascorbic acid, Picrorhiza kurroa and Lagotis 177 cashmeriana

LIST OF FIGURES Figure # Title Page # Figure 1 Calibration curves of Quercetin and Gallic acid 26 Figure 2 HPLC chromatogram of Quercetin Standard 27 Figure 3 Calibration curve of Ascorbic acid Standard 29 Figure 4 Marketing chain for trade of Medicinal plants in Pakistan 30 Figure 5 (a) Nigella sativa ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; 45 (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 6 (a) Allium cepa ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; (e) 46 Epidermal cells and stomata (LM-abaxial: 20X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 7 Total Phenolic and Flavonoid contents 52 Figure 8 DPPH radical scavenging activity (%) 52 Figure 9 HPLC chromatogram of Nigella sativa 53 Figure 10 HPLC chromatogram of Allium cepa 53 Figure 11 (a) Lallemantia royleana ; (b) Dried seeds; (c) SEM of pollen; (d) Exine 64 sculpturing; (e) Epidermal cells and trichome (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 12 (a) Ocimum basilicum ; (b) Dried seeds; (c) SEM of pollen; (d) Exine 65 sculpturing; (e) Epidermal cells and trichomes (LM-abaxial: 40X); (f) Stomata and trichome (LM-adaxial: 40X) Figure 13 Total Phenolic and Flavonoid contents 71 Figure 14 DPPH radical scavenging activity (%) 71 Figure 15 HPLC chromatogram of Lallemantia royleana 72 Figure 16 HPLC chromatogram of Ocimum basilicum 72 Figure 17 (a) Atropa acuminata ; (b) Dried leaves; (c) SEM of pollen; (d) Exine 83 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM-adaxial: 40X) Figure 18 (a) Solanum nigrum ; (b) Dried leaves; (c) SEM of pollen; (d) Exine 84 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 19 Total Phenolic and Flavonoid contents 90 Figure 20 DPPH radical scavenging activity (%) 90 Figure 21 HPLC chromatogram of Atropa acuminata 91 Figure 22 HPLC chromatogram of Solanum nigrum 91 Figure 23 (a) Datura stramonium ; (b) Dried leaves; (c) SEM of pollen; (d) Exine 101 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM-adaxial: 40X) Figure 24 (a) Xanthium strumarium ; (b) Dried leaves; (c) SEM of pollen; (d) Exine 102 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM-adaxial: 40X) Figure 25 Total Phenolic and Flavonoid contents 108 Figure 26 DPPH radical scavenging activity (%) 108 Figure 27 HPLC chromatogram of Datura stramonium 109 Figure 28 HPLC chromatogram of Xanthium strumarium 109 Figure 29 (a) Swertia cordata ; (b) Dried aerial parts; (c) SEM of pollen; (d) Exine 119 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 30 (a) Swertia paniculata ; (b) Dried aerial parts; (c) SEM of pollen; (d) Exine 120 sculpturing; (e) Epidermal cells (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X) Figure 31 Total Phenolic and Flavonoid contents 126 Figure 32 DPPH radical scavenging activity (%) 126 Figure 33 HPLC chromatogram of Swertia cordata 127 Figure 34 HPLC chromatogram of Swertia paniculata 127 Figure 35 (a) Crocus sativus ; (b) Dried styles and stigmas; (c) SEM of pollen; (d) 137 Exine sculpturing; (e) Epidermal cells (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X) Figure 36 (a) Carthamus tinctorius ; (b) Dried styles and stigmas; (c) SEM of pollen; 138 (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 37 Total Phenolic and Flavonoid contents 144 Figure 38 DPPH radical scavenging activity (%) 144 Figure 39 HPLC chromatogram of Crocus sativus 145 Figure 40 HPLC chromatogram of Carthamus tinctorius 145 Figure 41 (a) Althaea officinalis ; (b) Dried roots; (c) SEM of pollen; (d) Exine 154 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichomes (LM-adaxial: 40X) Figure 42 (a) Hibiscus rosa-sinensis ; (b) Dried roots; (c) SEM of pollen; (d) Exine 155 sculpturing; (e) Trichome and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X) Figure 43 Total Phenolic and Flavonoid contents 161 Figure 44 DPPH radical scavenging activity (%) 161 Figure 45 HPLC chromatogram of Althaea officinalis 162 Figure 46 HPLC chromatogram of Hibiscus rosa-sinensis 162 Figure 47 (a) Picrorhiza kurroa ; (b) Dried roots; (c) SEM of pollen; (d) Exine 171 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM-adaxial: 40X) Figure 48 (a) Lagotis cashmeriana ; (b) Dried roots; (c) SEM of pollen; (d) Exine 172 sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X) Figure 49 Total Phenolic and Flavonoid contents 178 Figure 50 DPPH radical scavenging activity (%) 178 Figure 51 HPLC chromatogram of Picrorhiza kurroa 179 Figure 52 HPLC chromatogram of Lagotis cashmeriana 179

LIST OF ABBREVATIONS

µg/mL Microgram/milliliter µL Microliter DPPH 2, 2-diphenyl-1-picrylhydrazyl DW Dry weight GA Gallic acid GAE Gallic acid equivalent gm Gram HPLC High pressure liquid chromatography LM Light microscopy ME Methanolic extract mg/g Milligram per gram mL Milliliter mM Millimole OD Optimal density QE Quercetin equivalent Rt Retention time SEM Scanning electron microscopy SI Stomatal index TF Total flavonoids TP Total phenolics UV Ultra violet WHO World Health Organization L x W Length x Width P/E ratio Polar equatorial diameter ratio

ACKNOWLEDGEMENT

I want to regard all my admirations and humble gratitude to Almighty ALLAH, Who blessed me with prospective ability and strength to complete this project and all respects are for His last Prophet “MUHAMMAD” (PBUH) who is an everlasting source of guidance and awareness for mankind.. I pay my humble gratitude to my supervisor Dr. Muhammad Zafar, Herbarium Botanist, Quaid-i-Azam University, Islamabad for his inspirational guidance, timely suggestions and encouragement throughout my studies. I would like to express my heartiest thanks to my co-supervisor Dr. Mushtaq Ahmad, Associate Professor, Quaid-i-Azam University, Islamabad for his valuable guidance, precious suggestion, keen interest, generous help, positive criticism, and timely correction of the present manuscript. Thanks are also extended to Dr. Mir Ajab Khan and Dr. Shazia Sultana for their fruitful suggestions. I offer my cordial and profound thanks Dr. Tariq Mehmood, Chairman, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, for providing all the possible research facilities during the research work. I gratefully acknowledge the Higher Education Commission (HEC) Pakistan, for providing financial support for this project under indigenous fellowship program. I offer my thanks to Prof. Dr. Muhammad Gulfraz and their lab researchers, PMAS Arid Agriculture University Rawalpindi for their cooperation and guidance during my research work. I extend sincere thanks to Prof. Dr. Mohammad Shahid, Head of Department, Material Engineering, National University of Science of Technology, Islamabad for access to Scanning Electron Microscopy (SEM). I am really thankful to Dr. Manzoor Hussain, Hazara University and Dr. Farooq Ahmad Lone, Associate Professor, SKUAST K, Srinagar for providing plant material for research work. I am thankful to Dr. Rizwana Aleem Qureshi and Hakeem Haroon Azam Niazi for giving kind suggestions and help during my research work. Special thanks are extended to my dear friends and lab fellows Abida Bano, Haleema Sadia, Sidra Nisar, Sadaf Kiayani, Mehwish Jamil, Tahira Bibi, Gulam Yaseen, Muhammad Pukhtoon Zada Khan, Zain ul Abideen and Niaz Tareen for their sincere cooperation and moral support. Words are inadequate to express my sincere thanks to my husband for his cooperation, motivation and patience during my research work. Last but not the least, I express my deepest gratitude to my dearest mother, sisters, brothers, father in law and whole family for their encouragement, affection and constant support.

SOFIA RASHID

ABSTRACT

This research work presents a detailed account of systematics and phytochemical studies of eight taxonomically problematic herbal drugs. The study aimed to authenticate the original sources of herbal drugs from their adulterants used in traditional medicines. These herbal drugs are traded in Pakistan under the name of Tukhm-e-kalonji (Nigella sativa L.), Tukhm-e- balango ( Lallemantia royleana Benth.), Belladona ( Atropa acuminata Royle.), Dhatura ( Datura stramonium L.), Chiraita ( Swertia cordata (G. Don) Clarke), Zafran ( Crocus sativus L.), Resha khatmi ( Althaea officinalis L.) and Kaur (Picrorhiza kurroa Royle ex Benth.). The study based on various techniques including morphological (qualitative & quantitative), palynological (LM & SEM), anatomical, pharmacognostic and chemical analysis. The data generated by using above mentioned techniques is useful to authenticate the original source of herbal drug Nigella sativa from its adulterant Allium cepa , Lallemantia royleana from Ocimum basilicum , Atropa acuminata from Solanum nigrum , Datura stramonium from Xanthium strumarium , Swertia cordata from Swertia paniculata , Crocus sativus from Carthamus tinctorius , Althaea officinalis from Hibiscus rosa-sinensis and Picrorhiza kurroa from Lagotis cashmeriana . It is stated that some specific diagnostic features such as fluorescence analysis is very useful in case of Nigella sativa , physicochemical analysis in case of Lallemantia royleana , antioxidant analysis in case of Atropa acuminata , foliar epidermal anatomy in case of Datura stramonium , morphological clarification in case of Swertia cordata , phytochemical characterization in case of Crocus sativus , organoleptic evaluation in case of Althaea officinalis and microscopic features of pollen (LM & SEM) in case of Picrorhiza kurroa . The findings of such projects are useful especially to develop monographs on herbal drugs and their commercial used adulterants. Furthermore, this study also recommends to use such classical parameters in addition to advance techniques in future to develop rules and laws for herbal drugs at national and global level as per WHO guidelines.

Introduction Chapter 1

1.1 MEDICINAL PLANTS AS A SOURCE OF HERBAL MEDICINE

Medicinal plants constitute an effective source of raw materials for both traditional (e.g. Ayurvedic, Unani, Chinese, Siddha and Homeopathy) and modern medicine. Nowadays, medicinal plants are employed throughout the developing and industrialized world as home remedies and ingredients for the pharmaceutical, nutraceutical and cosmaceutical industries. In recent era, medicinal plants have gained great consideration among the scientific societies. In spite of great advancements in the modern medicine, plants still make a significant contribution to health care (Calixto et al., 2000). Rising prices of synthetic drugs have enhanced the interest of use of medicinal plants as a re- emerging health aid and synthesis of new plants derived drugs (Hoareau and DaSilva, 1999). According to World Health Organization (WHO) about 80% population of developing countries relies on the local medicines derived exclusively from plants for primary health care and livelihood improvement (Calixto, 2005; WHO, 2002). Herbal drugs assumed commercial significance as medicines for the treatment of a wide range of diseases. In the Western world, people have started using plant based medications; this is due to the adverse side effects linked with synthetic drugs. Herbal drugs play a significant role in primary health care system of developing countries (Thomas et al., 2008). It is estimated that more than 10% of higher plants are used to cure various types of ailments (Shinwari, 2010).

Medicinal plants are widely used as alternative therapeutic tools for the treatment of many health problems and diseases throughout the world. In recent years, demand for plant derived drugs is increasing rapidly in developed countries. A number of phytomedicines are extracted from different parts of plants such as barks, leaves, roots, flowers, seeds and fruits (Cragg and Newman, 2001). Different phytochemical components present in plants such as carbohydrates, tannins, alkaloids, terpenoids, steroids, phenolic compounds and flavonoids are responsible for various pharmacological activities of plants (Shah

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 1 Introduction Chapter 1

et al., 2011; Zaman et al., 2012; Abbas et al., 2012). For the treatment of diseases and maintenance of public health the usage of medicinal plants is highly prevalent in many countries and cultures of different nations (Asgary et al., 2014; Bahmani et al., 2014). Nowadays, phytochemicals found in medicinal plants are getting more attention than ever among the food industries as these compounds prevent microbial deterioration, retard the oxidative lipids degradation, improve the food quality and nutritional value (Osorio et al., 2010; López et al., 2005). Several natural bioactive compounds are found in medicinal plants that are responsible for considerable antioxidant and other activities (Kaur et al., 2011). Thus, medicinal plants have become a focal point to improve the present and future health care needs.

1.2 MEDICINAL PLANTS DIVERSITY IN PAKISTAN

Pakistan has rich diversity of medicinal plants due to its unique geography and wide range of ecological zones (Bano et al., 2014). Local community of Pakistan living in rural areas frequently practiced herbal remedies due to lack of modern healthcare facilities. Among such areas, plants are most appropriate solution for many of the health problems (Jamal et al., 2012). About 6000 species of flowering plants are found in Pakistan. Out of which about 600-700 species are used extensively in traditional health care system (Shinwari, 2010). About 12% flora of country is used for medicinal purposes and a number of medicinal plants are being exported (Shinwari and Qaiser, 2011). According to Hussain et al. (2009), Pakistan is one of the leading countries exporting the medicinal plants. Huge herbal drug (Pansara) market system is available in country that relies on wild medicinal plant. Medicinal herbs are used for the treatment of human as well as animal ailments. Besides, hakims and the rural area dweller use the medicinal herbs on their own knowledge or acquired through their ancestors. Hundreds of medicinal plants are collected from wild habitats and traded to international market (Olsen and Bhattarai, 2005; Sher et al., 2012). The harvest and sale of these medicinal plants provide source of income to a number of rural households

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 2 Introduction Chapter 1

(Ali et al., 2012). About 900 monoherbal and 500 polyherbal preparations of medicinal plants has been listed in Pakistani Tibbi Pharamacopoeia. In Pakistan 39,584 hakim, 130,000 homoeopaths and 455 vaids are registered and about 457 clinics and Tibbi dispensaries offer medication to the local people. There are around 300-350 herbal manufacturing companies and about 300 homeopathic medicines manufacturing companies. Approximately, 25% of all medicinal prescription comes from plants or plant derived synthetic analogs (Vitalini et al., 2009). About 60 % of the population is served by traditional healers, especially those living in the rural areas (Haq, 1983). People who collect the plants from the wild are mostly untrained and use their inherited knowledge for identification of medicinal plants. In many cases morphologically similar species are collected and sold under the same local name at the drug stores. This intentional or unintentional adulteration of medicinal plants does not provide desire results. Therefore it is very important that medicinal plant must be correctly identified and should not be confused with other morphologically similar species. Unfortunately, with the passage of time the number of taxonomists and herbaria is decreasing rapidly all over the world as well as in Pakistan (Shinwari and Qaiser, 2011). In past medicinal plants have been used to extract a number of drugs which as compare to synthetic drugs have less side effects. Interest in herbal medicines has increased enormously over the last few decades. Now medicinal plants have become a target for the search by the multinational drugs companies and research institutes for new drugs.

1.3 MEDICINAL PLANTS TRADE

Traded herbal medicines and their production are gaining significant importance in almost all the countries. World Health Organization reported that about 70-80% of the world populace use folk medicines for their primary health care due to poverty and lack of access to modern health facilities (Calixto, 2005). Since 1978 World Health Organization is showing considerable interest to traditional medicine (WHO, 1978), with the new WHO traditional medicine

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 3 Introduction Chapter 1

strategy 2014-2023 (WHO, 2013). WHO’s strategy strongly emphasizes on development of traditional medicines, instead of a focus on traditional medicinal products. Herbal markets are necessary to describe the importance of medicinal plants for country’s inhabitant. These markets provide a picture of a country’s medicinal flora and reflect distinct cultural preferences (Schippmann et al., 2006; Van-Andel et al., 2012). In addition, medicinal plants are harvested, transported and traded in local markets, generating economic opportunities for vulnerable groups, especially women and farmers facing less agriculture income (Vodouhé et al., 2008). Approximately 50-75 thousand plants are used in traditional and modern medicines worldwide (Schippmann et al., 2006).

Plants are significant source of medicines and presently about 25% of pharmaceutical prescriptions in the United States contain at least one plant– derived ingredient (Verma and Singh, 2008). In the last century, about 121 pharmaceutical products were formulated on the basis of folk knowledge gathered from different sources (Samy and Gopalkrishnakone, 2007). WHO survey reported that local healers treat 90% patients in Bangladesh, 85% in Burma, 80 % in India, 75% in Nepal, 66% in Pakistan, 65% in Sri Lank, 60% in Indonesia (Ullah and Hussain, 2007).

Herbal medicines are lucrative globally and they represent a market value of about US$ 43 billion a year (Christie, 2001). The global market for herbal medicines currently stands at over $ 60 billion annually. The scale of herbal medicines is expected to get higher at 6.4% an average annual growth rate (Inamda et al., 2008). Europe alone annually imports about US$ 1 billion in medicinal plants from Africa and Asia (Sher and Hussain, 2009). It is estimated that by 2050 the global medicinal plants business will reach upto $ 5 trillion (US) (Shinwari, 2010). Estimates suggest that ayurvedic medicines market is expanding at 20% annually. In India, nearly 25% sales of medicinal plants have grown during 1987-96, which is the world highest growth rate (Masood, 1997). China and India are two largest users of medicinal plants. About 5000 plant

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species are used in Traditional Chinese Medicines; India uses about 7000 plant species. But in developed countries with the passage of time the percentage of population depending on traditional medicine decreased i.e. in Germany 40-50%, in the USA 42%, in Australia 48% and in France 49% (Titz, 2004). Disappearing of medicinal plant in the wild and unsustainable collection methods are basic reasons for this decrease. During 1991-2003 about 0.467 m tons, medicinal plants worth US $1.2 B were traded annually at global scale (Lange, 2006). China is at the top, regarding the medicinal plants markets, followed by France, Italy, Germany, Spain, Japan, the UK and the USA. Based on consumption of botanical medicines in the world, Japan is leading among the per capita (Laird, 1999). In 1999, the herbal medicines market was US $ 19.4 billion, Europe was at the top (US $ 6.7 billion) followed by Asia (US $ 5.1 billion), North America (US $ 4.0 billion), Japan (US $ 2.2 billion) and the rest of the world (US $ 1.4 billion) (Laird and Pierce, 2002). In 2002, the sale of herbal drugs was approximately US $ 60 billion globally (Anonymous, 2003). During 1991-2003 based on average import trade volume among the top 12 countries, Pakistan is at 9th position with 10.65 thousand tons, worth US $9.8138 m (Lange, 2006).

According to the Population Census Organization of Pakistan (2010), population of Pakistan is 16.98 million with a growth rate of 2.69. Besides this health awareness in people is also increasing day–by-day (Aamir and Zaman, 2011). Pakistan does not have sufficient herbal materials to meet its growing requirements and has to import major part from China, Nepal, Sri Lanka, Iran, India, Kenya and Uganda. About 600 plant species are used as medicines, out of which only 300 were available in the local markets (Shinwari et al., 2002). In the year 2001-2002 Pakistan spent Rs. 13988 million and 1040 million on import of pharmaceutical products and species respectively (Anonymous, 2002a). To estimate the annual production, consumption and export of crude drugs found naturally or cultivated, a survey of different pansara markets of country (i.e., Peshawar, Mangora, Dir, Rawalpindi, Faisalabad, Lahore, Sukkhar, Multan, Hyderabad and Karachi) was conducted during 1980-1983. Information revealed

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that a worth 119.5 million rupees is consumed by trade of crude drug in the country. A directory about the traded medicinal plants was prepared for medicinal plant collector, cultivators, dwakhanas, pharmaceuticals industries and the scientists working on medicinal plants (Khan, 1985). Information on consumption of crude drugs by manufacturing laboratories, pharmaceutical industries and (pansara) markets of Pakistan and average price lists for several hundred traditional medicines has been provided by Saeed (1997).

1.4 STANDARDIZATION OF HERBAL MEDICINES

Since times immemorial, herbal plants have been used as a remedy for a number of diseases because they have therapeutically active compounds (Cohen, 2002). Physicians significantly prescribe plant derived drugs (Cowan, 1999). Due to the presence of remedial natural products having no side effects demand of medicinal plants is increasing (WHO, 2001), these natural products as a crude drug can be included in herbal pharmacopoeia after establishment their pharmacological standards (Mahendra and Vaikos, 2009). Fan et al. (2012) presented a extensive overview of different global regulations for Chinese herbal medicines, demonstrating that different global regulations applicable to traditional medicines in the European Union, China, Japan, South Korea, Taiwan, Russian Federation, Australia, Canada, United States and in Brazil can be divergent, have different approaches and are following classifications, which at first sight are difficult to harmonized. Nevertheless a global approach towards regulations of traditional medicines seems to be the preferable way, not at least because these products already are used all over the world.

There is a renewed interest in natural drugs because they are considered as green medicine and green medicines are always safe, but the major problem with the green medicines is adulteration. Standardization may define as a process of describing a set of standards or constant parameters, definitive qualitative and quantitative values and inherent characteristics to ensure the quality, safety,

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efficacy and reproducibility of herbal drugs. Process involves the developing of technical standards and agreeing upon them. Standardization is a basic tool in the quality assurance process. To describe a set of constant parameters exhibited by the particular herbal drug, definite standards are worked out by experimentations and observations. In order to justify the acceptability of crude herbal drugs and their compound preparations in modern system of medicine; quality assurance is of considerable importance. But the major problem of herbal industry is the lack of rigid quality assurance profile for herbal drugs and their preparations. The benefit of traditional medicines with respect to quality, efficacy and safety could outcome in a healthier use of these alternative systems of medicine.

Globally, several pharmacopoeias have given monographs describing standards and parameters of many medicinal herbs and their products. Different pharmacopoeias are: • Pharmacopoeia Committee • Chinese Herbal Pharmacopoeia • The Ayurvedic Pharmacopoeia of India (API) • United States Herbal Pharmacopoeia • Japanese Standards for Herbal Medicine • British Herbal Pharmacopoeia • British Herbal Compendium

These Pharmacopeias provided monograph for medicinal herbs and their formulation to maintain their quality and efficacy. Ayurvedic Pharmacopoeia of India (API) provides basic quality parameters and standards for eighty common Ayurvedic herbal drugs (Verma and Singh, 2008). As the demand of natural drug is correlated to its effectiveness, so the more effective the natural drug more is its demand and the chances of its non-availability also increases. In order to meet the growing demand, the natural drug is mostly adulterated with inferior quality material. In the global distribution of herbal products, adulteration or mixing of raw material is one of the major problems (Khatoon et al., 1993; Dubey et al.,

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2004). Adulteration or substitution is intentionally or unintentionally replacement of genuine plant material with another plant material or mixing any foreign matter to enhance the weight or potency of the product or to decrease its cost. Therefore a proper procedure for the authentication of crude herbal drugs is needed to maintain their safety, efficacy and quality. Quality and quantity of chemical constituents are major factors on which the therapeutic efficacy of medicinal plants depends. Wrong identification of natural products or herbal medicines is the basis of misuse of herbal medicine. Identification, evaluation and standardization of crude herbal drug is based on macroscopical and microscopical features. To check the adulteration of herbal drugs, traditional methods of authentication depend greatly on morphological/anatomical description, organoleptic markers (color, odor, texture etc.) and chemical testing (Shaw et al., 2002). Chemical or biochemical methods are used for detection of adulteration in traded raw materials and authentication of food and agricultural commodities such as high performance liquid chromatography (HPLC) have also proven useful for the detection of adulterants and component identification in traded commodities of plant origin (Mandl et al., 1999; Wenzl et al., 2002; Kurz et al., 2008). These modern techniques are of extensive value to detect adulterant in certain instances but for routine sample analysis they are not convenient. Likewise, for detection of adulterants such as phytochemicals from unwanted plant material or synthetic drugs, chemical profiling is very useful (Joshi et al., 2005a). Phytochemical profiles of plant material depend on processing of plant parts or specific environmental conditions required for plant growth.

Currently, the World Health Organization (WHO) is emphasizing over the use of indigenous medicinal plants and alternative systems of traditional herbal medicines. In current scenario, the revival of immense interest in indigenous systems of medicine has been initiated all over the world. WHO encourages using traditional herbal medicines in national health care system as the traditional drugs are generally nontoxic, easily affordable, effective at low concentrations and environment friendly. WHO also emphasized much more the need to authenticate

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the quality and purity of traditional medicines by applying recent modern techniques, using suitable instruments and standards (WHO, 2002). It has issued specific guidelines for the evaluation of herbal medicines (WHO, 1996). These guidelines provide basic criteria for the assessment of quality, safety and efficacy of herbal medicines with the goal of assisting national regulatory authorities and manufacturers in assessing documentation, submission and dossiers in respects of such products. These guidelines emphasized the need for assessment of efficacy including the determination of pharmacological and clinical effects of the active ingredients and labeling which includes a quantitative list of active ingredient(s), dosage and concentrations.

1.5 RISK ASSESSMENT APPROACH OF HERBAL DRUGS

Misidentification: Crude medicinal plants and their parts are often adulterated or substituted in herbal market due to their improper identification by the herbal plant sellers and consumers, due to some morphological similarities of the plant parts and due to lack of a standard identification system, which may alter their efficacy and safety to toxicity. For the safe use of herbal drugs and products, the correct identification is very necessary. Without proper identification, the efficacy and safety of quality products cannot be achieved. Correct identification and detection of adverse effects of herbal ingredients is a difficult practice, as four different ways are used for plant naming-- the common English name, the Local name, the Scientific name and the Pharmaceutical name (But et al., 1993). It is necessary that binomial Latin names for genus and species should be used for plants naming; mostly, usage of other names leads to the misidentification problems. Misidentification of plant material can occur when wild plants are picked or at the time of the manufacturer's bulk purchase. There is a need at industry and government level to protect access and selection by consumers. At the same time, regarding the purity, safety and efficacy of traded herbal drugs consumers have a right to expect that these drugs can be used with confidence without having toxic effects (Ahmad et al., 2009).

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Lack of standardization: Herbal drugs describe plants or parts of plants converted into phytomedicines by simple processes including harvesting, drying, and storage (EMEA, 2002). All medicines should follow the basic criteria of being pure, safe and effective, whether they are of plant origin or synthetic (EMEA, 2005; WHO, 2005). Depending on the plant part used, geographic region, ripening stage and storage conditions of medicinal plants, their therapeutic and toxic components vary. Presently, for herbal drugs and formulations, no official standards and parameters are available. Currently, some companies or manufacturers are doing testing for their herbal drugs and preparations, have their own parameters many of which are very preliminary in nature. Presences of all the ingredients in a formulation as claimed are very difficult to identify. Hence it is need of time to evolve standards and parameters to identify the presence of the entire ingredients. To identify the presence of different ingredients in herbal drugs different spectrophotometric/ chromatographic methods and physicochemical properties can be used. For quantitative evaluation of bioactive compounds like alkaloids, polyphenolic components, flavonoids or evaluation of particular compound these methods can be applied (Wani, 2007)

Substitution: A drug is considered to be a substitute that exhibits similar therapeutic effects as that of genuine drug. The herbal drugs are mainly substituted due to non-availability of the drug, doubtful identity of the drug, cost of the drug and the adverse reaction of the drug. Substitution may be with totally different drug, different species, species belonging to same family and different parts of the plant.

Adulteration: It is a practice of intentionally or unintentionally replacement of genuine drug with other substances partially or fully which is either free from or low in therapeutic and chemical properties or addition of spoiled, inferior quality or entirely different drug having morphological resemblance to that of genuine drug with a purpose of enhancement of income (Mukherjee, 2002; Kokate et al., 2007). Adulteration may also expressed as the substituting or mixing the genuine

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drug with inferior, spurious, spoiled, defective, ineffective parts of same or different plant or harmful drugs or substances which are not confirmed by the official standards and parameters. A drug is considered to be adulterated if it contains, in whole or in part, of any filthy, putrid or decomposed substance (Anonymous, 2003).

Incorrect preparation/dosage: The processing of crude herbal material is carried out by a manufacturer. A major determinant of the pharmacological activity of the finished product is the patient. The source, purity and quality of crude material, high-quality agricultural practices and preparation processes are very crucial steps for the quality assurance of herbal drugs and play an important role in guaranteeing the quality and stability of herbal formulations (Roberts and Tyler, 1999; Blumenthal et al., 1998). Another important point to consider is that the activity of crude plant material may vary from that of the purified constituents of genuine drug, as some constituents may change the toxicity of others (De Smet, 1997).

Inappropriate labeling/advertising: The main source of information for consumers about herbal products is the product label. Presently, no government body or organization is available that certifies the correct labeling of an herb or a supplement. It is common practice that herbal products labels cannot be belief to reveal what is actually in the jar. Herbal products investigation have confirmed that consumers are actually getting less than a 50% probability of what is mentioned on the product label. Considerable differences between what is mentioned on the product label and what is in the container have been found through published analysis of herbal supplements. Mostly, consumers decide by themselves what is safe and effective for them and sometimes they are frustrated due to lack of consistent labeling on herbal products. Certain important information such as pharmacopoeia standards followed for manufacturing of products, active ingredients, serving quantity (dosage), and directions for use of drugs must be included on the product labels of all herbal drugs and pac

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1.6 AUTHENTICATION OF CRUDE HERBAL DRUGS

Authentication and quality assurance for safety and efficacy of herbal drugs is of great importance (WHO, 1999). Quality is a status of a drug determined by its identity, purity and by its physical, chemical or biological characterizations or by the manufacturing processes. Quality assurance is the processes involved in purity, quality and validity maintenance of a manufactured drug. For the quality control of herbal medicines the traditional information and the documents about the identity, quality and purity are interpreted in terms of modern assessment. All the medicines, whether they are of plant origin or synthetic should accomplish the basic criteria of being safe and efficacious. This criteria applies both to the multinational pharmaceutical company conducting a study with herbal extracts and to the local herbalist in a rural area who recommends a locally made herbal preparation. The process of standardization and quality assurance of herbal drugs is based on physicochemical evaluation of crude herbal drug covering aspects, such as proper identity, purity of crude material, efficacy, safety and stability assessment of end product (WHO, 1996a; 1996b).

Any herbal medicine must be botanically authenticated as well as chemically and biologically standardized. Currently, many analysis are employed for authentication and quality evaluation of crude herbal drugs. Most common are: Taxonomic evaluation Pharmacognostic evaluation Phytochemical evaluation Chromatographic evaluation

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1.7 TAXONOMIC EVALUATION

Taxonomic evaluation includes morphological, palynological and foliar epidermal anatomical features of herbal drugs. The morphological characters play an important role to delimit closely related taxa by variations in habit, habitat, vegetative and floral morphology (Shaheen et al ., 2014). Proper identification and differentiation of medicinal plants could not be achieved due to similarities in their morphological characters. In such cases palynological and leaf anatomical features could be utilized for evaluation of medicinal plants (Gilani et al., 2002). In order to delimit the closely related taxa shape of pollen is one of the significant parameter (Perveen, 2006). For authentication of marketed floral parts used as herbal drugs, scanning electron microscopy of pollen grains is a very useful tool (Zafar et al., 2011; Khan et al., 2011). Similarly, foliar epidermal features such as types of stomata, epidermal cells and trichomes play a significant role to delimit the taxa of many plant families (Hameed et al., 2010). Microscopic evaluation allows the detailed and quantitative examination of entire drug or in powder forms with the help of microscope (Kokate et al., 2005; Ansari, 2006). For qualitative microscopic evaluation, shape of epidermal cell, stomata types, trichome types, pollen types and pollen sculpturing are the commonly used features. Quantitative microscopic evaluation includes size, length, width and diameter of epidermal cells and subsidiary cells, size, length, width of trichomes, stomata number, stomatal index, polar diameter, equatorial diameter, exine thickness, length and width of spines, colpi and P/E ratio.

1.8 PHARMACOGNOSTIC EVALUATION

Pharmacognostic evaluation includes various tests that would be determined for crude herbal drugs; these include organoleptography, fluorescence analysis and physicochemical analysis. For correct identification and detection of adulterants, the crude herbal drugs are subjected to macroscopic examination which comprised of organoleptic characters of the crude drug viz., color, odor,

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taste, size, appearance, texture, fracture etc. Organoleptic evaluation means assessment of crude drug by using various sense organs (skin, tongue, eye, nose and ear). It is also called macroscopical evaluation. It is simplest technique of qualitative assessment based on morphological characterization and sensory profile of crude herbal drugs, commonly practiced by the health practitioners, herbalists and local herb sellers.

For pharmacognostic evaluation of crude drugs, fluorescence analysis is an important parameter. Crude drugs are often identified from their adulterants and assessed qualitatively on basis of fluorescence study (Zhao et al., 2011). Physicochemical analysis includes moisture contents, total ash, acid insoluble ash, water soluble ash, moisture contents, water soluble extractive and alcohol soluble extractive. Ash value measures the purity and quality of the crude drug; it determines the amount of various contaminations like silicate and carbonate in the crude drug. The amount of inorganic compounds present in crude drug is estimated through water soluble ash and amount of silica particularly in the form of sand and siliceous earth is determined by acid insoluble ash. To inhibit the growth of bacteria, fungi or yeast during the storage process, the moisture content of herbal drugs should be at minimal level. Moisture content is stricken by the environmental humidity and may affect the quality of the herbal drugs during storage.

