Chemotaxonomical Characterization of Solanum nigrum and its Varieties AYESHA MOHY-UD-DIN SESSION 2004-2008 20-PhD-CHEM-04 DEPARTMENT OF CHEMISTRY, GC UNIVERSITY LAHORE, PAKISTAN Chemotaxonomical Characterization of Solanum nigrum and its Varieties Submitted to GC University Lahore in partial fulfillment of the requirements for the award of degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by AYESHA MOHY-UD-DIN SESSION 2004-2008 20-PhD-CHEM-04 DEPARTMENT OF CHEMISTRY, GC UNIVERSITY LAHORE, PAKISTAN “I dedicate my thesis work to my beloved Parents, to my dear Sister, Brothers & Niece to my most respectable Teachers and to every person who taught me even a single word. I respect them from the core of my heart and am indebted to these people, as I would never have been able to complete my research work without their motivation and mentoring.” CONTENTS CONTENTS List of Tables v List of Figures & Schemes vi List of Chromatograms vii Acknowledgements viii-ix Abstract x-xii List of Publications xiii-xv CHAPTER 1: INTRODUCTION 1-36 1.1. Chemistry of Natural Products: Science of All Times 1 1.1.1. Historic Books on Herbal Medicines 1 1.1.2. Famous Historic Medicinal Systems 2 1.1.3. Herbal Medicine and Islam 3 1.2. Taxonomy of Plants 4 1.2.1. Causes of Taxonomic Complexity 4 1.3. Chemotaxonomy 5 1.3.1. Applications of Chemotaxonomy 6 1.4. Family Solanaceae 7 1.4.1. Importance of Solanaceae 7 1.5. Genus Solanum 9 1.6. Solanum nigrum: Taxonomic Complications 10 1.7. Botanical Aspects of the Investigated Taxa 12 1.7.1. Solanum americanum Miller 13 1.7.2. Solanum chenopodioides Lam. 14 1.7.3. Solanum nigrum L. 15 1.7.4. Solanum retroflexum Dunal 16 1.7.5. Solanum villosum Miller 17 1.7.6. Key to the Investigated Taxa of S. nigrum Complex 18 1.8. Importance of S. nigrum Complex 18 1.8.1. Medicinal Uses 18 1.8.2. Source of Alkaloids 19 1.8.3. Nutritional Value 19 1.8.4. Commercial Value 20 i Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS 1.9. Secondary Metabolites 21 1.9.1. Alkaloids 21 1.9.2. Flavonoids 26 1.9.3. Epicuticular Wax 29 1.10. Biological Evaluation 31 1.10.1. Antimicrobials 31 1.10.2. Antioxidants 32 1.11. Aims and Objectives 35 CHAPTER 2: LITERATURE SURVEY 37-55 2.1. Chemical Constituents from S. nigrum Complex 37 2.2. Investigations on S. nigrum (A Brief Review) 39 2.3. Chemotaxonomy and Secondary Metabolites 51 2.3.1. Alkaloids 51 2.3.2. Flavonoids 53 2.3.3. Epicuticular Waxes 54 CHAPTER 3: EXPERIMENTAL WORK 56-83 3.1. General Experimental Conditions 56 3.2. Plant Material 57 3.2.1. Dewaxing with n-Hexane 57 3.3. Phytochemical Examination of Plant Material 58 3.4. Analysis of Alkaloids 59 3.4.1. Estimation of Total Glycoalkaloid Content 59 3.4.2. Alkaloid Extraction and Detection 60 3.4.3. TLC Procedure for SGA 61 3.4.4. HPLC Analysis of SGA 61 3.4.5. Acid Hydrolysis of SGA 62 3.4.6. Derivatization of SGAA 62 3.4.7. GC-MS Analysis of SGAA Derivatives 62 3.5. Analysis of Flavonoids 63 3.5.1. Estimation of Total Flavonoid Content 63 3.5.2. Flavonoids Extraction and Detection 65 3.5.3. TLC Procedure for Flavonoid Glycosides 65 ii Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS 3.5.4. HPLC Analysis of Flavonoid Glycosides 66 3.5.5. Acid Hydrolysis of Flavonoid Glycosides 67 3.5.6. GC-MS Analysis of Flavonoid Aglycones 67 3.6. Analysis of Epicuticular Waxes 68 3.6.1. Extraction of Epicuticular Wax 68 3.6.2. Physicochemical Analysis of Wax 68 3.6.3. Detection of Epicuticular Wax Components 71 3.6.4. TLC Procedure for Epicuticular Wax 71 3.6.5. GC-MS Analysis of Epicuticular Wax 71 3.7. Proximate Analysis of the Plant Material 72 3.8. Antibacterial Study 77 3.9. Antioxidant Study 79 3.9.1 Preparation of Reagents 79 3.9.2. Total Phenolic Content Assay 80 3.9.3. Lipid Peroxidation Assay 81 3.9.4. FRAP Assay 81 3.9.5. ABTS•+ Assay Protocol 82 3.9.6. DPPH Radical Scavenging Capacity Assay 82 3.9.7. Metal Chelating Activity 83 CHAPTER 4: RESULTS AND DISCUSSION 84-149 4.1. Overview of the Work 84 4.2. Species Delimitation 84 4.3. Phytochemical Examination of Plant Material 87 4.4. Analysis of Alkaloids 87 4.4.1. Estimation of Total Glycoalkaloid Content 87 4.4.2. TLC Analysis 88 4.4.3. HPLC Analysis 90 4.4.4. GC-MS Analysis 92 4.5. Analysis of Flavonoids 94 4.5.1. Colorimetric Analysis 94 4.5.2. TLC Analysis 96 4.5.3. HPLC Analysis 97 4.5.4. GC-MS Analysis 99 iii Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS 4.6. Analysis of Epicuticular Waxes 101 