Chemotaxonomical Characterization of Solanum Nigrum and Its Varieties

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

CHEMISTRY

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

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. He, whose praise has no bounds. He has been with me through thick and thin of life and enabled me in tiding over the difficulties. His perpetuate patronage is treasure of my life.

Many respects and praises for HIS last PROPHET MUHAMMAD (Salal-La Ho Alaiyehe Was-sullum) Who himself has been sent as a teacher for the whole world to enable us to recognize our Creator and understand the philosophy of life. He remains the torch of knowledge and guidance for humanity.

I am lucky enough to have the prerogative to express my deep gratitude towards my research supervisor Dr. M. Akram Kashmiri (Chairman, Board of Intermediate and Secondary Education Lahore, former Dean of Sciences & Chairperson Department of Chemistry GCU Lahore), for his guidance, encouragement and counseling towards me throughout the course of this session. He provided me with all possible facilities and supervision and his principle “work from dawn to dusk” was thought provoking and source of inspiration for me.

I owe a special debt of cordial gratitude to my co-supervisor Dr. Mushtaq Ahmad (Professor GCU Lahore, Retd. Director General PCSIR Laboratory Quetta) whose studious guidance, keen interest and encouragement have been an innate source of inspiration for me. He has unfolded new and hitherto unexplored dimensions of Chemistry and groomed my vision of research as well as my life.

I am obliged to have the auspices of Dr. Islam Ullah Khan, (Chairperson Department of Chemistry GCU Lahore), for his kind behavior and special interest in mentoring and helping me in completing my research.

I am lucky enough, to have my exposure to the worthy guidance of Dr. Zaheer-ud-din Khan (Chairman Department of Botany GCU Lahore). I’ll always acknowledge his guidance, encouragement and affection towards me through out the course of this session.

I acknowledge the financial contribution of Higher Education Commission (HEC) Pakistan. As an initiative of Dr. Atta-ur-Rehman, it aims to provide all the possible help for students from graduate to post doctoral level as well as to faculty members to see the country an educational success.

A special feeling of gratitude to my respected teacher Mr. Amin Javaid who has played a key role in developing my interest and understanding in Chemistry. I am grateful to the teaching faculty of viii

different departments of GCU specially Dr. Ashraf Mirza, Dr. Mohsin Iqbal, Mr. Ehsan Elahi, Dr. Athar Abbasi, Dr. Iftikhar Ahmad, Dr. Adnan, Mr. Ayoub, Ms. Saba Qaiser, Ms. Samia Firdous, Dr. Shazia, Mr. Zaffar Siddique, administration officials Mr. Sharif Mughal (Treasurer), Dr. AZ Din (Instrument engineer), Mr. Bilal Moazam (Director purchase), Engr. Bilal (Campus engineer), Mr. Babar (Deputy registrar), Ms. Huma (Controller office), Mr. Mubbashir (HEC scholar desk), and people from other institutions Dr. N.M. Butt (Scientist Emeritus, PINSTECH), Mr. Waqar A. Butt (Head HRD PINSTECH), Mr. Talib (Instrument expert LCWU), Ms. Saima (Instrument expert UVAS), Mr. Naveed (Technical expert Technology Links) who were always there to help me whenever I needed them.

My warm regards are especially for my friend, labmate and Ph.D. Scholar Sammia Yasmin, whose help, encouragement and motivating personality kept me moving despite certain desperation stops. My jovial thanks to my friends Shagufta, Gulshan, Lubna, Khadija & Ambreen whom sincere prayers were with me. My fellow Ph.D. scholars Ishfaq, Nadeem, Akmal, Waqar and colleagues at Lahore Cantt Public High School, Leads Academy & Crescent Higher Secondary School have been very nice and cooperative.

I acknowledge the kind help of the non teaching staff of GCU specially Mr. Abdul Ghafoor, Mr. Hanif, Mr. Rehmat, Mr. Saajid, Mr. Manzoor, Mr. Usman, Mr. H. Athar, Mr. G. Mohy-ud-din & Mr. Farasat.

I sincerely pray for the excel of my students Saboor, Zain, Ali, Rizwan Sana, Hina, Sadaf, Sidra & Rabia, who contributed in my research work in any way and their prayers supported me a lot.

I can never pay the reward to my loving father Inayat Mohy-ud-Din who has always given me encouragement and motivation & my caring mother Nagina Inayat, who is so affectionate and has made my educational foundation. Without the persistent encouragement and untiring support of my dear sister Fayeza, brother-in law Sharjeel, so caring and helping brothers Fawad, Ammad & Shehzad beloved niece Amina, dear Uncles, Auntis & Cousins, I could never have been able to make even a single day successful. I can never pay the debt of their love and affection.

Finally, I bow my head before ALMIGHTY ALLAH, for giving me reverence to be a Ravian, to be a part of GC University Lahore, most privileged and prestigious University for eight years & for my selection as Young Scientist in 56th Meeting of Nobel Laureates, Lindau, Germany. This is the World of Opportunities, for those who are willing to explore new dimensions, and have ―Courage to Know‖.

AYESHA MOHY-UD-DIN. ix

ABSTRACT

Solanum nigrum Complex is the name given to a group of closely related plants whose taxonomic status is an International controversy among different taxonomists. But no chemotaxonomic relationship has so far been studied due to lack of comprehensive investigation of chemical composition of the individual plant taxa. The present chemotaxonomic studies, alongwith nutritional and biological evaluation, of the five locally available plant taxa of S. nigrum Complex viz.: S. americanum Mill., S. chenopodioides Lam., S. nigrum L., S. retroflexum Dunal and S. villosum Mill. were carried out in order to resolve the controversy on their taxonomic status and to reveal the medicinal importance of the individual taxa. In these studies the morphologically different plant taxa were grown under controlled conditions in the Botanic Garden, GC University Lahore, Pakistan, and third accession of each taxon was taken for the chemotaxonomic investigations. Comparative analyses of these plant samples were undertaken with respect to the Alkaloids, Flavonoids and Epicuticular wax as potential characters. The HPLC and GC-MS analyses of these constituents had not been reported previously. Also, with the exception of S. nigrum, literature is silent on the detailed chemical analysis of the taxa under study. Statistical analyses of results grouped taxa into different clusters. The comparison of alkaloidal profile of the five taxa was used to establish the boundaries among close taxonomic groups. Yield of total glycoalkaloids in the five taxa ranged from 68.9±0.6 to 25±0.8% as determined by Titrimetric method. Glycoalkaloids analysed by HPLC demonstrated that the concentration of β-Solamargine was much higher in S. villosum (9.8 mg/g) than other samples but that of α-Solamargine was relatively higher in S. nigrum (5.03 mg/g). There was a gradual change in Solasonine concentration ranging from 2.01 mg/g (S. villosum) to 5.8 mg/g (S. nigrum). α-Solanine concentration was maximum in S. retroflexum. GC-MS of the aglycones depicted that percentage of Solanidine in the samples varied from 8.85-20.31% (being highest in S. retroflexum) while that of Solasodine from 66.99-85.67% (being highest in S. americanum). Significant distances were shown between S. chenopodioides and S. villosum as well as in

x Chemotaxonomical Characterization of Solanum nigrum and its Varieties ABSTRACT americanum and S. nigrum in their respective clusters. However S. retroflexum did not show such a marked difference with respect to S. nigrum. In order to further compare the differences among the five plant taxa, their flavonoid compositions were investigated. Total flavonoid contents in the taxa were calculated by two complementary colorimetric methods and ranged from 0.883±0.020 to 2.116±0.032. From HPLC, it was found that S. americanum had the highest concentration of both Quercetin-3-glucoside (0.03520 mg/100mL) and Quercetin-3-galactoside (0.00750 mg/100mL) as well as of Quercetin aglycon (6.46±0.01 mg/100g) when determined by GC-MS. Percentage of quercetin in the samples varied from 7.28±0.33 to 92.92±0.45%. Statistical analyses of the results showed marked distances among S. americanum, S. chenopodioides, S. nigrum and S. villosum but indicated similarity between S. nigrum and S. retroflexum. Epicuticular wax, a complex mixture of different constituents, is considered another important parameter for chemotaxonomic studies. The yields and physicochemical characters like Colour, Melting point, Refractive index, Saponification value, Acid value and Ester value of waxes extracted were compared. TLC indicated the presence of different classes of compounds in the waxes. GC-MS analysis showed the presence of Squalene, Phytol, Palmitic acid, Linolenic acid, ester of Palmitoleic acid along with a variety of hydrocarbons as the chemical constituents of these epicuticular waxes. The hydrocarbons, alcohols, some of the esters, acids, aldehyde and ketone identified had been reported for the first time in S. nigrum. The cluster analysis indicated significant differences between S. chenopodioides and S. villosum as well as in between S. americanum and S. nigrum in their respective clusters. Again S. retroflexum depicted great resemblance with S. nigrum in its epicuticular wax composition. The similarity index and the Euclidean distance among the clusters formed by Multivariate cluster analysis of the above discussed parameters helped drawing the conclusion that S. americanum, S. chenopodioides, S. nigrum and S. villosum are distinct species of genus Solanum but S. retroflexum might be regarded as a variety/subspecies of S. nigrum. The nutritional potentials of the plants were assessed through their proximate and mineral analyses. The results of this research indicated that the plants had nutritional qualities that could provide the users with additional

xi Chemotaxonomical Characterization of Solanum nigrum and its Varieties ABSTRACT nutrients. Comparatively, because of the relatively high contents of total protein, total ash and crude fibres, the taxon S. nigrum could be good source of nutrients. Antibacterial and antioxidant activities were carried out to evaluate the medicinal value of the plants. The methanolic extracts of the five taxa had shown significant antibacterial activity against the Gram +ve and Gram –ve bacteria used. Infact, the methanolic extracts of S. villosum showed a higher MIC value against Proteus mirabilis compared to standard Benzyl penicillin. The extracts of S. nigrum and S. retroflexum demonstrated matching results which supported our conclusion that S. retroflexum may be considered as a variety of S. nigrum. S. chenopodioides and S. villosum also gave comparable but less coordinated results. Antioxidant activity of the plant extracts was evaluated using six different antioxidant assays. Results suggested that all taxa have moderate effects on scavenging DPPH free radical. Total Phenolic Contents of the five samples showed slight variations, ranging from 20.31-26.58 mg of GAE/100 g DW. In ABTS assay, S. retroflexum had highest antioxidant capacity (33.88 mM/100 g DW). The effect of the S. chenopodioides (70.37%) on metal chelation was found to be more than all the other taxa. These activities were attributed to the appreciable amounts of alkaloids, flavonoids and phenolic components present in these plant samples. The incredible morphological and chemical diversity, fundamental economic importance and worldwide distribution make the Solanaceae one of the most fascinating groups of flowering plants.

xii Chemotaxonomical Characterization of Solanum nigrum and its Varieties PUBLICATIONS

LIST OF PUBLICATIONS

1. Mohy-ud-din, A.; Khan, Z.; Ahmad, M.; Kashmiri, M.A.; Yasmin, S.; Asghar, M.N. & Ahmad, S.R.; Epicuticular waxes from Solanum nigrum Complex: Chemotaxonomic implications. Asian J. Chem., 22 (4), (2010) 2919-2927.

2. Mohy-ud-din, A.; Khan, Z.; Ahmad, M. & Kashmiri, M.A.; Chemotaxonomic Value of Alkaloids in Solanum nigrum Complex. Pak. J. Bot., 42(1), (2010) 653-660.

3. Mohy-ud-din, A.; Khan, Z.; Ahmad, M.; Kashmiri, M.A.; Yasmin, S. & Mazhar, H.; Chemotaxonomic significance of flavonoids in the Solanum nigrum Complex. J. Chil. Chem. Soc., 54 (4), (2009) 289-293.

4. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, M.; Khan, Z. & Yasmin, S.; Comparative studies on medicinal value of Solanum nigrum Complex using antibacterial activity. Accepted for presentation in 2nd ISMP 2010 at LCWU Pakistan, proceedings will be published in Bioscience Research. (Acceptance letter attached on p. xvi)

5. Mohy-ud-din, A.; Khan, Z.; Kashmiri, M.A.; Ahmad, M. & Yasmin, S.; Role of chemotaxonomy in classification of plants. Proceedings of International Seminar/workshop on preservation & enhancement of biodiversity, GC University Lahore, Pakistan. (October, 2009).

6. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, M.; Yasmin, S. & Ahmad, S.R.; Comparative Studies on Composition of Non-Polar Extracts in Solanum nigrum Complex. 11th International Symposium on Medicinal Plants, International Centre for Chemical and Biological Sciences Karachi,Pakistan. (October,2008) Abstract:p. 195.

xiii Chemotaxonomical Characterization of Solanum nigrum and its Varieties PUBLICATIONS

7. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, I.; Ahmad, M.; Yasmin, S. & Ahmad, S.R.; Comparative Studies on composition of non-polar extracts in Solanum nigrum Complex using GC-MS. Proceedings of 1st International seminar on Medicinal Plants Lahore College for Women University, Lahore, Pakistan. (May, 2008) pp. 225-226.

8. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, M.; Yasmin, S. & Ahmad, S.R.; Comparative Studies on composition of non-polar extracts in Solanum nigrum Complex using GC-MS. 18th National Chemistry Conference, Punjab University (February, 2008) Abstract: p. 166.

9. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, I.; Ahmad, M. & Ahmad, S.R.; Comparison of composition of non-polar extracts in Solanum nigrum Complex. 1st International Conference, Recent Advances in Chemistry, Lahore College for Women University, Lahore, Pakistan. (November, 2007) Abstract: p. 75.

10. Mohy-ud-din, A.; Kashmiri, M.A.; Ahmad, I.; Ahmad, M. & Ahmad, S.R.; Analysis of Antioxidant Activities of Solanum nigrum Complex employing six different Assays: A Comparative Study. (manuscript in process of submission)

xiv Chemotaxonomical Characterization of Solanum nigrum and its Varieties PUBLICATIONS

ACCEPTANCE LETTER

xv Chemotaxonomical Characterization of Solanum nigrum and its Varieties

Chap.1: INTRODUCTION

INTRODUCTION

1.1. CHEMISTRY OF NATURAL PRODUCT: SCIENCE OF ALL TIMES Natural products, as the term implies, are those chemical compounds derived from living organisms and the study of natural products is the investigation of their structure, formation, use and purpose in the organism. Drugs derived from natural products are usually secondary metabolites and their derivatives. Today those must be pure and highly characterized compounds. Since prehistoric times, the humans have relied on natural products as a primary source of medicine. Plants and animals were used to bring back the health of sick and frail. Plant were found to be beneficial as food, fodder, medicine etc. but also harmful as being poisonous and toxic (Fuller and Hemrick, 1985). The application of herbs for external and internal use has always been a major factor in practice of medicine (Steiner, 1986). The experience and knowledge gained in using the traditional medicines in different regions over the millennia resulted in the complex science of modern medication.

The various approaches to drug discovery from nature are:  Ethnobotanical: Ethnic and traditional medicine  Random screening: Bioassay guided routes  Chemotaxonomic: Screening of relatives

1.1.1. HISTORIC BOOKS ON HERBAL MEDICINES The use of drugs can be divided broadly into five periods. The early periods covers the Indians, Chinese, Sumerian, Egyptian and Assyrian civilization followed by the Greco-Roman, Arabian, Medieval and modern periods. Rig Veda, the claimed oldest religious book, had mentioned the medicinal use of plants. Records from as early as 2700 B.C. from China, traced to the Emperor Shennung, indicate the usefulness of plants for treating disease and the Ebers papyrus, written in about 1550 B.C., includes many of the plants used in Egyptian medicine. First Materia Medica of the world was developed by the Greeks. Ibn al-Baitar (1197-1248) listed over 1400 drugs and medicinal plants in his Corpus of Simples. Al-Qanun fi al-Tibb (The

1 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

Canon of Medicine) is a 14-volume Arabic medical encyclopedia written by a Persian scientist and physician Ibn Sina (Avicenna) and completed in 1025 (Stanley, 1994). Famous books Firdous al Hikmat, Deen-e-Doulat and Hifz al-Shehhat of Ali ibn Rabban are kept at European libraries for their importance (Jacquart, 2008). In Europe, after the tenth century, much of the medicinal lore was based in the church, particularly the monastic orders, but by the 1500‘s, after the invention of the printing press, herbals available to the general public were popular, particularly in England. By the late 1700‘s, studies like William Withering‘s An Account of the Foxglove and its Medicinal Uses (1785) began to appear. These studies included case histories as well as specific doses and administration instructions for herbal remedies. In the United States, before the advent of specific pharmaceuticals, herbal medicine was relied upon to treat many illnesses. Development of drugs based on natural products has had a long history in the United States, and in 1991, almost half of the best selling drugs were natural products or derivatives of natural products. There has recently been a resurgence of interest in herbal remedies and the U.S. market for natural supplements is increasing by as much as 10% per year (courtesy of TCM Physicians Clinic website visited on 20-05-2009).

1.1.2. FAMOUS HISTORIC MEDICINAL SYSTEM The traditional Chinese medicine system and Ayurveda (traditional medicine system of India) were fully as sophisticated and as documented system as western medicine systems. The Sushruta Samhita and the Charaka Samhita were influential works on traditional medicine during this era (Sahu and Mishra, 2003). The extensive records of Chinese medicine about response to Artemisia preparations for malaria also provided the clue to the novel antimalarial drug artemisinin. The therapeutic properties of the opium poppy (active principle morphine) were known in Ancient Egypt and those of the Solanaceae plants in ancient Greece (active principles atropine and hyoscine). The snakeroot plant was well regarded in India (active principle reserpine), and herbalists in medieval England used extracts from the willow tree(salicin) and foxglove (active principle digitalis - a mixture of compounds such as digitoxin, digitonin, digitalin) for cure of malaria. The Aztec and Mayan cultures of Mesoamerica used extracts from a variety of bushes and trees including the ipecacuanha root (active principle emetine), coca bush (active principle cocaine), and cinchona bark (active principle quinine) (Bensky et al., 2003).

2 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.1.3. HERBAL MEDICINE AND ISLAM ALLAH Almighty has repeatedly directed and encouraged people to ponder on and investigate thoughtfully the happenings in this universe through Holy Quran. For example:  Verily! It is Allah Who causes the seed-grain and the fruit-stone (like date- stone) to split and sprout. He brings forth the living from the dead, and it is He Who brings forth the dead from the living. Such is Allah, then how are you deluded away from the truth? (Al-Anaam, Verse: 95)  And We cause therein the grain to grow and grapes and cloverplants (i.e.green fodder for the cattle) and olives and date-palms(Surah Abasa, Verses: 27- 29).  And whatsoever He has created for you on the earth of varying colours [and qualities from vegetation and fruits (botanical life) and from animals (zoological life)] Verily! In this is a sign for people who remember. (An-Nahl, Verse: 13) In Islamic tradition, the first Muslim physician is believed to have been Prophet Mohammad (Sull-Allah-ho-Alaihe-Wasallum) himself, as a significant number of Hadiths (His sayings) concerning medicine are attributed to him. Several Sahaba (his companions who has seen him) are said to have been successfully treated of certain diseases by following his medical advice of. The three methods of healing known to have been mentioned by him were honey, fire cupping and cauterization, though he was generally opposed to the use of cauterization unless it "suits the ailment" and Mohammad (Sull-Allah-ho-Alaihe-Wasallum) also appears to have been the first to suggest that there is always a cause and a cure for every disease (Deuraseh, 2003; Borchardt, 2002):  Make use of medical treatment, for Allah has not made a disease without appointing a remedy for it, with the exception of one disease, namely old age Sunan Abi Dawood, 28:3846.

There are many sayings of Prophet Mohammad (Sull-Allah-ho-Alaihe- Wasallum) describing medicinal uses of the plants like olives and dates used by Arabs that time (Farooqi, 1998; Ghaznavi, 1991). The belief that there is a cure for every disease encouraged early Muslims to engage in biomedical research and seek out a cure for every disease known to them. The works of ancient Greek and Roman physicians Hippocrates (father of medicine),

3 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

Dioscorides, Soranus, Celsus and Galen had a lasting impact on Islamic medicine (Saad et al., 2005). From the 9th century, Muslim physicians soon began making many of their own significant advances and contributions to medicine, including all the fields of medicines and natural sciences. Responding to circumstances of time and place, Islamic physicians and scholars developed a large and complex medical literature exploring and synthesizing the theory and practice of medicine. Muslim physicians set up the earliest dedicated hospitals in the modern sense which were establishments where the ill were welcomed and cared for by qualified staff, and which were clearly distinguished from the ancient healing temples, which were more concerned with isolating the sick and the mad (insane) from society "rather than to offer them any way to a true cure (Morelon and Rashed, 1996).

1.2. TAXONOMY OF PLANTS Biological classification or scientific classification, is a method by which taxonomists group and categorize plants by biological type, such as genus or species. The classification of plants by grouping data according to morphological similarities is probably the oldest and most widely-used of all the approaches (Quinlan, 1993). However many approaches evolved over time towards the taxonomy of plants.

Scheme 1: Approaches towards the classification of plants

1.2.1. CAUSES OF TAXONOMIC COMPLEXITY Morphological variants of a species results because of different factors. In addition, many species exhibit considerable genetic variation, both florally and vegetatively. This variation may occur in different populations of the same species, or may characterize different infraspecific categories of a species. Sometimes, the

4 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION character may be genetically controlled in one species, but phenotypically plastic in another. Quaternary climatic changes have had a profound impact on speciation, structuring of genetic diversity and the shaping of the present-day distributions of plant and animal taxa (Avise, 2000; Hewitt, 1996, 2000, 2004; Vuilleumier, 1971). Oscillations of population sizes, bottle necks, founder events and other population historical events associated with climatic shifts have further contributed to differentiation among regional population groups. As a combined effect of range shifts and population differentiation, divergent lineages have occasionally formed contact zones, leading to reticulate speciation by means of hybridization and polyploidization (Grant, 1981; Stebbins, 1984). Polyploid speciation has long been recognized as an important process in plant evolution (Müntzing, 1936; Stebbins 1950; Grant, 1981). Recent genomic studies have made it clear that angiosperms possess genomes with considerable gene redundancy, indicating that ―most (if not all) plants have undergone one or more episodes of polyploidization‖ (Soltis et al., 2003).

1.3. CHEMOTAXONOMY Chemotaxonomy is also called chemosystematics or biochemical systematics. The science of chemical taxonomy is used on the classification of plants on the basis of their chemical constituents which are deeply concerned with the molecular characteristics. The method of chemical taxonomy is simple in principal and is based on the investigations of the distribution of chemical compounds or groups of biosynthetically related compounds in series of related plants. Different plants sometimes contain substances which although belong to different chemical compounds appear to be biosynthetically analogous. Such plants may contain similar enzyme systems, and the compounds produced by such enzymes are indicative of the relationships that exist between the plants. However, the chemotaxonomic studies include the investigation of the patterns of the compounds existing in plant. Climatic conditions have a major influence on the distribution of plants containing certain substances e.g. fats, volatile oils, alkaloids, flavonoids etc. It is well known that for tropics, and perhaps for all climates, the chemical products are highly organized. According to Reichert (1919), it is possible to identify many plants by their starch grains. Stress has been given on the importance of β-Cyanins and β-Xanthins in plant

5 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION taxonomy. β-Cyanins are commonly met within the families of order centrospermae. The other chemicals are also found specifically in particular orders or families of flowering plants e.g. Isoquinoline (Alkaloids) is found in the families of Ranales; retanone in the families of leguminales, biflavonoils in casuarinas equisetifolia (casuarinaceae). The presence of such chemicals in different groups of plants has great taxonomic significance (Stuessy, 2008). Taxonomic studies for various plant taxa by using different parameters have been successfully carried out in Pakistan including that of cereals (Ashraf et al., 2003), Legumes (Ahmad et al., 2007) and Maize (Nawaz and Ashraf, 2007).

1.3.1. APPLICATION OF CHEMOTAXONOMY (Stuessy, 2008) There are a few angiospermic taxa which are characterized by specific compounds of general occurrence. For example leaving aside the family of caryophyllaceae, the rest of the families such as chenopodiaceae, amaranthaceae, aizoacaceae etc. of the taxon caryophyllales (centrospermea) contain β-cyanin a colored substance but differs from anthocyanins. It appears that, with the exception caryophyllaceae, these families are closely related and therefore caryophyllaceae may be isolated. β-cyanin also occurs in cactaceae and therefore, the members of caryophyllales are phylogenetically related. There are certain other chemical connections between cactaceae and members of caryophyllales e.g. common presence of isoquinole alkaloids in Salsola of Chenopodiaceae and cactaceae. Another example that may be cited is in the family crucifereae, where unsaturated acid erucic acid is prominent and also in Tropaelum erucic acid is present; it indicates the relationship between Geraniales and Rhocadales. In umbellifereae and Araliaceae petroselinic acid (a structural isomer of Oleic acid) occurs and these two families are related and belong to the same order. The other examples are from Magnoliales Ranals taxa, where it is shown that magnoliaceae, lauraceae, Ranulculaceae, Annoraceae, the alkaloid isoquinoline is present, this supports that these families are loosely related. On the other hand Asclepiadaceae and Gentianaceae are allied due to the common occurrence of pyridine. The lilliaceae and Amarylldeceae are closely associated and this is supported by the presence of Isoquinoline in both. A number of citations regarding chemotaxonomy and secondary metabolites are given in Chapter 2.