1.9 PHYTOCHEMICAL EVALUATION

Most of drugs have specific pharmacological and biological activities which are employed for their evaluation. Specific types of chemical constituents found in the plant extracts are responsible for such activities. Qualitative and quantitative characterization of the chemical constituents should be analyzed for quality and purity assessment of herbal drugs. Strength and potency of drug in its formulation can be evaluated with the help of bioassays (Williamson et al., 1996; Kokate et al., 2005; Ansari, 2006).

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1.10 CHROMATOGRAPHIC EVALUATION

High Performance Liquid Chromatography (HPLC) is a technique used to identify, separate, purify and quantify the individual components of the mixture, based on differential passage of the compounds through the column (solid stationary phase). In the case of herbal drugs, the separation mechanisms are based on chemically modified silica as the stationary phase and polar solvents as the mobile phase. Almost all the compounds present in the herbal medicines can be analyzed by HPLC. The main goal of the quantitative analytical evaluation is to offer validated methods used to quantify the compounds mostly linked with pharmacological activities of drugs (Wani, 2007).

1.11 BACKGROUND JUSTIFICATION OF THE PRESENT PROJECT

Pakistan has a rich floral diversity because of its distinctive geography with the Karakorum, Hindu-kush and Himalayas (Shinwari and Qaiser, 2011). For the treatment of various ailments, people in the mountainous areas of Pakistan still rely on medicinal plants and for long time they have been utilizing the plants for food, health, shelter, fuel and other purposes (Alam et al., 2011). There are huge economic benefits in the use and commercialization of traditional medicinal plants for the treatment of various ailments (Azaizeh et al., 2003). Traditional medicines mainly employ the use of plant parts either in crude, fresh or dried form. It is very difficult to identify the parts of herbal medicines sold in the market in dried form, as during drying several useful diagnostic characteristics are lost. Herbal drug industry use medicinal plants, majority of which are collected from wild (Handa, 2004). The crude medicinal plants and their parts are often adulterated or substituted in market because of improper identification by the consumers and herbal plant sellers caused by some morphological similarities of the plant parts and lack of a standard identification system. The major problem in herbal drug industry is due to confusion and controversy of certain synonyms employed for more than one or two drugs. Sometimes under the same common or

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local name entirely different taxa are being sold in herbal market of India and Pakistan (Ahmad et al., 2010a; Khan et al., 2011). Correct and proper identification of medicinal plants is very crucial to ensure the safety and efficacy of herbal medicines, as many medicinal plants are intentionally or unintentionally adulterated with similar species or varieties (Kiran et al., 2010). In standardization of herbal drugs adulteration is the main hindrance (Shinde et al., 2009).

Unfortunately, in Pakistan little attention had been paid on standardization and quality control of medicinal plants used as a source of raw material for herbal drugs. Based on taxonomic techniques, few efforts have been made on authentication of traded herbal drugs after 1998 (Khan et al., 2002). Nasreen (1998) used morphology and micromorphology of pollen techniques for the correct identification and detection of adulterant in market samples of 16 different genera of medicinal plants. Based on morphological characterization, Akram (2004) reported 08 Unani crude herbal drugs to differentiate the genuine drug from their adulterant. Ahmad (2008) reported 10 cases of problematic traditional medicines based on multidimensional authentication techniques. Zafar (2011) reported chemotaxonomic authentication of 07 cases of problematic medicinal plants. Sultana (2012) used taxonomic and pharmacognostics parameters for differentiation of 07 cases of medicinal plants in comparison with their adulterants.

Present study is the continuation of the previously conducted studies on authentication of problematic medicinal plants in Pakistan. Present study deals with the authentication of medicinal plants traded as herbal drugs by using systematics and phytochemical characterization. 8 cases of problematic medicinal plants were selected during the field surveys of different herbal markets of Pakistan (Table 1). While selecting the problematic medicinal plants many herbalist and traditional health practitioners were also consulted. In the study, the selected problematic medicinal plants for differentiation of genuine drug from its adulterated, substituted or unpurified material are:

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i. Tukhm-e-Kalonji ( Nigella sativa L. by Allium cepa L.) ii. Tukhm-e-balango ( Lallemantia royleana Benth. by Ocimum basilicum L.) iii. Belladona ( Atropa acuminata Royle. by Solanum nigrum L.) iv. Datura ( Datura stramonium L. by Xanthium strumarium L.) v. Chiraita ( Swertia cordata (G. Don) Clarke by Swertia paniculata Wall.) vi. Zafran ( Crocus sativus L. by Carthamus tinctorius L.) vii. Resha khatmi ( Althaea officinalis L. by Hibiscus rosa-sinensis L.) viii. Kaur (Picrorhiza kurroa Royle ex Benth. by Lagotis cashmeriana (Royle) Rupr.)

1.12 OBJECTIVES

• To identity the original source of traded medicinal plants and solve the adulteration issues using systematics and phytochemical characterization. • To use morphological, palynological and anatomical characterizations as taxonomic markers for authentication of genuine herbal drugs and their adulterants. • To evaluate the pharmacognostic properties including organoleptography, fluorescence analysis and physicochemical parameters for rapid, easy and correct identification of herbal drugs. • To carry out phytochemical investigation, HPLC analysis and antioxidant potential of selected herbal drugs. • To document the indigenous knowledge of selected medicinal plants for treatment of various ailments.

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Materials and Methods Chapter 2

2.1 COLLECTION AND PRESERVATION OF PLANT MATERIAL

The study was conducted to authenticate the medicinal plants traded as herbal drugs by using systematics and phytochemical characterization in Plant Systematics and Biodiversity Lab, Department of Plant Sciences, Quaid-i-Azam University Islamabad. Various field trips were arranged to collect the fresh plant material from resource based areas of the country (Plate 1 & 2). Seeds of medicinal plants were purchased from different herbal shops. To ensure the thorough sampling of plant material, plants were collected in both blooming and fruiting conditions during the field surveys in different seasons. Plant material was collected in sufficient amount to perform different analysis. The plants specimens were properly dried, pressed, labeled and mounted on herbarium sheets. Standard taxonomic methods were followed for the authentication of collected plant species, based on floral morphological characters, analytical keys and using available herbarium samples for comparison. For correct identification of medicinal plants field photographs were taken by using digital camera (Sony DSC- W510). The collected plant specimens were deposited to the Herbarium of Pakistan (ISL), Quaid-i-Azam University Islamabad for future record.

2.2 TAXONOMIC EVALUATION

2.2.1 MORPHOLOGICAL DESCRIPTION

Both herbarium and fresh specimens were used to investigate the qualitative and quantitative morphological characteristics. 5 specimens of each species were studied under the binocular light microscope (Bausch and Lomb model W, New York). For detailed observations, different magnifiers of 10X, 20X and 40X were used. Study was based on macro and microscopic features of plant, habit, root, stem, leaf, flower and seed (Annexure 1) by using a ruler and binocular stereo zoom light microscope (SZF Kyowa, Japan). The morphological characteristics were further confirmed from Flora of Pakistan (Nasir and Ali,

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1970-1989; Ali and Qaiser, 1993-2007). Plant names and their citations were verified from International Plant Names Index.

Table 1. List of problematic medicinal plants of the study S. # Species Names/ Voucher # Family Collector’s Name

01 Nigella sativa L. Ranunculaceae Mushtaq Ahmad ISL-SR-201 M. Zafar Sofia Rashid 02 Allium cepa L. Alliaceae M. Zafar ISL- SR-202 Mir Ajab Khan Sofia Rashid 03 Lallemantia royleana Benth. Lamiaceae Tahira Tareen ISL-SR-203 M. Zafar Sofia Rashid 04 Ocimum basilicum L. Lamiaceae M. Zafar ISL- SR-204 Sofia Rashid 05 Atropa acuminata Royle. Solanaceae Manzoor Hussain ISL-SR-205 Sadaf Kiyani Sofia Rashid 06 Solanum nigrum L. Solanaceae Haleema Sadia ISL-SR-206 Sofia Rashid 07 Datura stramonium L. Solanaceae Mushtaq Ahmad ISL-SR-207 Sadaf Kiyani Sofia Rashid 08 Xanthium strumarium L. Asteraceae Shaukat Hussain ISL-SR-208 Sofia Rashid 09 Crocus sativus L. Iridaceae Farooq Ahmad ISL-SR-209 Lone Sofia Rashid 10 Carthamus tinctorius L. Asteraceae Mushtaq Ahmad ISL- SR-210 Sofia Rashid 11 Swertia cordata (G. Don) Clarke Gentianaceae M. Zafar ISL- SR-211 Pukhtoon Zada Sofia Rashid 12 Swertia paniculata Wall. Gentianaceae Mushtaq Ahmad ISL- SR-212 M. Zafar Sofia Rashid 13 Althaea officinalis L. Malvaceae Mushtaq Ahmad ISL-SR-213 M. Zafar Sofia Rashid 14 Hibiscus rosa-sinensis L. Malvaceae M. Zafar ISL-SR-214 Mushtaq Ahmad Sofia Rashid 15 Picrorhiza kurroa Royle ex Benth. Scrophulariaceae Mushtaq Ahmad ISL-SR-215 M. Zafar Sofia Rashid 16 Lagotis cashmeriana (Royle) Rupr. Scrophulariaceae Abida Bano ISL-SR-216 Sofia Rashid

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2.2.2 PALYNOLOGICAL STUDIES a) Light Microscopy (LM)

For palynological studies (LM), pollen samples were acetolysed by following the standard procedure (Erdtman, 1960). Pollen grains were mounted in glycerin jelly and after covering with cover slips the slides were permanently sealed with transparent nail varnish. Morphological observations and measurements were made using the light microscope (Meiji Techno MX5200H). The measurements were based on 5 to 7 readings from each specimen. The qualitative and quantitative pollen features studies are presented in Annexure 2. Microphotographs of the pollen grains were taken with digital camera (Infinity 1- 5 C-MEI, Canada) fixed to the Leica light microscope (DM 1000) (Plate 11). Terminologies for pollen morphology were described according to Barthlott (1984), Erdtman (1960) and Ronald (2000). b) Scanning Electron Microscopy (SEM)

For scanning electron microscopy (SEM), the pollen were dehydrated in a drop of ethanol (96%) and dissected using a needle. These pollen grains were subjected to acetolysis following the procedure described by Avetissian (1950). The acetolysed pollen grains were dried at room temperature and affixed to metal or aluminium stubs with cellophane tape. By using a gold sputter (JFC-1500), the stubs were coated with thin layer of gold. Then these specimens were examined using analytical low vacuum scanning electron microscope (JSM-6490LA) and SEM images were taken (Plate 12).

2.2.3 FOLIAR EPIDERMAL ANATOMY (LM)

For foliar epidermal anatomy, Shultze’s technique of maceration with some modifications was followed ( Subrahmanyam, 1996; Zafar et al., 2011).

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Leaves were kept in a test tube containing 4mL of 30% nitric acid, 1mL of distilled water and 2gm of potassium chloride. This mixture was boiled carefully for few seconds. When thin layer of epidermis was separated, the contents were poured in a petri plate partly filled with water and washed with distill water twice. Epidermis of both adaxial and abaxial surface was peeled off with the help of fine forceps and kept in 60% potassium hydroxide solution for 1 to 2 hours. Finally, epidermal peelings were suspended in 1-2 drops of 88% lactic acid on glass slide and sealed with transparent nail varnish after covering with cover slip. The epidermal peelings were prepared for qualitative and quantitative features of adaxial and abaxial surfaces by using light microscope (Meiji Techno MX5200H). Important foliar epidermal features studied are presented in Annexure 3. Leica light microscope (DM 1000) fitted with CCD digital camera (DK 5000) was used for microphotographs. Terminologies described by Prat (1932) and Metcalfe (1960) were used for qualitative features of foliar epidermal anatomy. Following the method described by Stace (1965) stomatal index was calculated. SI= S/(S+E) ×100 Where S represents the number of stomata for a unit area and E is the number of epidermal cells of the same area.

2.3 PHARMACOGNOSTIC EVALUATION

Pharmacognostic studies include various tests such as organoleptic study, fluorescence analysis, solubility tests (cold and hot) and physicochemical tests, which were carried out for herbal parts of medicinal plants. For pharamacognostic studies powder of dried plant material was made by using an electric grinder and sieved through 10-mesh sieve. These powdered samples were placed in labeled plastic zip envelopes and stored in desiccators at room temperature until further analysis (Plate 13 & 14).

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2.3.1 ORGANOLEPTIC EVALUATION

Organoleptic evaluation is the simplest as well as quickest technique to ensure identity and quality of a crude herbal drug. Organoleptic characters such as size, shape, odor, color, taste and fracture of the samples are evaluated (Anonymous, 2002b). The macroscopic description of the herbal parts is done by Zeiss binocular light microscope.

2.3.2 FLUORESCENCE AND SOLUBILITY ANALYSIS

Fluorescence analysis is an important parameter of pharmacognostical evaluation to assess the quality of crude drugs. On the basis of fluorescence analysis the genuine drugs may be identified from their adulterants (Rai et al., 2010). This analysis was carried out by treating powder sample with different chemical reagents to observe various colors instances (Trease and Evans, 2009). Fluorescence behaviors and solubility tests of powdered drugs were evaluated by following the standard procedures (Chase and Pratt, 1949; Harborne, 1973; Sultana et al., 2012). Nine different analytical grade reagents including acids, alkalis and alcohol both in concentrated and dilute forms were used. For cold method, powdered drugs (1gm) were treated with various acidic and basic solvents (5mL) at room temperature (25-30°C) and then observed under visible light and UV (365 nm) chamber while for hot method the mixtures were slightly heated on a burner in test tubes and then observed under visible light and UV (365 nm) chamber. The colors of powdered drugs with different chemical reagents were also observed on filters papers. Color scheme from Nippon Company (Pakistan) was used for color comparison.

2.3.3 PHYSICOCHEMICAL EVALUATION

Various physicochemical parameters of powdered drugs such as total ash, acid insoluble ash, water insoluble ash, water soluble ash and moisture contents

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were evaluated by following the standard methods as described by Association of Official Analytical Chemists (AOAC, 2000) and Anonymous (2002b).

2.3.3.1 DETERMINATION OF MOISTURE CONTENT

Sample powder (5gm) was weighed on an electric balance and kept in electric drying oven (Memmert, ULE-500) at 105°C for 12-18 hours (Plate 15). Dried sample powder was cooled in desiccator and reweighed. Loss in weight of powder was expressed as moisture contents. % of moisture content = Loss in weight of powder sample/ Weight of powder sample × 100

2.3.3.2 DETERMINATION OF ASH VALUES a) Total ash

Dried sample powder (2gm) was weighed in a cleaned and tared crucible and incinerated at 550°C in muffle furnace (Neycraft, JFF-2000) for 8-10 hours until ash was obtained. When constant value of ash was obtained, the crucible was shifted to desiccator, cooled to room temperature and weighed (Plate 16). Percentage of ash was calculated by using formula: % of ash value = Weight of residual ash/ Weight of powder sample × 100 b) Acid insoluble ash

Ash obtained from total ash was mixed with 25mL of 2N HCL and boiled for 5 min. The resultant material was filtered through ash less filter paper and washed with hot water. The residual material collected on filter paper was transferred into tarred silica crucible and subjected to ignition at 550°C in muffle furnace for 30 min. The crucible was cooled to room temperature and weighed. Percentage of acid insoluble ash was calculated by using formula:

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 23 Materials and Methods Chapter 2

% of Acid insoluble ash = Weight of residual ash/Weight of powder sample × 100 c) Water insoluble ash

Ash obtained from total ash was mixed with 25mL of water and boiled for 5 min. The resultant material was filtered through ash less filter paper. The residual material collected on filter paper was transferred into tarred silica crucible and subjected to ignition in muffle furnace at 550°C for 30 min. The crucible was cooled to room temperature and weighed. Percentage of water insoluble ash was calculated by using formula: % of water insoluble ash= Weight of residual ash/Weight of powder sample × 100 d) Water soluble ash

The weight of the water insoluble ash was subtracted from the weight of total ash to obtain the weight of water soluble ash. Percentage of water insoluble ash was calculated by using formula: % of water soluble ash= Weight of residual ash/Weight of powder sample × 100

2.4 PHYTOCHEMICAL EVALUATION a) Preparation of plant extracts

Collected plant material was washed with tap water and air dried in shade. Dried material was grinded to fine powder by using electric blender. Cold maceration method was used for extraction purpose. 5 grams of ground plant material was soaked in 50 mL of methanol in a tightly sealed vessel for 7 days at room temperature and stirred for 30 minutes daily during the maceration. The extract was filtered through Whatman filter paper No. 1 and the filtrate was concentrated on rotary evaporator (Laborata 4000, Heidolph) at reduced pressure. Analytical grade reagents were used for analysis.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 24 Materials and Methods Chapter 2

b) Determination of Total Phenolic Contents (TPC)

The total phenolic contents (TPC) of methanolic extracts of medicinal plants were determined by following the procedure described by Lin et al. (2011). To 0.5 mL of extract, 2.5 mL of Folin-ciocalteu reagent (10 fold diluted) and 2.5 mL of sodium carbonate (7.5%) were added and then this mixture was incubated at room temperature for 90 minutes. The absorbance was measured at 765 nm spectrophotometrically (MAPADA UV-Vis UV1100) (Plate 18). Calibration curve was calculated for standard Gallic acid and results were expressed as Gallic acid equivalents (GAE) (Figure 1). c) Determination of Total Flavonoid Contents (TFC)

The total flavonoid contents were determined by following the method described by Moreno et al. (2000). To 0.5 mL of the extract, 0.1 mL of aluminum chloride (10%), 0.1 mL of 1 M potassium acetate and 1.5 mL of methanol were added. The mixture was further diluted by addition of 2.8 mL distilled water and incubated at room temperature for 40 min. The absorbance was measured at 415 nm. The calibration curve was calculated using quercetin as standard and the results were expressed as quercetin equivalents (QE) (Figure 1).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 25 Materials and Methods Chapter 2

2.5 y = 0.007x + 0.429 R² = 0.963 Quercetin 2 Gallic acid

1.5 y = 0.005x + 0.409 R² = 0.923 1

Absorbance (nm) (nm) Absorbance 0.5

0 0 100 200 300 Concentration (µg/mL)

Figure 1. Calibration curves of Quercetin and Gallic acid

2.5 HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC) a) Chemicals and Preparation of Standard solution

Chemicals used for the development of HPLC fingerprints included the Standard Quercetin (99% pure), HPLC gradient grade water (99.8% pure) and acetonitrile (98.9% pure) from Sigma-Aldrich. All the glassware (Pyrex) was thoroughly washed with distilled water and oven dried. HPLC-grade methanol was used for preparation of standard and sample stocks. Serial dilutions of standard were prepared by using the stock of 1mg/mL in strength. Each day all the dilutions were prepared afresh before starting the calibration procedure. To avoid the risk of contamination and solvent evaporation, the stock standards were kept in sterile glass vials in refrigerator.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 26 Materials and Methods Chapter 2

b) HPLC conditions and procedure

For HPLC analysis procedures described by Iqbal et al. (2013) and Kamal et al. (2015) were followed. The HPLC conditions were optimized by running a series of standard dilutions for a number of times and the conditions were observed for three consecutive days to validate optimization. The optimum conditions were as: injection volume 20 μL, pressure 250 bars, the mobile phase consisted of acetonitrile and HPLC gradient grade water at a ratio of 70:30 eluted at a flow rate of 0.8 mL/min with a run time of 15 min. The temperature of column was set at 40°C and wavelength was at 368 nm. Prior to HPLC analysis all extracts were filtered through the 0.02 μL syringe filters. 20 μL of sample was injected into the HPLC Shimadzu, furnished with the SPD-20 A UV-vis detector, the LC-20AT pump and the DGU-20A5 degasser. The C-18 column was used for the analysis purposes (Plate 17). The peak area corresponding to the retention time of standard (quercetin) was recorded and concentration was calculated from the regression equation obtained from calibration curve. Quercetin in the samples was identified by comparison of their retention times with the standard quercetin (Figure 2).

Figure 2. HPLC chromatogram of Quercetin Standard

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 27 Materials and Methods Chapter 2

2.6 ANTIOXIDANT ACTIVITY

For evaluation of antioxidant assay the stock solutions were prepared by dissolving plant extracts and standard in 95% methanol at a concentration of 4mg/mL and then series of concentration ranging from 25-250 µg/mL were prepared by dilution of these stock solutions. a) DPPH Radical Scavenging Assay

The DPPH (1,1-diphenyl-1-picrylhydrazyl) scavenging activity of the medicinal plants was evaluated by following the protocol described by Torey et al. (2010) with slight modifications. Briefly, 2mL of DPPH solution (0.1 mM in methanol) was added in 200µL of sample extracts at varying concentrations (25- 250 µg/mL). The mixtures were vortex vigorously and allowed to incubate in dark at room temperature for 30 min. The optimal density (OD) was measured at 517 nm against blank. Ascorbic acid was used as positive reference (standard) and methanol as blank. Results were expressed as Ascorbic acid equivalents and percentage inhibition was estimated as:

Scavenging ability (%) = ( A Control – A Sample ) / A Control × 100

IC 50 value (concentration providing 50% inhibition of DPPH radicals) was calculated on the basis of linear regression analysis.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 28 Materials and Methods Chapter 2

120 y = 0.201x + 49.57 100 R² = 0.970

0 DPPH radical scavenging (%) DPPH 0 50 100 150 200 250 300 Concentration (µg/mL)

Figure 3. Calibration curve of Ascorbic acid Standard

2.7 DOCUMENTATION OF INDIGENOUS KNOWLEDGE OF MEDICINAL PLANTS

Data regarding the traditional uses of medicinal plants among local communities and herbalists was collected through field surveys following the procedure of Martin (1995). In order to achieve the authenticated approach about the plant resources and their medicinal utilization by the local inhabitants questionnaire method was adopted during the survey (Annexure 4). For documentation of data on therapeutic uses of medicinal plants, semi structured interviews were conducted with a number of local informants (Plate 5 & 6). Ethnobotanical inventory was developed that comprised botanical name, family, English name, local name, trade name, part(s) used, voucher specimen number, therapeutic uses and toxicity.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 29 Materials and Methods Chapter 2

2.8 ASSESSMENT OF HERBAL MARKETS

In order to collect the information about the market samples of crude herbal medicines fifteen largest and most popular herbal markets of the country in Lahore (Punjab), Rawalpindi, Abbotabad, Peshawar (Khyber Pakhtoon Khwa), Swat, Quetta (Baluchistan), Skardu (Gilgit Baltistan), Kherpur (Sindh), were surveyed (Plate 7 & 8). A total of 25 herb sellers and herbalist were interviewed to collect the relevant data of medicinal plants by the means of questionnaire (Annexure 5) (Plate 9 & 10). Marketing chain for collection and trade of medicinal plants by local people were investigated (Figure 3). The data collected during the surveys was compared with available literature on the herbal markets.

Collectors

Herb sellers Middle Men

Consumers Wholesale Traders

Manufacturers Hakeems/Traditional Healers Export

Figure 4. Marketing chain for trade of Medicinal plants in Pakistan

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 30 Materials and Methods Chapter 2

2.9 ANNEXURE – 1 KEY TO MORPHOLOGICAL STUDY 1. Life form : ……………………………………………….. 2. Stem Type: ……………………………………………………... Texture: ………………………………...... Height: …………………………………………………… 3. Leaf Size: ……………………………………………………… Shape: ……………………………………………………. Phylotaxy: ……………………………………………….. Texture: ………………………………………………….. Leaf margin: ……………………………………………… Leaf apex: ………………………………………………… Leaf base: ………………………………………………… Stipule type: ……………………………………………… Petiole length: ……………………………………………. 4. Flower Inflorescence………………………………………………. Position: …………………………………………………… Color: ……………………………………………………… 5. Calyx Bracts: ……………………………………………………. No. of sepals: ……………………………………………... Size: ………………………………………………………. Color: ……………………………………………………... Shape: …………………………………………………….. 6. Corolla No. of petals: ……………………………………………… Shape: ……………………………………………………… Color: ………………………………………………………. 7. Androecium No. of stamens: ……………………………………………. Arrangement: ………………………………………………. Anther: ……………………………………………………… Filaments: ………………………………………………….. 8. Gynoecium No. of carpels: ………………………………………………. Style length: …………………………………………………. Stigma type: …………………………………………………. Ovary type 9. Fruit & seed Shape: ……………………………………………………….. Size: …………………………………………………………. Color: ………………………………………………………...

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 31 Materials and Methods Chapter 2

2.10 ANNEXURE – 2 KEY TO POLLEN STUDY (LM & SEM) Botanical Name: …………………………………… Family: ………………………………………………

Pollen Morphology: (a) Qualitative features of pollen morphology Type: ……………………………………………………. Shape in polar view: …………………………………….. Shape in equatorial view: ………………………………... Colpi: Present/Absent: …………………………………… Spines: Present/Absent: ………………………………….. Sculpturing: ……………………………………………… Other features: …………………………………………….

(b) Quantitative features of pollen morphology Polar diameter: …………………………………………… Equatorial diameter: ………………………………………. P/E ratio: ………………………………………………….. No. of colpi: ………………………………………………. Colpi length: ………………………………………………. Colpi width: ……………………………………………….. Exine thickness: …………………………………………….

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 32 Materials and Methods Chapter 2

2.11 ANNEXURE – 3 KEY TO FOLIAR EPIDERMAL ANATOMY (LM) Botanical Name: …………………………………… Family: ………………………………………………

(a) Foliar Epidermal Anatomy: Adaxial and Abaxial sides

Type of Epidermal cells: ………………………………. Shape of Epidermal cells: ……………………………… Size of Epidermal cells (L×W): ……………………….. Pattern of wall: ………………………………………… Stomata type: ………………………………...... Size of Stomata (L×W): ……………………………….. Trichome Present/Absent: ……………………………… Trichome type: Glandular/Non-glandular: ……………… Size of Trichome (L×W): ……………………………….. Macrohairs Present/Absent: …………………………...... Stomatal index: ………………………………………….. Other epidermal features: …………………………………

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 33 Materials and Methods Chapter 2

2.12 ANNEXURE – 4 QUESTIONNAIRE FOR ETHNOBOTANICAL DATA COLLECTION Demographic data of local informants:

Gender: ………………………………………………………………. Age: ………………………………………………………………….. Education: ………………………………….. Date: …………………

Ethnobotanical data of medicinal plants:

Voucher number: …………………………………………………… Locality: ……………………………………………………. ……… Local name: ………………………………………………………… Drug name: …………………………………………………………. Habit: ………………………………………………………………… Flowering season: …………………………………………………… Harvesting season: …………………………………………………… Medicinal uses: ………………………………………………………. Part used: …………………………………………………………...... Method of utilization: ………………………………………………… Mode of administration: ……………………………………………… Traditional folk recipes: ………………………………………………

Remarks: Botanical name: ……………………………………………………… Family: ……………………………………………………………….

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 34 Materials and Methods Chapter 2

2.13 ANNEXURE – 5 QUESTIONNAIRE FOR MARKET ASSESSMENT OF HERBAL DRUGS Demographic data of Herb sellers (Pansars):

Vendor name: …………………………………….………………… Age: ………………………………………………………………… Education: ………………………………………… Date…………. Total number of traded species……………………………………… Number of employees………………………………………………. Number of suppliers…………………………………………………

Information about crude herbal drugs:

Local name: …………………………………………………………. Botanical name: ……………………………………………………… Part used: ……………………………………………………………. Price/Kg Rs: …………………………………………………………... Purchase Rate: …………………………………….………………….. Sale Rate: ……………….………………………….………………….. Source of supply: ….…………………………………………………... Availability: …………………………………………………………… Demand: ……………………………….……………………………… Condition of Plants: …………..………………………………………. Number of people treated: ………………………………….……...... Types of people treated: ……………………………………………… Trend in use of medicinal plants: ……………………………………… Problems associated with herbal medicines…………………………… Side effects or toxicity: …………………………………………………

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 35 Materials and Methods Chapter 2

Plate 1 & 2: Collection sites of problematic medicinal plants

Plate 3 & 4: Collection of medicinal plants in the field

Plate 5 & 6: Collection of traditional knowledge of medicinal plants

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 36 Materials and Methods Chapter 2

Plate 7 & 8: Observation of herbal drugs during market surveys

Plate 9 & 10: Data Collection from herbalists and hakims

Plate 11& 12: Light and Scanning electron microscopy (LM & SEM)

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 37 Materials and Methods Chapter 2

Plate 13 & 14: Powdered sample for analysis and Desiccator for constant weight

Plate 15 & 16: Determination of Moisture content and Ash analysis

Plate17 & 18: HPLC and Spectrophotometer for Phytochemical analysis

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 38

Results and Discussion Chapter 3

In this project eight cases of problematic herbal drugs used in traditional medicines in Pakistan are authenticated from their adulterants by using various techniques such as morphological characterization, microscopic features of pollen (LM & SEM), foliar epidermal anatomy, physicochemical tests, fluorescence and solubility analysis, phytochemical characterization, antioxidant analysis and HPLC screening. These techniques are used for authentication of original source of herbal drug Tukhm-e-kalonji (Nigella sativa ) from its adulterant Allium cepa , Tukhm-e-balango (Lallemantia royleana ) from Ocimum basilicum , Belladona (Atropa acuminata ) from Solanum nigrum , Dhatura (Datura stramonium ) from Xanthium strumarium , Chiraita (Swertia cordata ) from Swertia paniculata , Zafran (Crocus sativus ) from Carthamus tinctorius , Resha khatmi (Althaea officinalis ) from Hibiscus rosa-sinensis and Kaur (Picrorhiza kurroa ) from Lagotis cashmeriana . The results are provided in detail as diagnostic features to differentiate the genuine drugs from their closely related adulterants. Pictorial guide of genuine sources of herbal drugs and their adulterants have been provided for correct identification. Complete authentication profiles of eight cases obtained by using above mention techniques are presented in Tables.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 39 Case 1:

Table 2: Authentication of herbal drug Tukhm-e-kalonji ( Nigella sativa L.) in comparison with its adulterant

Characters Nigella sativa L. Allium cepa L.

English Names Black seed, Fennel flower, Black cumin Common onion, Bulb onion

Local Name Kalvanji Piaz

Trade Names Tukhm-e-Kalonji, Siyaia dana Tukhm-e-Piaz

Family Ranunculaceae Alliaceae

Flowering period February-March June-July

Phytogeography: In Pakistan Cultivated in different parts of Pakistan. Cultivated in almost all parts of Pakistan.

In World South Europe, South West Asia, North Africa, Arab South-East Asia, Central Asia, Mesopotamia, Egypt, countries, Pakistan, India, Bangladesh, Turkey, Middle Rome, Pakistan and India. East and the Mediterranean basin.

Morphological description Annual herb, short-lived, slender or stout, 20-60 cm tall, Perennial, bulbs clustered, fleshy, cylindrical to ovoid or pubescent, occasionally hairs are glandular. Stem stiff, almost rounded; coats white to brownish and papery. erect, simple or branched and finely striate. Leaves are Scape is 1 m tall, stout, fistular, terete, mostly inflated 2.5 cm wide, linear, acute, dark green, delicate and finely below the middle. Leaves 0.5-2 cm wide, fleshy, fistular divided by thread-like and wispy lobes with 0.8-2 mm and terete. Spathe persistent, 2- or 3-valved. Umbels

width. Flowers solitary, delicate and without an densely many flowered, globular. Flowers are stellate. involucre. Sepals are ovate, ± obtuse, pale blue or Pedicels are 3-4 times as long as the parianth. Parianth whitish, 5 in number, puberulous and having a distinct, segments are 4-5 mm long, oblong, obtuse and greenish- short stipe. Petals bearing a short, thick capitate white. Filaments equal or longer than parianth segments, appendix. Follicles tuberculate, inflated and coherent exserted, inside 2-toothed at the base. Seeds 2-3 mm throughout the length. Seeds triquetrous, 1-5 mm long, long, ovate, black, wall are ± straight, depressed; strip- black or grayish black in color with an oily white like, wall ornamentation is densely granulose. interior, rugose, ends slightly curved and tapered. Trade Part Seeds are traded at herbal shops of Pakistan under the Seeds are traded in herbal shops of Pakistan and also name of Tukhm-e-Kalonji. mixed as adulterant in Kalonji seeds.

Organoleptography In aerial dried parts branches are rigid and light green in In aerial dried parts leaves are pale green in color. They color. Leaves grayish green, pinnatisect, segments are fan-shaped, fleshy, hollow, cylindrical and with one oblong, 2-4cm long. Flowers are delicate, pale or bluish flattened side. Tip is blunt and base is flattened. Aroma white, star shaped, terminal and solitary. Seeds is very powerful and pungent and taste is bitter. Flowers compressed, black in color, trigonous and angular with are greenish white and in dense umbel. Seeds are black two sides are flat and one convex, aromatic odor and a in color, wide ovate, depressed, ± straight, tetra- spicy pungent taste. hexagonal and convex. Smell is mild and taste is bitter.