4.6.1. Physicochemical Analysis of Wax 101 4.6.2. TLC Analysis 102 4.6.3. GC-MS Analysis 103 4.7 Proximate Analysis and Mineral Composition 108 4.8. Antibacterial Study 111 4.9. Antioxidant Study 114 4.9.1. Antioxidant Activity of S. nigrum Complex 114 4.9.2. Total Phenolic Content Assay 114 4.9.3. Lipid Peroxidation Assay (Total Antioxidant Activity) 116 4.9.4. FRAP Assay 117 4.9.5. ABTS•+ Assay Protocol 118 4.9.6. DPPH Radical Scavenging Capacity Assay 119 4.9.7. Metal Chelating Activity 120 4.10. Conclusion 121 CHROMATOGRAMS 125-149 REFERENCES 150-172 ANNEX-I PUBLISHED PAPERS A1-A22 iv Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS LIST OF TABLES Table 1: Review of the Compounds reported from S. nigrum Complex 37 Table 2: List of plants investigated with voucher numbers 57 Table 3: Morphological comparison of five taxa of Solanum nigrum Complex 86 Table 4: TLC of alkaloids of five taxa of S. nigrum Complex 88 Table 5: Concentration of SGA in five taxa of S. nigrum Complex by HPLC 90 Table 6: SGAA concentration in five taxa of S. nigrum Complex by GC-MS 93 Table 7: Flavonoid contents of S. nigrum Complex by Colorimetric methods 96 Table 8: TLC of flavonoids of five taxa of S. nigrum Complex 97 Table 9: Concentration of Flavonoid Glycosides in S. nigrum Complex by HPLC 98 Table 10: Quercetin concentration in five taxa of S. nigrum Complex by GC-MS 100 Table 11: Physicochemical analysis of the waxes of S. nigrum Complex 102 Table 12: Wax yield and the distribution of different classes of compounds 103 Table 13: Composition of Epicuticular wax extracted from S. nigrum Complex 104 Table 14: Proximate analysis of the five taxa of S. nigrum Complex 109 Table 15: Mineral element concentration of the five taxa of S. nigrum Complex 110 Table 16: Zones of inhibition of different concentrations of methanolic extracts of S. nigrum Complex and standards against different bacterial strains 112 Table 17: Minimum Inhibitory Concentration (MIC) of methanolic extracts from S. nigrum Complex against different bacterial strains 113 Table 18: EC50 and TEC50 values of methanolic extracts of S. nigrum Complex 119 v Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS LIST OF FIGURES & SCHEMES Fig. 1: Popularity of Solanum sp. over time 9 Fig. 2: S. americanum 13 Fig. 3: S. chenopodioides 14 Fig. 4: S. nigrum 15 Fig. 5: S. retroflexum 16 Fig. 6: S. villosum 17 Fig. 7: General Structure of Steroidal Alkaloids 24 Fig. 8: Basic Structure of Flavonoids 26 Fig. 9: Quercetin 27 Fig. 10: The structures of solanidine- and solasodine-based glycoalkaloids 89 Fig. 11: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of SGA analysed by HPLC 91 Fig. 12: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of SGAA analysed by GC-MS 93 Fig. 13: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of flavonoid glycosides analysed by HPLC 98 Fig. 14: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of flavonoid aglycones analysed by GC-MS 100 Fig. 15: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of epicuticular wax analysed by GC-MS 107 Fig. 16: Total phenolic contents (TPC) of S. nigrum Complex 115 Fig. 17: Total Antioxidant capacity evaluated by the Lipid peroxidation method 116 Fig. 18: Antioxidant capacity evaluated by the FRAP method 117 Fig. 19: Antioxidant capacity evaluated by the ABTS method 118 Fig. 20: Antioxidant capacity evaluated by DPPH method 120 Fig. 21: Metal chelating ability of methanolic extracts 121 Scheme 1: Approaches towards the classification of plants 4 Scheme 2: Classification of Alkaloids 23 Scheme 3: Analysis of the Plant Material 58 vi Chemotaxonomical Characterization of Solanum nigrum and its Varieties CONTENTS LIST OF CHROMATOGRAMS 1. HPLC chromatograms of steroidal glyco-alkaloids of five taxa of S. nigrum Complex 125-129 2. GC-MS chromatograms of steroidal glyco-alkaloid aglycones of five taxa of S. nigrum Complex 130-134 3. HPLC chromatograms of flavonoid glycosides of five taxa of S. nigrum Complex 135-139 4. GC-MS chromatograms of flavonoid aglycones of five taxa of S. nigrum Complex 140-144 5. GC-MS chromatograms of epicuticular waxes of five taxa of S. nigrum Complex 145-149 vii Chemotaxonomical Characterization of Solanum nigrum and its Varieties ACKNOWLEDGEMENT All my praises and love belong to ALLAH ALMIGHTY, the most Merciful, the most Beneficent, Ruler of the Universes, Who created everything, from the sub-particles of an atom to this universe.