6 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.4. FAMILY SOLANACEAE The Solanaceae, to which the genus Solanum belongs, is a cosmopolitan family which is widely distributed throughout tropical and temperate regions of the world, with centers of diversity occurring in Central and South America and Australia. It is composed of approximately 84 genera and 3000 species. The name of the family comes from the Latin word Solanum, meaning "the nightshade plant", but the further etymology of that word is unclear; it has been suggested it originates from the Latin verb ‗solari‘ meaning "to soothe". This would presumably refer to alleged soothing pharmacological properties of some of the psychoactive species found in the family. It is more likely, however, that the name comes from the perceived resemblance that some of the flowers bear to the sun and its rays, and in fact a species of S. nigrum Complex (Solanum retroflexum) is known as the sunberry. The family is also informally known as the nightshade or potato family (Yasin, 1985).

1.4.1. Importance of Solanaceae The Solanaceae family is characteristically ethnobotanical and is an important source of food, spice and medicine. The family includes variety of plants like important vegetables and fruits as well as some poisonous plants (some plants have both edible and toxic parts). Most common plants of this family are the Datura or Jimson weed, eggplant, mandrake, deadly nightshade or belladonna, capsicum (paprika, chili pepper), potato, tomato, Petunia, Schizanthus and Lycium species. It includes many species cultivated for their edible fruits or tubers, such as the tomato, potato, aubergine/eggplant and chilli pepper. The most important species of this family for the global diet is the potato (S. tuberosum) whose carbohydrate-rich tubers have been a staple food in many times and places, and which is one of the most grown crops today. In many genera of this, the fruits are the economically desirable item, for example, tomatoes (S. lycopersicum L.), eggplants (S. melongena L.), and peppers (Capsicum sp.). It also contains tobacco (Nicotiana tabacum L.), one of the most harmful yet economically important plants in the world. It has a documented record of causing heart, lung, and circulatory problems as well as cancer and other health problems (Tso, 1977; Zitnak, 1977). Paradoxically, while we usually think of members of the

7 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

family as essential and familiar foods, many other members of the Solanaceae are famed for their alkaloid content and have been used throughout history for their medicinal, poisonous, psychotropic effects; examples include tobacco, jimson weed, henbane, and belladonna. Solanaceae species are often rich in alkaloids that can range in their toxicity to humans and animals from mildly irritating to fatal in small quantities (Edmonds and Chweya, 1997). Several solanaceous plants and products are highly poisonous, such as deadly black nightshade, Atropa belladonna L., and Jimsonweed, Atropa stramonium L (Heiser,1987; Kingsbury, 1968). Tropane alkaloids that have always played a significant role in ethno medicine as well as orthodox medicine were also extracted from the genera Datura, Brugmansia, and Atropa of the Solanaceae family. Plants in the drug family, Solanaceae (nightshades) are an important causative factor in arthritis in sensitive people (Childers and Margoles, 1993). Some drugs from the Solanaceae are widely used in medicine, such as scopolamine, atropine, hyoscyamine, and belladonna (Lewis, 1977). Common ornamental plants such as Petunia, Schizanthus, Salpiglossis, Lycium and Browallia are members of this family. Tobacco, petunia, tomato and potato are used as model experimental organisms in examining fundamental biological questions in cell, molecular and genetic studies. Alkaloids and steroids in the Solanaceae are reported extensively in the literature. By examining the biosynthetic routes leading to different alkaloids, the pathways can be visualized as a spiral from which the various compounds can be derived. Arrangement of the genera of Solanaceae according to their chemical contents in relationship to this spiral supports traditional classifications of the family, but the Anthocercidoideae and Atropoideae must be recognized as new subfamilies due to their biochemical synthesis syndromes. Similarly, Solaninae and Physalinae must be accepted as separate subtribes of tribe Solaneae because of their differing and exclusive steroid synthesis. Acnistus and Dunalia must be allied with Jaborosa in tribe Jaboroseae (Tetenyi, 1987).

8 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.5. GENUS SOLANUM Within Solanaceae family, Solanum constitutes the largest, variable and most complex genus. It is consists of annual and perennial plants, forbs, vines, sub-shrubs, shrubs, and small trees. They often have attractive fruit and flowers. Together with many other plants of both poisonous and medicinal value Solanum constitutes the largest and most complex genus of the family. It is composed of more than 1500 species, many of which are also economically important throughout their cosmopolitan distribution (Ganapathi and Rao, 1986; Edmonds and Chweya, 1997). Solanum is one of the most commercially and economically important genra of Solanaceae which had been extensively studied. Fig. 1 show a plot of number of papers published in different years depicting its popularity.

Solanum sp.

Fig. 1: Popularity of Solanum sp. over time: [Plots of numbers of papers mentioning Solanum sp. (filled column histogram and left hand axis scale) and line of best fit, 1926 to 2006 (complete line, with equation and % variation accounted for, in box on the left hand side); Plots of a proportional micro index, derived from numbers of papers mentioning Solanum sp. as a proportion (scaled by multiplying by one million) of the total number of papers published for that year (broken line frequency polygon and right hand scale) and line of best fit, 1926 to 2006 (broken line, with equation and % variation accounted for, in broken line box on the right hand side)]. (Courtesy of Australian New Crops Web Site visited on 20-05-2009)

9 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.6. SOLANUM NIGRUM: TAXONOMIC COMPLICATIONS Solanum nigrum is the most variable species of the genus Solanum. The species related to S. nigrum have been reclassified innumerable times. Characters used by later taxonomists to separate and describe additional taxa often differed very slightly from those given for species by earlier workers. These Solanum species display varying amounts of phenotypic variation, particularly in their vegetative features such as plant habit, leaf size and form, and stem winging. In addition, senescence is often accompanied by smaller and fewer flowers and fruits (Ganapathi and Rao, 1986). S. nigrum was first delimited in four taxa with polynomials by Dillenius. Linnaeus subsequently modified Dillenius‘s work, describing these in six varieties under the binomial S. nigrum (Edmonds and Chweya, 1997). Since then, the plants morphologically related to S. nigrum have been reclassified many times. Over 300 post-Linnean specific and infraspecific names have now been published, and synonymy is extensive within the section. However, no satisfactory revision of the whole section has yet been devised. The boundaries between many of the species are still ill-defined, with many of the ‗new‘ taxa proving to be no more than slight morphological variants of those already described. The situation is further complicated by the researchers who either treated different members of the section as varieties of S. nigrum or considered them as different species on the basis of morphological differences (e.g. Edmonds and Chweya, 1997; Schilling and Andersen, 1990; Stebbins and Paddock, 1949; Symon, 1970). These Solanum species display varying amounts of phenotypic variation, particularly in their vegetative features such as plant habit, leaf size and form, and stem winging. In addition, senescence is often accompanied by smaller and fewer flowers and fruits than usual. Natural hybridization is probably more widespread in this section than generally supposed. It is now named as Solanum nigrum Complex because it is composed of a large number (about 30) of morphologically distinct taxa (Schilling and Andersen, 1990). Only during the revision of Solanum section Solanum appear in 1979 for Flora Europaea 3, drawn turned out that in Europe two different forms of the species coexist. The most widespread form was considered subspecies S. nigrum ssp.

10 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION nigrum, the second, rarely encountered species as S. nigrum ssp. schultesii classified (Edmonds and Chweya, 1997).

Deadly nightshade, identified as S. nigrum, causes belladonna poisoning (Hubbs, 1947), with symptoms including widely dilated pupils (characteristic of the atropine group, but either unexpressed, or expressed very mildly, in poisonings by plants whose major poisoning principle is of the solanine group). Deadly nightshade is identified as S. nigrum, but black nightshade is botanically unidentified and gives a strong cat's-eye test for atropine (Case, 1955). According to a research at University of Pennsylvania, unripe berries are said to be more toxic than ripe berries. Berries are more toxic than leaves which, in turn, are more toxic than stems or roots. Overall plant glycoalkaloid content is often higher in the autumn than in the spring. These problems clearly state the importance of proper identification and detailed composition analysis of each taxa of S. nigrum Complex.

The taxa of S. nigrum Complex are difficult to distinguish because 1. They are morphologically similar. 2. These species are all highly phenotypically plastic.

Three taxa belonging to S. nigrum Complex viz.: S. americanum Mill., S. nigrum L. and S. villosum Mill. had been reported in Pakistan (Schilling and Andersen, 1990). S. chenopodioides Lam. and S. retroflexum Dunal are two other species that were found growing wild in and around Botanic Garden, GC University, Lahore. Morphologically S. nigrum is different from S. villosum in the respect that the former has black matured berries with peduncles longer than pedicels while latter has orange/orange-red matured berries and peduncles shorter than or equal to the pedicels. Classification of S. nigrum and S. villosum as varieties or distinct species started taxonomic controversy between Linnaeus and Miller (Edmonds and Chweya, 1997). Though S. americanum Mill., S. chenopodioides Lam. and S. retroflexum Dunal have morphological resemblance with S. nigrum, yet no chemotaxonomic relationship has so far been established due to lack of a comprehensive study of their chemical composition.

11 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7. BOTANICAL ASPECTS OF THE INVESTIGATED TAXA

The five locally available taxa investigated were:

1. Solanum americanum 2. Solanum chenopodioides 3. Solanum nigrum 4. Solanum retroflexum 5. Solanum villosum

These Solanum species display varying amounts of phenotypic variation, particularly in their vegetative features such as plant habit, leaf size and form, and stem winging. In addition, senescence is often accompanied by smaller and fewer flowers and fruits than usual, while the gene for anthocyanin pigmentation in flowers seems to be dependent on light intensity and temperature for its expression, in some species. It is therefore often difficult to define the limits within which such features are genetically fixed (Baylis, 1958; Henderson, 1974; Edmonds, 1977).

Natural hybridization is probably more widespread in this section than generally supposed. Though this is probably followed by subsequent genetic breakdown in F1 or F2 generations (Edmonds, 1977), it may also be followed by back-crossing to the parental species. This would result in morphogenetically complex population variation: the collection of specimens from such populations would explain some of the difficulties encountered in the morphological differentiation of these species in the herbarium (Edmonds, 1979).

A brief overview of their individual botanical aspects (Edmonds and Chweya, 1997; Ganapathi and Rao, 1986; Karschon and Horowitz, 1985; Schilling and Anderson, 1992) is given below:

12 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.1. SOLANUM AMERICANUM MILLER 1.7.1.1. Morphological Features S. americanum (Fig. 2), the American nightshade, is an herbaceous flowering plant native to the Americas. It grows up to 1-1.5 m tall and is an annual or short-lived perennial. Stem with edentate to inconspicuously dentate ridges. Leaves ovate-lanceolate to lanceolate, lower surfaces glabrescent to moderately or densely pilose; margins entire to sinuate, rarely sinuate-dentate. Inflorescence simple, umbellate cymes, 3 to 6 flowered. Berries globose, black, rarely dark green, with shiny opaque cuticles, falling from calyces when ripe.

1.7.1.2. Distribution: Its habitat is Rocky or dry open woods, thickets, shores or openings, often on cultivated or waste ground. This plant is native to the Americas, from the south and west of the United States south to Paraguay and Peru; it also occurs in Hawaii, where it is considered possibly indigenous or may be a Polynesian introduction. It is used as a medicinal in Cameroon, Kenya, Hawaii, Panama, Sierra Leone, and Tanzania, and as a wild or cultivated pot herb in Cameroon, Ghana, Guatemala, Kenya, Madagascar, Mauritius, Hawaii and other Pacific Islands, Nigeria, Papua New Guinea, Peru, Sierra Leone, the Seychelles, South Africa, Tanzania, and Uganda.

1.7.1.3. Vernacular names: Argentina: Arachichu Australia: Glossy nightshade New Zealand: Small-flowered nightshade

Fig. 2: S. americanum

13 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.2. SOLANUM CHENOPODIOIDES LAM. 1.7.2.1. Morphological Features S. chenopodioides (Fig. 3), is a species of nightshade known by the common name forked nightshade. It is native to South America. Plants sprawling herbs to 1m high, often grayish-green in color. Stem usually smooth with edentate ridges. Leaves elliptic to lanceolate, obtuse to acute, distinct lobes usually absent. Inflorescence simple, umbellate cymes, 4 to 6 flowered. Berries globose to broadly ovoid, purple, with dull opaque cuticles.

1.7.2.2. Distribution: It is native to Southern America and is present in Brazil, Argentina and Uruguay. It is also naturalized in Australia, Europe, New Zealand, South Africa, & United States. It grows in gullies and creeks; widespread in coastal districts, west to Mt Tomah area. It is usually found on waste land, but appears to be fairly tolerant and may turn up in most situations.