Part used Seeds Seeds, bulb, leaves

Ethnomedicinal Uses Indigestion, diarrhea, stomach pains, spasms, dropsy, Cholera, nausea, vomiting, fever, cold, abscesses, menstrual disorders, insufficient lactation, bronchial earache, heat diseases, oral infection, tooth decay, complaints, intestinal worms and skin eruptions. wounds and fungal skin complaints.

Traditional folk recipes Seeds tincture is used in diarrhea, indigestion, loss of Onion bulb extract is mixed with equal amount of mint appetite, dropsy, treatment of worms and skin eruptions. extract; one teaspoon of this mixture is given per hour to Seeds are grounded and mixed with water to make a treat cholera. Scales are roasted in mustard oil and 2 or 3 paste which is used for the treatment of boils. Seeds are drops of this oil are poured in the ear to relief the used to increases the flow of milk in nursing mothers. earache. Warmed scales are used as poultice for the Roasted seeds are effective to stop vomiting. Powdered treatment of abscesses. Fresh leaves are used in culinary. seeds are used to remove lice from the hairs. Grounded Seeds powder mixed with egg yolk is used to enhance seeds mixed with sesame oil are applied externally to the sexual power. Seeds powder mixed with lemon juice treat abscesses and haemorrhoids. Seed oil is used or vinegar applied on daad twice a day is very effective externally as an antiseptic and local anesthetic. remedy.

Table 3: Comparative qualitative pollen morphology of Nigella sativa and Allium cepa

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Nigella sativa Tricolporate Circular Prolate-spheroidal Present Scabrate

2 Allium cepa Monocolporate Sub-angular Prolate Present Subpsilate

Table 4: Comparative quantitative pollen morphology of Nigella sativa and Allium cepa

Sr # Species name Polar diameter Equatorial diameter P/E Colpi length Colpi width Exine thickness (μm) (μm) ratio (μm) (μm) (μm)

1 Nigella sativa 45.5 (43.5-47.5) 51.75 (47.7-56.5) 0.87 1.25 (1.2-1.5) 3.5 (3.35-3.85) 3.22 (3.12-3.45)

2 Allium cepa 37.5 (33.5-40.5) 40.25 (37.6-42.5) 0.93 1.5 (1.25-1.85) 2.75 (2.5-3.12) 0.78 (0.62-0.97)

Table 5. Comparative qualitative characters of foliar epidermal anatomy of Nigella sativa and Allium cepa

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Nigella sativa Irregular Weakly Anomocytic Non-glandular Irregular Weakly Anomocytic Non-glandular undulate undulate

Allium cepa Elongated Straight Paracytic Absent Elongated Straight Paracytic Absent

Table 6. Comparative quantitative characters of foliar epidermal anatomy of Nigella sativa and Allium cepa

Abaxial epidermis Adaxial epidermis

Name of Size of cells Size of stomata Stomatal Trichomes ( μm) Size of cells ( μm) Size of stomata Stomatal Trichomes ( μm) species (μm) L ×W (μm) L ×W index (%) L×W L×W (μm) L×W index (%) L×W

Nigella sativa 117.5 (85.2- 46.5 (45.3-47.2) 18.5 217.5 (185.2- 121.2 (84.7-147.3) 43.3 (41.5-45.8) 12.81 225.2 (189.7- 150.6) × 45.5 × 39.75 (37.5- 250.6) × 24.5 × 46.7 (39.2-52.2) × 38.25 (35.5- 247.3) × 27.7 (40.5-50.3) 40.2) (10.5-37.3) 39.5) (15.2-32.2)

Allium cepa 168 (87.5-250.3) 37.5 (35.8-38.3) 21.6 Absent 174 (89.5-253.8) × 35.5 (33.8-37.2) 17.5 Absent × 24.5 (22.5- × 24 (22.5-26.5) 27 (25.5-28.6) × 20.5 (19.5- 27.5) 22.3)

Results and Discussion Chapter 3

Figure 5 (a) Nigella sativa ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 45 Results and Discussion Chapter 3

Figure 6 (a) Allium cepa ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 20X); (f) Epidermal cells and stomata (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 46

Table 7: Fluorescence and solubility analysis of powdered drug of Nigella sativa (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Black Purplish black - - - 2 Sample powder + 5% KOH Grayish black Brownish black Antique white Ash white Partially soluble

3 Sample powder + 10% aq. FeCl 3 Purplish black Brownish black Halo Ash white Partially soluble

4 Sample powder + dH 2O Grayish black Purplish gray Satin white Snow bell Semi soluble 5 Sample powder + HCL Conc. Reddish black Purplish black Rose white Light mulberry Semi soluble 6 Sample powder + HCL 50% Yellowish black Purple Orchid shadow Lavender white Partially soluble

7 Sample powder + H 2SO 4 Conc. Reddish black Purplish brown Antique white Light chocolate Soluble

8 Sample powder + H 2SO 4 50% Yellowish black Purplish brown Halo Light brown Partially soluble

9 Sample powder + HNO 3 Conc. Reddish black Brownish black Ash white Lavender white Soluble

10 Sample powder + HNO 3 50% Brownish black Brownish purple Antique white Snow bell Soluble

11 Sample powder + CH 3OH Conc. Grayish black Ash white Satin white Ash white Partially soluble

12 Sample powder + CH 3OH 50% Grayish black Sky gray Onyx Orchid shadow Partially soluble

13 Sample powder + CHCl 3 Conc. Purplish black Purplish brown Antique white Snow bell Slightly soluble

14 Sample powder + CHCl 3 50% Brownish black Snow mountain Lavender white Snow mountain Slightly soluble

15 Sample powder + C 2H5OH Conc. Orion Lavender white Satin white Light mulberry Partially soluble

16 Sample powder + C 2H5OH 50% Light chocolate Ash white Sky gray Orchid shadow Partially soluble

17 Sample powder + CH 3COOH Conc. Grayish black Orion Ash white Halo Soluble

18 Sample powder + CH 3COOH 50% Brownish black Light chocolate Greenish purple Ash white Slightly soluble

19 Sample powder + C 6H6 Conc. Yellowish gray Halo Satin white Brilliant white Partially soluble

20 Sample powder + C 6H6 50% Milky gray Orion Brilliant white Ash white Partially soluble

Table 8: Fluorescence and solubility analysis of powdered drug of Nigella sativa (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Black Purplish black - - - 2 Sample powder + 5% KOH Dark gray Grayish brown Light brown Orchid shadow Semi soluble

3 Sample powder + 10% aq. FeCl 3 Purplish gray Blackish gray Halo Ash white Semi soluble

4 Sample powder + dH 2O Grayish black Blackish gray Satin white White ice Partially soluble 5 Sample powder + HCL Conc. Dark chocolate Purplish gray Abbey cream Shingle Soluble 6 Sample powder + HCL 50% Brownish black Purplish brown Peach silk Abbey cream Soluble

7 Sample powder + H 2SO 4 Conc. Dark chocolate Purplish gray Black Dark purple Soluble

8 Sample powder + H 2SO 4 50% Dark brown Blackish brown Brownish black Dark mulberry Soluble

9 Sample powder + HNO 3 Conc. Brownish black Reddish brown Orion Classic ivory Soluble

10 Sample powder + HNO 3 50% Light brown Purplish brown Halo Dark mulberry Soluble

11 Sample powder + CH 3OH Conc. Dark Gray Brownish gray White ice Shimmery sky Partially soluble

12 Sample powder + CH 3OH 50% Brownish gray Greenish brown Satin whit Snow bell Partially soluble

13 Sample powder + CHCl 3 Conc. Grayish black Purplish brown Antique white Lavender white Semi soluble

14 Sample powder + CHCl 3 50% Greenish black Brownish gray Satin white Shimmery sky Semi soluble

15 Sample powder + C 2H5OH Conc. Brownish black Ash white Lavender white Orchid shadow Semi soluble

16 Sample powder + C 2H5OH 50% Brownish black Onyx Satin white Orchid shadow Semi soluble

17 Sample powder + CH 3COOH Conc. Dark gray Light chocolate Snow mountain White ice Soluble

18 Sample powder + CH 3COOH 50% Brownish gray Greenish brown Sky gray Orchid shadow Soluble

19 Sample powder + C 6H6 Conc. Brownish black Purplish brown Satin white Lavender white Soluble

20 Sample powder + C 6H6 50% Blackish gray Light gray Satin white Lavender white Soluble

Table 9: Fluorescence and solubility analysis of powdered drug of Allium cepa (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Grayish black Brownish black - - - 2 Sample powder + 5% KOH Grayish brown Brown Lavender white Snow bell Partially soluble

3 Sample powder + 10% aq. FeCl 3 Light gray Greenish brown Ash white Abbey cream Partially soluble

4 Sample powder + dH 2O Grayish brown Light gray Antique white White ice Slightly soluble 5 Sample powder + HCL Conc. Blackish brown Yellowish gray Lavender white Ash white Semi soluble 6 Sample powder + HCL 50% Blackish gray Blackish brown Light mulberry Lavender white Semi soluble

7 Sample powder + H 2SO 4 Conc. Chocolate brown Purplish gray Halo Light brown Soluble

8 Sample powder + H 2SO 4 50% Light gray Brownish gray Satin white Antique white Soluble

9 Sample powder + HNO 3 Conc. Dark brown Purplish brown Orchid shadow Light mulberry Semi soluble

10 Sample powder + HNO 3 50% Brownish black Brown Orion Snow bell Partially soluble

11 Sample powder + CH 3OH Conc. Grayish black Grayish black Ash white Orchid shadow Slightly soluble

12 Sample powder + CH 3OH 50% Brownish gray Purplish gray Orion White ice Slightly soluble

13 Sample powder + CHCl 3 Conc. Brownish black Purplish black Antique white Orion Partially soluble

14 Sample powder + CHCl 3 50% Light gray Brownish gray Satin white Ash white Partially soluble

15 Sample powder + C 2H5OH Conc. Blackish gray Dark brown Halo Classic ivory Semi soluble

16 Sample powder + C 2H5OH 50% Brownish gray Greenish brown Snow bell Light mulberry Partially soluble

17 Sample powder + CH 3COOH Conc. Light gray Greenish gray Brilliant white Lavender white Semi soluble

18 Sample powder + CH 3COOH 50% Blackish gray Chocolate brown Ash white Light mulberry Partially soluble

19 Sample powder + C 6H6 Conc. Brownish black Brownish black Satin white Antique white Partially soluble

20 Sample powder + C 6H6 50% Blackish gray Greenish gray Orchid shadow Brilliant white Partially soluble

Table 10: Fluorescence and solubility analysis of powdered drug of Allium cepa (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Grayish black Brownish black - - - 2 Sample powder + 5% KOH Blackish brown Chocolate brown Light mulberry Abbey cream Semi soluble

3 Sample powder + 10% aq. FeCl 3 Grayish brown Grayish green Satin white Orchid shadow Semi soluble

4 Sample powder + dH 2O Grayish black Blackish gray Satin white Brilliant white Semi soluble 5 Sample powder + HCL Conc. Dark chocolate Dark chocolate Abbey cream Shingle Soluble 6 Sample powder + HCL 50% Brownish black Purplish brown Peach silk Abbey cream Soluble

7 Sample powder + H 2SO 4 Conc. Dark chocolate Purplish gray Black Dark purple Soluble

8 Sample powder + H 2SO 4 50% Chocolate brown Blackish brown Brownish black Dark mulberry Soluble

9 Sample powder + HNO 3 Conc. Blackish brown Reddish brown Orion Purplish brown Soluble

10 Sample powder + HNO 3 50% Light brown Purplish brown Halo Purple Soluble

11 Sample powder + CH 3OH Conc. Gray Brownish gray White ice Lavender white Semi soluble

12 Sample powder + CH 3OH 50% Brownish gray Greenish brown Satin white Orchid shadow Semi soluble

13 Sample powder + CHCl 3 Conc. Dark gray Purplish brown Antique white Lavender white Partially soluble

14 Sample powder + CHCl 3 50% Greenish black Brownish gray Satin white Shimmery sky Partially soluble

15 Sample powder + C 2H5OH Conc. Brownish gray Ash white Satin white Orchid shadow Semi soluble

16 Sample powder + C 2H5OH 50% Brownish black Onyx Antique white Orchid shadow Semi soluble

17 Sample powder + CH 3COOH Conc. Dark gray Light chocolate Snow mountain Ash white Soluble

18 Sample powder + CH 3COOH 50% Brownish green Greenish brown Sky gray White ice Partially soluble

19 Sample powder + C 6H6 Conc. Brownish black Purplish brown White ice Lavender white Partially soluble

20 Sample powder + C 6H6 50% Blackish gray Light gray Lavender white Lavender white Partially soluble

Results and Discussion Chapter 3

Table 11: Physicochemical Parameters of Nigella sativa and Allium cepa

S. No. Physicochemical parameters Nigella sativa Allium cepa (%) i Total ash 4 4.33 ii Acid insoluble ash 0.67 1 iii Water soluble ash 2.66 2.67 iv Water insoluble ash 1.34 1.66 v Moisture content 6 8

Table 12: Antioxidant activity of Ascorbic acid, Nigella sativa and Allium cepa

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic acid Nigella sativa Allium cepa 25 50.91 5.58 37.16 50 60.47 12.35 38.23 100 72.39 21.48 39.74 150 81.06 25.13 40.70 200 92.8 30.29 43.17 250 96.02 35.76 44.79 IC 50 value 2.14 µg/mL 353 µg/mL 413 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 51 Results and Discussion Chapter 3

45 40 35 Total Phenols 30 Total Flavonoids 25 20 15 10

Concentration (mg /g) (mg Concentration 5 0 Nigella sativa Allium cepa Plant extracts

Figure 7 . Total Phenolic and Flavonoid contents

120

100 25µg/mL 50µg/mL 80 100µg/mL 60 150µg/mL 40 activity (%) activity 200µg/mL 20 250µg/mL DPPH radical scavenging radical DPPH 0 Ascorbic acid Nigella sativa Allium cepa Plant extracts and standard at different concentrations

Figure 8 . DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 52 Results and Discussion Chapter 3

Figure 9. HPLC chromatogram of Nigella sativa

Figure 10. HPLC chromatogram of Allium cepa

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 53 Results and Discussion Chapter 3

3.1 Authentication of Herbal Drug Tukhm-e-kalonji (Nigella sativa L.)

Nigella sativa (Ranunculaceae) has been used extensively for a long time in traditional folk medicines as a protective and health remedy for numerous human diseases (Chopra et al., 1956; Nadkarni, 1976). Seeds of herb have great medicinal importance in Unani-Tibb/Greco-Arab and Ayurveda systems of traditional medicine. In recent years, many studies have been attributed that seeds of N. sativa have many medicinal properties such as anthelmenthic, diaphoretic, antineoplastic, antibacterial and antifungal activities (Hanafy and Hatem, 1991; Ramadan, 2007). Seeds of N. sativa are sold at herbal shops under the trade name of Tukhm-e-kalonji. Commonly in herbal market, the seeds are intentionally or accidently adulterated with Allium cepa seeds due to their morphological resemblance. Generally the raw material of many medicinal plants procured through markets is found substituted or adulterated like N. sativa . Standardization of the raw material is very crucial for the global acceptance of herbal medicines. In this study a systematic approach was adopted to differentiate the seeds of N. sativa from its morphologically similar seeds of A. cepa by using taxonomic, pharmacognostic and phytochemical techniques.

Taxonomic Clarification

Taxonomic features play a vital role to differentiate closely related taxa by showing variations in vegetative and floral morphology (Shaheen et al ., 2014). Morphologically, N. sativa is an annual herb with linear, finely divided leaves and seeds are triquetrous and rugose having white oily interior (Figure 5a & 5b). This species can be distinguished morphologically from its trade adulterant A. cepa which is a perennial herb with fleshy leaves and seeds are wide ovate and depressed with straight walls (Figure 6a & 6b). Detailed morphological characterization for differentiation of N. sativa and A. cepa is presented in Table 2. In previous studies, Margout et al. (2013) used morphological and microscopic features of seeds for differentiation of N. sativa and N. damascene .

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 54 Results and Discussion Chapter 3

Sometimes, certain plants show resemblance in their morphological appearance which may cause confusions in plant identification and differentiation (Shaheen et al., 2014). In such cases palynological characters and foliar epidermal features can be used as an aid to differentiate problematic taxa (Gilani et al., 2002). In literature it is reported that palynological features play a crucial role to differentiate the closely related taxa (Zafar et al., 2007). By using palynological features N. sativa can be distinguished from A. cepa in having tricolporate pollen and scabrate exine sculpturing (Figure 5c & 5d) while in A. cepa pollen are monocolporate with subpsilate exine sculpturing (Figure 6c & 6d). Comparative palynological features of N. sativa and A. cepa are presented in Table 3 & 4. In literature many workers cited the use of palynological features to differentiate problematic taxa such as Azadirachta indica from its morphologically similar species Melia azedarach (Sultana et al., 2011).

The other useful characters that can be used to differentiate similar taxa are foliar epidermal features such as shapes of epidermal cells, type of hairs and stomata (Naz et al., 2009; Riaz et al., 2010). Role of trichomes at a microscopic level to differentiate similar species has been reported by many authors (Adedeji, 2004; Adedeji et al., 2007). According to Metcalfe and Chalk (1979) particular types of trichomes are found in species, genera or even in whole families. Valuable intergeneric and interspecific variations in foliar epidermal cells has been found by Ahmad et al. (2010b) that can be used as an important taxonomic tool for identification and differentiation of many closely related species. While using foliar epidermal features, N. sativa can be distinguished from A. cepa by presence of irregular epidermal cells with anomocytic stomata and non-glandular trichomes (Figure 5e & 5f). However in case of A. cepa epidermal cells are elongated with paracytic stomata and trichomes are absent (Figure 6e & 6f). Detailed foliar epidermal features of N. sativa and A. cepa are presented in Table 5 & 6. According to Yousaf et al. (2008) leaf epidermal anatomy can be used as significant tool for the resolution of taxonomic confusions among the Allium

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 55 Results and Discussion Chapter 3

species. Jing-Hua and Liang-Qian (2003) reported that leaf epidermal anatomy serve as an important criteria for distinguishing the Clematis species in family Ranunculaceae.

Pharmacognostic Evaluation

Pharmacognostic parameters are useful for quality and purity assessment of crude herbal drugs (Kadam et al., 2012). Fluorescence analysis and solubility tests of N. sativa and A. cepa with various chemical reagents are presented in Table 7, 8, 9 & 10. In literature many workers like Kokoski et al. (1958), Badami et al. (2002) and Sunita et al. (2010) have used such pharmacognostic features for authentication of various genuine drugs of plant origin. In standardizing the herbal medicines physicochemical parameters play a very important role. Physicochemical parameters of N. sativa and A. cepa evaluated in the study are presented in Table 11. In A. cepa slightly increased value of ash and moisture content was recorded as compared to N. sativa . In herbal drugs moisture content should not be more than 14% w/w (Ilanchezhian et al., 2011). During storage of crude drugs the growth of fungi and yeast is enhanced in excess moisture which may cause the breakdown of crucial bioactive compounds. Antara (2012) employed such physicochemical parameters for correct identification and standardization of powdered drugs.

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

The quantitative estimation of secondary metabolites is essential for extraction, separation, purification, crystallization and identification of various phytocompounds (Geetha and Geetha, 2014). Earlier studies proved that phenolic and flavonoids compounds found in plants are potent antioxidants with reported anticarcinogenic and antimutageneic effects (Middleton and Kandaswami, 1994). Quantitative investigation of phenols and flavonoids indicated that higher

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 56 Results and Discussion Chapter 3

concentration of total phenolic contents (9.165 mg GAE/g, DW) was found in N. sativa as compared to A. cepa (6.54 mg GAE/g, DW) whereas flavonoid contents were higher in A. cepa (43.85 mg QE/g, DW) as compared to N. sativa (36.54 mg QE/g, DW) (Figure 7). These results are in accordance with previous reported literature (Al-Bishri and Danial, 2013). Ahmad et al. (2014) carried out the phytochemical standardization of N. sativa seeds to establish the standard parameters of this miracle herb.

HPLC Screening

The phytochemicals present in herbal medicines are analyzed with the help of High pressure liquid chromatography (Farnsworth et al., 1966; Bilia et al., 2002). During last few decades HPLC has got most extensive importance in the analysis of the bioactive chemical compounds in herbal drugs. The HPLC chromatograms of N. sativa and A. cepa are presented in Figure 9 & 10. These chromatograms can be used as reference for the identification and authentication of genuine drug. Results indicated that higher concentration of quercetin was found in A. cepa (40.53 mg/g, DW) as compared to N. sativa (31.91 mg/g, DW). Quercetin concentration of A. cepa recorded in this study is in accordance with previous reported literature (Lu et al., 2011).

Antioxidant analysis

Findings regarding antioxidant activity of N. sativa and A. cepa seeds revealed that methanolic extracts showed varying degrees of scavenging abilities at different concentrations. DPPH scavenging activity (%) of N. sativa and A. cepa is shown in Table 12 & Figure 8. N. sativa seeds extract exhibited good antioxidant activity in comparison with its adulterant A. cepa . Al-Bishri and Danial (2013), conducted a comparative study on the antioxidant activity of selected seeds from Saudi Arabia. They reported that N. sativa seeds showed strong scavenging activity, while A. cepa seeds were found as weak antioxidants.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 57 Results and Discussion Chapter 3

Results of scavenging activity of N. sativa recorded in this study are in accordance to those previously reported in literature by Singh et al. (2005), Şen et al. (2010) and Meziti et al. (2012).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 58 Case 2:

Table 13: Authentication of herbal drug Tukhm-e-balango (Lallemantia royleana Benth.) in comparison with its adulterant

Characters Lallemantia royleana Benth. Ocimum basilicum L.

English Names Black psyllium seeds/Salvia seeds Basil /Common basil/ Sweet basil/ Holy basil

Local Name Balangu Nyazbo

Trade Name Tukhm-e- balango Tukhm-e-rehan

Family Lamiaceae Lamiaceae

Flowering period March-June Throughout the year

Phytogeography: In Pakistan Baluchistan, Sindh, Layyah, Bhakkar, Hasilpur, Cultivated in all most all parts of Pakistan. Bahawalpur and Chishtian.

In World Afghanistan, Iran, Turkestan, Kazakhstan, Kyrgyzstan, Subtropical and tropical Asia, Africa, Central South Tajikistan, Uzbekistan, Pakistan, India, Russia, China, America, European countries like France, Italy and Spain. south West Asia and Europe.

Morphological description Annual, branched or unbranched herb. Stem is erect, 5- Annual or perennial aromatic herb, stem 60 cm long, 30 cm, quadrangular, leafy, having dense, short, erect, four edged, profusely branched with retrorse simple eglandular and retrorse hairs. Leaves 15-20 x 7-15 mm, or eglandular spreading hairs. Leaves stalked, not divided, simple, blade oblong-obovate, margin crenate, base up to 5 x 2 cm, opposite, ovate-lanceolate, acuminate and

cuneate, apex obtuse having short eglandular hairs. attenuate-serrate. Inflorescence condensed raceme, Petiole up to 15 (-20) mm. Inflorescence is verticillaster verticillasters, flowers 6 to 10, white to purplish in color in axils of leaves, 6-8 flowered, shortly petiolate or with bell-shaped calyx and corolla. Bracts small, sessile, distant or contiguous, cuneate, with 2-4 mm long deciduous and clearly different from leaves. Caly 5-9 mm marginal awns, bracts are linear-oblong, several, length long, ovoid-campanulate, bilabiate, upper lobe is broader same or greater than calyx. Calyx tubular, prominently than lower. Lower lobe has four pointed narrow teeth, 2 veined or ribbed, 6-7 mm long having sessile oil globules lateral teeth ovate and 2 lower teeth lanceolate-acuminate, and short spreading eglandular hairs. Upper lip having 3 curved upwardly in fruit. Corolla 7-9 mm long, white, lobes that are ovate-obtuse and lower lip with 2 narrow pale purple or violet, bilabiate, upper lip 4-lobed and lobes, all lobes are shortly acuminate. Corolla is whitish lower lip flat, entire, spreeding. Style forked, stamens four pink to blue, pale lilac, slightly longer than calyx; 6-8 and didynamous. Fruit with four nutlets, 2 x 1.3 mm, and mm long, upper lip is 2 mm long. Lower lip is longer enclosed inside the calyx. Nutlets 2 mm long, rugulose, than upper lip. Nutlets dark brown, 2.5 x 1 mm, apically rounded, oblong-ellipsoid, glabrous and triquetrous having a short attachment scar and on wetting mucilaginous on wetting. are clearly mucilaginous. Trade Part Seeds are traded at herbal shops under the trade name Seeds are sold under the name Tukhm-e-rehan at herbal Tukhm-e-balango. shops and also mixed with seeds of Tukhm-e-balango as adulterant.

Organoleptography Dried branches are white, quadrangular and pubescent. Dried branches are smooth, without distinct ring, Leaves are simple, oblong-ovate, sparsely pubescent and pubescent, terete and purplish green in color. Leaves whitish green in color. Flowers are blue or pale lilac in deltoid, with dark green upper surface and light green color. Nutlets dark brown, triquetrous, narrowly oblong, lower surface, venation not distinct. Flowers are arranged glabrous, adaxially ribbed having a small globular, white in terminal spikes and white in color. Nutlets are ellipsoid, pore at thin position and other end is convex having a black in color and texture is pitted. Aroma is very strong, notch. On soaking in water the seeds produce a turbid,

sticky and tasteless liquid. pungent and sweet. Taste is somewhat like anise.

Part used Leaves, seeds Leaves, flowers, seeds

Ethnomedicinal Uses Gastrointestinal problems, rheumatism, joint Headache, nausea, diarrhea, dysentery, cough, bronchitis, inflammation, abscesses inflammations, osteoarthritis, sore throat, stomach cramps, joints pain, earache, fever, common cold, hepatic, nervous and renal diseases. toothache, heart diseases, kidneys and worms infections.

Traditional herbal recipes Locally seeds are dipped in water or milk in a clay pot Leaves juice is an excellent remedy for the treatment of and next day seeds with mucilage are taken orally as ringworm and bruised leaves for scorpion stings. Leaves refrigerant and to cure dyspepsia, jaundice, high blood are used to cure sore throat. Hot basil tea is a good pressure and chest pain. Seed poultice is applied topically remedy for treating nausea, dysentery and flatulence. to cure abscesses, boils and inflammation. Seeds in Leaves decoction or coffee is used as digester and to cure combination with cumin are used for the treatment of inflammation. The juice of plant is effective to cure menstrual problems, dysmenorrheal and menorrheal hearing problems. Dried leaves powder is used in conditions. It is used by many ladies to reduce the size of culinary. Flowers are used as stimulant, diuretic and stomach after child birth in combination with different demulcent. Seeds infusion is mucilaginous, cooling and herbs, like neem leaves, calamus and raw sugar. Along given in fever, gonorrhea, chronic dysentery and diarrhea. with opium it is used for prolapsed uterus. To relieve the after pains of parturition a cold infusion of seeds is recommended. In case of snake and scorpion bites the seeds are chewed and also applied to the bitten parts. Seeds poultice is effective for unhealthy sore and sinuses.

Table 14: Comparative qualitative pollen morphology of Lallemantia royleana and Ocimum basilicum

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine view /Absent Sculpturing

1 Lallemantia royleana Tricolporate, Sub-angular Suboblate Present Reticulate Hexacolporate

2 Ocimum basilicum Tricolporate, Circular Prolate-spheroidal Present Reticulate Hexacolporate

Table 15: Comparative quantitative pollen morphology of Lallemantia royleana and Ocimum basilicum

Sr # Species name Polar diameter Equatorial diameter P/E Colpi length Colpi width Exine thickness (μm) (μm) ratio (μm) (μm) (μm)

1 Lallemantia royleana 19.5 (17.5-22.5) 22.5 (20-25.5) 0.86 0.75(0.65-0.86) 2.5 (2.25-2.9) 2 (1.85-2.25)

2 Ocimum basilicum 37.5 (33.7-42.5) 32.75 (28.7-37.5) 1.14 9.5 (8.5-10) 3.5 (3-3.75) 2.35 (2.25-2.75)

Table 16. Comparative qualitative characters of foliar epidermal anatomy of Lallemantia royleana and Ocimum basilicum

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Lallemantia Irregular Weakly Diacytic Non-glandular Irregular Weakly Diacytic Non-glandular royleana Undulate & unicellular Undulate & unicellular

Ocimum Polygonal Heavily Diacytic Both types, Polygonal Heavily Diacytic Both types, basilicum Undulate Non-glandular Undulate Non-glandular trichomes are trichomes are unicellular unicellular

Table 17. Comparative quantitative characters of foliar epidermal anatomy of Lallementia royleana and Ocimum basilicum

Abaxial epidermis Adaxial epidermis

Lallemantia 52.2 (41.5-62.5) 21.5 (19.2-23.7) 22.5 363 (287.5-440) × 53.6 (42.3-65) × 19.5 (17.3-22.5) 17.4 313.25 (238-389.5) royleana × 33.9 (32.7- × 12.6 (10.5-15) 30.5 (15.2-45.5) 32.5 (31.5-34.3) ×13.25 (11.3-14) × 34.4 (16.4 × 35.2) 52.7)

Ocimum 65 (57.7-72.2) × 25 (22.5-27.5) × 18.9 265.3 (140.5- 58.8 (50-66.5) × 21.5 (19.3-23) × 15.5 283 (111.4-454.7) basilicum 21.5(19.2-24.7) 17.5 (15.3-19.7) 390.8) × 39 (22.5- 20.2 (18.5-22.3) 14.8 (12.2-16) × 28.25 (18.2- 55.6) 38.5)

Results and Discussion Chapter 3

Figure 11 (a) Lallemantia royleana ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and trichome (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 64 Results and Discussion Chapter 3

Figure 12 (a) Ocimum basilicum ; (b) Dried seeds; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and trichomes (LM-abaxial: 40X); (f) Stomata and trichome (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 65

Table 18: Fluorescence and solubility analysis of powdered drug of Lallemantia royleana (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Brownish black Purplish brown - - - 2 Sample powder + 5% KOH Reddish black Purplish black Lavender white Orchid shadow Partially soluble

3 Sample powder + 10% aq. FeCl 3 Purplish black Purplish black Orchid shadow Light mulberry Insoluble

4 Sample powder + dH 2O Brownish black Purplish gray Satin white Orion Partially soluble 5 Sample powder + HCL Conc. Purplish brown Brownish purple Rose white Light mulberry Slightly soluble 6 Sample powder + HCL 50% Yellowish black Brownish black Orchid shadow Snow bell Slightly soluble

7 Sample powder + H 2SO 4 Conc. Reddish black Purplish brown Brilliant white Light chocolate Soluble

8 Sample powder + H 2SO 4 50% Maroonish black Light chocolate Halo Light brown Slightly soluble

9 Sample powder + HNO 3 Conc. Reddish black Brownish black Ash white Lavender white Soluble

10 Sample powder + HNO 3 50% Brownish black Light chocolate Antique white Snow bell Semi soluble

11 Sample powder + CH 3OH Conc. Yellowish black Lavender white Sky gray Orchid shadow Partially soluble

12 Sample powder + CH 3OH 50% Grayish black Sky gray Onyx Light mulberry Semi soluble

13 Sample powder + CHCl 3 Conc. Purplish black Purplish brown Antique white Halo Semi soluble

14 Sample powder + CHCl 3 50% Brownish black Lavender white Satin white Snow mountain Semi soluble

15 Sample powder + C 2H5OH Conc. Milky gray Snow mountain Brilliant white Snow bell Slightly soluble

16 Sample powder + C 2H5OH 50% Grayish black Ash white Sky gray Orchid shadow Slightly soluble

17 Sample powder + CH 3COOH Conc. Brownish black Orion Ash white Brilliant white Semi soluble

18 Sample powder + CH 3COOH 50% Grayish black Lavender white Greenish purple Ash white Semi soluble

19 Sample powder + C 6H6 Conc. Light chocolate Snow mountain Ash white Light mulberry Semi soluble

20 Sample powder + C 6H6 50% Brownish black Orion Brilliant white Orchid shadow Slightly soluble

Table 19: Fluorescence and solubility analysis of powdered drug of Lallemantia royleana (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Brownish black Purplish brown - - - 2 Sample powder + 5% KOH Blackish brown Purplish gray White ice Lavender white Semi soluble

3 Sample powder + 10% aq. FeCl 3 Reddish black Purplish red Orchid shadow Light mulberry Semi soluble

4 Sample powder + dH 2O Marronish black Purplish black Satin white Orion Partially soluble 5 Sample powder + HCL Conc. Purplish brown Dark brown Peach silk Classic ivory Soluble 6 Sample powder + HCL 50% Yellowish black Brownish black Orchid shadow Shimmering sky Soluble

7 Sample powder + H 2SO 4 Conc. Reddish brown Reddish brown Off white Light chocolate Soluble

8 Sample powder + H 2SO 4 50% Maroonish black Light chocolate Orion Halo Soluble

9 Sample powder + HNO 3 Conc. Red oxide Deep black Lavender white Ash white Soluble

10 Sample powder + HNO 3 50% Orange red Dark brown Antique white Halo Soluble

11 Sample powder + CH 3OH Conc. Yellowish black Ash white Snow mountain Sky gray Semi soluble

12 Sample powder + CH 3OH 50% Greenish black Snow mountain Sky gray Orion Partially soluble

13 Sample powder + CHCl 3 Conc. Purplish brown Purplish brown Orchid shadow Light mulberry Sparingly soluble

14 Sample powder + CHCl 3 50% Brownish black Brilliant white Satin white Snow mountain Sparingly soluble

15 Sample powder + C 2H5OH Conc. Milky gray Shimmering sky Brilliant white Snow bell Partially soluble

16 Sample powder + C 2H5OH 50% Brownish gray Sky gray Shimmering sky Light mulberry Partially soluble

17 Sample powder + CH 3COOH Conc. Brownish black Halo Satin white Satin white Semi soluble

18 Sample powder + CH 3COOH 50% Purplish gray Lavender white Sky gray Ash white Semi soluble

19 Sample powder + C 6H6 Conc. Light chocolate Lavender white White ice Light mulberry Semi soluble

20 Sample powder + C 6H6 50% Brownish black Antique white Satin white Light mulberry Semi soluble

Table 20: Fluorescence and solubility analysis of powdered drug of Ocimum basilicum (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Grayish black Brownish gray - - - 2 Sample powder + 5% KOH Brownish black Purplish gray Satin white Classic ivory Slightly soluble

3 Sample powder + 10% aq. FeCl 3 Brownish gray Greenish gray Light mulberry Light brown Slightly soluble

4 Sample powder + dH 2O Brownish black Grayish black White ice White ice Insoluble 5 Sample powder + HCL Conc. Blackish gray Purplish gray Halo Classic ivory Semi soluble 6 Sample powder + HCL 50% Grayish brown Dark brown Antique white Snow bell Semi soluble

7 Sample powder + H 2SO 4 Conc. Light chocolate Brownish gray Orion Brown Soluble

8 Sample powder + H 2SO 4 50% Light gray Blackish gray Antique white Abbey cream Soluble

9 Sample powder + HNO 3 Conc. Dark brown Brownish purple Orchid shadow Light brown Partially soluble

10 Sample powder + HNO 3 50% Blackish gray Dark brown White ice Lavender white Partially soluble

11 Sample powder + CH 3OH Conc. Light gray Grayish black Satin white Orchid shadow Semi soluble

12 Sample powder + CH 3OH 50% Blackish gray Light chocolate Brilliant white Lavender white Semi soluble

13 Sample powder + CHCl 3 Conc. Brownish black Greenish gray Light mulberry Orion Semi soluble

14 Sample powder + CHCl 3 50% Dark brown Brownish gray Satin white Orchid shadow Slightly soluble

15 Sample powder + C 2H5OH Conc. Blackish gray Light mulberry White ice Light mulberry Semi soluble

16 Sample powder + C 2H5OH 50% Dark brown Greenish gray Lavender white Brilliant white Partially soluble

17 Sample powder + CH 3COOH Conc. Light gray Greenish brown Brilliant white Light mulberry Semi soluble

18 Sample powder + CH 3COOH 50% Blackish gray Light chocolate White ice Snow bell Semi soluble

19 Sample powder + C 6H6 Conc. Brownish black Brownish gray Satin white Orchid shadow Semi soluble

20 Sample powder + C 6H6 50% Dark brown Purplish gray Lavender white Lavender white Partially soluble

Table 21: Fluorescence and solubility analysis of powdered drug of Ocimum basilicum (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried seeds powder Grayish black Blackish gray - - - 2 Sample powder + 5% KOH Grayish black Greenish gray Sky gray Classic ivory Semi soluble

3 Sample powder + 10% aq. FeCl 3 Brownish gray Brownish gray Orchid shadow Light brown Semi soluble

4 Sample powder + dH 2O Blackish brown Brownish black Ash white Light mulberry Slightly soluble 5 Sample powder + HCL Conc. Blackish gray Purplish gray Orion Abbey cream Soluble 6 Sample powder + HCL 50% Grayish black Grayish brown Off white Shimmering sky Soluble

7 Sample powder + H 2SO 4 Conc. Dark brown Dark brown Halo Dark brown Soluble

8 Sample powder + H 2SO 4 50% Blackish gray Grayish black Brilliant white Classic ivory Soluble

9 Sample powder + HNO 3 Conc. Dark brown Brownish purple Orchid shadow Light brown Semi soluble

10 Sample powder + HNO 3 50% Blackish gray Dark gray White ice Satin white Semi soluble

11 Sample powder + CH 3OH Conc. Light gray Grayish black Antique white Light mulberry Semi soluble

12 Sample powder + CH 3OH 50% Grayish black Chocolate brown White ice Satin white Semi soluble

13 Sample powder + CHCl 3 Conc. Blackish brown Blackish gray Orchid shadow Antique white Semi soluble

14 Sample powder + CHCl 3 50% Dark brown Brownish gray Lavender white Orchid shadow Partially soluble

15 Sample powder + C 2H5OH Conc. Blackish gray Grayish black Shimmering sky Light mulberry Semi soluble

16 Sample powder + C 2H5OH 50% Chocolate brown Greenish gray White ice Lavender white Semi soluble

17 Sample powder + CH 3COOH Conc. Dark gray Brownish gray Brilliant white Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Blackish gray Light chocolate Ash white Snow bell Semi soluble

19 Sample powder + C 6H6 Conc. Grayish black Grayish black Satin white Halo Partially soluble

20 Sample powder + C 6H6 50% Dark brown Purplish gray Orchid shadow Light mulberry Partially soluble

Results and Discussion Chapter 3

Table 22: Physicochemical Parameters of Lallemantia royleana and Ocimum basilicum

S. No. Physicochemical parameters Lallemantia Ocimum (%) royleana basilicum i Total ash 6 21.33 ii Acid insoluble ash 0.67 11.66 iii Water soluble ash 2.33 1.67 iv Water insoluble ash 3.67 19.67 v Moisture content 6 10

Table 23: Antioxidant activity of Ascorbic acid, Lallemantia royleana and Ocimum basilicum

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic Lallemantia Ocimum acid royleana basilicum 25 50.91 36.09 25.02 50 60.47 36.41 25.34 100 72.39 40.60 26.42 150 81.06 41.99 27.71 200 92.8 45.11 28.46 250 96.02 47.69 29.43 IC 50 value 2.14 µg/mL 298 µg/mL 1277 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 70 Results and Discussion Chapter 3

20 Total Phenols 15 Total Flavonoids 10

5 Concentration (mg /g) (mg Concentration 0 Lallemantia Ocimum basilicum royleana Plant extracts

Figure 13 . Total Phenolic and Flavonoid contents

100 90 25µg/mL 80 70 50µg/mL 60 100µg/mL 50 150µg/mL 40 (%) 30 200µg/mL 20 250µg/mL 10 0 Ascorbic acid Lallemantia Ocimum

DPPH radical scavenging activityscavenging radical DPPH royleana basilicum Plant extracts and standard at different concentrations

Figure 14 . DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 71 Results and Discussion Chapter 3

Figure 15. HPLC chromatogram of Lallemantia royleana

Figure 16. HPLC chromatogram of Ocimum basilicum

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 72 Results and Discussion Chapter 3

3.2 Authentication of Herbal Drug Tukhm-e-balango (Lallemantia royleana Benth.)

Lallemantia royleana (Lamiaceae) is naturally occurring mucilaginous herb. Its seeds have great medicinal potential and are commonly used for the relief of rheumatism, joint inflammation, abscesses inflammations, osteoarthritis, fever and common cold (Moghaddam et al., 2011). Plant processes considerable medicinal properties and is an excellent source of oil, fiber and protein. Its mucilage is effective for gastrointestinal disorders and used for the treatment of hepatic, renal and nervous diseases (Naghibi et al., 2005). In herbal market, seeds of Tukhm-e-balango are generally confused with the seeds of Tukhm-e rehan (Ocimum basilicum ) due to their similar seed morphology, in turn decreasing the quality and therapeutic value of genuine drug. Sapna et al. (2008) reported that the main problem encountered by herbal industry in herbal drug preparation is the correct identification of raw material. During market surveys in Pakistan it is observed that under the trade name Tukhm-e-balango the seeds of Ocimum basilicum are sold and commonly mixed with genuine drug. We have differentiated the seeds of L. royleana from its adulterant O. basilicum by using morphological, palynological, foliar epidermal anatomy, pharmacognostic and phytochemical characterization. While in literature such botanical characters were used by number of researchers to differentiate the genuine herbal drugs from their closely related adulterants such as Valeriana wallichii from its market adulterant Acorus calamus (Ahmad et al., 2009).

Taxonomic Clarification

In interpreting plant identification and nomenclature related problems the morphological studies have proved to be immense aid. Morphologically L. royleana is characterized by oblong-obovate, obtuse, crenate leaves having short egalndular hairs and triquetrous nutlets (Figure 11a & 11b). While in O. basilicum leaves are ovate-lanceolate, acute, entire or toothed, nutlets are oblong-ellipsoid,

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 73 Results and Discussion Chapter 3

apically rounded and glabrous (Figure 12a & 12b). Detailed morphological features for differentiation of L. royleana and O. basilicum are presented in Table 13. In literature Jayaweera (1982) reported morphological description of L. royleana and Bihari et al. (2011) reported morphological characterization of O. basilicum. While the other workers used macro and micro morphological nutlets characteristics to differentiate various closely related taxa of family Lamiaceae from Saudi Arabia (Hassan and Al-Thobaiti, 2015).

Pollen morphological characters can also be used as microscopic tool to differentiate L. royleana and O. basilicum . Palynological features provide immense aid in establishing the systematic position of taxa within their respective classification by providing additional taxonomic characters. The palynological variations among L. royleana and O. basilicum are presented in Table 14 & 15. L. royleana can be distinguished from O. basilicum in having suboblate pollen (Figure 11c & 11d). While in O. basilicum the pollen are prolate-spheroidal (Figure 12c & 12d). According to Erdtman (1945) shape of pollen and number of apertures have a significance importance in family Lamiaceae. Dinç et al. (2009) reported considerable variations in exine sculpturing among the Lallemantia species. In previous studies, Ahmad (2008) used microscopic pollen features to distinguish authentic source of herbal drug Ount Katara ( Echinops echinata ) from its commercial adulterant Silybum marianum .

Similarly, foliar epidermal features can also be used to differentiate the L. royleana from O. basilicum . Foliar epidermal features of L. royleana and O. basilicum are shown in Table 16 & 17. Epidermal cells are irregular and trichomes are non-glandular and unicellular in L. royleana (Figure 11e & 11f). Whereas in case of O. basilicum the epidermal cells are polygonal and both glandular and non-glandular types of trichomes are found (Figure 12e & 12f). Adtani et al. (2014) reported that both glandular and non-glandular trichomes are found in O. basilicum . In family Lamiaceae the interspecific and intrageneric classification of some genera is based on trichome micromorphology (Navarro

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 74 Results and Discussion Chapter 3

and El Oualidi, 1999; Grubeši ć et al., 2007; Dınç, Öztürk, 2008). Several studies reported that different types of trichomes in terms of number and size of cell component are found in family Lamiaceae (Kahraman et al., 2010; Prabhu et al., 2009). According to Thanomchat and Paopun (2013) in family Lamiaceae trichomes structures and stomata types are frequently used for species identification. Similarly, based on micromorphology of trichomes Xiang et al. (2010) delimited seventeen taxa of family Lamiaceae and reported that trichomes can be used as microscopic taxonomic features for differentiation of closely related species of family Lamiaceae.

Pharmacognostic Analysis

Pharmacognostic tests such as physicochemical analysis can be used as important parameters for quality control and standardization of herbal drugs (Ashish and Sharma, 2011). World Health Organization (WHO) highlighted the role of pharmacognostic analysis for the standard herbal drug formulations at global perspective (Sharma and Kumar, 2011). Based on pharmacognostic tests seeds of L. royleana can be distinguished from seeds of O. basilicum. Fluorescence analysis and solubility tests of the powdered drugs with different chemical reagents are presented in Table 18, 19, 20 & 21. Use of pharmacognostic analysis is well established in previous literature such as Bihari et al. (2011) used pharmacognostical characterization as important tool for the standardization and botanical identity of various Ocimum species available in South Eastern Odisha. Results of physicochemical analysis presented in Table 22 shows that in O. basilicum ash value and moisture contents are very high as compare to L. royleana. Similarly, Brummer et al. (2003) evaluated the physicochemical characterization of L. royleana for its correct identification.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 75 Results and Discussion Chapter 3

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

Phytochemical characterization can furnish a basis of judging the authenticity of genuine drug and also to differentiate the drug from its allied species and adulterants. Quantitative investigation of phenols and flavonoids showed that L. royleana had higher concentration of total phenolic contents (27.21 mg GAE/g, DW) as compared to O. basilicum (15.33 mg GAE/g, DW). Whereas, flavonoid contents were higher in O. basilicum (18.16 mg QE/g, DW) as compared to L. royleana (16.03 mg QE/g, DW) (Figure 13). According to Aydemir and Becerik (2011) total phenolic contents in O. basilicum ranged from 74 to 92 mg GAE/100g in different solvent extracts. Variations in phenolic contents of different solvent extracts are attributed to polarities of different compounds present in seeds. Such variations in phenolic contents for other seeds have been cited in the literature by many researchers (Jayaprakasha et al., 2001).

HPLC Screening

On the basis of HPLC screening L. royleana can be differentiated from its adulterant. Results showed that slightly higher concentration of quercetin (15 mg/g, DW) was found in O. basilicum as compared to L. royleana (12.18 mg/g, DW). The HPLC chromatograms of L. royleana and O. basilicum are presented in Figure 15 & 16. Naidu et al. (2015) carried out the HPLC analysis of methanolic extracts of O. basilicum and Mentha spicata and recorded lower concentration of quercetin in aerial parts of O. basilicum . Similarly, Vlase et al. (2014) evaluated polyphenols in O. basilicum by HPLC analysis and found two free flavonoids i.e. luteolin and quercetin in small quantities (6.06 and 3.39 mg/g respectively).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 76 Results and Discussion Chapter 3

Antioxidant analysis

Based on antioxidant analysis the genuine drug can be distinguished from its adulterant. Results indicated that L. royleana showed higher DPPH scavenging effect (47.69%) as compared to O. basilicum (29.43%) at a concentration 250 µg/mL (Table 23 & Figure 14). Amanzadeh et al. (2011) analyzed the antioxidant activity of essential oil of L. iberica by DPPH radical scavenging assay and reported that essential oil of aerial parts showed highest radical scavenging potential. Aydemir and Becerik (2011) evaluated the antioxidant activity of different extracts from O. basilicum, Lepidium sativum and Apium graveolens seeds and recorded 56.4% scavenging activity in methanolic extract of O. basilicum seeds at a concentration 250 µg/mL. In literature antioxidant activity of O. basilicum leaves (60.75%) has also been reported by Zakaria et al. (2008).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 77 Case 3:

Table 24: Authentication of herbal drug Belladona (Atropa acuminata Royle.) in comparison with its adulterant

Characters Atropa acuminata Royle. Solanum nigrum L.

English Names Deadly nightshade, Belladona Common nightshade, Black nightshade

Local Names Angur-e-Shifa, Mako Siyah Mako, Kach-Mach

Trade Names Belladona, Luffah Mako

Family Solanaceae Solanaceae

Flowering period June-July March-December

Phytogeography In Pakistan Azad jammu and Kashmir, Hazara, Muree hill, Ayubia, Rawalpindi, Gujranwala, Mianwali, Attock, Hazara, Swat and Dir. Mansehra, Peshawar, Gilgit, Chitral, Quetta, Bannu,

Sibbi, Dera Nawab Khan, Mirpur and Muzaffarabad.

In World South central Europe, Southwestern Asia, Northwestern Cosmopolitan Africa, Mediterranean region, Canada, The United States East Afghanistan, East Iran, eastwards to Kashmir, India, Pakistan and Mongolia.

Morphological description Branched perennial herb, up to 1.8 m tall. Stem erect, Annual herb, 20-60 cm tall, base suffrutescent, shoots fistular and purplish in color. Young branches are pubescent to glandular-villous or subglabrous, hairs

puberulous. Leaves alternate, 8-17 x 5-8.0 cm, ovate- patent or appressed. Leaves 3-10 x 2-5 cm, glabrous to lanceolate to elliptic-lanceolate, cuneate and acuminate. puberulous-pubescent or glandular, sinuate to irregularly Petiole up to 20 mm, purplish at base. Calyx 9-15 mm, ± dentate. Lamina ± decurrent. Petiole 8-40 mm, glandular cupular, puberulous, up to 20 mm in fruit, lobes 6-10 or appressed pubescent. Peduncle 10-22 mn, shorter or mm, unequal, ovate-acute and persistent. Corolla 20-23 longer than the pedicle. Calyx lobes glabrous to mm, purplish yellow in color, lobes are obtuse. Anthers 3 pubescent, 1-1.5 mm long. Corolla longer than the calyx, mm long, filaments oblong, 10-11 mm long. Berry 10 lobes are 3-6 mm long, triangular-acute, 2 apical pores mm broad, globose, black when ripe. Seeds brown, 2 mm involved in dehiscing of anther. Filaments pilose, shorter long subreniform, reticulate and foveolate. than or as long as the anthers. Style base is pubescent. Stigma capitate and ovary glabrous. Berries 5.5-8 mm broad, globose to subovoid, orange-red or black. Seeds are reticulate-foveolate and discoid . Trade Part Leaves and roots are sold at the herbal shops of Pakistan. Leaves are used as adulterant of Atropa acuminata at herbal shops.

Organoleptography Aerial parts consist of branches, leaves and flowers. Dried parts consist of branches, leaves and flowers. Branches are erect, purplish to green, densely covered Branches are light green in color, angular, pubescent, with short, fine hairs. Leaves simple, broad, dark green ridges and furrows are present. Leaves ovate and dark having narcotic odor and disagreeably bitter taste Veins green in color. Veins are prominent on lower surface of are prominent on lower surface and depressed at upper leaves. Odor and taste are pleasant. Flowers are cup surface. Flower purplish with pale base, bell shaped and shaped and white in color. Berries are globose and dull solitary. Berries are dark black in color and full of sweet black in color. juice.

Part used Leaves, roots Leaves, young stem, fruits

Medicinal Uses Asthma, whooping cough, hay fever, peptic ulcer, Digestive problems, fever, burns, rheumatic pains and diarrhea, joints pain, boils, inflammation, Parkinson's skin disorders. disease, heart diseases, eye infections and skin problems.

Traditional herbal recipes Leaves are applied externally to treat boils. Dried leaves Leaves are chewed to treat mouth ulcers. Leaves extract are smoked as an antispasmodic. Extract prepared from is applied topically for painful eyes. Tea made from leaves causes dilation of pupil of the eyes and used in younger leaves is used for curing fever, cough and flue. ophthalmic surgery. Paste of crushed roots is applied The extract of plant is given thrice a day for the externally for the treatment gout and rheumatism. treatment of chronic enlargement of liver, piles and Belladonna is very poisonous plant and should be used dysentery. Fresh leaves are cooked as vegetable and are only under the supervision of a qualified practitioner. recommended to control diabetes. Leaves and young Belladonna is traditionally thought to be safe in smaller twigs are boiled in water for 10-15 minutes and then doses, but may cause frequent side effects such as dry seasoned with roasted garlic and condiments in ghee. mouth, blushing of the skin, dilated pupils, rapid Poultice of leaves is effective for skin burns and heartbeat, nervousness, and hallucinations. High doses wounds. can cause death.

Table 25: Comparative qualitative pollen morphology of Atropa acuminata and Solanum nigrum

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Atropa acuminata Tricolporate Circular Prolate Present Striate

2 Solanum nigrum Tricolporate Semi-angular Prolate-spheroidal Present Scabrate

Table 26: Comparative quantitative pollen morphology of Atropa acuminata and Solanum nigrum

Sr # Species name Polar diameter Equatorial diameter P/E ratio Colpi length Colpi width Exine thickness (μm) (μm) (μm) (μm) (μm)

1 Atropa acuminata 27 (24.5-30.2) 29.5 (27-32.5) 0.91 0.85 (0.75-1) 4.2 (3.8-4.75) 1.2 (1-1.5)

2 Solanum nigrum 24 (22.5-26) 22.3 (20.5-24.2) 1.07 0.72 (0.62-0.85) 1.45 (1.25-1.75) 1.72 (1.6-1.95)

Table 27. Comparative qualitative characters of foliar epidermal anatomy of Atropa acuminata and Solanum nigrum

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Atropa Irregular Heavily Anisocytic Glandular Irregular Heavily Anisocytic Glandular acuminata undulate Peltate undulate Peltate

Solanum Irregular Heavily Anomocytic Non-glandular, Irregular Heavily Anomocytic Non-glandular, nigrum undulate Unicellular undulate Unicellular

Table 28. Comparative quantitative characters of foliar epidermal anatomy of Atropa acuminata and Solanum nigrum

Abaxial epidermis Adaxial epidermis

Atropa 59.3 (55.5-63.5) 39.2 (37.5-40.5) 23 49.3 (47.5-51) × 61.5 (57.8-65.5) × 36.5 (35.8-37.6) 19.8 46.5 (45-48.5) × 20 acuminata × 37.5 (34.5- × 24 (22.7-25.5) 18.5 (17.5-19.3) 39.25 (37.3-41.4) × 21.4 (20-23.2) (19-21.5) 39.9)

Solanum 37.6 (35-40.5) × 21.8 (19.7-24.2) 22.5 120 (55-187.5) × 43.35 (41.2-45.7) 23.25 (21.3-25) × 17.4 129.2 (69.25-185) nigrum 25.2 (22.5-28) × 11.7 (11.25- 17.4 (12.5-22.5) × 23.7 (20.5-27) 12.4 (11.75-13.2) × 19 (16.8-21.3) 12.5)

Results and Discussion Chapter 3

Figure 17 (a) Atropa acuminata ; (b) Dried leaves; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 83 Results and Discussion Chapter 3

Figure 18 (a) Solanum nigrum ; (b) Dried leaves; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 84

Table 29: Fluorescence and solubility analysis of powdered drug of Atropa acuminata (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Muddy green Leaf green - - - 2 Sample powder + 5% KOH Pine forest Brownish black Golden brown Yellowish brown Partially soluble

3 Sample powder + 10% aq. FeCl 3 Leaf green Black Pinkish brown Dark brown Partially soluble

4 Sample powder + dH 2O Leaf green Brownish black Orchid shadow Light mulberry Semi soluble 5 Sample powder + HCL Conc. Pine forest Black green Yellowish brown Golden brown Soluble 6 Sample powder + HCL 50% Greenish black Greenish brown Orchid shadow Pinkish brown Semi soluble

7 Sample powder + H 2SO 4 Conc. Blackish brown Blackish brown Dark Brown Dark brown Soluble

8 Sample powder + H 2SO 4 50% Brownish black Brownish black Brownish black Reddish brown Semi soluble

9 Sample powder + HNO 3 Conc. Reddish brown Dark brown Golden brown Brown Soluble

10 Sample powder + HNO 3 50% Red oxide Dark brown Pinkish brown Reddish brown Semi soluble

11 Sample powder + CH 3OH Conc. Leaf green Greenish black Yellow Yellow Partially soluble

12 Sample powder + CH 3OH 50% Grapes green Leaf green Pinkish brown Light mulberry Partially soluble

13 Sample powder + CHCl 3 Conc. Leaf green Leaf green Brownish green Pinkish green Soluble

14 Sample powder + CHCl 3 50% Leaf green Signal green Pinkish brown Pinkish green Partially soluble

15 Sample powder + C 2H5OH Conc. Golden glimmer Golden glimmer Orchid shadow Light mulberry Soluble

16 Sample powder + C 2H5OH 50% Pine forest Black Brownish green Pinkish brown Partially soluble

17 Sample powder + CH 3COOH Conc. Leaf green Leaf green Orchid shadow Pinkish brown Partially soluble

18 Sample powder + CH 3COOH 50% Leaf green Signal green Brown Reddish brown Soluble

19 Sample powder + C 6H6 Conc. Light green Leaf green Orchid shadow Pinkish yellow Soluble

20 Sample powder + C 6H6 50% Light green Leaf green Yellowish brown Pinkish yellow Partially soluble

Table 30: Fluorescence and solubility analysis of powdered drug of Atropa acuminata (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Muddy green Leaf green - - - 2 Sample powder + 5% KOH Dark green Greenish black Golden glimmer Yellowish brown Semi soluble

3 Sample powder + 10% aq. FeCl 3 Pine forest Reddish brown Pinkish brown Brown Semi soluble

4 Sample powder + dH 2O Light green Deep green Light Pink Golden brown Semi soluble 5 Sample powder + HCL Conc. Greenish black Dark black Yellowish brown Pinkish brown Soluble 6 Sample powder + HCL 50% Pine forest Blackish green Brownish pink Brownish black Soluble

7 Sample powder + H 2SO 4 Conc. Brownish black Brownish black Brownish black Dark brown Soluble

8 Sample powder + H 2SO 4 50% Dark black Dark brown Brownish black Reddish brown Soluble

9 Sample powder + HNO 3 Conc. Orange brown Blackish brown Golden brown Dark brown Soluble

10 Sample powder + HNO 3 50% Orange green Orange brown Pinkish brown Reddish brown Soluble

11 Sample powder + CH 3OH Conc. Rado green Reddish brown Pinkish Yellow Yellowish brown Semi soluble

12 Sample powder + CH 3OH 50% Golden glimmer Leaf green Pinkish brown Pinkish black Semi soluble

13 Sample powder + CHCl 3 Conc. Light green Brownish green Brownish green Pinkish green Soluble

14 Sample powder + CHCl 3 50% Leaf green Reddish green Pinkish brown Pinkish brown Soluble

15 Sample powder + C 2H5OH Conc. Spring green Capitol’s gold Pinkish red Dark Pink Soluble

16 Sample powder + C 2H5OH 50% Leaf green Brownish green Brownish green Pinkish brown Semi soluble

17 Sample powder + CH 3COOH Conc. Leaf green Reddish brown Light pink Brownish green Soluble

18 Sample powder + CH 3COOH 50% Dark brown Brownish black Dark Brown Reddish brown Soluble

19 Sample powder + C 6H6 Conc. Leaf green Reddish brown Dark Pink Brownish green Soluble

20 Sample powder + C 6H6 50% Golden glimmer Pine forest Brownish yellow Pinkish yellow Partially soluble

Table 31: Fluorescence and solubility analysis of powdered drug of Solanum nigrum (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Dull green Blackish green - - - 2 Sample powder + 5% KOH Golden grren Dark green Yellowish brown Golden yellow Partially soluble

3 Sample powder + 10% aq. FeCl 3 Golden brown Blackish green Swiss almond Dark yellow Partially soluble

4 Sample powder + dH 2O Dark green Charcoal Pinkish yellow Pinkish brown Partially soluble 5 Sample powder + HCL Conc. Pine forest Greenish black Yellowish green Yellowish pink Soluble 6 Sample powder + HCL 50% Cameo/Cream Spring green Off white Light mulberry Partially soluble

7 Sample powder + H 2SO 4 Conc. Brownish black Black Brownish Green Deep black Soluble

8 Sample powder + H 2SO 4 50% Black Black Brownish green Dark green Semi soluble

9 Sample powder + HNO 3 Conc. Golden brown Blackish brown Greenish brown Yellowish brown Soluble

10 Sample powder + HNO 3 50% Reddish brown Reddish gray Pinkish brown Dark Pink Semi soluble

11 Sample powder + CH 3OH Conc. Dark green Greenish brown Light mulberry Light mulberry Semi soluble

12 Sample powder + CH 3OH 50% Golden glimmer Leaf green Light mulberry Pinkish brown Slightly soluble

13 Sample powder + CHCl 3 Conc. Light green Yellowish pink Pinkish yellow Pinkish brown Semi soluble

14 Sample powder + CHCl 3 50% Leaf green Lake green Light mulberry Light mulberry Slightly soluble

15 Sample powder + C 2H5OH Conc. Spring green Reddish green Yellowish pink Light mulberry Partially soluble

16 Sample powder + C 2H5OH 50% Leaf green Lake green Pinkish brown Pinkish brown Partially soluble

17 Sample powder + CH 3COOH Conc. Light mustard Reddish brown Off white Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Mustard Black Pinkish yellow Pinkish brown Partially soluble

19 Sample powder + C 6H6 Conc. Pistachio green County cream Yellow Light mulberry Slightly soluble

20 Sample powder + C 6H6 50% Leaf green Yellowish brown Light mulberry Pinkish yellow Slightly soluble

Table 32: Fluorescence and solubility analysis of powdered drug of Solanum nigrum (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Dull green Blackish green - - - 2 Sample powder + 5% KOH Golden glimmer Blackish brown Yellowish brown Golden glimmer Semi soluble

3 Sample powder + 10% aq. FeCl 3 Dark brown Deep black Light yellow Dark yellow Semi soluble

4 Sample powder + dH 2O Leaf green Leaf green Pinkish yellow Yellowish pink Slightly soluble 5 Sample powder + HCL Conc. Dark green Greenish black Greenish yellow Swiss almond Soluble 6 Sample powder + HCL 50% Dark brown Brownish black Dark yellow Light mulberry Soluble

7 Sample powder + H 2SO 4 Conc. Blackish brown Brownish black Golden glimmer Deep black Soluble

8 Sample powder + H 2SO 4 50% Blackish green Blackish green Golden glimmer Dark green Soluble

9 Sample powder + HNO 3 Conc. Pale cream Beige Greenish brown Yellowish brown Soluble

10 Sample powder + HNO 3 50% Reddish brown Daisy chain Pinkish brown Dark Pink Soluble

11 Sample powder + CH 3OH Conc. Leaf green Leaf green Orchid shadow Light mulberry Soluble

12 Sample powder + CH 3OH 50% Pine forest Leaf green Pinkish green Pinkish brown Semi soluble

13 Sample powder + CHCl 3 Conc. Reddish black Pine forest Pinkish yellow Pinkish brown Semi soluble

14 Sample powder + CHCl 3 50% Spring green Leaf green Pinkish green Light mulberry Semi soluble

15 Sample powder + C 2H5OH Conc. Spring green Foliage Orchid shadow Yellowish pink Sparingly soluble

16 Sample powder + C 2H5OH 50% Dark green Dark green Pinkish brown Pinkish brown Sparingly soluble

17 Sample powder + CH 3COOH Conc. Mustard brown Black Yellowish pink Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Golden brown Brownish black Pinkish yellow Pinkish brown Soluble

19 Sample powder + C 6H6 Conc. Spring green Golden glimmer Orchid shadow Light mulberry Soluble

20 Sample powder + C 6H6 50% Leaf green Leaf green Pinkish green Pinkish yellow Semi soluble

Results and Discussion Chapter 3

Table 33: Physicochemical Parameters of Atropa acuminata and Solanum nigrum

S. No. Physicochemical parameters Atropa acuminata Solanum (%) nigrum i Total ash 12 15 ii Acid insoluble ash 0.67 2.33 iii Water soluble ash 2 3.66 iv Water insoluble ash 10 11.33 v Moisture content 6 7

Table 34: Antioxidant activity of Ascorbic acid, Atropa acuminata and Solanum nigrum

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic acid Aropa acuminata Solanum nigrum 25 50.91 28.89 28.14 50 60.47 52.09 29.64 100 72.39 54.35 33.51 150 81.06 56.28 37.80 200 92.8 57.57 38.23 250 96.02 61.33 42.42 IC 50 value 2.14 µg/mL 4 µg/mL 372 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 89 Results and Discussion Chapter 3

70 60 Total Phenols 50 Total Flavonoids 40 30 20 10 Concentration (mg /g) (mg Concentration 0 Atropa acuminata Solanum nigrum Plant extracts

Figure 19 . Total Phenolic and Flavonoid contents

100 90 25µg/mL 80 50µg/mL 70 100µg/mL 60 50 150µg/mL 40 200µg/mL 30 250µg/mL 20 10 0 Ascorbic acid Atropa Solanum acuminata nigrum DPPH radical scavenging activity (%) activityscavenging radical DPPH Plant extracts and standard at different concentrations

Figure 20 . DPPH radical scavenging activity (%)

______Authentication of M edicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 90 Results and Discussion Chapter 3

Figure 21. HPLC chromatogram of Atropa acuminata

Figure 22. HPLC chromatogram of Solanum nigrum

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 91 Results and Discussion Chapter 3

3.3 Authentication of Herbal Drug Belladona ( Atropa acuminata Royle.)

Atropa acuminata (Solanaceae) has a significant importance for its use in medicines and cosmetics. Although it is poisonous but has a long history of medicinal use. Plant is used in traditional medicines for the treatment of headache, inflammation, menstrual problems, motion sickness and peptic ulcer disease as well as in homeopathic medicines (Chevallier, 1996). Belladonna drops are used to dilate pupil and eyes (Evans and Albert, 1979; Bousta et al., 2001). In herbal market the leaves of A. acuminata are sold under the trade name belladona, but the plant is commonly confused with Solanum nigrum due to similar morphology of leaves and berries. S. nigrum is commonly known as black nightshade and used for the treatment of boils, wounds and leucoderma (Moerman, 1998).