1.7.2.3. Vernacular names Australia: White tip nightshade Brazil: Liaghe New Zealand: Velvety nightshade

Fig. 3: S. chenopodioides

14 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.3. SOLANUM NIGRUM L. 1.7.3.1. Morphological Features S. nigrum (Fig. 4), or The Black nightshade is a fairly common plant, found in many wooded areas, as well as disturbed habitats. It has a height of 30-120 cm (12-48"). Herb, shrubs or small trees, often prickly or hairy, berries in small hanging clusters, remaining on plants or falling from calyces when ripe. Flowers rarely solitary. Stem decumbent to erect. Leaves ovate, ovate-lanceolate, and ovate- rhombic to lanceolate. Berries usually broadly ovoid, dull purple to blackish or yellowish-green, remaining on plants or falling from calyces when ripe.

1.7.3.2. Distribution: The Black Nightshade is an annual plant, common and generally distributed in the South of England, less abundant in the North and somewhat infrequent in Scotland. It is one of the most cosmopolitan of wild plants, extending almost over the whole globe. It is sometimes called the Garden Nightshade, because it so often occurs in cultivated ground.

1.7.3.3. Vernacular names: Australia: Black or black berry nightshade Ethiopia: ―Dime people eat‖ Europe: Black Nightshade, annual nightshade, common nightshade South Africa: Nightshade

Fig. 4: S. nigrum

15 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.4. SOLANUM RETROFLEXUM DUNAL 1.7.4.1. Morphological Features S. retroflexum (Fig. 5), is spreading, erect, pubescent herb upto 70 cm tall. Stem with smooth or inconspicuously dentate ridges. Leaves rhomboidal to ovate- lanceolate, broad, margins usually deeply lobed with 3 to 5(7) obliquely triangular lobes on each margin. Inflorescence simple, umbellate erect cymes, 3 to 6(7)- flowered; Berries spherical, purple, dull with opaque cuticles, falling from calyces when ripe.

1.7.4.2. Distribution: It is widely distributed in Northeast Tropical Africa in Ethiopia, Somalia, Sudan and In East Tropical Africa in Tanzania. In West Tropical Africa, Mauritania; Nigeria; Sierra Leone, in South Tropical Africa, Angola; Malawi; Mozambique; Zambia; Zimbabwe and also in Southern Africa, Botswana; Lesotho; Namibia; South Africa Cape Province, Natal, Orange Free State, Transva; Swaziland. It is sparingly cultivated in North America & naturalized in South Australia

1.7.4.3. Vernacular names North America: Sunberry, Wonderberry South Africa: Nastergal

Fig. 5: S. retroflexum

16 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.5. SOLANUM VILLOSUM MILLER 1.7.5.1. Morphological Features S. villosum (Fig. 6), is sub glabrous to villous annuals, up to 50 cm high. Stem rounded to angled, almost glabrous to pubescent with appressed hairs. Leaves rhombic to ovatelanceolate. Inflorescence simple, umbellate to slightly lax solitary cymes, 3 to 5 lowered, rarely 10 flowered. Berries usually longer than wide, occasionally globose, red, orange or yellow.

1.7.5.2. Distribution: S. villosum is believed to have originated in Eurasia, and is sometimes considered to have a southern European origin. It is widespread, but absent in Central and South America, and New Guinea. It has been introduced in North America and Australia. In Africa it is recorded from Tunisia, Algeria and South Africa, and from many countries of tropical Africa, e.g. Burundi, Sudan, Ethiopia, Somalia, Kenya, Uganda, Tanzania, Zambia and Angola. It can grow on a wide range of soils, but prefers soils that are rich in organic matter and land covered with ash of recently burnt vegetation. In the wild along the edges of agricultural fields.

1.7.5.3. Vernacular names: Great Britain: Red-fruited nightshade Kenya: Soiyot-Ap-Poinet Uganda: Eswiga

Fig. 6: S. villosum

17 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.7.6. KEY TO THE INVESTIGATED TAXA OF S. NIGRUM COMPLEX S. americanum Miller: Plants glabrescent to moderately pilose with appressed eglandular hairs; flowers small, fruiting pedicels usually erecto-patent; berries spherical, black and usually shiny when mature; seeds 1-1.5 mm long.

S. chenopodioides Lam.: Plants somewhat tomentose; umbellate cyme inflorescence with 4,6 sometimes 8 flowers; fruiting peduncles strongly deflexed from the base; berries globose to ovoid, purple with dull opaque cuticle.

S. nigrum L.: Plants subglabrous to pubescent usually with appressed, eglandular- headed multicellular hairs; berries black.

S. retroflexum Dunal: Plants pubescent with appressed, eglandular-headed multicellular hairs; flowers white with distinct purple vein to outer surface of petals; berries usually spherical, purple with opaque cuticles.

S. villosum Miller: Plants villous, covered with glandular headed and often patent multicellular hairs; stems usually terete, with smooth ridges; berries red, orange or yellow.

1.8. IMPORTANCE OF S. NIGRUM COMPLEX

1.8.1. MEDICINAL USES Many varieties of S. nigrum Complex have been reported to have medicinal properties although most of the reported work does not throw light on the taxonomy of varieties used. S. nigrum is antiperiodic, antiphlogistic, diaphoretic, diuretic, emollient, febrifuge, narcotic, purgative and sedative (Emboden, 1979; Lust, 1983; Grieve, 1985; Singh and Kachroo, 1985). The leaves, stems and roots are used externally as a poultice; wash etc. in the treatment of cancerous sores, boils, leucoderma and wounds (Lust, 1983). Extracts of the plant are analgesic, antispasmodic, anti-inflammatory and vasodilator. The plant has been used in the manufacture of locally analgesic ointments.

18 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

The juice of the fruit has been used as an analgesic for toothaches. S. nigrum mixed with other herbal medicines has hepatoprotective effect in cirrhotic patients, anti-oxidative and immunomodulating properties of the commonest herbs it also protects against hepatitis B virus infection. In Hawaii plants conspecific with S. americanum are used in disorders of the respiratory tract, skin eruptions, cuts, wounds and trachoma. While in the Mauritius, a poultice of the plant is used to relieve abdominal pain and Inflammation of the urinary bladder (Grieve, 1985). In Tanzania, ground and soaked leaves of S. villosum were reportedly placed on swellings and fruit juice squeezed into sore eye (Ganapathi and Rao, 1986).

1.8.2. SOURCE OF ALKALOIDS The berries of Solanum contain the alkaloid solasodine. It is a glycol which is used by pharmaceutical companies for the preparation of many important drugs. It is a nitrogen analogue of diosyenin and is a good source of sapogenin. Sapogenin is used as a base for the preparation of cortisone and allied product. The synthetic substituents are not known for these drugs; hence the importance of this source thus increases. Cortisone, a steroidal harmone prepared from solasodine is found to be effected in treatment of acute stages of rheumatoid arthritis, chronic cases of asthma, leukemia (Panday, 2004).

1.8.3. NUTRITIONAL VALUE Several studies have been conducted to investigate the nutritive value of the vegetable black nightshades. The leaves can provide appreciable amounts of protein and amino acids, minerals including calcium, iron and phosphorus, vitamins A and C, fat and fiber, as well as appreciable amounts of methionine, an amino acid scarce in other vegetables (Fawusi, 1983). They are also widely used in pies and preserves, and sometimes as a substitute for raisins in plum puddings, particularly in North America. They can also make a delightful jam, with the ‗Wonderberry‘ making an excellent preserve for tea with bread and butter. Leaves and tender shoots are widely used as vegetables throughout the world and have provided a food source since early times.

19 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

S. nigrum being used as an ancient famine plant and the Chinese S. villosum are frequently eaten as fruits, particularly in parts of Africa. However, S. chenopodioides does not seem to be utilized as a food source, with the only reference to its use as ‗spinach‘-plant being Natal in South Africa. S. retroflexum is believed to be the ‗Sunberry‘ promoted by the plant breeder Luther Burbank as ―a new food plant from a poisonous family‖ at the beginning of this century in North America (Edmonds and Chweya, 1997).

1.8.4. COMMERCIAL VALUE It was a widespread practice in Argentina, S. chenopodioides together with wool waste was used as manure. It has also been extensively spread on light agricultural sandy soils and has been a major source of South American adventives which have become established as weeds in Europe. Both the leaves and berries are used as a source of dyes. Leaves are macerated to extract a dye used to color sisal baskets (Khanna and Rathore, 1977). The black berries of S. americanum are reportedly used as a source of ink. Seeds of fresh fruit rubbed on cheeks to remove freckles and improve the complexion. The species are apparently used as fodder and browse by various animals, especially in Africa. Herbarium records showed that plants tentatively identified as S. villosum are eaten by sheep and goats in the Sudan, and by bush-buck and browsed by goats (Grieve, 1985). The plants of both S. americanum and S. nigrum persisting as weeds of cultivation in Australia were known to be alternative hosts for insects attacking crops such as tobacco, for plant viruses transmitted by insects, and for pathogenic bacteria attacking commercial strains of ginger (Lust, 1983). This specie group has been found to be effective in removing PCB's from the soil and detoxifying them. The plant is more effective in doing this if it is infected with the bacterial parasite Agrobacterium tumefaciens (Moerman, 1998).

20 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9. SECONDARY METABOLITES The term ―secondary metabolites‖ indicates compounds that are not required for plant growth and development but presumed to function in communication or defence (Luckner, 1990). Secondary metabolites, on the basis of their properties and functions, can be classified into different groups like Alkaloids, Flavonoids, Tannins, and Saponins etc (Mann, 1986).

1.9.1. ALKALOIDS: The name ―Alkaloids‖ is derived from the fact that these compounds in general behave similar to alkali bases (e.g. NaOH) in that they neutralize acids. Thus early workers coined the term alkaloid from ―Alkali‖ and ―Oid‖ meaning ―Alkali- like‖ (Mann, 1986). Most recently W. Pelletier suggested the following new definition for an alkaloid: ―An alkaloid is a cyclic organic compound containing nitrogen in a negative oxidation state, which is of limited distribution among living organisms‖ (Bhat et al., 2005). Alkaloids are a naturally occurring group of compounds containing heterocyclic nitrogen, having a more or less distinctly basic character, and a complex molecular structure. They possess recognizable physiological and pharmaceutical activity (Wilson and Gisvold, 1956). The most recent comprehensive survey had shown that a total of 1932 individual compounds have been reported as alkaloids by the investigators who isolated them from 158 botanical families (Swain, 1960). Not withstanding the many valuable synthetic medicinal and antibiotic agents that have been added to the list of weapons against diseases, the alkaloids still constitutes an indispensable and most potent group of substances for the treatment and migration of functional disturbances and relief from suffering (William and Schubert, 1961).

1.9.1.1. Studies on Alkaloids The history of alkaloid chemistry, in structural terms, began in 1804, when Sertürner (the Paderborn apothecary) discovered the so-called principium somniferum in opium (Trommsdorf, 1805), which he reported the following year in the Journal der Pharmacie (Sertürner, 1805). The attention of scientists, however, was aroused only twelve years later by a publication appearing in the Annalen der

21 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

Physik (Sertürner, 1817; Schmitz, 1983). There, Sertürner named his principium somniferum for the first time "morphium" (after Morpheus, the son or servant of sleep and creater of dream states in Ovid; altered to "morphinium" by the French physicist Gay-Lussac). Often, a very great deal of time would pass between the isolation of an alkaloid and the determination of both its structure and absolute configuration. In the case of strychnine, 138 years passed by and for morphine 150 years (Hesse, 2002). Today, it is usual to determine the structure of a substance in the year of its isolation, especially when it seems to possess pharmacological properties as promising as those of strychnine and morphine. In true alkaloids the basic units of biogenesis are amino acids. The non- nitrogen containing rings or side chains are derived from terpene units and/or acetate, while methionine is responsible for the addition of methyl groups to nitrogen atoms. Alkaloids are basic and form water-soluble salts. Most alkaloids are well-defined crystalline substances that react with acids to form salts. In plants they may exist in the free state, as salts or as N-oxides. The different criteria currently used for the classification of 243 alkaloids are biogenesis, structural relationship, biological origin and spectroscopic/spectrometric properties (chromophores in UV spectroscopy, ring systems in mass spectrometry) (Hesse, 2002). Based on amino acid precursor, alkaloids can be further subdivided. The principal precursors are ornithine, lysine, nicotinic acid, tyrosine, tryptophan, anthranilic acid and histidine. Ornithine gives rise to pyrrolidine and trypane alkaloids, lysine to piperidine, quinolizidine and indolizidine alkaloids and nicotinic acid to pyridine alkaloids. Tyrosine produces phenylethylamines, tetrahydroisoquinoline, benzyltetrahydroisoquinoline, phenethylisoquinoline, terpenoid tetrahydroisoquinoline and Amaryllidaceae alkaloids. Tryptophan gives rise to β- carboline, terpenoid indole, quinoline, pyrroloindole and ergot alkaloids. Anthranilic acid acts as a precursor to quinazoline, quinoline and acridine alkaloids, while histidine gives imidazole derivates (Dewick, 2002). Concerning the distribution of the main secondary metabolites in Rubiaceae (Robbrecht, 1988), indole alkaloids were the chemotaxonomic markers more intensely studied so far, aiming the establishment of phyllogenetic correlations between secondary metabolites and taxonomic data. Our chemical studies revealed several interesting correlations among tribes and subfamilies of Rubiaceae due to their structural variability and restrict distribution (Bolzani et al., 2001). In

22 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

Rubiaceae, the occurrence and distribution of iridoids, indole alkaloids and anthraquinones has provided valuable chemosystematic clues (Young et al., 1996). Terpenes, sterols and saponins are often found in association with alkaloids or as their precursors. Saponins are glycoside compounds often referred to as a ―Natural Detergent‖ because of their foamy texture. They are mainly of triterpenoidal type, being the oleanolic acid and the hedagenin in the main constituents. They are found in many plants and get their name from soapwort plant (Saponaria), the root of which was used historically as a soap (Latin sapo means soap). They are shown to be anticarcinogenic and antioxidant (Fink and Fusion, 1918). The name terpene is derived from English word ―Turpentine‖. The terpenes are generally colorless liquids, which are lighter than water and boil in 140-190 0C temperature range. They are insoluble in water, highly refractive and optically active and rotate the plane of polarized light. The terpenes are unsaturated hydrocarbons, which have distinct architectural and chemical relation to simple isoprene molecule

C5H8. They have the molecular formula C10H16, thus are constituted by two isoprene units combines by head to tail union (Pinder, 1960).