Taxonomic Clarification

A. acuminata can be differentiated from its morphologically similar adulterant S. nigrum in having erect, fistular, puberulous and purplish colored stem (Figure 17a). However in case of S. nigrum stem is rigid, pubescent and pale green in color (Figure 18a). Comparative description of A. acuminata and its adulterant is presented in Table 24. Morphological description of A. acuminata and S. nigrum has been reported in literature by many workers (Rita and Animash, 2011; Jani et al., 2012). Palynologically, A. acuminata can be differentiated from S. nigrum as in A. acuminata pollen are circular in polar view and prolate in equatorial view with striate exine sculpturing (Figure 17c & 17d) whereas in case of S. nigrum pollen are semi-angular in polar view and prolate- spheroidal in equatorial view with scabrate exine sculpturing (Figure 18c & 18d). On the basis of exine sculpturing A. acuminata can easily be distinguished from S. nigrum. Palynological variations between A. acuminata and S. nigrum are given in Table 25 & 26. According to Hesse (1981) and Meltsov et al. (2008) the size of pollen grain plays a significant role in delimiting some taxa within the family. The equatorial diameter of pollen grains in case of A. acuminata is larger as compared

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 92 Results and Discussion Chapter 3

to S. nigrum . Perveen and Qaiser (2007) used pollen morphological features to differentiate twenty species of family Solanaceae and reported that pollen morphological features of the family are significantly helpful at the generic and specific level. Similarly, Lashin (2011) on the basis of palynological variations delimited six species of Solanum from Saudi Arabia and reported that tricolporate pollen and scabrate tectum is more commonly found in family Solanaceae.

Regarding foliar epidermal features A. acuminata can be authenticated from S. nigrum by the presence anisocytic stomata and glandular peltate trichomes (Figure 17e & 17f) whereas in case of S. nigrum stomata are anomocytic and trichomes are non-glandular and unicellular (Figure 18e & 18f). Foliar epidermal features of A. acuminata and S. nigrum are presented in Table 27 & 28. Adedeji et al. (2007) reported that trichomes have considerable taxonomic importance in family Solanaceae. Nkem et al. (2007) on the basis of leaf epidermal variations distinguished S. nigrum from S. macrocarpum . Similarly, Nurit-Silva et al. (2012) employed the type and distribution of stomata along with their foliar features for differentiation of ten species of Solanum .

Pharmacognostic Authentication

Pharmacognostic analysis can also be used to authenticate the herbal drug belladonna from its adulterant. Fluorescence analysis is accurate and sensitive method for authentication of herbal drugs. The fluorescence analysis and solubility tests of belladonna and its adulterant are presented in Table 29, 30, 31 & 32. Results of physicochemical analysis indicated that high %age of total ash and moisture content was observed in S. nigrum as compared to A. acuminata (Table 33). Jani et al. (2012) conducted pharmacognostic study of S. nigrum to ensure correct botanical standardization. Our findings regarding physicochemical analysis of S. nigrum are in agreement with previous studies (Sreeramulu, 1982; Sebit, 1995; Jagtap et al., 2013).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 93 Results and Discussion Chapter 3

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

A. acuminata can be differentiated from S. nigrum on the basis of phytochemical characterization. Quantitative investigation of total phenolic and flavonoid contents indicated that higher concentration of phenolic (36.16 mg GAE/g, DW) and flavonoid contents (62.49 mg QE/g, DW) was found in leaves of A. acuminata as compared to S. nigrum where phenolic content were 13.59 mg GAE/g, DW and flavonoid contents were 23.86 mg QE/g, DW (Figure 19). Total phenols and flavanoids contents of five species of family Solanaceae from Iran have been reported by Khalighi-Sigaroodi et al. (2012), they recorded total phenols (11.32 mg GAE/g, DW) and total flavonoid contents (22.80 mg RE/g, DW) in S. nigrum leaves. Similarly, Padmashree et al. (2014) reported total phenolic and flavonoid contents in methanolic extract of S. nigrum and results are in accordance to our findings.

HPLC Screening

HPLC can be used to analyze almost all compounds in medicinal plants. In current study HPLC was used as one of the characterization methods to evaluate the quality of medicinal plants. For each family, the chemical profile as expressed by occurrence of the secondary metabolites is remarkably distinctive (Young et al., 1996). The plants of family Solanaceae contain bioactive compounds like alkaloids, steroids, flavonoids and saponins etc. (Nicoletti et al., 2008). The result of present study showed that concentration of quercetin was higher in A. acuminata (53.76 mg/g, DW) as compared to S. nigrum (14.10 mg/g, DW) (Figure 21 & 22). Lim (2012) also observed lower concentration of quercetin in S. nigrum as compared to other members of family Solanaceae.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 94 Results and Discussion Chapter 3

Antioxidant analysis

Results of antioxidant analysis indicated that A. acuminata showed higher

%age inhibition than S. nigrum (Figure 19). IC 50 value for methanolic extract of A. acuminata was 4.41 µg/mL while for S. nigrum its value was 372 µg/mL (Table 34 & Figure 20). It is observed that like other plants of family Solanaceae, A. acuminata has a strong antioxidant potential. Munir et al. (2014) reported that A. belladonna showed the maximum scavenging (62.64%) at highest concentration 8 μg/mL. According to Khalighi-Sigaroodi et al. (2012) four species of family Solanaceae including S. nigrum, S. dulcamara , S. incanum and D. innoxia presented high free radical scavenging activity. Our results of antioxidant activity of S. nigrum are comparable with previous reported literature (Balasuriya and Dharmaratne, 2007; Sharma et al., 2014a).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 95 Case 4:

Table 35: Authentication of herbal drug Dhatura ( Datura stramonium L.) in comparison with its adulterant

Characters Datura stramonium L. Xanthium strumarium L.

English Names Thorn apple, Jimson weed, Stramonium, Devil's apple Cocklebur, Common cocklebur, Rough cocklebur

Local Names Dhatura Muhabbat booti, Chota dhatura

Trade Names Dhatura/Gul-e-siya None

Family Solanaceae Asteraceae

Flowering period June-July July-August

Phytogeography: In Pakistan Chitral, Dir, Swat, gilgit, Hunza, Abbotabad, Thandiani, Northern parts of NWFP, Chitral, Astor, Swat, Ayubia, Muree, Nathiagali, Naran, Muzafarabad, Hazara Peshawar, Abbotabad, Haripur, Attock, Mansehra,

and Kohat. Rawalpindi, Jehlum and Multan.

In World Tropical regions of Central and South America, North Europe, Central Asia, Western Siberia, Western and America, Europe, Asia, New Zealand, Africa Canada and Eastern Mediterranean, Iran, Afghanistan, India, Australia. Pakistan, Ceylon, Mongolia, China, Northern America, Northern Africa, southern Canada, United States and Mexico. Morphological description Annual herb, 60-120 cm tall, stem stout, erect, branched, Annual herb, 30-120 cm tall, nodal spines absent, stem

pubescent, the branches are often pale green in color. purplish, short, stout, hairy. Petiole not winged, 4-10 cm, Leaves large, alternate, ovate, 8-17 x 4-13 cm, sinuately leaves 9-25 cm, cauline, papery, ovate-deltate, both dentate, puberulose and cuneate. Petiole 2-5 cm long. surfaces are densely scabrid, apex acute, base broadly Flowers are 8 cm, large, white or violet in color. Calyx 4- cuneate to shallowly cordate, margin 3-lobed and 5 cm, puberulous, tubular, 5-dentate and persistent. irregularly dentate. Monoecious capitula. Male capitula Lobes 7-10 mm, apiculate, strongly reflexed in fruit. in terminal umbels, phyllaries 2.2 mm, oblong- Corolla whitish, 7-10 cm, funnel shaped, limb 8 cm lanceolate, 1-seriate, inner paleae 2.2 mm, lanceolate, broad, shallowly, 5-lobed, triangular-acuminate. Anthers outer paleae oblong-lanceolate, corolla 2.5 mm, white, 5 mm, lobes oblong and narrow, usually white. Capsule 5-dentate, tubular. Female capitula axillary, phyllaries 3 3.5-4.5 cm, ovoid, erect, densely pubescent and spiny, 4- mm, oblong-lanceolate, 1-seriate, inner bracts connate valved and spines up to 5 mm long. Seeds 3 mm, with outer paleae. Burs 10-18 × 6-12 mm, oblong, reniform, black, reticulate-foveolate. ellipsoid or ovoid, sessile, 2-beaked and densely puberulent. Trade Part Dried leaves are sold in herbal market under the trade Dried leaves are mixed with leaves of dhatura at herbal name dhatura. shops. Organoleptography Dried branches are pale green in color, stout and hollow. Dried branches are purplish green in color, rigid, Dried leaves are wrinkled, stalked, unequal at the base, pubescent and angular. Dried leaves are shriveled, yellowish green in color and characterized by very coarse irregularly lobed, having stiff hairs and dull green in pointed teeth. Branching veins are plainly developed. color. Veins are prominent. Odor is very irritating and Odor is very unpleasant and taste is very bitter. Flowers unpleasant. Flowers are pale green to yellowish green in are white to light purple. Capsule ovoid and covered with color. Capsule is hard, woody, brown in color and prickle. Seeds are dull black or dark brown and wrinkled. covered with stout, hooked prickled.

Part used Leaves, seeds, roots Leaves, roots, seeds

Ethomedicinal Uses Asthma, bronchitis, joints pain, gouts, sinus infections, Diarrhea, constipation, flatulence leprosy, kidney

fever, skin disorders, dental infection, respiratory diseases, nasal infection, joints pain, cuts and scrapes. congestions, burns, ulcers and swellings.

Traditional herbal recipes Fresh green leaves are applied for the softening of the Leaves are effective for treatment of asthma. Leaf boils. Leaves powder mixed with mustard oil is useful in decoction is used in long standing malarial fever. Juice skin disorders. Leaves are smoked in form of cigarettes of leaves and fruits is very effective for small pox. Ash or in a pipe to get relief of bronchial congestion. Leaves of stem is used as pain killer. Root decoction is used in poultice is applied externally to treats swellings, ulcers leucorrhoea and high fever. Seeds decoction is effective and burns. Roasted leaves are applied externally to for bladder complains. The herb is reported to be used in relieve joints pain. Juice of fruits is applied to the scalp snake bite. According to Chinese Herbal Medicine for falling hairs and as antidandruff. Juice of flowers is Materia Medica, plant is slightly toxic Over dosage may effective for earache. Powder of seeds is mixed with cause diarrhea, nausea, vomiting, and abdominal pain. mustard oil and used externally to treat rheumatism. The toxic ingredients of herb are not significantly Seeds smoke is given to cure the toothache. Seed present after it has been decocted or subjected to high infusion is used as a tonic for horses. Plant is toxic in heat. nature, should be used under medical supervision. Over dosage may cause death, smaller dosage causes varying symptoms, like impaired vision, thirst, flushed skin leading to convulsions and coma.

Table 36: Comparative qualitative pollen morphology of Datura stramonium and Xanthium strumarium

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Datura stramonium Tricolporate Sub-angular Oblate-spheroidal Present Reticulate

2 Xanthium Tricolporate Circular Prolate-spheroidal Present Echinate strumarium

Table 37: Comparative quantitative pollen morphology of Datura stramonium and Xanthium strumarium

Sr # Species name Polar diameter Equatorial diameter P/E ratio Colpi length Colpi width Exine thickness (μm) (μm) (μm) (μm) (μm)

1 Datura stramonium 41.5 (39.5-43.3) 47.5 (44-50.5) 0.87 1.12 (1-1.25) 1.8 (1.75-2) 0.92 (0.625-1.25)

2 Xanthium strumarium 27.5 (25.5-28.5) 25 (23-26.5) 1.1 1.2 (0.75-1.25) 2 (1.75-2.5) 2.25 (2.5-2.75)

Table 38. Comparative qualitative characters of foliar epidermal anatomy of Datura stramonium and Xanthium strumarium

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Datura Polygonal Straight Anisocytic Glandular, Polygonal Straight Anisocytic Glandular, stramonium Peltate Peltate

Xanthium Irregular Heavily Anomocytic Non-glandular, Irregular Heavily Anomocytic Non-glandular, strumarium undulate Uniserriarte, undulate Uniserriarte, Unicellular Unicellular

Table 39. Comparative quantitative characters of foliar epidermal anatomy of Datura stramonium and Xanthium strumarium

Abaxial epidermis Adaxial epidermis

Datura 47.25 (45.25-49) 27.5 (25.8-29.4) 26.9 51 (49.5-53) × 49.5 (48-51.5) × 26 (24.5-27.7) × 23.7 46 (45-47.5) × 17.5 stramonium × 37.5 (35.7- × 22.45 (20.5- 16.5 (15-17.5) 35.5 (33.7-37.5) 22.75 (21.2-24.7) (16.5-19) 39.2) 24.5)

Xanthium 39.5 (37.5-42) × 25.6 (25-26.5) × 19.3 62.25 (37.5-87.5) 38.5 (35.5-41) × 25.5 (24-27.5) × 17.6 58.2 (42.5-73.5) × strumarium 23.6 (22.5-25) 22.7 (21.25-24.5) × 22.6 (17.5- 28) 24.3 (23.5-25.5) 21.5 (19.5-23.8) 23.5 (20-27.5)

100

Results and Discussion Chapter 3

Figure 23 (a) Datura stramonium ; (b) Dried leaves; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 101 Results and Discussion Chapter 3

Figure 24 (a) Xanthium strumarium ; (b) Dried leaves; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 102

Table 40: Fluorescence and solubility analysis of powdered drug of Datura stramonium (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Golden green Leaf green - - - 2 Sample powder + 5% KOH Dark brown Blackish brown Dark brown Golden glimmer Soluble

3 Sample powder + 10% aq. FeCl 3 Black brown Deep black Brownish black Golden mustard Semi soluble

4 Sample powder + dH 2O Dark green Blackish green Yellowish green Pinkish yellow Slightly soluble 5 Sample powder + HCL Conc. Pine forest Blackish green Orchid shadow Light mulberry Semi soluble 6 Sample powder + HCL 50% Yellowish green Greenish black Yellowish pink Yellowish brown Semi soluble

7 Sample powder + H 2SO 4 Conc. Blackish brown Chocolate brown Blackish brown Brown Soluble

8 Sample powder + H 2SO 4 50% Pine forest Greenish black Dark brown Yellowish brown Semi soluble

9 Sample powder + HNO 3 Conc. Reddish brown Chocolate brown Brown Golden yellow Soluble

10 Sample powder + HNO 3 50% Dark brown Dark brown Yellowish pink Pinkish yellow Semi soluble

11 Sample powder + CH 3OH Conc. Spring green Leaf green Dark yellow Pinkish yellow Soluble

12 Sample powder + CH 3OH 50% Leaf green Brownish green Pinkish yellow Golden yellow Semi soluble

13 Sample powder + CHCl 3 Conc. Dark green Pine forest Dark brown Light brown Soluble

14 Sample powder + CHCl 3 50% Light green Brownish green Light pink Dark pink Semi soluble

15 Sample powder + C 2H5OH Conc. Pine forest Dark green Yellow Golden yellow Soluble

16 Sample powder + C 2H5OH 50% Leaf green Grass green Pinkish yellow Pinkish yellow Semi soluble

17 Sample powder + CH 3COOH Conc. Greenish yellow Algal green Orchid shadow Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Pine forest Greenish black Brownish yellow Brownish yellow Partially soluble

19 Sample powder + C 6H6 Conc. Light green Chocolate brown Orchid shadow Light mulberry Soluble

20 Sample powder + C 6H6 50% Pine forest Blackish brown Pinkish yellow Pinkish yellow Soluble

103

Table 41: Fluorescence and solubility analysis of powdered drug of Datura stramonium (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Golden green Leaf green - - - 2 Sample powder + 5% KOH Brownish black Dark brown Deep black Golden mustard Soluble

3 Sample powder + 10% aq. FeCl 3 Dark brown Deep black Brownish black Golden glimmer Semi soluble

4 Sample powder + dH 2O Leaf green Bottle green Yellowish green Light mulberry Semi soluble 5 Sample powder + HCL Conc. Brownish black Greenish black Yellowish pink Pinkish yellow Soluble 6 Sample powder + HCL 50% Brownish green Greenish brown Orchid shadow Yellowish brown Soluble

7 Sample powder + H 2SO 4 Conc. Chocolate brown Blackish brown Dark brown Brown Soluble

8 Sample powder + H 2SO 4 50% Brownish black Deep black Blackish brown Yellowish brown Soluble

9 Sample powder + HNO 3 Conc. Orange red Brownish black Dark brown Golden yellow Soluble

10 Sample powder + HNO 3 50% Reddish orange Reddish brown Pinkish yellow Yellowish brown Soluble

11 Sample powder + CH 3OH Conc. Leaf green Dark green Dark yellow Pinkish yellow Soluble

12 Sample powder + CH 3OH 50% Spring green Yellowish green Pinkish yellow Golden yellow Semi soluble

13 Sample powder + CHCl 3 Conc. Glowing green Grass green Blackish brown Light brown Soluble

14 Sample powder + CHCl 3 50% Pine forest Chocolate brown Yellowish pink Yellowish pink Semi soluble

15 Sample powder + C 2H5OH Conc. Grass green Dark green Light yellow Golden yellow Soluble

16 Sample powder + C 2H5OH 50% Leaf green Gold dust Pinkish yellow Pinkish yellow Semi soluble

17 Sample powder + CH 3COOH Conc. Greenish brown Greenish brown Pinkish green Greenish pink Soluble

18 Sample powder + CH 3COOH 50% Golden green Brownish green Orange yellow Brownish yellow Semi soluble

19 Sample powder + C 6H6 Conc. Leaf green Algal green Orchid shadow Pinkish yellow Soluble

20 Sample powder + C 6H6 50% Dark green Leaf green Pinkish yellow Light mulberry Soluble

104

Table 42: Fluorescence and solubility analysis of powdered drug of Xanthium strumarium (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Dark green Light lichen _ _ _ 2 Sample powder + 5% KOH Pine forest Greenish purple Brownish green Satin white Partially soluble

3 Sample powder + 10% aq. FeCl 3 Leaf green Grayish green Spiny green Light mulberry Partially soluble

4 Sample powder + dH 2O Marsh green Celery seed Yellowish green Light brown Partially soluble 5 Sample powder + HCL Conc. Blackish brown Brownish black Brownish black Classic ivory Soluble 6 Sample powder + HCL 50% Capitols gold Orange brown Satin white Light brown Semi soluble

7 Sample powder + H 2SO 4 Conc. Brownish green Medford green Light mulberry Chocolate brown Soluble

8 Sample powder + H 2SO 4 50% Mansion gold Light lichen Antique white Snow bell Semi soluble

9 Sample powder + HNO 3 Conc. Orange red Reddish brown Light mulberry Spiny green Soluble

10 Sample powder + HNO 3 50% Dark orange Copper Light orange Pinkish green Semi soluble

11 Sample powder + CH 3OH Conc. Marsh green Brownish green Light leaf Caramel cream Slightly soluble

12 Sample powder + CH 3OH 50% Yellowish green Rust Satin white Abbey cream Slightly soluble

13 Sample powder + CHCl 3 Conc. Bright green Reddish brown Spiny green Pinkish green Semi soluble

14 Sample powder + CHCl 3 50% Yellowish green Golden dust Light orange Greenish blue Slightly soluble

15 Sample powder + C 2H5OH Conc. Yellowish green Plum frost White ice Abbey cream Semi soluble

16 Sample powder + C 2H5OH 50% Leaf green Grass green Brilliant white Abbey cream Semi soluble

17 Sample powder + CH 3COOH Conc. Greenish golden Orange brown Yellowish orange Pinkish green Soluble

18 Sample powder + CH 3COOH 50% Yellowish green Leaf green Brilliant white Abbey cream Semi soluble

19 Sample powder + C 6H6 Conc. Pine forest Reddish brown Yellowish green Pinkish green Slightly soluble

20 Sample powder + C 6H6 50% Blackish green Dirty red Lavender white Pinkish green Slightly soluble

105

Table 43: Fluorescence and solubility analysis of powdered drug of Xanthium strumarium (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried leaves powder Dark green Light lichen _ _ _ 2 Sample powder + 5% KOH Dark green Purplish green Brownish green Satin white Semi soluble

3 Sample powder + 10% aq. FeCl 3 Leaf green Brownish green Spiny green Light mulberry Semi soluble

4 Sample powder + dH 2O Marsh green Yellowish green Spinney green Light brown Partially soluble 5 Sample powder + HCL Conc. Brownish green Blackish green Chocolate brown Classic ivory Soluble 6 Sample powder + HCL 50% Blackish maroon Chocolate brown Copper bell Deep black Soluble

7 Sample powder + H 2SO 4 Conc. Dark brown Light brown Blackish green Dark purple Soluble

8 Sample powder + H 2SO 4 50% Woven slates Willow branch Light leaf Caramel cream Soluble

9 Sample powder + HNO 3 Conc. Fresh orange Blackish green Glinted white Ash white Soluble

10 Sample powder + HNO 3 50% Orange red Red oxide Light orange Snow bell Soluble

11 Sample powder + CH 3OH Conc. Rado green Brownish green Spiny green Snow bell Sparingly soluble

12 Sample powder + CH 3OH 50% Yellowish green Celery seed Leaf green Abbey cream Sparingly soluble

13 Sample powder + CHCl 3 Conc. Pine forest Reddish brown Gardenia Pinkish green Semi soluble

14 Sample powder + CHCl 3 50% Yellowish green Greenish brown Light orange Pinkish green Slightly soluble

15 Sample powder + C 2H5OH Conc. Marsh green Lavender white Light leaf Snow bell Semi soluble

16 Sample powder + C 2H5OH 50% Glinted white Celery seed Brilliant white Caramel cream Semi soluble

17 Sample powder + CH 3COOH Conc. Golden green Mansion gold Orange yellow Abbey cream Soluble

18 Sample powder + CH 3COOH 50% Golden glimmer Brown Yellowish green Snow mountain Soluble

19 Sample powder + C 6H6 Conc. Blackish green Reddish brown Yellow green Pinkish green Semi soluble

20 Sample powder + C 6H6 50% Hidden green Capitol’s gold Green grapes Pinkish green Semi soluble

106

Results and Discussion Chapter 3

Table 44: Physicochemical Parameters of Datura stramonium and Xanthium strumarium

S. No. Physicochemical parameters Datura Xanthium (%) stramonium strumarium i Total ash 16.66 17.33 ii Acid insoluble ash 0.67 4.33 iii Water soluble ash 6 2.67 iv Water insoluble ash 10.66 14.66 v Moisture content 4 9

Table 45: Antioxidant activity of Ascorbic acid, Datura stramonium and Xanthium strumarium

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic Datura Xanthium acid stramonium strumarium 25 50.91 40.81 28.46 50 60.47 44.89 29.75 100 72.39 47.58 33.72 150 81.06 52.20 36.84 200 92.8 53.06 41.46 250 96.02 56.06 41.99 IC 50 value 2.14 µg/mL 145 µg/mL 354 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 107 Results and Discussion Chapter 3

20 Total Phenols Total Flavonoids 15

5 Concentration (mg /g) (mg Concentration 0 Datura stramonium Xanthium strumarium Plant extracts

Figure 25 . Total Phenolic and Flavonoid contents

100 90 80 25µg/mL 70 50µg/mL 60 100µg/mL 50 40 150µg/mL

activity (%) activity 30 200µg/mL 20 10 250µg/mL DPPH radical scavenging DPPH 0 Ascorbic acid Datura Xanthium stramonium strumarium Plant extracts and standard at different concentrations

Figure 26 . DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 108 Results and Discussion Chapter 3

Figure 27. HPLC chromatogram of Datura stramonium

Figure 28. HPLC chromatogram of Xanthium strumarium

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 109 Results and Discussion Chapter 3

3.4 Authentication of Herbal Drug Dhatura ( Datura stramonium L.)

Datura stramonium (Solanaceae) is well known medicinal plant for its use as indigenous cure in folklore or traditional system of medicines. It possesses both medicinal and poisonous properties (Devi et al., 2011). It is a natural source of phytochemicals processing antimicrobial and antioxidant activities (Akharaiyi, 2011). Herb is used in ayurvedic medicines for treating various human ailments such as ulcer, wounds, gouts, rheumatism, swellings, bronchitis, asthma, fever and toothache (Pretorius and Marx, 2006). In herbal market the demand for seeds of D. stramonium is very limited but the leaves are commonly traded under the trade name Dhatura. Due to careless collection and improper identification the dried leaves of dhatura are commonly mixed with leaves of Xanthium strumarium commonly known as cocklebur , which has spiny though smaller fruits. X. strumarium is used in traditional medicines of Europe, Indo-Pakistan and China to treat various human ailments.

Taxonomic Clarification

Morphologically, D. stramonium can be differentiated from its adulterant in having pale green colored stem and sinuately dentate leaves covered with very fine minute hairs (Figure 23a). Whereas in case of X. strumarium stem is purplish in color having rough, scaly and irregularly dentate leaves (Figure 24a). Morphological characterization for differentiation of D. stramonium and X. strumarium is presented in Table 35. Detailed morphological description of D. stramonium and X. strumarium is also reported in literature (Bhogaonkar and Ahmad, 2012; Gaire and Subedi, 2013).

Pollen morphological features are helpful to differentiate D. stramonium from X. strumarium. Pollen of D. stramonium are sub-angular in polar view and oblate-spheroidal in equatorial view with reticulate sculpturing having distinct lumina and columella (Figure 23c & 23d). However in X. strumarium pollen grains are circular in polar view and prolate-spheroidal in equatorial view with

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 110 Results and Discussion Chapter 3

echinate sculpturing having broad base spines (Figure 24c & 24d). Both qualitative and quantitative palynological variations between D. stramonium and X. strumarium are shown in Table 36 & 37. The results of pollen morphology of X. strumarium are in consistent with data previously reported by Mazari et al. (2012). According to Erdtman (1952) the pollen morphology of Solanaceae is heterogenous. However the pollen are tricolporate universally.

By using foliar epidermal features, D. stramonium can be distinguished from its adulterant by presence the of straight walled polygonal cells with anioscytic stomata and glandular peltate trichomes (Figure 23e & 23f) while in X. strumarium epidermal cells are irregular with heavily undulate walls, anomocytic stomata and non-glandular unicellular trichomes (Figure 24e & 24f). Foliar epidermal features of D. stramonium and X. strumarium are presented in Table 38 & 39. Albert and Sharma (2013) employed foliar micromorphological features as aid in identification of problematic taxa. Similarly, Khan et al. (2013) studied the significance of trichomes diversity in identification of various taxa from tropical flora of Pakistan and reported that trichomes of D. stramonium are glandular and that of X. strumarium are non-glandular and uniserriate.

Pharmacognostic Authentication

Pharmacognostic evaluation such as fluorescence tests can be used as a tool to differentiate D. stramonium from its adulterant. Organoleptography particularly smell and taste can help to differentiate the powdered drugs. Fluorescence analysis and solubility tests of powdered drugs of D. stramonium and X. strumarium are shown in Table 40, 41, 42, & 43. Present findings of fluorescence analysis of D. stramonium are in line with previous studies (Ananth, 2013). Physicochemical findings of D. stramonium and X. strumarium are shown in Table 44. In literature many workers have reported the importance of such pharmacognostic tests for the authentication of various problematic taxa of herbal drugs (Khatoon et al., 2009; Sultana and Zafar, 2013).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 111 Results and Discussion Chapter 3

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

Phytochemical characterization can be utilized as substantial tool for identification and standardization of genuine drug. Quantitative investigation of phenols and flavonoids indicated that higher concentration of flavonoid contents (22.45 mg QE/g, DW) was found in D. stramonium as compared to X. strumarium (8.83 mg QE/g, DW) whereas phenolic contents were slightly higher in X. strumarium (17.19 mg GAE/g, DW) as compared to D. stramonium (15.67 mg GAE/g DW) (Figure 25). Tripathi et al. (2014a) investigated the total polyphenolic contents of selected traditional plants of family Solanaceae and reported that D. stramonium possess highest polyphenolic contents. Similarly, Scherer and Godoy (2014) reported the total phenolic contents in X. strumarium leaves. Results were affected by method of extraction and solvent used and the best results were obtained with the methanolic extract.

HPLC Screening

For the quantitative analysis of flavonoids in plants reverse-phase HPLC has been used in many occasions, it is used to distinguish species based on quantitative variations of flavonoids among them (Kerhoas et al., 2006; Rajalakshmi and Senthil, 2009). Zheng and Wang (2001) used HPLC analysis for identification and quantification of quercetin, kaempferol and rosmarinic acid in selected medicinal herbs. In present study the reverse phase HPCL analysis was carried out to quantify the amount of quercetin in the D. stramonium and X. strumarium. The amount of quercetin in samples was achieved by comparison of retention time of the samples with those obtained for external standard quercetin. The HPLC chromatograms of samples are presented in Figure 27 & 28. Based on quantification of quercetin D. stramonium can be distinguished in having higher

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 112 Results and Discussion Chapter 3

concentration of quercetin (21.62 mg/g, DW) as compared to X. strumarium (6.07 mg/g, DW).

Antioxidant analysis

Nowadays, a great effort being expended to find effective antioxidants for the prevention or treatment of free radical-mediated deleterious effects (Hussein et al., 2011; Olofintoye et al., 2011). Results of antioxidant analysis indicated that

D. stramonium showed higher scavenging activity with IC 50 145 µg/mL as compared to X. strumarium with IC 50 354 µg/mL (Table 45 & Figure 26). In previous studies it is reported that methanolic extract of D. stramonium leaves showed maximum scavenging activity (Sharma et al., 2014b; Tripathi et al., 2014a; Chintem and Chukwuemeka, 2015). Antioxidant activity of D. stramonium has been reviewed by various workers such as kumar et al. (2010) and Akharaiyi (2011) and it was observed that leaf exhibit higher antioxidant activity than bark. The present results of antioxidant activity of X. strumarium are in accordance with those of Kamboj et al. (2014).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 113 Case 5:

Table 46: Authentication of herbal drug Chiraita ( Swertia cordata G. Don) in comparison with its adulterant

Characters Swertia cordata (G. Don) Clarke Swertia paniculata Wall.

English Name Chiretta Chiretta

Local Name Chirata Chirata

Trade Name Chiraita Chiraita

Family Gentianaceae Gentianaceae

Flowering period August- October July – November

Phytogeography: In Pakistan Swat, Gilgit, Hunza, Karimabad, Murree, Nathiagali, Hazara, Abottabad, Swat, Bagh, Murree hills, Dunga Ayubia, Hazara, Thandiani, Kaghan valley, Shogran, Gali, Bhagnather, Bunn, Karor and Charra Pani.

Balakot, Muzaffarabad, Baltistan, Skardu, Kurram

valley, Kohat and South Waziristan.

In World Pakistan, Kashmir, India, Nepal, Bhutan, Assam, Sikkim Pakistan, India, Kashmir, Nepal, Bhutan and Burma. and Burma.

Morphological description Annual herb, 25-70 cm tall, stem erect, quadrangular, Annual 30-90 cm tall, stem erect, quadrangular, striate, branched or unbranched. Leaves 20-35 x 15-25 mm, branched or unbranched, winged or not. Leaves 10-40 x opposite, cordate-ovate, sessile, apex acute, base cordate, 2-12 mm, pubescent or ciliate, glabrous, linear-oblong,

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margin scabrous. Inflorescence panicles of cymes. obtuse or sub-acute. Inflorescence paniculate, dense, Flower pentamerous. Pedicle striate, 3-9 mm. Calyx many-flowered. Pedicle 0.8-1.5 cm, erect, striate. lobes 5-8 x 2-4 mm, free, lanceolate to ovate-lanceolate, Flowers pentamerous. Calyx lobes 2-10 x 1-5 mm, free, margin scabrous, apex acute. Corolla bluish white to pale ovate to ovate-lanceolate, apex acute, equal or unequal, purple with dark purple veins, tube 0.5-1 mm, lobes 4-7 x pubescent. Corolla bluish white with a purple band or 2-3 mm, ovate and acute. Glands naked, one per lobe. blotches above each nectary, tube 1-1.5 mm, lobes 2-5 x Filaments 3-6 mm, subulate to filiform, anthers obtuse, 1-2 mm, ovate, apex acute, margin entire or eroded. ovate. Ovary 3-5 mm, stigmas orbicular, style short, Gland 1 per lobe, naked, horse-shoe shaped. Filaments distinct. Capsule 5-8 mm, ovoid-ellipsoid. Seeds smooth, 4-5 mm, free, anther purple, ovate-oblong, obtuse. corrugate-cristate. Ovary 5-7 mm, style slender, distinct, stigmas linear to oblong. Capsule 6-9 mm. Seeds pale yellow, rounded and warted.

Trade Part Aerial parts (leaves and branches) are sold in herbal Aerial parts are sold under the same common name markets of Pakistan under the trade name chiraita. chiraita at herbal shops of Pakistan.

Organoleptography Dried braches are angular, hollow and light brown in Dried branches are quadrangular, striate and yellowish color. Ridges and furrows are visible on the branches. green. Leaves pubescent or ciliate, linear-oblong with Leaves are sessile, ovate or elliptic, apex acute, margin acute apex. Ridges and furrows are prominent. Odor is entire. Vein are not prominent. Flowers are greenish mild and taste is bitter. Flowers are yellow-green in yellow in color. Smell is mild and taste is very bitter. color. Seeds are rounded and pale yellow in color. Seeds are smooth and many angled.

Part used Aerial parts, roots Aerial parts, roots

Ethnomedicinal Uses Malarial fever, dyspepsia, diarrhea, nausea, cough, cold, Digestive, diabetes, nausea, cough, cold, asthma, asthma, headache, hypertension, convulsion, skin

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diseases, liver and kidney diseases. headache, malarial fever and liver diseases.

Traditional herbal recipes Whole plant is soaked in water overnight and the extract Decoction of the plant is used as tonic and laxative. is taken in the morning to cure malarial fever. Plant Juice of herb is effective to cure the malarial fever. decoction is taken twice daily for one week to treat boils. Shoots powder is used to cure eye diseases. Leaves paste prepared with mustard oil is effective for boils and scabies. Roots juice is taken to cure liver infections. Roots paste is applied over joints for quick pain relief.

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Table 47: Comparative qualitative pollen morphology of Swertia cordata and Swertia paniculata

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial view Exine Sculpturing

1 Swertia cordata Tricolporate Subangular Prolate-spheroidal Reticulate

2 Swertia paniculata Tricolporate Circular Subprolate Reticulate

Table 48: Comparative quantitative pollen morphology of Swertia cordata and Swertia paniculata

Sr # Species name Polar diameter Equatorial diameter P/E Colpi length Colpi width Exine thickness (μm) (μm) ratio (μm) (μm) (μm)

1 Swertia cordata 22.3 (21.5-23.25) 20.75 (20.5-21.25) 1.07 0.5 (0.25-0.75) 1 (0.75—1.25) 1.87 (1.5-2)

2 Swertia paniculata 24.5 (23.4-25.5) 21.25 (20.5-22.5) 1.15 1.5 (1.2-1.7) 0.5 (0.25-0.75) 1 (0.75-1.5)

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Table 49. Quantitative characters of foliar epidermal anatomy of Swertia cordata and Swertia paniculata

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Swertia cordata Irregular Heavily Anisocytic Non-glandular Irregular Heavily Anisocytic Non-glandular undulate & unicellular undulate & unicellular

Swertia Hexagonal Straight Anisocytic A-shaped Hexagonal Straight Anisocytic A-shaped paniculata

Table 50. Quantitative characters of foliar epidermal anatomy of Swertia cordata and Swertia paniculata

Abaxial epidermis Adaxial epidermis

Name of Size of cells Size of stomata Stomatal Trichomes ( μm) Size of cells ( μm) Size of stomata Stomatal Trichomes (μm) species (μm) L ×W (μm) L ×W index (%) L×W L×W (μm) L×W index (%) L×W

Swertia 62.5 (53-69.5) × 32.5 (23-41.7) × 24.5 268.5 (248.4-288) 58.75(57.5-60) × 29.5 (28.25-30.5) 19.8 279 (265.8-292.5) cordata 45 (35-54) 20 (13-27.3) × 25.5 (23-29) 41 (39.3-42.6) × 19.5 (14-25.7) × 32.5 (27.5-38)

Swertia 61.25 (52-71.5) 29.5 (24-35.50) 22.3 17.25 (15.2-19.5) 65.3 (58.5-69.7) × 25.7 (20.5-31.2) 18.7 15.5 (13.5-17.7) × paniculata × 37.5 (28-45.7) × 21.5 (19.5-23) × 13 ( 12.5-14.25) 32.5 (25.5-38.25) × 18.25 (16.5-20) 13.2 (11.8-14.5)

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Results and Discussion Chapter 3

Figure 29 (a) Swertia cordata ; (b) Dried aerial parts; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 119 Results and Discussion Chapter 3

Figure 30 (a) Swertia paniculata ; (b) Dried aerial parts; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 120

Table 51: Fluorescence and solubility analysis of powdered drug of Swertia cordata (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried aerial parts powder Celery seed Light lichen - 2 Sample powder + 5% KOH Marsh green Hidden green Spinney green Spinney green Partially soluble

3 Sample powder + 10% aq. FeCl 3 Blackish green Blackish green Spinney green Brownish green Partially soluble

4 Sample powder + dH 2O Marsh green Marsh green Gardenia Gardenia Slightly soluble 5 Sample powder + HCL Conc. Brownish green Marsh green Hidden green Blackish brown Slightly soluble 6 Sample powder + HCL 50% Capitol’s gold Mansion gold Gardenia Celery seed Slightly soluble

7 Sample powder + H 2SO 4 Conc. Brownish green Medford green Blackish green Brownish black Soluble

8 Sample powder + H 2SO 4 50% Woven slates Willow branch Light leaf Light lichen Semi soluble

9 Sample powder + HNO 3 Conc. Rock alyssum Blackish green Glinted white Comfy tan Soluble

10 Sample powder + HNO 3 50% Capitol’s gold Brownish green Gardenia Caramel green Partially soluble

11 Sample powder + CH 3OH Conc. Light lichen Celery seed Gardenia Phantom violet Semi soluble

12 Sample powder + CH 3OH 50% Celery seed Light lichen Hidden green Felicia rose Partially soluble

13 Sample powder + CHCl 3 Conc. Marsh green Capitol’s gold Light leaf Warm toast Partially soluble

14 Sample powder + CHCl 3 50% Celery seed Brownish green Gardenia Silk knot Partially soluble

15 Sample powder + C 2H5OH Conc. Spinney green Light lichen Gardenia Silk knot Slightly soluble

16 Sample powder + C 2H5OH 50% Glinted white Celery seed Blackish green Glinted white Partially soluble

17 Sample powder + CH 3COOH Conc. Celery seed Mansion gold Gardenia Autumn white Semi soluble

18 Sample powder + CH 3COOH 50% Comfy tan Golden grain Gardenia Gardenia Partially soluble

19 Sample powder + C 6H6 Conc. Light lichen Golden grain Light leaf Gardenia Slightly soluble

20 Sample powder + C 6H6 50% Hidden green Capitol’s gold Gardenia Egg shell Slightly soluble

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Table 52: Fluorescence and solubility analysis of powdered drug of Swertia cordata (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried aerial parts powder Celery seed Light lichen - - - 2 Sample powder + 5% KOH Marsh green Dark green Brownish green Spinney green Partially soluble

3 Sample powder + 10% aq. FeCl 3 Greenish black Greenish black Spinney green Blackish green Partially soluble

4 Sample powder + dH 2O Light lichen Willow branch Blackish brown Blackish brown Partially soluble 5 Sample powder + HCL Conc. Brownish green Willow branch Glinted white Blackish brown Soluble 6 Sample powder + HCL 50% Blackish brown Blackish brown Glinted white Celery seed Semi Soluble

7 Sample powder + H 2SO 4 Conc. Brownish black Blackish green Dark black Brownish black Soluble

8 Sample powder + H 2SO 4 50% Brownish black Marsh green Golden grain Tracking tan Soluble

9 Sample powder + HNO 3 Conc. Marsh marigold Mansion gold Green whimsy Tracking tan Soluble

10 Sample powder + HNO 3 50% Rock alyssum Spinney green Glinted white Sunset light Soluble

11 Sample powder + CH 3OH Conc. Marsh green Brownish green Glinted white Demure Semi soluble

12 Sample powder + CH 3OH 50% Marsh green Brownish green Gardenia Faded violet Partially soluble

13 Sample powder + CHCl 3 Conc. Hidden green Capitol’s gold Hidden green Silk knot Semi soluble

14 Sample powder + CHCl 3 50% Woven slates Capitol’s gold Gardenia Caramel cream Semi soluble

15 Sample powder + C 2H5OH Conc. Marsh green Brownish green Glinted white Silk knot Partially soluble

16 Sample powder + C 2H5OH 50% Capitol’s gold Brownish green Silk knot Silk knot Partially soluble

17 Sample powder + CH 3COOH Conc. Rock alyssum Marsh marigold Gardenia Demure Soluble

18 Sample powder + CH 3COOH 50% Capitol’s gold Tracking tan Faded violet Faded violet Semi soluble

19 Sample powder + C 6H6 Conc. Spinney green Comfy tan Gardenia Gardenia Partially soluble

20 Sample powder + C 6H6 50% Light lichen Sunshine tea Gardenia Egg shell Partially soluble

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Table 53: Fluorescence and solubility analysis of powdered drug of Swertia paniculata (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried aerial parts powder Golden green Leafy green - - - 2 Sample powder + 5% KOH Brownish green Marsh green Glinted white Light leaf Semi soluble

3 Sample powder + 10% aq. FeCl 3 Golden brown Marsh green Mustard yellow Golden yellow Partially soluble

4 Sample powder + dH 2O Celery seed Light leaf Creamy white Silk knot Partially soluble 5 Sample powder + HCL Conc. Mustard green Celery seed Move pearl Ramie Semi soluble 6 Sample powder + HCL 50% Capitol’s gold Comfy tan Creamy white Caramel cream Semi soluble

7 Sample powder + H 2SO 4 Conc. Woven slates Willow branch Cotton canvas Harrison grey Semi soluble

8 Sample powder + H 2SO 4 50% Spinney green Willow branch Windmill white Grey stone Semi soluble

9 Sample powder + HNO 3 Conc. Golden grain Golden green Egg shell Rocky nook Soluble

10 Sample powder + HNO 3 50% Rock alyssum Orange green Glinted white Tracking tan Semi soluble

11 Sample powder + CH 3OH Conc. Willow branch Orange green Green whimsy Egg shell Semi soluble

12 Sample powder + CH 3OH 50% Glinted white Green whimsy Gardenia Autumn white Slightly soluble

13 Sample powder + CHCl 3 Conc. Leafy green Orange green Green whimsy Phantom violet Semi soluble

14 Sample powder + CHCl 3 50% Comfy tan Tracking tan Egg shell Caramel cream Semi soluble

15 Sample powder + C 2H5OH Conc. Leafy green Orange green Creamy white Shy sky Slightly soluble

16 Sample powder + C 2H5OH 50% Ramie Celery seed Autumn white Bow bells Slightly soluble

17 Sample powder + CH 3COOH Conc. Celery seed Reddish green Egg shell Caramel cream Semi soluble

18 Sample powder + CH 3COOH 50% Comfy tan Creamy white Gardenia Demure Slightly soluble

19 Sample powder + C 6H6 Conc. Hidden green Faded violet Gardenia Shy sky Slightly soluble

20 Sample powder + C 6H6 50% Spinney green Orange green Gardenia Caramel cream Partially soluble

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Table 54: Fluorescence and solubility analysis of powdered drug of Swertia paniculata (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried aerial parts powder Golden green Leafy green 2 Sample powder + 5% KOH Mustard green Golden green Celery green Spring cut Semi soluble

3 Sample powder + 10% aq. FeCl 3 Dark brown Marsh green Greenish yellow Brownish yellow Partially soluble

4 Sample powder + dH 2O Celery seed Spring cut Gardenia Glinted white Partially soluble 5 Sample powder + HCL Conc. Blackish brown Marsh green Cotton canvas Windmill white Semi soluble 6 Sample powder + HCL 50% Capitol’s gold Brownish green Peach pinch Sunset light Semi soluble

7 Sample powder + H 2SO 4 Conc. Dark black Dark green Dark black Dark black Soluble

8 Sample powder + H 2SO 4 50% Dark brown Marsh green Cotton canvas Harrison grey Soluble

9 Sample powder + HNO 3 Conc. Marsh marigold Mansion gold Gardenia Cotton canvas Soluble

10 Sample powder + HNO 3 50% Mustard yellow Orange green Mobe pearl Ships gate Soluble

11 Sample powder + CH 3OH Conc. Willow branch Orange green Gardenia Brownish pink Soluble

12 Sample powder + CH 3OH 50% Celery seed Light lichen Egg shell Lullaby Partially soluble

13 Sample powder + CHCl 3 Conc. Spinney green Reddish green Glinted white Faded violet Semi soluble

14 Sample powder + CHCl 3 50% Woven slates Orange green Gardenia Sunset light Semi soluble

15 Sample powder + C 2H5OH Conc. Spinney green Reddish green Light approach Demure Partially soluble

16 Sample powder + C 2H5OH 50% Comfy tan Creamy green Egg shell Fragile pink Partially soluble

17 Sample powder + CH 3COOH Conc. Woven slates Reddish brown Glinted white Faded violet Soluble

18 Sample powder + CH 3COOH 50% Lacquered maple Foamy green Creamy white Felicia rose Partially soluble

19 Sample powder + C 6H6 Conc. Spinney green Fragile pink Creamy white Phantom violet Soluble

20 Sample powder + C 6H6 50% Celery seed Pinkish green Glinted white Silk knot Partially soluble

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Results and Discussion Chapter 3

Table 55: Physicochemical Parameters of Swertia cordata and Swertia paniculata

S. No. Physicochemical parameters Swertia cordata Swertia (%) paniculata i Total ash 4.66 5.33 ii Acid insoluble ash 1 2 iii Water soluble ash 1 1 iv Water insoluble ash 3.66 4.33 v Moisture content 5 6

Table 56: Antioxidant activity of Ascorbic acid, Swertia cordata and Swertia paniculata

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic Swertia cordata Swertia acid paniculata 25 50.91 27.71 11.49 50 60.47 33.51 11.60 100 72.39 36.51 12.45 150 81.06 44.79 17.50 200 92.8 46.40 23.30 250 96.02 56.39 25.13 IC 50 value 2.14 µg/mL 208 µg/mL 624 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 125 Results and Discussion Chapter 3

90 80 Total Phenols 70 Total Flavonoids 60 50 40 30 20

Concentration (mg /g) (mg Concentration 10 0 Swertia cordata Swertia paniculata Plant extracts

Figure 31 . Total Phenolic and Flavonoid contents

100 90 80 25µg/mL 70 50µg/mL 60 50 100µg/mL 40 150µg/mL

activity (%) activity 30 20 200µg/mL 10 250µg/mL DPPH radical scavenging DPPH 0 Ascorbic acid Swertia Swertia cordata paniculata Plant extracts and standard at different concentrations

Figure 32 . DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 126 Results and Discussion Chapter 3

Figure 33. HPLC chromatogram of Swertia cordata

Figure 34. HPLC chromatogram of Swertia paniculata

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 127 Results and Discussion Chapter 3

3.5 Authentication of Herbal Drug Chiraita (Swertia cordata G. Don)

Swertia cordata (Gentianaceae) is one of the potential medicinal plants extensively used in Eastern traditional medicine such as Unani, Ayurveda, Siddha and also in traditional Tibetan and Chinese medicine. Herb is collected from wild habitat and has an established market both domestic as well as globally. It is used for the treatment of malarial and chronic fever, bronchial asthma, hepatitis, liver disorders, dyspepsia, constipation, epilepsy, worms, ulcers and diabetes (Karan et al., 1999; Banerjee et al., 2000; Airi et al., 2002; Rai, 2003; Saha et al., 2004). In Ayurveda this herb is described as bitter in taste and cooling in thermal action (Joshi and Dhawan, 2005). Herb with dried brownish stem and leaves is sold at herbal shops under the trade name chiraita. Due to immense medicinal importance S. cordata is being harvested indiscriminately from its wild habitat. In the trade of chiraita adulteration with other closely related species of Swertia is very common. However during market survey it is observed that S. cordata is most dominant in trade due to its superior quality in comparison to its adulterant species which are considered to be inferior in quality. In Nepal about nine species of Swertia have been reported under trade with common name chiraito (Barakoti, 2002). In general practice it is observed that Swertia paniculata is the common adulterant of S. cordata at herbal shops. S. paniculata is also used in number of herbal formulations (Anonymous, 1966; Joshi and Joshi, 2008).

Taxonomic Clarification

In this study by using taxonomic features S. cordata can be differentiated from S. paniculata based on distinct morphology of leaves and flowers. The leaves of S. cordata are cordate-ovate and flowers are bluish white to pale purple with dark purple veins (Figure 29a). While in S. paniculata leaves are linear- oblong and flowers are bluish white with dark purple band above each gland (Figure 30a). Our taxonomic findings for differentiation of S. cordata are in accordance with Bisht et al. (2011). The detailed morphological description for

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 128 Results and Discussion Chapter 3

authentication of S. cordata in comparison with its adulterant is presented in Table 46.

Palynological characters can also used to distinguish S. cordata from S. paniculata . As in S. cordata the pollen are subangular in polar view and prolate- spheroidal in equatorial view (Figure 29c & 29d). While pollen grains of S. paniculata are circular in polar view and subprolate in equatorial view (Figure 30c & 30d). Shape of pollen is one of the significant features which could help in delimiting the closely related species (Perveen, 2006). Based on exine sculpturing Hedberg (1957) and Jonsson (1973) divided the African species of Swertia into three groups viz., Kilimandscharica-type (finely striato-reticulate), Crassiuscula type (spinulose) and and Volkensii-type (coarsely striate pattern). Depending on exine sculpturing four groups of pollen are found in Gentianaceae from Pakistan and Kashmir viz., striate, scabrate, reticulate and striato-reticulate (Omer, 1991).

During taxonomic investigation, it is observed that leaf epidermal features can be used as important parameter to distinguish genuine drug from its adulterant. Foliar epidermal investigation presented in Table 50 shows that in S. cordata the epidermal cells are irregular, walls are heavily undulate and trichomes are long, thin with pointed tips (Figure 29e & 29f). Whereas in S. paniculata epidermal cells are hexagonal, walls are straight and trichomes are A-shaped (Figure 30e & 30f). According to Ahmad et al. (2010a) types and shapes of epidermal cells, stomata, trichomes and glands are important anatomical features and commonly serve as an aid in taxonomic verification. Similarly, Renobales et al. (2008) on basis of secretory hairs delimited eleven species of Gentiana and one species of Swertia (Gentianaceae) from the Iberian Peninsula .

Pharmacognostic Authentication

Pharmacognostic analysis can also be helpful in identification of genuine drug S. cordata from its adulterant. Organoleptic, physicochemical, fluorimetric, and spectroscopic parameters can be used for authentication of medicinal plants

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 129 Results and Discussion Chapter 3

and detection of their adulterants (Nair et al., 1997). The fluorescence behaviors of S. cordata and S. paniculata with various chemical reagents under visible and UV lights and solubility tests are presented in Table 51, 52, 53 & 54. Latif and Rehman (2014) used fluorescence analysis for standardization of herbal drug S. chirayita. Bisht et al. (2011) employed pharmacognostic analysis for comparative evaluation of three species of Swertia . Results of physicochemical analysis of present study showed that S. cordata contain low %age of total ash and moisture contents as compared to S. paniculata (Table 55). These results are in accordance with previous reported studies (Mukherjee et al., 1997; Sayyed et al., 2013).

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

Quantitative analysis of phenols and flavonoids showed that aerial parts of S. cordata contained higher phenolic (84.83 mg GAE/g, DW mg) and flavonoid contents (70.26 mg QE/g, DW) while in aerial parts of S. paniculata phenolic contents were 54.16 mg GAE/g, DW and flavonoid contents were 35.7 mg QE/g, DW (Figure 31). Kshirsagar et al. (2015) reported phytochemical composition of different Swertia species to authenticate the genuine source of drug. According to them highest amount of total phenolic contents (102.04 mg GAE/g) and total flavonoid contents (104.21 mg RE/g) was found in S. cordata . In previous studies many workers reported different concentrations of phenolic and flavonoid contents in Swertia species (Mahendran and Bai, 2013; Jassal et al., 2014). By reviewing various studies it was noted that total phenolic and flavonoids contents showed variations depending on the method of extraction and solvent system used. The influence of solvent system used on extraction of total phenolic and flavonoids contents of Swertia species have also been reported by different workers (Kalt et al., 2001; Jagtap et al., 2010).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 130 Results and Discussion Chapter 3

HPLC Screening

For standardization of herbal medicines HPLC is the most popular analytical method due to its precision and versatility (Tsao and Yang, 2003). In this study HPLC screening of S. cordata and S. paniculata was performed for the development of characteristic profile, which may be used as a marker for quality evaluation and standardization of genuine drug. HPLC screening showed that higher concentration of quercetin (65.17 mg/g, DW) was found in S. cordata as compared to S. paniculata (30.09 mg/g, DW). Therefore based on the results of this analysis S. cordata can be a potential source of quercetin. The HPLC chromatograms of S. cordata and S. paniculata are presented in Figure 33 & 34. In literature other workers also used HPLC screening for correct identification of genuine drugs such as cassia bark (Cinnamomum cassia ) from seven closely related adulterants Cinnamomum species frequently found in the market (He et al., 2005).

Antioxidant analysis

The results of antioxidant analysis presented in Table 56 indicated that S. cordata showed higher scavenging activity (59.39%) as compared to S. paniculata (25.13%) at a concentration 250 µg/mL (Figure 32). Comparative screening of Swertia species for their possible antioxidant activities justify that S. cordata is the main source of chiraita in herbal market. In previous studies many other researchers have also reported the highest scavenging activity in S. cordata as compared to other closely related species (Chen et al., 2011; Kumar et al., 2011). Ahirwal et al. (2014) evaluated the antioxidant potential of methanolic extract of S. chirayita and reported that highest free radical scavenging activity

(88.4%) was shown at a concentration 500 µg/mL with an IC 50 222.74 µg/mL.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 131 Case 6:

Table 57: Authentication of herbal drug Zafran ( Crocus sativus L.) in comparison with its adulterant

Characters Crocus sativus L. Carthamus tinctorius L.

English Name Saffron Safflower

Local Names Zafran Tukhmigartum//Kusum

Trade Name Kesar Kusumba

Family Iridaceae Asteraceae

Flowering period October–November May-August

Phytogeography: In Pakistan Kashmir, Quetta and Deosai. Sindh, Baluchistan and Punjab.

In World Mediterranean Region and South Western Asia, native to Mexico, Kazakhstan, Ethiopia, Australia, USA, India, Iran and Greece, cultivated in Southern Europe, India, Bangladesh and Afghanistan. Tibet and Kashmir.

Morphological description Perennials, 10 cm tall, corms 2-3.5 cm in diameter, Annual thistle-like herb, 50-100 cm tall, glabrous. Stem flattened at the base, depressed globose, tunica fibrous, branched, erect, branches white, glabrous, smooth. Leaves the fibers finely reticulated and very slender. Cataphylls cauline, leathery, rigid, glabrous with numerous spines. white, 3–5, membranous. Leaves 5–11, 1.5–2.5 mm, Lower and middle leaves simple, 7-15 × 2.5-6 cm, sessile, radical, linear, synanthous, green, glabrous and enclosed elliptic, lanceolate or ovate-lanceolate, apex acute, base

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in membranous sheath. Flowers 1–4, sterile, deep lilac- semi-amplexicaul and attenuate, margin entire or spinosely purple with a darker violet stain and darker veins in the toothed, teeth apically with 1-1.5 mm spinules. Upper throat, throat pubescent, white or lilac. Prophyll present. leaves lanceolate, decreasing in size upward, spinosely Bract and bracteole present, white, unequal, toothed margin, teeth apically with 3 mm spine. Capitula membranous, tip long-tapering rather flaccid. Perianth few to many. Involucre 2.5 cm in diameter, ovoid. tube yellow, 4–5 cm long, segments 3–6 cm long, 2–3 Phyllaries in 5 rows, outer phyllaries leaf like, 2-4 × 1 cm, spiny, ovate-lanceolate, middle phyllaries 6-20 × 4-7 mm, cm wide, subequal, obtuse, obovate or oblanceolate. leaf like, inner phyllaries 22 × 5 mm, oblanceolate-elliptic, Filaments 6.5–10 mm long, glabrous, yellow or pale scarious, apex attenuate. Corolla 2.8 cm, red to orange. orange, anther yellow, 15–22 mm long. Style has three Achene 5.5 mm, 4-angled, ovoid to ellipsoid. Pappus deep red clavate branches, each branch 22–32 mm long, absent. much exceeding the anthers. Seeds are not produced.

Trade Part Styles and stigmas are sold at herbal shops under the Styles and stigmas are sold under the trade name wars and trade name zafran. also mixed with zafran as adulterant.

Organoleptography Dried corms are flattened at base, globose and tunica is Dried branches are cylindrical, woody and greenish white fibrous. Leaves are yellowish green in color; grass like in color. Leaves are oblong or ovate-lanceolate, sessile, with sharp edges and tapering to a slender point. Flowers sharp pointed, spiny and pale green in color. Flowers are are pale purple in color. Stigmas are three, delicate, globular, terminal and yellow orange in color. Taste is very fragile, deep orange red in color, rolled at the edges. pleasant and aroma is mild but sweat and smoky. Seeds are Taste is pleasantly bitter, slightly metallic and warming. four sided, shiny and white in color. Fragrance is iodoform or hay-like.

Part used Flowers Flowers, roots, seeds

Ethnomedicinal Uses Measles, jaundice, cold, cough, asthma, arthritis, cholera, Measles, fever, skin inflammations, skin problems, dysentery, indigestion, insomnia, skin diseases, eye wounds, rheumatism, liver inflammation and coronary

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diseases, blood purification and menstruation problems. heart diseases.

Traditional folk recipes Saffron paste is applied to treat the skin diseases like acne. Safflower tea is used for colds and cough. Poultice of Its powder is mixed with honey and rubbed on gums to powdered seeds is used to treat inflammation of the relieve the gum pains in children. Infusion of saffron is womb after child birth. Locally the color bread is made effective for cough cold. Saffron along with milk or its tea is by using petals as dye in Eid festival days. Oil is used to very effective for painful menstruation. Powder of stigma is lower the cholesterol level. used to cure night blindness, cataracts and poor visions. It is an excellent tonic for nervous and heart system and for smoothing menstruation. It is also used as blood purifier and to cure skin eruptions internally.

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Table 58: Comparative qualitative pollen morphology of Crocus sativus and Carthamus tinctorius

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Crocus sativus Tricolporate Circular Subprolate Present Echinate

2 Carthamus Tricolporate Circular Oblate-spheroidal Present Echinate tinctorius

Table 59: Comparative quantitative pollen morphology of Crocus sativus and Carthamus tinctorius

Sr # Species name Polar diameter Equatorial diameter P/E Colpi length Colpi width Exine thickness (μm) (μm) ratio (μm) (μm) (μm)

1 Crocus sativus 105.2 (95-115.2) 82.02 (76.5-87.5) 1.28 Not visible Not visible 6.3 (5.25-7.5)

2 Carthamus tinctorius 52.5 (48.2–56.94) 54.56 (52.88–56.13) 0.96 10.25(8.35–12.44) 6.17 (5.4–7.42) 3.05 (2.9-3.28)

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Table 60. Comparative qualitative characters of foliar epidermal anatomy of Crocus sativus and Carthamus tinctorius

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Crocus sativus Rectangular Straight Paracytic Absent Rectangular Straight Paracytic Absent

Carthamus Polygonal Straight Anisocytic Absent Polygonal Straight Anisocytic Absent tinctorius

Table 61. Comparative quantitative characters of foliar epidermal anatomy of Crocus sativus and Carthamus tinctorius

Abaxial epidermis Adaxial epidermis

Name of Size of cells Size of stomata Stomatal Trichomes Size of cells ( μm) Size of stomata Stomatal Trichomes ( μm) species (μm) L×W (μm) L ×W index (%) (μm) L ×W L×W (μm) L ×W index (%) L×W

Crocus 181.5 (137.5- 15.75 (13.5-17.5) 26.3 Absent 184.75 (134.5-235.2) 12.5 (10.25-15.2) 19.8 Absent sativus 225) × 25.2 × 14.2 (12.5- × 21.2 (19-23.2) × 11.2 (9.5-13.2) (22.5-27.5) 15.5)

Carthamus 56.5 (43.5-67.5) 35 (33.5-37.5) × 18.2 Absent 51.5 (41.4-62.5) × 34 (31.2-36.5) × 15.8 Absent tinctorius × 37.45 (32.5- 17.25 (15.5-18.5) 35.45 (30.2-40.5) 14.35 (13.5-15.3) 42.5)

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Results and Discussion Chapter 3

Figure 35 (a) Crocus sativus ; (b) Dried styles and stigmas; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 137 Results and Discussion Chapter 3

Figure 36 (a) Carthamus tinctorius ; (b) Dried styles and stigmas; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 138

Table 62: Fluorescence and solubility analysis of powdered drug of Crocus sativus (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried stigmas powder Deep red Reddish black - - - 2 Sample powder + 5% KOH Reddish black Brownish black Abbey cream Light mulberry Partly soluble

3 Sample powder + 10% aq. FeCl 3 Deep red Purplish red Bone white Swiss almond Partly soluble

4 Sample powder + dH 2O Reddish orange Milky red Light orange Kitten white Semi soluble 5 Sample powder + HCL Conc. Reddish black Cora reef Light brown Ash white Freely soluble 6 Sample powder + HCL 50% Reddish orange Fresh orange Lemon yellow Sun shower Soluble

7 Sample powder + H 2SO 4 Conc. Purplish black Rado green Rose white Goose wing Freely soluble

8 Sample powder + H 2SO 4 50% Red oxide Purple Choco malt Light brown Soluble

9 Sample powder + HNO 3 Conc. Reddish black Deep black Dull brown Ash white Soluble

10 Sample powder + HNO 3 50% Blackish red Coral reef Peach silk Classic ivory Partly soluble

11 Sample powder + CH 3OH Conc. Reddish orange Orange red Desert dawn Orchid shadow Semi soluble

12 Sample powder + CH 3OH 50% Orange red Fresh orange Apricot Light mulberry Semi soluble

13 Sample powder + CHCl 3 Conc. Purplish red Milky orange Lemon yellow Snow bell Soluble

14 Sample powder + CHCl 3 50% Brownish red Snow mountain Sweet jewel Sea shell Soluble

15 Sample powder + C 2H5OH Conc. Reddish brown Purple Sugar cane Tea rose Semi soluble

16 Sample powder + C 2H5OH 50% Brownish red Roof tile Coral reef Orchid shadow Partly soluble

17 Sample powder + CH 3COOH Conc. Grayish red Terracotta Abbey cream Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Blackish red Tea rose Light mulberry Light purple Soluble

19 Sample powder + C 6H6 Conc. Grayish red Choco malt Roof tile Autumn stone Semi soluble

20 Sample powder + C 6H6 50% Deep red Tea rose Shingle Light mulberry Partly soluble

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Table 63: Fluorescence and solubility analysis of powdered drug of Crocus sativus (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light

1 Dried stigmas powder Deep red Reddish black - - - 2 Sample powder + 5% KOH Blood red Brownish red Abbey cream Light mulberry Soluble

3 Sample powder + 10% aq. FeCl 3 Reddish black Deep black Light brown Dark brown Soluble

4 Sample powder + dH 2O Reddish orange Orange red Desert dawn Light mulberry Soluble 5 Sample powder + HCL Conc. Deep black Tea rose Deep black Lavender white Highly soluble 6 Sample powder + HCL 50% Roof tile Pine forest White ice Orchid shadow Highly soluble

7 Sample powder + H 2SO 4 Conc. Brownish black Purplish brown Dull brown Dark purple Freely soluble

8 Sample powder + H 2SO 4 50% Deep black Purple Abbey cream Night magic Soluble

9 Sample powder + HNO 3 Conc. Deep black Rose white Peach silk White ice Highly soluble

10 Sample powder + HNO 3 50% Reddish black Orange red Ash white Light mulberry Soluble 11 Sample powder + CH OH Conc. Purplish red Dark purple Lavender white Orchid shadow Soluble 3 12 Sample powder + CH 3OH 50% Bright red Orange red Deep orange Snowbell Soluble