Following are some important classes of alkaloids:

Scheme 2: Classification of Alkaloids

23 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9.1.2. Solanum Alkaloids Steroidal glycoalkaloids derived from a cholestane skeleton are mainly found in the Solanaceae family and therefore termed Solanum glycoalkaloids. Besides S. nigrum complex, there are also other plants in the Solanaceae that contain glycoalkaloids: e.g. potato (Solanum tuberosum), tomato (Lycopersicon esculentum Mill.) and eggplant (Solanum melongena L.). Examples: Solanidine, Solanine, Chaconine, Solasodine, (Solanum Alkaloids) Veratramine, Muldamine; Samandarin (Fire Salamander Alkaloids)

Fig. 7: General Structure of Steroidal Alkaloids

1.9.1.3. Bioactive Properties of Alkaloids The isoquinoline alkaloid emetine obtained from the underground part of Cephaelis ipecacuanha, and related species, has been used for many years as amoebicidal drug as well as for the treatment of abscesses due to the spread of Escherichia histolytica infections. Another important drug of plant origin with a long history of use is quinine. This alkaloid occurs naturally in the bark of Cinchona tree. Apart from its continual usefulness in the treatment of malaria, it can also used to relieve nocturnal leg cramps. Currently widely prescribed drugs are analogs of quinine such as chloroquinine .Some strains of malarial parasites have become resistant to quinines,therefore, antimalarial drugs with novel mode of action are required. Similarly, higher plants have made important contributions in the areas beyond anti-infective, such as cancer therapies. Early examples include the anti- leukaemic alkaloids, vinblastine and vincristine, which were both obtained from the Madagascan periwinkle (de-Pavia, 2003). Other cancer therapeutic agents include taxol, homoharringtonine and several derivatives of camptothein. For example, a well- known benzyl isoquinoline alkaloid, papaverine, has been shown to have a potent inhibitory effect on the replication of several viruses including cytomegalovirus, measles and HIV. Most recently, three new atropisomeric naphthyl

24 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION isoquinoline alkaloid dimmers, michellamines A, B, and C were isolated from a newly described species tropical liana Ancistrocladus korupensis from the rain forest of Cameroon. The three compounds showed potential anti-HIV with michellamine B being the most patent and abundant member of the series. These compounds were capable of complete inhibition of the cytopathic effects of HIV-1 and HIV-2 on human lymphoblastoid target cell in vitro (Robert, 1985). Toxicity: Glycoalkaloids are toxic compounds. An official safety or acceptable limit of total glycoalkaloid content for human consumption in tubers is 200 mg/kg fresh weight (fw) (1000mg/kg dry weight, dw) or 1 mg/kg body weight (bw). Glycoalkaloid contents below this guideline are not thought to represent health risks for humans. However, lower acceptable levels for different plant types have been recommended because of the variation in glycoalkaloid concentrations between years and growth locations. According to estimates based on poisoning cases reported, a toxic dose of glycoalkaloids in human consumption can vary between 2–5 mg/kg bw and a lethal dose about 3–6 mg/kg bw (Morris & Lee 1984) or it can be even as low as from 1 to 2 mg/kg bw (reviewed in Friedman & McDonald 1997). Symptoms of poisoning caused by glycoalkaloids have been reported to be gastrointestinal, causing vomiting, diarrhoea, abdominal pain, neurological resulting in restlessness, confusion, delirium, stuporose, drowsiness, hallucination, and others such as nausea, malaise and skin lesions (van Gelder 1991). It is worth noting that glycoalkaloids are stable and are not destroyed by cooking, except during frying where a minor reduction in glycoalkaloid levels has been reported (Bushway & Ponnampalam 1981). Glycoalkaloids are toxic compounds for all mammals. However, animals are generally less susceptible to glycoalkaloids than humans. The effects of glycoalkaloids on animals have been reviewed by Morris and Lee (1984) and Friedman and McDonald (1997). Their major toxic properties are due to: i) The ability of glycoalkaloids to bind with membrane 3β-hydroxy sterols and to disrupt membrane function. ii) The ability to inhibit acetylcholinesterase. In producing toxic effects, glycoalkaloids may act synergistically, i.e. a mixture of glycoalkaloids has greater/different toxicity than could be expected from their individual effects. To be concluded, different glycoalkaloids and aglycones possess various, specific biological activities and toxicity depends on their structural characteristics.

25 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9.2. FLAVONOIDS The term flavonoids refer to a class of plant secondary metabolite. They are the largest group of naturally occurring phenolic compounds, which occur in different plants parts both in the free state and as glycosides (Harborne, 1974). The term flavonoids has been derived from a Latin word ―FLAVUS‖ meaning yellow as a large no of flavonoids are yellow in color. Flavonoids are also known as plant pigment or co pigment. The presence of these pigments is responsible for color and combination of colors exhibited by bark, leaves, flower, fruits and seeds of plants. Flavonoids are commonly referred to as bioflavonoid in the media; the terms are largely equivalent and interchangeable for most flavonoids are biological in origin (Ray Sahelian, 2005). They are polyphenolic compounds with general structure possessing 15 carbon atoms, two benzene rings joined by a linear three carbon chain. The skeleton below can be represented as the C6 - C3 - C6 system.

(a) (b) Fig. 8: Basic Structure of Flavonoids: (a) Carbon skeleton (b) Chromane ring

The chemical structure is based on a C15 skeleton with a chromane ring bearing a second aromatic ring B in position 2, 3 or is replaced, in a few cases, where the six-membered heterocyclic ring C occurs in an isomeric open form or by a five membered ring.

The major classes of Flavonoids are  Flavone  Flavonol  Flavanone  Flavanol  Isoflavone  Anthocyanin

26 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9.2.1. Studies on Flavonoids Flavonoids first came into the spotlight in the 1930‘s when Szent-Gyorgyi and his colleagues extracted two flavonoids from citrus fruit. They discovered that a crude form of vitamin C which contained a flavonoids fraction worked better for treating bleeding gums than did a more refined form of vitamin C. They investigated the effects of the flavonoids and found they decreased the fragility and permeability of human capillaries. This is why flavonoids were then called "vitamin P" [P for permeability] (Ray Sahelian, 2005).

1.9.2.2. Solanum Flavonoids Many glycosides of quercetin, kaempferol and myricetin had been reported from various Solanum species (da Silva et al., 2003). Schilling (1984) isolated 10 flavonoids from leaf extract of 11 species belonging to the section Solanum; including coumarins (such as scopolrtin). Flavonols and Anthocyanidins for S. scabrum in Nigeria (Gbile and Adesine, 1984) and the anthocyanin pigments were also found in European samples of this species (Francis and Harborne 1966). Quercetin is the most commonly occurring flavonol aglycone detected in S. nigrum Complex. It forms many glycosides like quercitrin, isoquercitrin and rutin together with rhamnose and glucose as sugar moieties attached in different patterns.

Fig. 9: Quercetin

1.9.2.3. Bioactive Properties of Flavonoids Research suggests that flavonoids may have diverse benefits including antioxidant, antiviral, anti-allergic, antimicrobial, anti-platelet, anti-inflammatory, and anti-tumor effects. In vivo research has demonstrated that quercetin can increase

27 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

the anti-tumor activity of cisplatin and busulfan and can be used in conjunction with doxorubicin and etoposide without interfering with their therapeutic action. Flavonoids are some of the most powerful and effective antioxidant compounds available to humans and since we are unable to produce flavonoids ourselves, we must get them from the food we eat and from supplements. Flavonoids exert these antioxidant effects by neutralizing all types of oxidizing radicals (Robak and Gryglewski, 1988) including the superoxide (Husain et al., 1987) and hydroxyl radicals (So et al., 1996) and by chelation. Certain flavonoids may even have antihistamine, memory and mood enhancing properties. Most flavonoids have anti- germ activity. Immuno-Shield is an immune system product formulated by Dr. Sahelian that has flavonoids and several immune herbs and nutrients (Ray Sahelian, 2005). While they are not considered essential nutrients, some flavonoids support health by strengthening capillaries and other connective tissue, and some function as antihistaminic and antiviral agents. Rutin and several other flavonoids may also protect blood vessels. Flavonoids have been referred to as ―nature‘s biological response modifiers‖ because of strong experimental evidence of their inherent ability to modify the body‘s reaction to allergens, viruses and carcinogens. They show antimicrobial and anticancer activity (Bhat et al., 2005). Quercetin is found to be the most active of the Flavonoids and many medicinal plants owe much of their activity to their high quercetin content. It has demonstrated significant anti-inflammatory activity because of direct inhibition of several initial processes of inflammation. For example, Quercetin inhibits both the production and release of histamine and other allergic, inflammatory mediators. In addition, it exerts potent antioxidant activity and vitamin C –sparing action. It can be found in the herbal products based on Hawthorn, which are used for acute symptoms of Congestive Heart Failure. It has been reported to be used for the treatment of Diabetes, Peptic ulcer & Edema Allergies, Atherosclerosis & Cataracts Capillary fragility & Hay fever (So et al., 1981).

28 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9.3. EPICUTICULAR WAX The term ‗wax‘ is derived from the Anglo-Saxon word ‗Weax‘ which was applied to the natural material obtained from the honeycomb of the bee in beeswax. When similar substances were found in plants, they were also called ‗Weax‘ or Wax. Wax can be defined as ‗a substance belonging to a specific group of organic thermoplastics (as a rule, opaque or translucent but not transparent) with melting point between 50o and 90oC, exceptionally high to relatively low viscosity liquids or semisolids or solids which do not exhibit thread spinning phenomenon, compatible with other waxes, forming pastes of gels with organic or non-polar solvents, having water repellent properties, imparting gloss, and possessing-in principle-illuminating power. Chemically waxes are esters of fatty acids and monohydric fatty alcohols. These are widely distributed in nature with commercially important representatives in each of the following classification, viz. animal (and insect), vegetable and mineral (Thorpe, 1937).

―The cuticle of terrestrial vascular plants and some bryophytes is covered with a complex hydrophobic mixture of lipids, usually called epicuticular waxes.‖

Self-assembly processes of wax molecules lead to crystalline three- dimensional micro- and nano structures that emerge from an underlying wax film. Wax crystals form a hydrophobic water-repellent surface due to their chemistry and micro structure. Such surfaces often display a self-cleaning property, called the ‗lotus-effect‘, by increased water repellency and reduced adhesion of the contaminating particles (Barthlott, and Neinhuis, 1997). Epicuticular waxes form thin films or thick crustsand often superimposed three-dimensional structures on an underlying wax film (Kolattukudy, 1980; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998). The varying shape of the wax crystals is determined by their chemical composition. In some types of crystals, the ultrastructure is determined by one predominating wax component (Jeffree et al., 1975; Jeffree et al., 1976). Nevertheless, due to their small size, the chemical composition of an individual wax crystal is still hypothetical

29 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.9.3.1. Studies on Epicuticular Waxes The first person to describe waxes on plant surfaces, using light microscopy, and Term them ‗kristalloids‘ (crystalloids) was De Bary (1871). More recently, the crystalline Nature of the wax of many species has been verified by X-ray powder Diffractograms and electron diffraction (Reynhardt, 1997; Reynhardt and Riederer, 1988, 1994; Meusel et al., 1994, 1999, 2000). The epicuticular waxes had often been considered as a potential character for chemotaxonomy of the plants in general (Evans et al., 1975; Griffiths et al., 1999; Tsydendambaev et al., 2004).