13 Sample powder + CHCl 3 Conc. Reddish purple Milky orange Purplish brown White ice Highly soluble

14 Sample powder + CHCl 3 50% Reddish brown Lilac time Abbey cream Orchid shadow Soluble

15 Sample powder + C 2H5OH Conc. Brownish red Light purple Dark brown White ice Soluble

16 Sample powder + C 2H5OH 50% Purplish red Roof tile Tea rose Silk stone Partly soluble

17 Sample powder + CH 3COOH Conc. Reddish gray Terracotta Light brown Light mulberry Soluble

18 Sample powder + CH 3COOH 50% Blackish red Dark Purple Bone white Shingle Soluble 19 Sample powder + C H Conc. Deep red Dark purple Snow mountain Brilliant white Freely soluble 6 6 20 Sample powder + C 6H6 50% Reddish gray Tea rose Satin white Light mulberry Freely soluble

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Table 64: Fluorescence and solubility analysis of powdered drug of Carthamus tinctorius (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light

1 Dried stigmas powder Orange red Deep orange - - - 2 Sample powder + 5% KOH Reddish orange Rado green Goose wing White ice Partly soluble

3 Sample powder + 10% aq. FeCl 3 Orange red Lilac time Sky gray Snow mountain Partly soluble

4 Sample powder + dH 2O Fresh orange Golden glimmer Satin white Ash white Semi soluble 5 Sample powder + HCL Conc. Maroonish black Lilac time Coral reef Sweet jewel Soluble 6 Sample powder + HCL 50% Orange Golden glimmer Silk stone Tea rose Partly soluble

7 Sample powder + H 2SO 4 Conc. Brownish black Leaf green Sky gray Sea mist Freely soluble

8 Sample powder + H 2SO 4 50% Orange brown Candy pink Goose wing Moorland Soluble

9 Sample powder + HNO 3 Conc. Brownish red Purple Dove gray Portland Soluble

10 Sample powder + HNO 3 50% Reddish brown Rado green Goose wing Silk gray Semi soluble 11 Sample powder + CH OH Conc. Orange red Leaf green Sky gray Brilliant white Soluble 3 12 Sample powder + CH 3OH 50% Fresh orange Pine forest Orchid shadow Light mulberry Partly soluble

13 Sample powder + CHCl 3 Conc. Brownish black Leaf green Onyx Snow bell Semi soluble

14 Sample powder + CHCl 3 50% Brownish red Lilac time Shimmering sky White ice Semi soluble

15 Sample powder + C 2H5OH Conc. Grayish orange Light mulberry Snow mountain Lavender white Slightly soluble

16 Sample powder + C 2H5OH 50% Brownish red Candy pink Halo Antique white Slightly soluble

17 Sample powder + CH 3COOH Conc. Blackish orange Roof tile Satin white Lavender white Soluble

18 Sample powder + CH 3COOH 50% Dark orange Lake green Peach silk Shingle Semi soluble 19 Sample powder + C H Conc. Brownish black Leaf green Rose white Classic ivory Semi soluble 6 6 20 Sample powder + C 6H6 50% Brownish red Golden glimmer Satin white Orchid shadow Partly soluble

141

Table 65: Fluorescence and solubility analysis of powdered drug of Carthamus tinctorius (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried stigmas powder Orange red Deep orange - - - 2 Sample powder + 5% KOH Orange Pine forest Tea rose Lavender white Partly soluble

3 Sample powder + 10% aq. FeCl 3 Dark orange Lilac time Roof tile Orchid shadow Semi soluble

4 Sample powder + dH 2O Bright orange Golden green Satin white Ash white Semi soluble 5 Sample powder + HCL Conc. Blackish brown Lilac time Silk gray Shingle Freely soluble 6 Sample powder + HCL 50% Blackish orange Rado green Goose wing Abbey cream Soluble

7 Sample powder + H 2SO 4 Conc. Dark chocolate Pine forest Sky gray Sea mist Highly Soluble

8 Sample powder + H 2SO 4 50% Light brown Lilac time Smoke gray Moorland Soluble

9 Sample powder + HNO 3 Conc. Reddish black Dark Purple Sky gray Moorland Soluble

10 Sample powder + HNO 3 50% Maroonish red Pine forest Silk gray Sea mist Soluble

11 Sample powder + CH 3OH Conc. Reddish orange Lichen green Goose wing Silk gray Semi soluble

12 Sample powder + CH 3OH 50% Deep orange Golden glimmer Roof tile Light mulberry Semi soluble

13 Sample powder + CHCl 3 Conc. Purplish black Lilac time Chocomalt Coral reef Soluble

14 Sample powder + CHCl 3 50% Orange brown Light chocolate Clay Fawn Semi soluble

15 Sample powder + C 2H5OH Conc. Brownish gray Light mulberry Smoke gray Lavender white Soluble

16 Sample powder + C 2H5OH 50% Dark brown Passion Snow mountain Antique white Semi soluble

17 Sample powder + CH 3COOH Conc. Brownish black Tile red Off white Lavender white Highly soluble

18 Sample powder + CH 3COOH 50% Orange black Forest green Sea shell Sugar cane Soluble

19 Sample powder + C 6H6 Conc. Blackish red Rado green Tea rose Magnolia Semi soluble

20 Sample powder + C 6H6 50% Brownish red Pine forest Satin white Light mulberry Semi soluble

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Results and Discussion Chapter 3

Table 66: Physicochemical Parameters of Crocus sativus and Carthamus tinctorius

S. No. Physicochemical parameters Crocus sativus Carthamus (%) tinctorius i Total ash 7.21 17.5 ii Acid insoluble ash 1 2.5 iii Water soluble ash 1.54 3.83 iv Water insoluble ash 5.67 13.67 v Moisture content 7 9

Table 67: Antioxidant activity of Ascorbic acid, Crocus sativus and Carthamus tinctorius

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic Crocus sativus Carthamus acid tinctorius 25 50.91 49.51 48.76 50 60.47 52.95 51.77 100 72.39 55.96 54.24 150 81.06 58.75 56.28 200 92.8 60.04 60.36 250 96.02 62.51 60.68 IC 50 value 2.14 µg/mL 6 µg/mL 29 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 143 Results and Discussion Chapter 3

120

100 Total Phenols 80 Total Flavonoids 60

20 Concentration (mg /g) (mg Concentration 0 Crocus sativus Carthamus tinctorius Plant extracts

Figure 37. Total Phenolic and Flavonoid contents

100 90 25µg/mL 80 50µg/mL 70 60 100µg/mL 50 150µg/mL 40 200µg/mL activity (%) activity 30 20 250µg/mL 10 DPPH radical scavenging DPPH 0 Ascorbic acid Crocus sativus Carthamus tinctorius Plant extracts and standard at different concentrations

Figure 38. DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 144 Results and Discussion Chapter 3

Figure 39. HPLC chromatogram of Crocus sativus

Figure 40. HPLC chromatogram of Carthamus tinctorius

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 145 Results and Discussion Chapter 3

3.6 Authentication of Herbal Drug Zafran ( Crocus sativus L.)

Crocus sativus (Iridaceae) commonly known as saffron is widely cultivated in different parts of the world, particularly in Spain and Iran (Esmaeili et al., 2011). Saffron name is derived from Arabic word za-faran meaning “be yellow” (Rios et al., 1998; Winterhalter and Straubinger, 2000). The common use of saffron is in food industry as a food coloring and flavoring agent but it is also used in folk medicines as stimulant, carminative, stomachic, expectorant, antispasmodic, aphrodisiac and cardio tonic (Goli et al., 2012). Saffron is reported for many pharmacological effects such as antidepressant, antitumor, anti- inflammatory, anticonvulsant and antioxidants (Hadizadeh et al., 2003; Melnyk et al., 2010). The trade product saffron is the dark red, dried stigmas of C. sativus flowers and is the world most expensive spice. About 76 % of the world saffron is produced by Iran. In international markets Iranian saffron has distinctive qualities for its fragrance, flavor and color in comparison with those produced in other parts of the world (Saffron gold, 2006). Due to high value and high price in herbal market saffron is most frequently subjected to adulteration (Fernández, 2004). Generally in herbal market the stigmas of quite different herbs such as Carthamus tinctorius are fraudulently mixed with genuine stigmas of C. sativus and traded at herbal shops. C. tinctorius is a famous traditional medicine and for a long time has been used as a source of natural dye and crude drugs in China, Iran, India, Pakistan, Eastern Asia and Japan. Plant is reported for antibacterial and anti- inflammatory actions (Kasahara et al., 1994) .

Taxonomic Clarification

Morphologically, Crocus sativus can be distinguished from C. tinctorius having linear, glabrous leaves that are enclosed in membranous sheath and deep lilac-purple, sterile flowers (Figure 35a). While in C. tinctorius leaves are elliptic with numerous spines, flowers are arranged in capitulum and enclosed by bracts (Figure 36a). In literature many workers reported morphological description of C.

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 146 Results and Discussion Chapter 3

sativus and C. tinctorius (Singh and Nimbkar, 2006; Saxena, 2010; Gautam et al., 2014). Detailed morphological differentiation between C. sativus and C. tinctorius is presented in Table 57.

Palynologically, the qualitative features such as pollen class, shape and exine sculpturing of C. sativus and C. tinctorius are almost similar. So these palynological characters cannot be depend upon to delimit these two species. However the difference in pollen morphology of two species lies in the quantitative parameters such as polar diameter, equatorial diameter and exine thickness. The polar and equatorial diameter of C. sativus is greater as compared to C. tinctorius. Colpi are present in both species but are not visible in C. sativus. In C. sativus the spines are short with broad base and blunt tip (Figure 35c & 35d). While in C. tinctorius the spines are broad with circular base and mucronate tip (Figure 36c & 36d). Palynological variations between C. sativus and C. tinctorius are presented in Table 58 & 59. Caiola (2000) reported that among the Crocus species the pollen of C. sativus has highest dimensions and size variations. Chichiriccò (1999) conducted a comparative pollen study in C. sativus and allied species and reported that exine in Crocus species is atectate, echinate and pollen grains are with apertures of various types . In C. sativus the colpi are more are less extensive and apertures both in shape and dimensions may vary within the species (Mathew, 1977). According to I şık and Dönmez (2006) exine structures of Crocus were found as echinate (spinulate)-microperforate. In C. tinctorius exine ornamentation is echinate with microperforations between spines (Bülbül et al., 2013). Results documented in this study correlate to all previous reports.

Foliar epidermal features of C. sativus and C. tinctorius showed significance degree of differentiation in terms of epidermal cells and stomata (Table 60 & 61). In case of C. sativus epidermal cells are rectangular and stomata are paracytic (Figure 35e & 35f). While in C. tinctorius the epidermal cells are polygonal and stomata are anisocytic (Figure 36e & 36f). Based on anatomical characters Özdemir et al. (2004) differentiated two endemic species of Crocus

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 147 Results and Discussion Chapter 3

from Turkey. Özdemir et al. (2006) reported that in Crocus species the stomata are present only on the groove parts of leaf and are sunken between the epidermal cells. Same results have been observed in the leaf of C. aerius (Özyurt, 1978).

Pharmacognostic Authentication

For the first line standardization of crude drug fluorescence analysis is an essential parameter. In fluorescence analysis the ultraviolet light produces fluorescence in many substances which do not visibly fluoresce in daylight (Ashok and Upadhyaya, 2013). Results of fluorescence and solubility analysis of stigmas of C. sativus in comparison with its adulterant C. tinctorius are presented in table 62, 63, 63 & 64. Results of physicochemical properties presented in Table 66 shows that total ash and moisture contents are higher in C. tinctorius stigmas as compared to C. sativus. Our findings of physicochemical analysis of C. sativus are in accordance with Khan (2007). Jamna et al. (2012) used such pharmacognostic parameters for differentiation of Inula racemosa from its adulterant Coffea travancorensis . Similarly, Ashok and Upadhyaya (2013) employed physicochemical parameters for differentiation of Artemisia absinthium and A. annua .

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

Flavonoids found in flowerings parts are important part of the diet because of their effects on human nutrition (Frankel, 1997; Larson, 1988). The most vital function of flavonoids is their antioxidant activity, as they are highly effective scavengers of many types of oxidizing molecules (Buettner, 1993; Bravo, 1998). Results of quantitative phytochemical analysis showed that C. sativus stigma had higher phenolic (7.61 mg GAE/g, DW) and flavonoid contents (116.4 mg QE/g, DW) as compared to C. tinctorius where the phenolic contents were 3.33 mg GAE/g, DW and flavonoid contents were 18.51 mg QE/g, DW (Figure 37). In

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 148 Results and Discussion Chapter 3

previous studies Karimi et al. (2010) reported total phenolic contents (6.55 mg GAE/g, DW) in C. sativus stigmas.

HPLC Screening

Quantification of quercetin by high pressure liquid chromatography showed that high level of quercetin was found in C. sativus (111.72 mg/g, DW) as compared to C. tinctorius (16.20 mg/g, DW). HPLC chromatograms of C. sativus and C. tinctorius are presented in Figure 39 & 40. Jaimand et al. (2012) also reported highest quercetin concentration in C. sativus stigma by using heat soxlet and ultrasonic extraction. Rios et al. (1998) and Xi and Qian (2006) isolated carotenoids, monoterpenoids, crocins and flavoniods from the stigmas, leaves, petals and pollen of C. sativus . Montoro et al. (2008) determined the qualitative and quantitative profile of flavonoids from petals of C. sativus. Authors reported that the major flavonoid compounds isolated from C. sativus were derivatives of quercetin. The quercetin profile of C. tinctorius in current study is in agreement with previous reported studies (Lee et al., 2002; Sun et al., 2003).

Antioxidant analysis

C. sativus can be distinguished from C. tinctorius by showing high %age of scavenging activity (Table 67 & Figure 38). In literature antioxidant activity of C. sativus has been reported by many workers (Chatterjee et al., 2005; Sengul et al., 2009; Goli et al., 2012). The antioxidant activity of C. sativus documented in this project is in accordance with the range reported by Karimi et al. (2010). In previous studies many workers reported the antioxidant activity of C. tinctorius (Ebadi et al., 2014; Salem et al., 2014). The variation in antioxidant activity encountered in different studies might be attributed to genotypic and environmental differences within species, the time of year samples were taken, the parts of plants studied and analytical method used (Shan et al., 2005).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 149 Case 7:

Table 68: Authentication of herbal drug Resha khatmi ( Althaea officinalis L.) in comparison with its adulterant

Characters Althaea officinalis L. Hibiscus rosa-sinensis L.

English Names Althaea, Marshmallow, Sweet weed Shoe flower, Chinese hibiscus, China rose

Local Names Khatmi, khatma Gudhal, Gulhar

Trade Name Resha-khatami Gurhal

Family Malvaceae Malvaceae

Flowering period July-August Throughout the year

Phytogeography: In Pakistan Himalayan region from Kashmir to Punjab. Cultivated as ornamentals in all parts of the country.

In World Northern Africa, Europe, United States, Asia, Pakistan. China, Pakistan, India and East Africa.

Morphological description Perennial herb, 1 m tall, stem erect, densely stellate Glabrate shrub, 1-5 m tall. Stem profusely branched. hirsute. Petiole upto 1-4 cm, stellate tomentose, leaves Leaves ovate, 4-8 × 2-5 cm, serrate-dentate, lobed or cordate or ovate-orbicular, 3-8 × 1.5-6 cm, papery, 3- not, stipules linear, 5-10 mm long, petiole 0.8-2 cm. lobed or not lobed, base rounded or nearly cordat, apex Flowers solitary, axillary, single or double, acute, margins bluntly dentate, both surfaces densely subpendulous or erect, pedicel 2-7 cm, articulate near stellate tomentose. Epicalyx segments 4 mm, lanceolate, the top. Epicalyx segments linear or linear-lanceolate, 5- densely stellate strigose. Calyx persistent, 5-parted, cup- 10 mm. Calyx tubular-campanulate, 1-2 cm, lobes

150

shaped, densely stellate hirsute, longer than epicalyx, triangular to lanceolate, 8-15 mm. Corolla pinkish to red, lobes lanceolate. Corolla 2.5 5-10 cm across, petals 5-10 cm long, 4-8 cm broad, cm in diameter, pink, petals 1.5 cm, obovate-oblong. oblong or obovate, apex irregularly lobed or obtuse. Staminal column 8 mm. Ovary 15-25-loculed. Fruit 8 Staminal tube exserted, antherifierous at the top. Capsule mm in diameter, a disk-shaped schizocarp, puberulent, 2 cm long, ovoid, glabrous. enclosed by calyx. Seeds reniform. Trade Part Roots are sold in herbal market under the trade name Roots are mixed with the roots of Althaea officinalis in resha khatmi. herbal market.

Organoleptography Dried branches are dark brown with stellate hairs. Leaves Dried branches are rigid and greenish brown in color. are dark green. Flowers are pale rose in color. Capsule is White dots are visible on the branches. Leaves are light circular with reniform seed. Roots are whitish yellow or green and veins are prominent. Flower is pale purple in light yellow, slenderly tapering, externally longitudinally color. Capsule is ovoid. Roots branched, reddish yellow furrowed, spirally twisted and covered with loosened in color, tasteless and aroma mild. bast-fibers. Odor is slight and taste is sweetish.

Part used Flower, leaves, roots Flowers, roots

Ethnomedicinal Uses Coughs, urinary and respiratory organs irritation, Piles, vomiting, inflammation, gonorrhea and diarrhea, dysentery and bronchitis. menorrhagia.

Traditional folk recipes Infusion of root is effective for coughs and respiratory Flowers are effective for gonorrhea and the powder of problems. Infusion or decoction of the peeled root is used roots is given for menorrhagia. Hot water extract of for skin problems and taken orally for mouth ulcers and flowers and roots is effective for fever and sore throat. To cure diarrhea powdered roots are boiled in emmenagogue. Petals along with coconut oil are applied milk and taken orally. externally for alopecia.

151

Table 69: Comparative qualitative pollen morphology of Althaea officinalis and Hibiscus rosa-sinensis

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Althaea officinalis Pantoporate Circular Spheroidal Present Echinate

2 Hibiscus rosa- Pantoporate Globose Oblate-spheroidal Present Echinate sinensis

Table 70: Comparative quantitative pollen morphology of Althaea officinalis and Hibiscus rosa-sinensis

Sr # Species name Polar diameter Equatorial diameter P/E Colpi/Spines Colpi width Exine thickness (μm) (μm) ratio length ( μm) (μm) (μm)

1 Althaea officinalis 110.5 (107-120.5) 103 (97.5-110) 1.07 11.5 (10.5-12) 3.8 (3-4.5) 8.5 (7.4-9.5)

2 Hibiscus rosa-sinensis 162 (159-168.5) 156.5 (152.5-164.5) 1.03 25 (22.5-30) 13.5 (12.5-15) 6.25 (5.7-7.5)

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Table 71. Comparative qualitative characters of foliar epidermal anatomy of Althaea officinalis and Hibiscus rosa-sinensis

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of wall Stomata Trichome type Shape of cell Pattern of wall Stomata type Trichome type species type

Althaea Polygonal Straight Anisocytic Stellate Polygonal Straight Anisocytic Stellate officinalis

Hibiscus rosa- Polygonal Straight or Anisocytic Non-glandular- Polygonal Straight or Anisocytic Non-glandular- sinensis slightly undulate unicellular slightly undulate unicellular

Table 72. Comparative quantitative characters of foliar epidermal anatomy of Althaea officinalis and Hibiscus rosa-sinensis

Abaxial epidermis Adaxial epidermis

Althaea 62.5 (54.5-70) × 23.6 (23-24.5) × 25.7 375 (250.5-515) × 57.5 (50.5-65) × 25.5 (25-26.5) × 21.5 390 (275-550) × officinalis 24.5 (20-28.5) 16.5 (15.5-18) 21 (18.5-24) 24 (20.5-27.5) 16 (15.25-17) 20 (15-25.5)

Hibiscus 45 (40.5-52.5) × 33 (31.25-35.5) 27.2 228.2 (200-254.8) 39.5 (36.5-42.8) × 31.5 (28.5-33.75) 23.9 210.5 (186.5-237) rosa-sinensis 25.5 (22.5-29.9) × 23.5 (20-24.5) × 14 (12-15.5) 23 (20.6-26.3) × 20 (18.5-22.6) × 16.5 (14.5-19.25)

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Figure 41 (a) Althaea officinalis ; (b) Dried roots; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 154 Results and Discussion Chapter 3

Figure 42 (a) Hibiscus rosa-sinensis ; (b) Dried roots; (c) SEM of pollen; (d) Exine sculpturing; (e) Trichome and stomata (LM-abaxial: 40X); (f) Epidermal cells and stomata (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 155

Table 73: Fluorescence and solubility analysis of powdered drug of Althaea officinalis (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried root powder Brownish yellow Yellowish green _ _ _ 2 Sample powder + 5% KOH Marsh green Copper bell Yellowish orange Golden grain Sparingly soluble

3 Sample powder + 10% aq. FeCl 3 Light brown Greenish brown Light brown Filtered light Slightly soluble

4 Sample powder + dH 2O Golden grain Celery seed Golden Demure Slightly soluble 5 Sample powder + HCL Conc. Fire brush Greenish brown Light brown Filtered light Slightly soluble 6 Sample powder + HCL 50% Golden grain Greenish brown Orange brown Chocolate brown Slightly soluble

7 Sample powder + H 2SO 4 Conc. Deep black Marsh green Golden Comfy tan Semi soluble

8 Sample powder + H 2SO 4 50% Golden grain Chocolate brown Deep brown Dark brown Slightly soluble

9 Sample powder + HNO 3 Conc. Copper bell Copper bell Light brown Golden grain Sparingly soluble

10 Sample powder + HNO 3 50% Mansion gold Chocolate brown Light orange Copper bell Semi soluble

11 Sample powder + CH 3OH Conc. Comfy tan Spiny green Light brown Summer day Slightly soluble

12 Sample powder + CH 3OH 50% Golden grain Copper bell Light orange Marsh green Slightly soluble

13 Sample powder + CHCl 3 Conc. Comfy tan Spiny green Light brown Golden grain Slightly soluble

14 Sample powder + CHCl 3 50% Capitols gold Celery seed Light yellow Comfy tan Slightly soluble

15 Sample powder + C 2H5OH Conc. Golden grain Glinted white Light yellow Golden grain Semi soluble

16 Sample powder + C 2H5OH 50% Capitols gold Spiny green Light brown Comfy tan Partially solube

17 Sample powder + CH 3COOH Conc. Comfy tan Light leaf Deep brown Golden grain Semi soluble

18 Sample powder + CH 3COOH 50% Mansion gold Spring cut Light brown Creamy white Sparingly soluble

19 Sample powder + C 6H6 Conc. Glinted white Celery seed Deep brown Creamy white Slightly soluble

20 Sample powder + C 6H6 50% Mansion gold Light leaf Light brown Comfy tan Slightly soluble

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Table 74: Fluorescence and solubility analysis of powdered drug of Althaea officinalis (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Brownish yellow Yellowish green _ _ _ 2 Sample powder + 5% KOH Copper Greenish brown Golden Orange yellow Soluble

3 Sample powder + 10% aq. FeCl 3 Dark copper Dark brown Golden orange Golden green Slightly soluble

4 Sample powder + dH 2O Capitols gold Light lichen Light golden Filtered light Sparingly soluble 5 Sample powder + HCL Conc. Golden grain Woven slats Light brown Chocolate brown Slightly soluble 6 Sample powder + HCL 50% Capitols gold Light lichen Dark brown Dark brown Slightly soluble

7 Sample powder + H 2SO 4 Conc. Deep black Brownish black Deep black Chocolate brown Soluble

8 Sample powder + H 2SO 4 50% Deep black Deep black Brownish black Dark brown Sparingly soluble

9 Sample powder + HNO 3 Conc. Golden orange Dark copper Light brown Whitish yellow Soluble

10 Sample powder + HNO 3 50% Red oxide Reddish black Light brown Light orange Sparingly soluble

11 Sample powder + CH 3OH Conc. Capitols gold Light lichen Light golden Ash white Slightly soluble

12 Sample powder + CH 3OH 50% Marsh green Silk knot Brownish gray Hidden green Slightly soluble

13 Sample powder + CHCl 3 Conc. Celery seed Celery seed Brownish green Light orange Partially soluble

14 Sample powder + CHCl 3 50% Capitols gold Light lichen Light golden Golden grain Slightly soluble

15 Sample powder + C 2H5OH Conc. Woven slats Forest green Light brown Orchid shadow Slightly soluble

16 Sample powder + C 2H5OH 50% Capitols gold Celery seed Light brown Light golden Slightly soluble

17 Sample powder + CH 3COOH Conc. Leaf green leafy green Orange green Ash white Semi soluble

18 Sample powder + CH 3COOH 50% Grayish brown Brownish green Light gray Orchid shadow Semi soluble

19 Sample powder + C 6H6 Conc. Blackish brown Brownish purple Lavender white Orchid shadow Slightly soluble

20 Sample powder + C 6H6 50% Grayish black Light gray Satin white Lavender white Slightly soluble

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Table 75: Fluorescence and solubility analysis of powdered drug of Hibiscus rosa-sinensis (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Yellowish brown Dark brown _ _ _ 2 Sample powder + 5% KOH Yellow Brownish orange Orange yellow Capitols gold Sparingly soluble

3 Sample powder + 10% aq. FeCl 3 Blackish green Dark brown Dark orange Woven slats Sparingly soluble

4 Sample powder + dH 2O Golden grain Leafy green Golden brown Leafy green Slightly soluble 5 Sample powder + HCL Conc. Dark orange Greenish yellow Brownish black Kiwi crush Sparingly soluble 6 Sample powder + HCL 50% Light green Yellowish green Dark brown Spring meadow Sparingly soluble

7 Sample powder + H 2SO 4 Conc. Dark brown Marsh green Chocolate brown Deep black Semi soluble

8 Sample powder + H 2SO 4 50% Mansion gold Brownish green Brownish black Ocean ripple Sparingly soluble

9 Sample powder + HNO 3 Conc. Light orange Golden brown Golden brown Capitols gold Soluble

10 Sample powder + HNO 3 50% Mansion gold Mansion gold Yellowish brown Golden grain Sparingly soluble

11 Sample powder + CH 3OH Conc. Golden grain Forest green Dark brown Light orange Slightly soluble

12 Sample powder + CH 3OH 50% Golden grain Leafy green Chocolate brown Lemon spirit Slightly soluble

13 Sample powder + CHCl 3 Conc. Mansion gold Marsh green Light brown Soft apple Slightly soluble

14 Sample powder + CHCl 3 50% Brownish black Golden green Orange brown Vanilla shake Slightly soluble

15 Sample powder + C 2H5OH Conc. Golden grain Celery seed Light yellow Orchid shadow Slightly soluble

16 Sample powder + C 2H5OH 50% Dark brown Golden grain Orange yellow Spiny green Slightly soluble

17 Sample powder + CH 3COOH Conc. Yellowish golden Spiny green Light brown Light green Sparingly soluble

18 Sample powder + CH 3COOH 50% Light lichen Forest green Orange yellow Light blue Slightly soluble

19 Sample powder + C 6H6 Conc. Golden grain Marsh green Apple white Lavender white Slightly soluble

20 Sample powder + C 6H6 50% Yellowish brown Light lichen Lemon tropics Ash white Slightly soluble

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Table 76: Fluorescence and solubility analysis of powdered drug of Hibiscus rosa-sinensis (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Yellowish brown Dark brown _ _ _ 2 Sample powder + 5% KOH Dark yellow Reddish yellow Light yellow Yellowish brown Sparingly soluble

3 Sample powder + 10% aq. FeCl 3 Dark copper Brownish green Golden brown Light brown Sparingly soluble

4 Sample powder + dH 2O Mansion gold Golden brown Light golden Leafy green Sparingly soluble 5 Sample powder + HCL Conc. Mansion gold Greenish brown Light brown Spiny green Sparingly soluble 6 Sample powder + HCL 50% Capitols gold Dark orange Chocolate brown Ocean ripple Slightly soluble

7 Sample powder + H 2SO 4 Conc. Deep black Blackish brown Orange brown Deep black Soluble

8 Sample powder + H 2SO 4 50% Blackish brown Blackish brown Chocolate brown Blackish brown Sparingly soluble

9 Sample powder + HNO 3 Conc. Red oxide Reddish brown Golden brown Capitols gold Soluble

10 Sample powder + HNO 3 50% Copper red Red orange Orange brown Golden grain Soluble

11 Sample powder + CH 3OH Conc. Golden grain Capitols gold Light golden Light golden Sparingly soluble

12 Sample powder + CH 3OH 50% Golden grain Golden grain Golden green Light yellow Slightly soluble

13 Sample powder + CHCl 3 Conc. Capitols gold Mansion gold Chocolate brown First frost Sparingly soluble

14 Sample powder + CHCl 3 50% Chocolate brown Copper Orchid shadow Light orange Sparingly soluble

15 Sample powder + C 2H5OH Conc. Capitols gold Mansion gold Light orange Light golden Sparingly soluble

16 Sample powder + C 2H5OH 50% Dark brown Blackish brown Orange brown Light lichen Slightly soluble

17 Sample powder + CH 3COOH Conc. Yellowish golden Leafy green Orchid shadow Light yellow Semi soluble

18 Sample powder + CH 3COOH 50% Woven slats Hidden green Light orange Light yellow Partially soluble

19 Sample powder + C 6H6 Conc. Yellowish brown Light lichen Ash white Ash white Slightly soluble

20 Sample powder + C 6H6 50% Golden grain Marsh green White ice Lavender white Slightly soluble

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Results and Discussion Chapter 3

Table 77: Physicochemical Parameters of Althaea officinalis and Hibiscus rosa-sinensis

S. No. Physicochemical parameters Althaea officinalis Hibiscus (%) rosa-sinensis i Total ash 5.33 7.66 ii Acid insoluble ash 0.33 0.67 iii Water soluble ash 2 2.66 iv Water insoluble ash 3.66 5 v Moisture content 4 6

Table 78: Antioxidant activity of Ascorbic acid, Althaea officinalis and Hibiscus rosa-sinensis

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic Althaea officinalis Hibiscus rosa- acid sinensis 25 50.91 21.16 13.4 50 60.47 27.49 19.97 100 72.39 32.97 26.1 150 81.06 39.2 29.75 200 92.8 42.21 34.04 250 96.02 54.67 35.76 IC 50 value 2.14 µg/mL 232 µg/mL 376 µg/mL

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 160 Results and Discussion Chapter 3

30 Total Phenols 25 Total Flavonoids 20 15 10 5

Concentration (mg /g) (mg Concentration 0 Althaea officinalis Hibiscus rosa- sinensis Plant extracts

Figure 43 . Total Phenolic and Flavonoid contents

100 90 25µg/mL 80 50µg/mL 70 100µg/mL 60 50 150µg/mL 40 200µg/mL

activity (%) activity 30 20 250µg/mL 10 DPPH radical scavenging DPPH 0 Ascorbic acid Althaea Hibiscus rosa- officinalis sinensis Plant extracts and standard at different concentrations

Figure 44 . DPPH radical scavenging activity (%)

______Authentication of M edicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 161 Results and Discussion Chapter 3

Figure 45. HPLC chromatogram of Althaea officinalis

Figure 46. HPLC chromatogram of Hibiscus rosa-sinensis

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 162 Results and Discussion Chapter 3

3.7 Authentication of Herbal Drug Resha khatmi ( Althaea officinalis L.)

Althaea officinalis (Malvaceae) is one of the important medicinal plants used therapeutically since ancient time. It is extensively used to cure cough, irritation of oral and pharyngeal mucosa, skin burns, insect bites, ulcers, inflammation, constipation and diarrhea (Shah et al., 2011). A. officinalis possessed anti-inflammatory, antiviral, antibacterial, antitumor, demulcent, smoothing and many other pharmacological effects (Al-Snafi, 2013). Roots of plant are used as medicine and sold in herbal market under the name of Resha khatmi. In herbal markets the roots of Hibiscus rosa-sinensis are occasionally encountered as medicinal marshmallow and sold under the trade name resha- khatmi. H. rosa-sinensis is used in alternative system of medicine as well as in conventional system of medicines (Houghton and Mukherjee, 2009). Plant is effective for fever, diabetes, epilepsy and leprosy (Kasture et al., 2000; Gilani et al., 2005).

Taxonomic Clarification

Morphologically, A. officinalis is characterized by branched velvety stem with broadly ovate leaves and roots are whitish yellow, spirally twisted and covered with loosened bast-fibers (Figure 41a & 41b). While H. rosa-sinensis have cylindrical and glabrate stem with lobed, heavily veined leaves and roots are brownish yellow with coarse surface (Figure 42a & 42b). The morphological description of A. officinalis and H. rosa-sinensis also reported in literature by several workers (Ng, 2006; Uzunhisarcikli and Vural, 2012; Divya et al., 2013). In this study detailed characterization for differentiation of A. officinalis and H. rosa-sinensis is presented in Table 68. Uzunhisarcikli and Vural et al. (2012) also used taxonomic characters for differentiation of seventeen species of Alcea and four species of Althaea from Turkey.