1.9.3.2. Solanum Wax Compounds found in the Epicuticular waxes (Osske and Schreiber, 1965; Jewers et al., 1969; Croteau and Fagerson, 1971; Streibl et al., 1974; Meusel et al., 2000) include:  Hydrocarbons  Ketones  Aldehydes  Fatty acids  Alcohols  Esters  Sterols  Flavonoids  terpenes

Hanna et al. (1996), although, had reported the presence of some fatty acids such as Palmitic, Stearic, Linolenic acids and Squalene without specifying the taxon of S. nigrum Complex, yet no detailed chemotaxonomic study based upon chemical constituents of epicuticular waxes of the complex has been undertaken.

1.9.3.3. Uses of Epicuticular Waxes Epicuticular waxes form the outermost boundary layer of the plant, representing a multifunctional interface between plant and environment. A major function is to serve as a barrier against uncontrolled water loss (Schönherr, 1976, 1982). In some cases waxes cause an increase in the reflection of solar radiation (Barnes and Cardoso-Vilhena, 1996; Holmes and Keiller, 2002).

30 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.10. BIOLOGICAL EVALUATION 1.10.1. ANTIMICROBIALS An antimicrobial is a substance that kills or inhibits the growth of microorganisms whereas Antimicrobial drugs either kill microbes (microbicidal) or prevent the growth of microbes (microbistatic). Antibiotics are now considered to be the specific chemical products or the derivatives of such products often produced from the microorganisms and having an inhibitory action against certain kind of microorganisms (Nandkarni, 1954). In today's common usage, the term antibiotic is used to refer to almost any drug that cures a bacterial infection. The history of antimicrobials begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. They did not know at that time that the reason one bacterium failed to grow was that the other bacterium was producing an antibiotic. There are several plants that are the source of antibiotics medicine, which are successively used against several diseases caused by bacteria and fungi. Antimicrobials include not just antibiotics, but synthetically formed compounds as well. The discovery of antimicrobials like penicillin and tetracycline paved the way for better health for millions around the world. Before 1941, the year penicillin was discovered, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds often had to have a wounded limb removed, or face death from infection. Now, most of these infections can be easily cured with a short course of antimicrobials. Main classes of antimicrobials include:  Antibiotics  Antivirals  Antifungals  Antiparasitics  Non-pharmaceutical antimicrobials However, the future effectiveness of antimicrobial therapy is somewhat in doubt. Microorganisms, especially bacteria, are becoming resistant to more and more antimicrobial agents. Bacteria found in hospitals appear to be especially resilient, and are causing increasing difficulty for the sickest patients–those in the hospital. Currently, bacterial resistance is combated by the discovery of new drugs. However, microorganisms are becoming resistant more quickly than new drugs are being

31 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION found. Thus, future research in antimicrobial therapy may focus on finding how to overcome resistance to antimicrobials, or how to treat infections with alternative means. When people from the remote communities get an infectious disease, they are usually treated by traditional healers because of their expertise in such procedures as making diagnoses, treating wounds, setting bones and making herbal medicines. Traditional healers claim that their medicine is cheaper and more effective than modern medicine. Patients of these communities have a reduced risk to get infectious diseases from resistant pathogens than people from urban areas treated with traditional antibiotics. However, if they are treated in a hospital the chance of contracting a nosocomial infection is increased (Ospina et al., 2002). One way to prevent antibiotic resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents (Shah, 2005). Traditional healers claim that some medicinal plants such as bixa spp. and bidens spp. are more efficient to treat infectious diseases than synthetic antibiotics. It is necessary to evaluate, in a scientific base, the potential use of folk medicine for the treatment of infectious diseases produced by common pathogens. Medicinal plants might represent an alternative treatment in non-severe cases of infectious diseases. They can also be a possible source for new potent antibiotics to which pathogen strains are not resistant (Fabricant and Farnsworth, 2001).

1.10.2. ANTIOXIDANTS Antioxidants are substances that may protect our cells against the effects of free radicals. Free radicals are molecules produced when our body breaks down food in oxidation reactions, or by environmental exposures like tobacco smoke and radiation. Chain reactions are stated that damage cells and may play a role in heart disease, cancer and other diseases (Medline plus, 2009). Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols (Sies, 1997). Although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of

32 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION antioxidants, or inhibition of the antioxidant enzymes, causes oxidative stress and may damage or kill cells. While the body has its defenses against oxidative stress, these defenses are thought to become less effective with aging as oxidative stress becomes greater. Research suggests there is involvement of the resulting free radicals in a number of degenerative diseases associated with aging, such as cancer, cardiovascular disease, cognitive impairment, Alzheimer‘s disease, immune dysfunction, cataracts, and macular degeneration. Certain conditions, such as chronic diseases and aging, can tip the balance in favor of free radical formation, which can contribute to ill effects on health. Consumption of antioxidants is thought to provide protection against oxidative damage and contribute positive health benefits. For example, the carotenoids Lutein and Zeaxanthin engage in antioxidant activities that have been shown to increase macular pigment density in the eye. Whether this will prevent or reverse the progression of macular degeneration remains to be determined. An increasing body of evidence suggests beneficial effects of the antioxidants present in grapes, cocoa, blueberries, and teas on cardiovascular health, Alzheimer‘s disease, and even reduction of the risk of some cancers (Allemann and Baumann, 2008). Antioxidants are present in foods as vitamins, minerals and carotenoids among others. Many antioxidants are often identified in food by their distinctive colors—the deep red of cherries and of tomatoes; the orange of carrots; the yellow of corn, mangos, and saffron; and the blue-purple of blueberries, blackberries, and grapes. The most well-known components of food with antioxidant activities are vitamins A, C, and E; β-carotene; the mineral selenium; and more recently, the compound lycopene (Allemann and Baumann, 2008). The increasing interest in naturally occurring antioxidants (polyphenols, vitamins) is attributed to their capability of scavenging free radicals that are formed in various biochemical processes. The reactive oxygen species like superoxide anion, hydrogen peroxide, and hydroxyl radicals cause an extensive oxidative damage to biomolecules such as nucleic acids, proteins, and lipids. These highly unstable radicals have been found to be related to oxidative stress-related diseases like cardiovascular diseases, cancer, inflammatory disorders, neurological degeneration (Parkinson's and Alzheimer's diseases), premature ageing, etc. Polyphenols (cinnamic acid derivatives, flavonols, anthocyanins) and vitamins are present in vegetables, fruits, berries, and herbs, which are the main

33 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION source of natural antioxidants in our daily diet. The basic structure of polyphenols is composed of one or more phenolic rings that are substituted with several hydroxyl groups and these are highly correlated with their strong antioxidant activity. Vitamins are structurally a heterogeneous group of compounds, which are essential in the diet for the maintenance of healthy growth and development. In general, vitamins are divided into two main categories, fat and water-soluble ones. Among vegetables, tomato (Solanum lycopersicum), eggplant (Solanum melongena), chilli pepper (Capsicum annuum), and potato (Solanum tuberosum), which belong to the Solanaceae family, are important for their richness in healthy components due to which they are also widely consumed. Tomato is rich in phenolic compounds (flavonoids, flavones, cinnamic acid derivatives), phytoalexins, protease inhibitors, glycoalkaloids, and carotenoids, but especially in lycopene and [beta]- carotene. In addition, vitamins C, E, and A have been determined in tomato. The main polyphenols found in eggplant are phenolic acids (chlorogenic acid, caffeic acid, p-coumaric p acid), but this vegetable is poor in provitamin A and vitamin E. However, the presence of vitamins C and B in eggplant has been established. It is also rich in anthocyanins like nasunin and delphinidin conjugates. Chilli pepper has been reported to contain flavones (luteolin, quercetin), flavonols (myricetin, quercetin), and capsaicinoids. Of phenolic compounds, chlorogenic and caffeic acid, catechins, and also glycoalkaloids have been reported to be the main compounds present in potato. Vitamin C has been also determined in potato (Sies, 1997). In the late 19th and early 20th century, extensive study was devoted to the uses of antioxidants in important industrial processes, such as the prevention of metal corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling of internal combustion engines (Matill, 1947). Early research on the role of antioxidants in biology focused on their use in preventing the oxidation of unsaturated fats, which is the cause of rancidity. Antioxidant activity could be measured simply by placing the fat in a closed container with oxygen and measuring the rate of oxygen consumption. However, it was the identification of vitamins A, C, and E as antioxidants that revolutionized the field and led to the realization of the importance of antioxidants in the biochemistry of living organisms (Jacob, 1996; Knight, 1998).

34 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.1: INTRODUCTION

1.11. AIMS AND OBJECTIVES

There are various approaches to the taxonomic studies of the plants based on the structural, cytological and chemical constituents. The ancient classification of the plants was mainly carried out on the comparative morphological and anatomical concepts of the natural plant flora. However with the rapid progress in the isolation, purification, identification, elucidation of structure and the configuration of natural plant products, the phytochemists and ethnobotanists believe that it is possible to characterize and classify the plants on the basis of their chemical constituents. The chemical constituents are formed within the plants by definite biosynthetic pathways aided by the specific enzymes.

Linnaeus classified S. nigrum in 2 varieties viz.: S. nigrum Var. nigrum (with black berries) and S. nigrum Var. villosum (with orange red berries). But the other taxonomists like Lamarck and Miller classified each of these as a separate species (Edmonds and Chweya, 1997). Since there is a controversy to assign S. villosum a separate species of the genus Solanum or a variety of S. nigrum, therefore it needed further clarification. S. nigrum is now named as S. nigrum Complex because it is composed of about 30 morphologically different taxa Meanwhile, in addition to the above mentioned two, three more morphological variants of S. nigrum viz.: S. americanum, S. chenopodioides and S. retroflexum were reported in the Botanic Garden of GC University Lahore, Pakistan. So the present study was designed on the chemotaxonomic evaluation of all these five taxa of S. nigrum Complex found in Pakistan. The purpose was to confirm their taxonomic status on the basis of their secondary metabolites, as they are more or less morphologically similar.

35 Chemotaxonomical Characterization of Solanum nigrum and its Varieties

Chap.1: INTRODUCTION

So far there had been very little and insufficient chemical analysis reported with respect to the three lately mentioned taxa in literature. Hence our specific aims and objectives for these studies were:

[1]. Phytochemical analysis to study the profiles of secondary metabolites in the five locally available taxa; [2]. Chemotaxonomic studies of all these taxa in order to eliminate/reduce the criticism/confusion on their taxonomic status; [3]. And finally the ethnopharmacological studies of the biologically active components of these taxa.

36 Chemotaxonomical Characterization of Solanum nigrum and its Varieties

Chap.2: LITERATURE SURVEY

2.1. CHEMICAL CONSTITUENTS FROM S. NIGRUM COMPLEX

A comprehensive literature review of S. nigrum was carried out covering a period of more than hundred years. S. nigrum had been investigated deeply specially for its steroidal alkaloids and flavonoids. However the reporting authors had not taken into account the morphological complexity and the proper taxonomy of the plant material investigated. Following is the summary of the compounds identified and isolated from different taxa of S. nigrum Complex.

Table 1: Review of the Compounds reported from S. nigrum Complex

Class of compound Compound name References Solasodine Schreiber, 1958; Mathe, 1974. 12β,27-dihydroxy- Doepke et al., 1987; Yoshida et al., solasodine 1987. 23-O-acetyl-12β- Doepke et al., 1988. hydroxysolasodine N- Methylsolasodine Doepke et al., 1987. Tomova, 1962; Waclaw-Rokrutowa, Solasonine(- Solanigrine) 1968; Aslanov, 1971.

α-Solamargine Schreiber, 1958; Aslanov, 1971;

(-Solanigrine) Aslanov and Novruzov, 1978. Solanigridine Schreiber, 1958. β-Solamargine Waclaw-Rokrutowa, 1968; Aslanov

Steroidal Glycoalkaloid Steroidal and Novruzov, 1978. Solanaviol Yoshida et al.,1987. Solanine (α,β) Abbas, 1998; El-Ashaal et al., 1999. Tomatidenol Doepke et al., 1987. Solanocapsine Doepke et al., 1987. Solasodi-3,5-ene Waclaw-Rokrutowa, 1968.

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Class of compound Compound name References

Diosgenin Khanna, 1977.

Tigogenin Pkheidze, 1967; Doepke et al.,

1987.

Desgalactotigonin Saijo et al., 1982; Ke et al., 1999. Nigrumnin I Ikeda et al., 2000.

Steroidal saponins Steroidal Nigrumnin II Ikeda et al., 2000. Cholestrol Bhatt and Bhatt, 1984.

Campesterol Bhatt and Bhatt, 1984.

Stigmasterol Bhatt and Bhatt, 1984; Hanna et al.,

Sterols β-Sitosterol Bhatt and Bhatt, 1984; Hanna et al., 1996. Quercetin-3-glucosyl Biard, 1974; Nawwar et al., 1989. (16) galactoside Quercetin-3- Biard, 1974; Nawwar et al., 1989 gentiobioside, Quercetin-3-galactoside Biard, 1974; Nawwar et al., 1989. Quercetin-3-glucoside Biard, 1974; Nawwar et al., 1989.

Quercetin-3-O- Schilling, 1984.

neohesperidoside

Isoquercitrin Nawwar et al., 1989. Gal Flavonoids Quercetin 3-O-(2 -- Nawwar et al., 1989. rhamnosyl)-- glucosyl(16)-- galactoside Quercetin3-O-- Nawwar et al., 1989. rhamnosyl (12)-- galactoside Carotenoids β-Carotene Manunta and Solinus, 1952; Rzhavitin, 1959. Vitamins Vitamin C Rzhavitin, 1959.

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Class of compound Compound name References

Palmitic acid Hanna et al., 1996.

Palmitoleic acid Hanna et al., 1996.

Stearic acid Hanna et al., 1996.

Fatty Acids Fatty Linoleic acid Hanna et al., 1996. Triterpenes Squalene Hanna et al., 1996. Uttronin-β-D- Sharma et al., 1983.

glucopyranosyl

Uttronin-β-D- Sharma et al., 1983.

xylopyranosyl

gofurostanosides

Ol Uttroside-β-D-glucosyl Sharma et al., 1983.

Vitamins Vitamin C Rzhavitin, 1959. Tricarboxylic Acid Citric acid Rzhavitin, 1959. Fructose Rzhavitin, 1959. Carbohydrates D-glucose Bull, 1958; Rzhavitin, 1959. L-Rhamnose Bull, 1958.

2.2. INVESTIGATIONS ON S. NIGRUM (A brief Review) It is difficult to sort out the literature according to various taxa grouped into S. nigrum Complex because of the tendency of researchers to call them all as S. nigrum or occasionally as different species of genus Solanum or sometimes as variety of S. nigrum.

Manoko et al., (2007) differentiated two species of Solanum by using AFLP markers. The relationship between S. americanum and S. nodiflorum was examined. 96 individuals representing 39 accessions of S. americanum sensu lato and related diploid species from the widest possible geographical range, and one accession of S. dulcamara (as outgroup) were used. The AFLP results suggested that American

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S. americanum differs from S. nodiflorum and that the material investigated in this study can be assigned to three different species. These species can be differentiated based on a combination of floral and fruit characteristics.

Kumar and Pushpangadan, (2005) studied molecular systematic study of variants of Solanum nigrum L. in India. Solanum nigrum L. is a widely distributed polyploidy species with naturally occurring diploid (n=12), tetraploid (n=24) and hexaploid (n=36) forms. Information concerning the extent of the relationship among different species of Solanum nigrum complex is a prime importance for the evolutionary and taxonomic points of view. An investigation of randomly amplified polymorphic DNA (RAPD) marker was made for the origin of Indian variant hexaploid S. nigrum and inter-relationship with diploid and tetraploid of the complex. Out of 60 random primers 45 were used to examine 362 bands for all 3 variants. Fifteen did not amplify or showed unclear amplification across all variants.

Dehmer et al., (2004) studied the taxonomic status and geographic provenance of germplasm accessions in S. nigrum L. complex. AFLPs were employed in order to characterize the genetic diversity present in S. nigrum L. complex in a collection of the Gatersleben Genebank, to classify taxonomically unknown material. Cluster analysis was used for comparison with other examined species of the complex.

Jainu and Devi, (2004) investigated the antioxidant effect of methanolic extract of Solanum nigrum berries on Aspirin induced Gastric mucosal injury. Oxygen free radicals were considered to be important factors in the pathogenesis of gastric ulcer. The level of lipid peroxides, which were elevated highly in rats with acute gastric mucosal injury was taken as an index of oxidative stress. The activities of antioxidant defense enzymes were also decreased considerably by oral gastric administration of aspirin. The decreased levels of antioxidant enzymes and increased mucosal injury were altered to near normal status upon pretreatment with (SBE) when compared to the ulcer induced rats. The results indicate that (SBE) may exert

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its gastroprotective effect by a free radical scavenging action. Their observations suggested that (SBE) may have considerable therapeutic potential in the treatment of gastric diseases.

Schmidt et al., (2004) studied S. nigrum as a model ecological expression system and its tools. Plants respond to environmental stresses through a series of complicated phenotypic responses. S. nigrum L. is a Solanaceae relative of potato and tomato for which many genomic tools are being developed and it was presented as model plant ecological expression system here. The analysis of secondary metabolites that function as direct and indirect defenses and growth and fitness for plants grown under field conditions was discussed. To facilitate manipulative ecological studies with S. nigrum, they described: (i) an Agrobacterium-based transformation system and illustrated its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, they demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

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Al Chami et al., (2003) analyzed toxicological effects of α-Solamargine in experimental animals isolated from the fresh fruits of S. americanum Miller.

Lethality studies in rats showed a dose-mortality relationship with a LD50 of 42 mg/kg body weight intraperitoneally. The chronic and subchronic toxicity investigations indicated that the size of the glycoalkaloid dose was more important than the total glycoalkaloid intake. No appreciable toxic effects were observed at doses<35 mg/kg body weight as indicated by blood parameters, enzyme levels, and histology section of kidney, liver, and cardiac muscle.

da Silva et al., (2003) studied occurrence of flavones, flavonols, and their glycosides in Solanum (Solanaceae). During the last decade several flavonoids of Solanum species have been isolated. The study described the flavones, flavonols and their glycoside present in Solanum species.

Campos et al., (2002) studied putative pathogenesis-related genes within S. nigrum L. var. americanum genome. Isolation of two genes coding for PR5-like proteins, phylogenetic and sequence analysis. Two PR5-like genes were isolated from black nightshade (S. nigrum L. var. americanum) genome, a solanaceous weed. Southern blot analyses revealed that osmotin-like proteins re-encoded by atleast eight members of a multigene family in S. nigrum. Phylogenetic analysis of Solanaceous PR-5 proteins was also carried out revealing three major groups with different characteristics.

Lim et al., (2002) studied antioxidative and antimicrobial effects of glycoprotein isolated from S. nigrum L. Glycoprotein was tested with hydroxyl radical (OH)-expressed NIH/3T3 and microorganisms such as Escherichia coli (JA221, XL1-Blue), and Listeria monocytogenes. From antioxidative test, two glycoproteins were revealed to have protective effects against hydroxyl radical.

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Miller et al., (2002) studied conservation in divergent Solanaceous species of the unique gene structure and enzyme activity of a gametophytically-expressed flavonol 3-O-galactosyltransferase. Flavonol 3-O galactosyltranferase (F3GalTase) is a pollen specific enzyme which glycosylates the flavonols required for germination in petunia. Together with enzyme assays and HPLC analyses of pollen extracts from tobacco, tomato and potato, these results confirmed that the unique F3galtase gene structure, enzyme activity and pollen-specific flavonol glucosyl galactaosides are conserved throughout the Solanaceae.

Polkowska et al., (2001) worked on the oxidative processes induced in cell suspensions of Solanum species by culture filterate of Phytophthora in festans. Solanum genotypes that differ in the level of polygenic resistance to the oomycete plant pathogen Phytophthora infestans were studied for their oxidative response to culture filterate (CF) of the pathogen. In both the resistant and susceptible cells the CF induced similar processes, but these varied with respect to the kinetics and intensity. It suggests that lack of stringent control of the oxidative processes and sensitivity to the pathogen toxins may be decisive for limited polygenic resistance in potato.

Xu, et al., (2001) studied proteinase inhibitor ІІ of S. americanum expressed in phloem. Although proteinase inhibitor proteins are known to confer insect resistance in transgenic plants, their endogenous roles remain undefined. Two cDNAs encoding PIN2, SaPIN2a and SaPIN2b, from a S. americanum cDNA library using a tomato PIN2 cDNA as hybridization problem. SaPIN2a shows 73.6% identity to SaPIN2b. Southern blot analysis confirmed that two genes occur in S. americanum. Northern blot analysis showed that both are wound inducible and were expressed in flowers. Immunohistochemical localization, using these antibodies, revealed SaPIN2a expression in external and internal phloem of stem. These results suggest that, other than a possible role in plant defense, SPIN2a could be involved in regulating proteolysis in the SE.

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Zhu et al., (2001) analyzed peculiar steroidal saponins with opened E-ring from solanum genera plants. Peculiar novel steroidal saponins with E-ring opened skeleton, a cholesten-3β, 16, 22, 26-tetraol 3, 26-di-O-glycoside corresponding to the precursor of the spirostanol, a 16, 22-dicarbonyl type steroid and a 20-22-seco- typesteroid have been obtained from the Solanum genera plants. The occurrence of these unusual compounds would propose new important biogenetic routes for steroidal glycosides.

Guntner et al., (2000) studied effect of phosphorus fertilization on S. nigrum. The authors reported the deterrent, toxic, and anti-reproductive effect of several solanum glycoalkaloids on the potato aphids, Macrosiphum euphorbiae, and discussed the structure-activity relationship of the tested compounds. The results indicated a structure dependent boil effect of the glycoalkaloids, suggesting that, while the structure of the aglycone defines a basal boil effect the carbohydrate moity is crucial for the overall boil effect.

Hashem and Eldin, (2000) studied lipid changes in S. nigrum calli grown under salt stress. Induced calli were removed and sub cultured on medium either without salt (control) or provided with either of NaCl concns. (50, 100, 150 and 200mM) then kept growing at 28◦ under low light intensity for 8 wk. The results obtained showed with the slight increase of NaCl (50, 100 mM), no significant changes detected in neutral or phospholipids, but at the higher NaCl conc. (150, 200) most of the individual classes of neutral, phospho and the total lipids had shown a significant decrease.

Hassanein and Soltan, (2000) revealed that S. nigrum is a model system in plant tissue and protoplast cultures. Shoots culture had been easily established from shoot cuttings of germinated seeds on Gamborg (B5), or Murashige and Skoog (MS) medium without phytohormones. The best culture condition was the culture of microshoot segments on half strength MS medium supplemented with 1 mg dm-3 isobutyric acid. Regeneration of protoplasts isolated from shoot tips and fully expanded leaves was also simple. Finally, the transfer of rooted plantlets to the soil was successful.

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Khan et al., (2000) studied response of black night shade (S. nigrum L.) to phosphorus application. A pot experiment was conducted to study the effect of P on the vegetative and reproductive growth of black nightshade (S. nigrum L.). Seeds were sown directly in pots and plant samples were taken at fortnightly intervals for recording growth and yield parameters. In addition the solasodine content in fruit and N, P and K levels in leaves were also estimated. The berries were suggested to be harvested between 160 and 190 days (days after sowing), preferably at 175 days for maximum fruit yield and solasodine production. Thus, if low leaf P content was noted at 40 days, corrective measures like foliar application or top dressing were adopted to increase the leaf P content to ensure maximum Solasodine at harvest.

Kucerova et al., (2000) studied metabolism of polychlorinated biphenyl by S. nigrum hairy root clone SNC-90 and analysis of transformation products. The study investigated the aspects of PCB conversion (22 individual PCB congeners examined in common mixture. The conversion products formed from three monochlorobiphenyls were monohydroxychlorobiphenyls and dihydroxychlorobiphenyls, while six dichlorobiphenyls yielded different monohydroxydichlorobiphenyl. Efficiency of the transformation of individual PCB congeners was evaluated together with phytotoxic effect on the clone SNC-90.

Ramos et al., (2000) gave the use of the crude extract of S. nigrum L fruits in chemical education. Anthocyanins, a class of flavonoid pigments, are the most important chromophors found in blue, red, and purple plant tissues whose color changes depending on pH. This class of compounds is of potential interest to students and teachers, for illustrating chemistry principles such as acid-base equilibrium pKa, light absorption, effects of changes in conjugated double bonds, etc. A crude extract of the fruit of S. nigrum L. was used to obtain molecular absorption spectra, to verify the Lambert-Beers law, and as an indicator in acid-base titration. The color changed from red to yellow between pH 4 and 10. Absorption spectra were obtained at different pH values to detect the maximum absorption wavelength and the changing shape of spectra as a function of acidity.

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Szczerbakowa et al., (2000) studied plant regeneration from the protoplast of Solanum tubersum, S. nigrum and S. bulbocastanum. The mesophyll protoplasts were isolated from the Solanum tubersum, S. bulbocastanum and two accessions of S.nigrum. The conditions of protoplast isolation as well as the media for their culturing and regeneration were selected and optimized for the studied genotypes. The shoots excised from the protoplast–derived calli developed into whole plants in all the studied potato clones but only in one accession of S.nigrum, i.e. s.ngr var. gigantean. The regenerative capacity of the protoplast isolated from leaves and cell suspensions is compared and discussed.

Xiao et al., (2000) isolated, purified and identified Polysaccharides from black nightshade (S. nigrum). Water soluble Polysaccharides SNL-3 SNL-4 was isolated from S. nigrum L. The carbohydrate components of these two polysaccharides were determined and their molecular weights were 23,700 and 47,700.