According to Perveen (1993) pollen are usually classified based on their shapes, size, aperture type, polarity, symmetry and exine sculpturing. Nair (1965)

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 163 Results and Discussion Chapter 3

and Lakshmi (2003) reported that variation in aperture type is considered to be primary importance in taxonomy, whereas exine sculpturing secondary and all others tertiary. Palynological features of A. officinalis and H. rosa-sinensis are presented in Table 69 & 70. It is noted that in A. officinalis pollen spines are with narrow base and gradually tapering tip (Figure 41c & 41d) while in H. rosa- sinensis pollen spines are conical, bifurcate and with blunt apex (Figure 42c & 42d). In accordance to our findings, Shaheen et al. (2010) recorded pantoporate pollen in A. officinalis . Hanif et al. (2013) recorded pantoporate pollen with echinate sculpturing in H. rosa-sinensis . Based on pollen morphology and degree of pollen variability Shaheen et al. (2009a) delimited fourteen species of Abutilon and Hibiscus of the family Malvaceae from Pakistan. Bibi et al. (2008) used palynological features to delimite four species and three cultivars of genus Hibiscus from North West Frontier Province (N.W.F.P.) Pakistan. Similarly, El Naggar (2004) described the pollen morphology of twenty one species of family Malvaceae and observed pantoporate pollen with straight, tapering or blunt apices spines in H. rosa-sinensis.

Foliar epidermal features of A. officinalis and H. rosa-sinensis showed significant degree of differentiation in term of trichomes. In A. officinalis trichomes are stellate (Figure 41e & 41f). While in H. rosa-sinensis the trichomes are non-glandular, forked and clearly demarked by twisted appearance of ray cells (Figure 42e & 42f). Comparative foliar epidermal features of A. officinalis and H. rosa-sinensis are presented in Table 71 & 72. Adedeji and Dloh (2004) conducted a comparative study of foliar anatomy of ten species of genus Hibiscus and observed that trichomes can be successfully used for delimitation of species within the genus. Shaheen et al. (2010) employed foliar epidermal anatomy as a significant taxonomic tool to differentiate four species of Alcea and Althaea from Pakistan. According to Shaheen et al. (2009b) variation in morphology and distribution of foliar trichomes emerged as an important supportive taxonomic tool in delimiting species of genus Hibiscus .

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 164 Results and Discussion Chapter 3

Pharmacognostic Authentication

Pharmacognostic analysis of powdered drug of A. officinalis along with its adulterant H. rosa-sinensis are presented in Table 73, 74, 75 & 76. Physicochemical parameters play important role in detection of adulterants and improper handling of drugs. Physicochemical parameters of A. officinalis and H. rosa-sinensis are presented in Table 77. Physicochemical analysis of A. officinalis and H. rosa-sinensis has been reported in literature by many workers (Soni et al., 2011; Al-Snafi, 2013). Standard of ASEAN herbal medicine (1993) and Pharmacopoeia of the People’s Republic of China (1997) described that roots of A. officinalis contain total ash not more than 6%, acid insoluble ash not more than 3% and moisture contents not more than 10%. Our findings regarding physicochemical characterization of A. officinalis are in accordance with these standards.

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

For ensuring the purity and efficacy of plant derived medicines phytochemical standardization considered to be a reliable tool to verify the identity of raw materials (Tripathi et al., 2014b). Results of phytochemical characterization showed that in A. officinalis roots higher phenolic (19 mg GAE/g, DW) and flavonoid contents (32.5 mg QE/g, DW) were found as compared to H. rosa-sinensis roots where phenolic contents were 11.5 mg GAE/g, DW and flavonoid contents were 17 mg QE/g, DW (Figure 43). Flavonoid contents in roots of A. officinalis have been reported by many workers (Gudej, 1991; Ninov et al., 1992; Ionkova, 1992). Benbassat et al. (2014) reported the phytochemical evaluation of the various extracts of A. officinalis flowers and observed that higher concentration of flavonoids was obtained by extraction with lower ethanol concentration. Sadighara et al. (2012) reported the flavonoid contents in the A. officinalis flowers based on their color. They observed that high

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 165 Results and Discussion Chapter 3

flavonoid contents were found in white flowers followed by reddish pink and pink. Ghaffar and El-Elaimy (2012) recorded total phenolic contents (48.4 mg GAE/g) and total flavonoid contents (24.26 mg QE /g) in H. rosa-sinensis crude extract. Similarly, Vankar and Srivastava (2008) assessed the comparative data of total phenol and flavonoid contents in H. rosa-sinensis and Canna indica .

HPLC Screening

The interest in identification and quantification of flavonols, especially quercetin, in different products, is growing owing to their antioxidant activity. HPLC is a preferred technique for both separation and quantification of phenolic compounds (Naczk and Shahidi, 2004). Reversed phase HPLC has been used to distinguished species based on quantitative variation of flavonoids among them (Harborne et al., 1985). It has been applied especially for identification of flavonoids derivatives (Kerhoas et al., 2006). HPLC analysis showed that slightly high amount of quercetin (15.03 mg/g, DW) was found in A. officinalis as compared to H. rosa-sinensis (14.78 mg/g, DW). HPLC chromatograms of A. officinalis and H. rosa-sinensis are presented in Figure 45 & 46.

Antioxidant analysis

Antioxidant analysis showed that A. officinalis has higher antioxidant activity (54.67%) as compared to H. rosa-sinensis (35.63%) at 250 µg/mL concentration (Table 78 & Figure 44). Benbassat et al. (2014) evaluated the antioxidant activity of various ethanolic extracts of A. officinalis roots. The authors concluded that higher antioxidant activity was obtained with low concentration ethanol solution. Divya et al. (2013) reported 63.5% antioxidant activity at 100 µg/mL concentration in methanolic extract of leaves of H. rosa- sinensis. In previous study, based on antioxidant activities Mak et al. (2013) differentiated flowers of Hibiscus rosa-sinensis and Senna bicapsularis .

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 166 Case 8:

Table 79: Authentication of herbal drug Kaur (Picrorhiza kurroa Royle ex Benth.) in comparison with its adulterant

Characters (Picrorhiza kurroa Royle ex Benth.) Lagotis cashmeriana (Royle) Rupr.

English Names Picrorrhiza, Gentian Lagotis, Hare’s ear

Local Names Kutki, Karroo, Kaur, Kutaki safed Kashmir lagotis

Trade Name Kaur None

Family Scrophulariaceae Scrophulariaceae

Flowering period June-August June – August

Phytogeography: In Pakistan Kashmir to Sikkim and Astore valley. Deosai plains, Hunza and Kaghan.

In World Western Himalayan region, Nepal. China, India and Himalayas, China, India, Bhutan and Pakistan. Pakistan. Morphological description Perennial herb, rhizome creeping, 6-12 cm, brown with Perennial fleshy herb, rhizome creeping. Leaves basal, thin rootlets, slender, longitudinally grooved. Leaves 5- 2- 15cm long, elliptic oblong, fleshy, stalked and crenate 15cm long, basal resette, elliptic to spatulate, margin margin. Stem leaves are bract like, stalk less, simple and serrate. Leaf base tapered into petiole. Inflorescence in small. Inflorescences capitate or narrowly spicate, terminal receme 10-15cm, bear few bracts like leaves. densely flowered, scapes 1 to many, bracts imbricate. Bracts hairy, elliptic, situated at fruit base. Sepals five, Flowers 8 mm long, borne in short, stout spikes 3-4 cm. persistent, hairy, lanceolate. Corolla 8 mm long, five Bracteoles absent. Corolla 2-lipped, Bracts acute and

167

lobed. Fruit capsule, brown, monolocular, pointed, split shorter than the corolla (about 5mm long). Stamens 2, along four lines. Capsule 6-10 mm long, ovoid atenuate, stigma 2-lobed, ovary 2-loculed. Seeds 1-5mm long and laterally sulcate surrounded by persistent sepals. Seeds surrounded by persistent calyx. 1.5 mm long, brownish and numerous. Trade Part Roots and rhizomes are sold in herbal market under the Roots and rhizomes are used as adulterant of Picrorhiza trade name kaur. kurroa .

Organoleptography Dried leaves are dull green in color and coarsely tooth. Dried leaves are pale green in color, oblong to elliptic, Flowers are small, pale purplish. Rhizomes are woody, stalked with rounded teeth on the margins. Flowers are cylindrical, slightly curved, texture coarse due to tubular, pale blue in color. Rhizomes are thick with longitudinal wrinkles, scales and scars are present, fragile brown rootlets. Roots are thin, and light brown in color. and grayish brown in color. Roots are thin, straight or Aroma is orange like and taste of roots and leaves is slightly curved with dotted scar and dusty gray. Aroma is bitter. slightly unpleasant and taste is very bitter.

Part used Roots, rhizomes Roots, rhizomes, leaves

Ethnomedicinal Uses Diabetes, chronic fever, dyspepsia, rheumatism, Fever, dyspepsia, diabetes, skin infections, stomachache stomachache, skin disorders and liver disorders. and liver infections.

Traditional folk recipes Roots decoction is taken for the treatment of Root decoction is effective for fever and dyspepsia. As stomachache, high fever, gastritis, liver ailments and the roots are as adulterant of Picrorhiza kurroa . So they rheumatism. Root decoction flavored with honey is given are used as the same way as that of Picrorhiza kurroa to cure stomachache in adults. Root powder mixed with and have similar properties. black piper and honey is very effective for fever. It also enhances the resistance of the body. Roots smoke is considered efficacious for asthma.

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Table 80: Comparative qualitative pollen morphology of Picrorhiza kurroa and Lagotis cashmeriana

Sr # Species name Type of Pollen Shape in Polar view Shape in Equatorial Colpi: Present Exine Sculpturing view /Absent

1 Picrorhiza kurroa Tricolporate Spheroidal Subprolate Present Psilate

2 Lagotis cashmeriana Tricolporate Spheroidal Prolate-spheroidal Present Reticulate

Table 81: Comparative quantitative pollen morphology of Picrorhiza kurroa and Lagotis cashmeriana

Sr # Species name Polar diameter Equatorial diameter P/E Colpi length Colpi width Exine thickness (μm) (μm) ratio (μm) (μm) (μm)

1 Picrorhiza kurroa 32.5 (29.5-35) 27 (24-30.5) 1.20 0.75 (0.5-1) 0.62 (0.30-0.90) 1.25 (1-1.75)

2 Lagotis cashmeriana 39 (37.5-41.2) 34 (32.5-37) 1.14 1.65 (1.5-1.82) 3.25 (3.2-3.75) 1.75 (1.5-2.25)

169

Table 82. Comparative qualitative characters of foliar epidermal anatomy of Picrorhiza kurroa and Lagotis cashmeriana

Abaxial epidermis Adaxial epidermis

Name of Shape of cell Pattern of Stomata Trichome type Shape of cell Pattern of Stomata type Trichome type species wall type wall

Picrorhiza Irregular Undulate Anisocytic Non-glandular Irregular Undulate Anisocytic Non-glandular kurroa multicellular, multicellular, Glandular Glandular Peltate Peltate

Lagotis Polygonal Straight Anomocytic Glandular Polygonal Straight Anomocytic Glandular cashmeriana

Table 83. Comparative quantitative characters of foliar epidermal anatomy of Picrorhiza kurroa and Lagotis cashmeriana

Abaxial epidermis Adaxial epidermis

Picrorhiza 85 (62.5-100) × 37.5 (35-42.5) × 16.36 480 (350-750) × 86 (75.5-95.8) × 28.5 (25-32.5) × 14.54 450 (280-625) × kurroa 34 (30-37.5) 17.5 (15.5-20) 31.5 (25-37.5) 37 (35-39.5) 13.5 (12.5-15) 26.5 (22.5-30)

Lagotis 56 (50-62.5) × 38.5 (37.5-39.25) 12.72 90 (44.5-135) × 54.5 (48.5-60.3) × 41.5 (39.25-43.5) 9.09 110.5 (65.5-157.5) cashmeriana 38.25 (35.6-41.8) × 26 (25-28.5) 12.5 (10.5-15.25) 40.5 (37.2-45) × 29 (28.5-30.5) × 21.5 (9.25-12.5)

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Results and Discussion Chapter 3

a b

c d

e f

Figure 47 (a) Picrorhiza kurroa ; (b) Dried roots; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells and trichome (LM- adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 171 Results and Discussion Chapter 3

a b

c d

e f

Figure 48 (a) Lagotis cashmeriana ; (b) Dried roots; (c) SEM of pollen; (d) Exine sculpturing; (e) Epidermal cells and stomata (LM-abaxial: 40X); (f) Epidermal cells (LM-adaxial: 40X).

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 172

Table 84: Fluorescence and solubility analysis of powdered drug of Picrorhiza kurroa (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Dark brown Chocolate brown _ _ _ 2 Sample powder + 5% KOH Light chocolate Dark brown Dull pink Glowing pink Slightly soluble

3 Sample powder + 10% aq. FeCl 3 Dark reddish brown Brown Dark brown Pinkish brown Slightly soluble

4 Sample powder + dH 2O Muddy brown Chocolate brown Light mulberry Tea rose Very slightly soluble 5 Sample powder + HCL Conc. Reddish black Lilac time Ash white Fawn Sparingly soluble 6 Sample powder + HCL 50% Light brown Golden glimmer Fawn Clay Sparingly soluble

7 Sample powder + H 2SO 4 Conc. Dark black Pine forest Sky gray Sea mist Soluble

8 Sample powder + H 2SO 4 50% Light chocolate Purple Light mulberry Tea rose Sparingly soluble

9 Sample powder + HNO 3 Conc. Copper bell Cranberry Shingle Abbey cream Sparingly soluble

10 Sample powder + HNO 3 50% Reddish black Copper bell Glinted white Brilliant white Sparingly soluble

11 Sample powder + CH 3OH Conc. Light brown Marsh green Clay Summer day Slightly soluble

12 Sample powder + CH 3OH 50% Golden brown Rado green Sand stone Autumn stone Slightly soluble

13 Sample powder + CHCl 3 Conc. Dark brown Light leaf Beige Magnolia Partially soluble

14 Sample powder + CHCl 3 50% Purplish brown Forest green Light brown Clay Partially soluble

15 Sample powder + C 2H5OH Conc. Deep brown Golden glimmer Cameo Beige Slightly soluble

16 Sample powder + C 2H5OH 50% Brown Golden glimmer Fawn Clay Slightly soluble

17 Sample powder + CH 3COOH Conc. Brownish black Spring cut Light mulberry Orchid shadow Partially soluble

18 Sample powder + CH 3COOH 50% Greenish brown Leaf green Magnolia Creamy white Sparingly soluble

19 Sample powder + C 6H6 Conc. Deep brown Pine forest Deep brown Tea rose Sparingly soluble

20 Sample powder + C 6H6 50% Light brown Golden glimmer Ash white Lavender white Slightly soluble

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Table 85: Fluorescence and solubility analysis of powdered drug of Picrorhiza kurroa (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Dark brown Chocolate brown _ _ _ 2 Sample powder + 5% KOH Dark chocolate Dark brown Dull pink Tea rose Partially soluble

3 Sample powder + 10% aq. FeCl 3 Brownish black Dark brown Dark brown Brownish pink Partially soluble

4 Sample powder + dH 2O Dark brown Chocolate brown Dull pink Light mulberry Partially soluble 5 Sample powder + HCL Conc. Deep black Lilac time Light brown Dark brown Soluble 6 Sample powder + HCL 50% Dark brown Leaf green Dark brown Clay Soluble

7 Sample powder + H 2SO 4 Conc. Deep black Charcoal Antique white Classic ivory Soluble

8 Sample powder + H 2SO 4 50% Blackish brown Dull purple Dark chocolate Dark brown Soluble

9 Sample powder + HNO 3 Conc. Red oxide Dark brown Peach silk Abbey cream Soluble

10 Sample powder + HNO 3 50% Copper oxide Chocolate brown Sea shell Sugar cane Partially soluble

11 Sample powder + CH 3OH Conc. Brown Spiny green Magnolia Beige Semi soluble

12 Sample powder + CH 3OH 50% Golden brown Golden glimmer Fawn Clay Semi soluble

13 Sample powder + CHCl 3 Conc. Chocolate brown Leaf green Autumn stone Sand stone Semi soluble

14 Sample powder + CHCl 3 50% Brownish purple Marsh green Brown Light mulberry Semi soluble

15 Sample powder + C 2H5OH Conc. Brownish black Rado green Silk stone Hopsack Semi soluble

16 Sample powder + C 2H5OH 50% Dark brown Pine forest Smoke gray Ash white Partially soluble

17 Sample powder + CH 3COOH Conc. Blackish brown Forest green Light mulberry Coral reef Semi soluble

18 Sample powder + CH 3COOH 50% Grayish brown Lichen green Abbey cream Classic ivory Partially soluble

19 Sample powder + C 6H6 Conc. Brownish black Pine forest Orion Ash white Partially soluble

20 Sample powder + C 6H6 50% Dark brown Golden glimmer Light mulberry Lilac time Partially soluble

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Table 86: Fluorescence and solubility analysis of powdered drug of Lagotis cashmeriana (Cold method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried roots powder Blackish brown Greenish brown _ _ _ 2 Sample powder + 5% KOH Dark copper Brownish orange Yellowish orange Capitols gold Slightly soluble

3 Sample powder + 10% aq. FeCl 3 Reddish black Chocolate brown Blackish orange Golden grains Slightly soluble

4 Sample powder + dH 2O Dark brown Dark brown Chocolate brown Spring meadow Slightly soluble 5 Sample powder + HCL Conc. Deep black Purplish black Brownish black Dark brown Semi soluble 6 Sample powder + HCL 50% Golden brown Blackish brown Orange brown Ash white Semi soluble

7 Sample powder + H 2SO 4 Conc. Dark brown Dark reddish brown Dark brown Lemon yellow Semi soluble

8 Sample powder + H 2SO 4 50% Chocolate brown Orange brown Lemon yellow Off white Semi soluble

9 Sample powder + HNO 3 Conc. Red oxide Golden brown Golden brown Orchid shadow Semi soluble

10 Sample powder + HNO 3 50% Reddish brown Dark brown Deep orange Light mulberry Partially soluble

11 Sample powder + CH 3OH Conc. Chocolate brown Dark brown Orange brown Halo Slightly soluble

12 Sample powder + CH 3OH 50% Light brown Reddish black Dark brown Lemon spirit Partially soluble

13 Sample powder + CHCl 3 Conc. Blackish brown Deep black Dark brown Ocean ripple Semi soluble

14 Sample powder + CHCl 3 50% Golden brown Brownish black Orange brown Lavender white Partially soluble

15 Sample powder + C 2H5OH Conc. Dark brown Reddish brown Orange yellow Snow bell Partially soluble

16 Sample powder + C 2H5OH 50% Chocolate brown Dark brown Orange brown Light green Partially soluble

17 Sample powder + CH 3COOH Conc. Greenish brown Chocolate brown Orange brown Light green Semi soluble

18 Sample powder + CH 3COOH 50% Mustard brown Maroonish brown Light yellow Soft apple Partially soluble

19 Sample powder + C 6H6 Conc. Golden brown Brownish black Orange brown Orchid shadow Partially soluble

20 Sample powder + C 6H6 50% Yellowish brown Light lichen Light yellow Off white Slightly soluble

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Table 87: Fluorescence and solubility analysis of powdered drug of Lagotis cashmeriana (Hot method)

S. No. Treatment Under visible light Under UV Filter paper under Filter paper under Solubility visible light UV light 1 Dried root powder Blackish brown Greenish brown _ _ _ 2 Sample powder + 5% KOH Deep Black Brownish black Creamy orange Off white Partially soluble

3 Sample powder + 10% aq. FeCl 3 Copper oxide Dark brown Chocolate brown Spring meadow Partially soluble

4 Sample powder + dH 2O Blackish brown Purplish brown Dark brown Light yellow Slightly soluble 5 Sample powder + HCL Conc. Deep black Purplish black Brownish purple Light mulberry Soluble 6 Sample powder + HCL 50% Dark brown Brownish red Orange black Ash white Soluble

7 Sample powder + H 2SO 4 Conc. Reddish black Reddish brown Chocolate brown Lemon yellow Soluble

8 Sample powder + H 2SO 4 50% Reddish brown Orange brown Light yellow Lavender white Soluble

9 Sample powder + HNO 3 Conc. Dark red Reddish brown Golden brown Satin white Soluble

10 Sample powder + HNO 3 50% Reddish brown Dark brown Orange brown Light purple Soluble

11 Sample powder + CH 3OH Conc. Chocolate brown Light brown Yellowish brown Antique white Semi soluble

12 Sample powder + CH 3OH 50% Light brown Orange brown Light brown First frost Slightly soluble

13 Sample powder + CHCl 3 Conc. Muddy brown Chocolate brown Orange yellow Light yellow Sparingly soluble

14 Sample powder + CHCl 3 50% Golden brown Brownish black Orange brown Lavender white Sparingly soluble

15 Sample powder + C 2H5OH Conc. Light brown Dark brown Lemon yellow Halo Semi soluble

16 Sample powder + C 2H5OH 50% Yellowish brown Light brown Orange yellow Light green Semi soluble

17 Sample powder + CH 3COOH Conc. Golden brown Blackish brown Dark brown Ocean ripple Soluble

18 Sample powder + CH 3COOH 50% Light brown Mustard brown Light mulberry Orchid shadow Semi soluble

19 Sample powder + C 6H6 Conc. Dark brown Brownish black Orange brown Light mulberry Partially soluble

20 Sample powder + C 6H6 50% Light brown Reddish brown Light brown Ash white Partially soluble

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Results and Discussion Chapter 3

Table 88: Physicochemical Parameters of Picrorhiza kurroa and Lagotis cashmeriana

S. No. Physicochemical parameters Picrorhiza kurroa Lagotis (%) chasmeriana i Total ash 3 6.67 ii Acid insoluble ash 0.33 0.67 iii Water soluble ash 2 1 iv Water insoluble ash 1 4.67 v Moisture content 7 9

Table 89: Antioxidant activity of Ascorbic acid, Picrorhiza kurroa and Lagotis cashmeriana

Concentration % Scavanging % Scavanging of % Scavanging of (μg/mL) of Ascorbic acid Picrorrhiza Lagotis kurroa cashmeriana 25 50.91 47.36 40.27 50 60.47 52.20 43.82 100 72.39 54.99 47.26 150 81.06 57.25 52.84 200 92.8 61.22 53.59 250 96.02 61.76 55.53 IC 50 value 2.14 µg/mL 34 µg/mL 146 µg/mL

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50 Total Phenols 40 Total Flavonoids 30

10 Concentration (mg /g) (mg Concentration 0 Picrorrhiza kurroa Lagotis cashmeriana Plant extracts

Figure 49. Total Phenolic and Flavonoid contents

100 90 25µg/mL 80 50µg/mL 70 60 100µg/mL 50 150µg/mL 40 200µg/mL

activity (%) activity 30 20 250µg/mL 10 DPPH radical scavenging DPPH 0 Ascorbic acid Picrorhiza Lagotis kurroa cashmeriana Plant extracts and standard at different concentrations

Figure 50. DPPH radical scavenging activity (%)

______Authentication of Medicinal p lants traded as herbal drugs by using Systematics and Phytochemical c haracterization 178 Results and Discussion Chapter 3

Figure 51. HPLC chromatogram of Picrorhiza kurroa

Figure 52. HPLC chromatogram of Lagotis cashmeriana

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 179 Results and Discussion Chapter 3

3.8 Authentication of Herbal Drug Kaur (Picrorhiza kurroa Royle ex Benth.)

Picrorhiza kurroa (Scrophulariaceae) is a small alpine herb, commonly known as Gentian. The roots and rhizomes of herb are used in medicinal formulations for treatment of constipation, flatulence, anorexia, burning sensation, fever, anemia, skin diseases, diabetes, liver and spleen problems, jaundice and hemorrhoids (Keshari et al., 2015). Plant is harvested manually and its roots and rhizomes are sold in herbal markets of Pakistan under the trade name kaur. It is a self regenerating herb but unsustainable use and harvesting has caused it to be threatened to near extinction. Due to high demand of P. kurroa for its increased therapeutic applications, there is extensive harvesting practice that leads the shortage of genuine source of this drug and its growing demand is fulfill by using morphologically similar roots of Lagotis cashmeriana. Roots of Lagotis cashmeriana are most common adulterant of P. kurroa roots and given in fever and dyspepsia (Handa, 2000; Gogte, 2000).

Taxonomic Clarification

Morphologically, P. kurroa can be differentiated from its adulterant L. cashmeriana in having elliptic and coarsely tooth leaves with purplish blue flowers and woody, cylindrical, fragile rhizomes (Figure 47a & 47b). While in L. cashmeriana leaves are oblong-elliptic with crenate margins, flowers are dark blue and rhizomes are thin with brown rootlets (Figure 48a & 48b). Kaul (1997) and Kletter and Kriechbaum (2001) also described the morphological features of P. kurroa and L. cashmeriana . Morphological differentiation between P. kurroa and L. cashmeriana is shown in Table 79. Similar to our project Joshi (2005) used taxonomic features for differentiation of Valeriana procera from other commercially important Valeriana species.

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By using palynological features, P. kurroa can be distinguished from L. cashmeriana . The pollen of P. kurroa are subprolate in equatorial view with psilate sculpturing (Figure 47c & 47d) while in L. cashmeriana pollen are prolate- spheroidal in equatorial view with reticulate sculpturing (Figure 48c & 48d). In P. kurroa sculpturing is psilate with complete smooth surface while in L. cashmeriana sculpturing is reticulate with large quadrangular lumina and thin elevated muri. Palynological characters of P. kurroa and L. cashmeriana are shown in Table 80 & 81. Similarly, other herbal drugs can also be authenticated from their adulterants by using palynological features such as herbal drug Jadwar (Delphinium denudatum ) can be distinguished from its adulterant ( Aconitum hetrophyllum ) on the basis of pollen sculpturing (Zafar et al., 2011).

In addition to palynological features, foliar epidermal anatomy can be used to differentiate P. kurroa and L. cashmeriana . In P. kurroa epidermal cells are irregular with undulate walls, anisocytic stomata and both glandular and non- glandular trichomes. Glandular trichomes are peltate and non-glandular trichomes are multicellular (Figure 47e & 47f). While in L. cashmeriana epidermal cells are polygonal, walls are straight, stomata are anomocytic and trichomes are glandular (Figure 48e & 48f). Comparative foliar epidermal features of P. kurroa and L. cashmeriana are presented in Table 82 & 83. Our taxonomic findings for differentiation of these two species are in accordance with those of Kaul (1997) and Kletter and Kriechbaum (2001). Similar to our study, Srilakshmi and Naidu (2014) employed foliar epidermal features for differentiation of Artemisia vulgaris , Chrysanthemum indicum and Comos sulphureus of the family Asteraceae and reported that epidermal features such as epidermal cells, type of stomata and trichomes are important parameters at generic and specific levels.

Pharmacognostic Authentication

Pharmacognostic analysis of powdered drugs provides proper authenticity for the quality of raw materials. Results of fluorescence analysis and solubility

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tests of powdered drugs are presented in Table 84, 85, 86 & 87. Colors fluorescence of P. kurroa obtained after treating the powder drug with different chemicals are in accordance with Keshari et al. (2015). Results of physicochemical analysis of P. kurroa and L. cashmeriana are shown in Table 88. According to Keshari et al. (2015) in P. kurroa the standard pharmacopeia limit for total ash is <7%, acid insoluble ash <1% and water soluble ash >20%. Meena et al. (2010) evaluated the physicochemical parameters for authentication of P. kurroa. Our findings of physicochemical analysis are in accordance with previous reported studies.

Phytochemical Characterization

Quantitative analysis of Phenols and Flavonoids

Phytochemical characterization can also be used as an aid to distinguish P. kurroa from L. cashmeriana. Results showed that higher concentration of phenolic contents (37.5 mg GAE/g, DW) and flavonoid contents (55.08 mg QE/g, DW) was found in P. kurroa roots as compared to L. cashmeriana roots where phenolic contents were 22.35 mg GAE/g, DW and flavonoid contents were 28.61 mg QE/g, DW (Figure 49). Our results of phenolic and flavonoid contents in P. kurroa are similar to those reported in literature (Kalaivani et al., 2010; Sinha et al., 2011). Several studies reported that quantification of phenols and flavonoids can also be helpful for correct identification and standardization of other herbal drugs such as Glycyrrhiza glabra roots (Husain et al., 2015).

HPLC Screening

Recently, chromatographic fingerprint techniques are attracting more attention in the quality control methods of herbal samples. These techniques emphasize on the integral characterization of compositions of samples with a quantitative degree of reliability and focus on identifying and assessing the stability of herbal plants (Shams et al., 2014). Results of HPLC screening showed higher concentration of quercetin in P. kurroa roots (52.46 mg/g, DW) as

______Authentication of Medicinal plants traded as herbal drugs by using Systematics and Phytochemical characterization 182 Results and Discussion Chapter 3

compared to L. cashmeriana (24.43 mg/g, DW). HPLC chromatograms of P. kurroa and L. cashmeriana are presented in Figure 51 & 52. Similarly, Hullatti and Sharada (2010) used HPLC chromatographs for differentiation of Cissampelos pareira , an ayurvedic drug Patha from its adulterants Cyclea peltata and Stephania japonica .

Antioxidant analysis

Recently, interest has increased greatly in finding natural antioxidants for use in foods because of their promising affects in the health promotion, disease prevention, high safety and consumer acceptability (Agnihotri et al., 2008). Results of antioxidant analysis showed that higher activity was shown by P. kurroa as compared to L. cashmeriana . Antioxidant activity of P. kurroa in comparison with adulterant is shown in Table 89 & Figure 50. In previous studies many workers reported the highest scavenging activity of P. kurroa (Bhandari et al., 2010; Kant et al., 2013; Gurelia et al., 2013). Based on antioxidant activity Shubha et al. (2013) differentiated the roots of P. kurroa from roots of Solanum xanthocarpum and reported that P. kurroa roots showed highest scavenging activity with IC 50 value 72.58 µg/mL.

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3.9 Conclusion

Global resurgence of interest in herbal medicines coupled with increased market demand and resource crises have led to the use of adulterated and spurious raw material in drug production. Purity of the plant material is the prerequisite for the safety and efficacy of the plant derived medicine. This study presents detailed account of authentication of medicinal plants traded as herbal by using systematic and phytochemical characterization. Eight cases of problematic medicinal plants were selected from the herbal markets of Pakistan. These medicinal plants are traded under the name of Tukhm-e-kalonji ( Nigella sativa ), Tukhm-e-balango ( Lallemantia royleana ), Belladona ( Atropa acuminata ), Dhatura ( Datura stramonium ), Chiraita ( Swertia cordata ), Zafran (Crocus sativus ), Resha khatmi ( Althaea officinalis ) and Kaur (Picrorhiza kurroa ). As there is no comprehensive information regarding systematics and phytochemical characterization of these eight herbal medicines. So the study was designed to develop standards parameters for the authentication of these problematic medicinal plants which could be helpful in identification of genuine drug. Systematics confined to morphology, palynology (LM & SEM) and foliar epidermal anatomy (LM) which play a significant role in identification of taxa at specific level. The results of qualitative and quantitative taxonomic descriptions are provided as diagnostic features to differentiate the genuine drugs from their closely related adulterants. Phytochemical standardization is considered to be a reliable tool for verification of identity of raw materials for ensuring the purity and efficacy of plant derived medicines. The pictorial profiles of genuine drugs and their adulterants can serve as an identification manual for researchers, herbalists, herbs collectors and traders. The outcome of this study could be helpful in identification of genuine drug and also contribute towards establishing pharmacopoeial standards. Study can also serve as an important source of information to achieve the authenticity and to evaluate the quality and purity of the plant material in accordance to WHO guidelines.

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4. Recommendations

Identification, collection, drying, storage and transportation process of raw material used in manufacturing of herbal drugs needs to be streamlined on scientific grounds as per WHO guidelines. Cultivation of medicinal plants should be encourage at large scale as it not only ensures the authentication but also maintains economic sustainability and easy availability of medicinal plants for better health of people. Herbal drug manufacturing companies should employ the services and skills of botanical experts for correct identification of medicinal plants. A short identification manual containing coloured pictures of medicinal plants in their natural habitats should be prepared for collectors so that they can easily identify the genuine source of drug. Strict laws should be implemented in the whole country regarding the sale of substandard and adulterated crude herbal drugs. There are still other problems of correct identification, which can be solved by using up to date modern techniques like HPTLC, GC-MS, FTIR, DNA finger prints etc. Hence attention should be paid on these critical issues, which has been ignored so far.

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