Hu et al., (1999) isolated three known steroidal glycosides by bioactivity- guided fractionation from Solanum nigrum L. (2-Solamargine, Solamargine and degalactotigonin) and elucidated their structure on the basis of chemical evidence and spectral analysis specially by 2D-NMR analysis which induced morphological abnormality in Pyricularia oryzae mycelia. The cytotoxic assay indicated that Solamargine is the main antneoplastic agent in S. nigrum.

Kang and Sun, (1999) examined Black nightshade derived from tissue culture with higher sensitivity to ozone than seedlings. The study is aimed to find or develop some super sensitive indicator plants for biomonitoring of ozone peroxyacetyl nitrate in Taiwan. Indicator plants, S. nigrum L. was cultured and propagated by tissue culture techniques. Results showed that Black nightshade could be efficiently and rapidly propagated from cotyledons, or leaves of adult plants. With this technique, the super sensitive indicator plants can be developed for biomonitoring photochemical pollutants in a more promising way in the future.

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Subroto, (1999) examined growth and steroidal alkaloid synthesis in liquid cultures of transformed roots of S. nigrum L. Transformed root cultures of S. nigrum previously to produce significantly higher level of steroidal alkaloids compared to the differentiated callus and suspension cultures of the same species. The best medium for growth as well as for steroidal alkaloids synthesis was half-strength MS medium supplemented with 3% sucrose without plant growth regulators. A maximum steroidal alkaloid concentration of 2.87 mg/g dry weight was achieved during stationary phase at day 30. Only trace amounts of steroidal alkaloid were released into the culture media.

Hashem and El-Din, (1998) analyzed nitrogen fractions and nucleic acids content in hairy roots and regenerated S. nigrum plants induced by Agrobacterium rhizogenes. Transformed hairy roots and regenerated plants of S. nigrum were investigated for nitrogen metabolism and nucleic acid contents. From the investigated amino acids, tryptophan was markedly increased and ornithine, lysine and histidine were decreased in transformed plant material. Other amino acids did not distinguish from the control plants.

Liang et al., (1997) determined the solasodine content in S. nigrum L. by a TLC method .Solasodine in the whole S. nigrum L. and its fruits were 0.0928 and 1.16% respectively ,RSD=3.4%. The average recovery was 97.13%.The regress equation was (y=5145.1+-6449.1x,r=0.9993(n= 5).

Hanna et al., (1996) determined the phytosterol and fatty acid constituents by GLC. The anitmicrobial activity was shown against B.subitilis and higher activity against E.coli and S. aeraus.

Price and Wrolstad, (1995) separated the 12 anthocyanins by HPLC and characterized them from their retention times and UV-Visible spectra of purified pigments and their hydrolyzed products. The major pigment was Petunidin 3-(p- coumaroylrutinoside)-5-glucoside(I). Other identified as Petunidin, delphinidin and malvidin with the same glycosidic substitution pattern as (I) and varying degrees of acylation with p-coumaric, ferulic and caffeic acids.

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Colceag, (1985) purified a lectin to homogeneity from the fruits of S. nigrum by precipitates with (NH4) 2SO 4 affinity chromatogram on chitin and gel filtration on Sephadex G-100 .Its molecular weight was estimated to be 76,000 by gel filteration. Some characteristics of the lectin are compared with those of other solanaceous lectins.

Bhatt and Bhatt, (1984) found significant negative correlation between sterol content and rate of sterol synthesis from [1-14C] acetate in organs of S. nigrum. Thus cholesterol inhibited conversion of acetate to mevalonate. This is taken as evidence of a negative feedback control on sterol synthesis.

Marini and Balestrieri, (1984) performed quantitative analysis of a commercial antirheumatism formulation comprising of S. nigrum components. The fractions were separated by Sephadex LH-20 column chromatography. The individual fractions were quantified by determination of glucoalkaloids (expressed as solasodine) and of total alkaloids (expressed as hyoscyamine) by gas chromatography.

Bhatt and Bhatt, (1983) examined the accumulation of steroids in S. nigrum the young leaf. Leaf accumulates a large amount of steroids and had allowed synthesis rate whereas in old leaves the converse was true suggesting a negative feedback control. studies with labeled acetate and labeled mevalonate suggested the inhibition at the level of mevalonate.

Khan and Ikram, (1983) reported that Solanum could be a source of steroidal drugs and determined the solasodine content in 5 species of Solanum including S. nigrum. The survey showed that the leaves contain 0.3-2.3% of the compound and the fruits 1.37-5.2%.

Carle, (1981) investigated plant material of 55 strains from 32 species of Solanum sect.Solanum ( = sect.Morella) for the content of steroidal alkaloids and sapogenins. The leaf-extracts of all species contained diosgenin resp. tigogenin or

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both together in considerable amount. On the contrary the characteristic steroidal alkaloid solasodine was absent in these samples. For 29 species the presence of steroidal sapogenins had been proved for the first time. In unripe fruits, however, solasodine could be detected in most of the species examined. New occurrences of solasodine are reported for seven further species. These chemical results may contribute to a better comprehension of the difficult genus Solanum. Ontogenetic studies showed the relationship between accumulation of steroidal compounds and plant maturity.

Khanna and Sharma, (1981) studied protein bounded amino acids in different plant species including S. nigrum. It was maintained for different time periods by frequently subculturings on revised Murashige and Skoog medium supplemented with 1 ppm of 2,4-D and 1% agar. These plant tissues thus grown were analyzed qualitatively and quantitatively for their soluble and protein bound amino acids. Twenty two types of soluble and 21 types of protein-bound amino acids were obtained in all plant species.

Bose, (1980) observed that the fatty ester and alkaloid contents of S. nigrum berries increased during ripening. The solasodine contents varied from 5 to 6% and from 4 to 5% in ripe and unripe berries respectively.

Matai et al., (1971) studied leaf protein from some plants including S. nigrum. Highest N extract ability was found in S. nigrum. The highest protein extractability in S. nigrum( 42.1% of total N).

Sen et al., (1970) determined paper chromatographic studies of Anthocyanins in some arid zone plants. Floral pigments of some arid zone plants, such as Petunia violacea and Hibiscus schizopatalus, were studied by paper chromatography with BuOH-HCl-H2O (7:2:5). The red cyathia of Euphorbia caducifolia and purple black fruits of S. nigrum contained Anthocyanins.

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Rzhavitin, (1959) analyzed S. nigrum for its nutrition value. Results showed high nutrition values. Berries contained glucose, fructose 3-4 times more vitamin C than lemon, orange and tangerines, and 0.36-0.55% carotene. Seeds contained from 20-30% oil.

Kareav et al., (1955) studied the chemical composition of S. nigrum growing in Azerbaidzhan and the effect of its preparations upon the cardiovascular system. The plant material contained alkaloids 0.04%, glycosides and other sugar components 1.8%, fatty matter1.6%, resinous substances5%, volatile oil traces and Vitamin C 120 mg. Galenic preparations (aqueous infusion and decoction), after transient stimulation, depress the central nervous system and the reflexes of spinal cord; small doses increase and large doses decreased cardiac activity,; in acute experiments an appreciable reduction in blood pressure is evident; in the isolated rabbit ear vasodilation is observed.

Manunta and Ida, (1952) studied metabolism of carotenoids pigment in the different Solanum species. Intermediate quantities were found in S. nigrum.

Murray, (1939) studied five suspected poisonous plants including S. nigrum. Negative results were obtained with wooly black nightshade (S. nigrum).

Henri, (1938) reported the pharmacology of S. nigrum L. the use as an analgesic and antispasmodic was based on its content of solanine. Indications were neuralgias, painful spasma of viscera, trembling in multiple sclerosis and Parkinsonism. It is also of value in anxieties and insomnia caused by disorders of the neuro-vegetative system. The dose was 0.05-0.2 g of the dry extract dissolved in dil. Glycerol or as a suppository.

Max, (1937) studied occurrence and distribution of saponins in drug plants including S. nigrum. Some 102 drug plants were examined of which 25 contain saponins including S. nigrum.

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2.3. CHEMOTAXONOMY AND SECONDARY METABOLITES Various morphological variants are now grouped as S. nigrum Complex by the botanists. To the best of my knowledge, there is not a single report on the chemotaxonomy of S. nigrum Complex with respect to its secondary metabolites. A great deal of work had been carried out in the area of chemotaxonomy, however few references are presented here for support.

2.3.1. ALKALOIDS: Dinchev et al., (2008) examined the distribution of steroidal saponins in Tribulus terrestris from different geographical regions. LC-MC analysis of samples of Tribulus terrestris from different geographical regions revealed significant differences in their chemical composition. The data was used to suggest the existence of one chemotype common to East South Europe and West Asia and the presence of other chemotypes in India and Vietnam.

Loaiza et al., (2008) carried out the analysis of pyrrolizidine alkaloids of the endemic Mexican genus Pittocaulon and assignment of stereoisomeric 1,2-saturated necine bases. The alkaloid profiles of Pittocaulon species which contain mixed macrocyclic 1,2-unsaturated pyrrolizidine alkaloids and angeloylesters of different 1,2-saturated necine bases was used for chemotaxonomic consideration.

Greinwald et al., (1995) studied the alkaloid pattern of Plagiocarpus axillaris (Fabaceae: Brongniartieae). The alkaloidal data support the idea that Plagiocarpus is more closely related to Templetonia incana, T. biloba and Lamprolobium than it is to Hovea and the remaining species of Templetonia. The presence of hydroxysparteines in both taxa could point to an especially close link of Plagiocarpus and T. incana.

Gresser et al., (1993) studied distribution and taxonomic significance of quinolizidine alkaloids in Leontice leontopetalum and L. ewersmannii (Berberidacea). The alkaloid pattern from various organs of L. leontopetalum L. and

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L. ewersmannii (Berberidaceae) was analyzed by capillary GC and GC-MS. The alkaloid pattern of L. leontopetalum is characterized by quinolizidine alkaloids of the lupanine-type with lupanine as main compound. In L. ewersmannii 15 quinolizidine alkaloids could be found; nine constituents are described for this species for the first time. In contrast to L. leontopetalum, L. ewersmannii accumulates quinolizidine alkaloids of the matrine-type and the α-pyridone-type as major compounds. The taxonomic meaning of alkaloid profiles in Leontice and Leonticeae was discussed.

Wyk et al., (1993) reported the taxonomic significance of alkaloids in the South American genus Anarthrophyllum. The discovery of α-pyridone alkaloids in Anarthrophyllum has important taxonomic implications. It provides evidence that the true affinities of the genus are with the Argyrolobium group (presently in the tribe Crotalarieae) and Lupinus (tribe Genisteae) with which it shares, in addition to the alkaloid pattern, circumcauline stipules, a trifid lower lip of the calyx and a similar chromosome number. The alkaloid data agree with morphological evidence that Anathrophyllum and Sellocharis will be better placed near Lupinus in the tribe Genisteae.

Stermitz et al., (1980) examined alkaloids and other constituents of Zanthoxylum williamsii, Z. monophyllum and Z. fagara. Z. williamsii was found to contain (+)-asaranin, (+)-sesamin, esculetin dimethyl ether, nitidine, chelerythrine, magnoflorine, laurifoline, skimmianine and edulinine. The quaternary alkaloid fraction of Z. monophyllum contained berberine, magnoflorine, chelerythrine and a 1,2,9,10-substituted dihydroxydimethoxy-N,N-dimethylaporphinium salt. Leaves of Z. fagara were found to contain synephrine. Leaves of each species were examined for the presence of bishordeninyl terpene alkaloids, but none was found. Some chemotaxonomic relationships among Zanthoxylum species are discussed.

Izaddoost, (1975) examined the alkaloid chemotaxonomy of the genus Sophora. Cytisine and matrine alkaloids detected in Sophora species have been used to classify the genus chemotaxonomically.

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2.3.2. FLAVONOIDS: Veitch et al., (2008) studied the flavonol pentaglycosides of Cordyla (Leguminosae: Papilionoideae: Swartzieae), their distribution and taxonomic implications. The flavonol pentaglycosides of quercetin and kaempferol that accumulate in leaflets of three species of Cordyla were identified and the recent transfer of two species of Cordyla s.l. to the genus Dupuya was objected. Flavonoid glycoside profiles are also used to reassess the generic relationship between Cordyla s.l. and Mildbraediodendron.

Citoglu et al., (2005) studied chemotaxonomy of Ballota species. Sixteen taxa of Ballota were investigated by analyzing the contents of diterpenoids and flavonoid compositions and the relationship were compared with their morphological properties. HPLC chromatograms of diterpenoids and flavonoids from acetone extracts of the taxa revealed the presence of thirteen compounds. In general, morphologic, anatomic characters, distributions, and habitats were not concordant with the clusters.

Svehlikova et al., (2002) explained chemotaxonomic significance of flavonoids and phenolic acids in the Hieracium rohacsense group (Hieracium sect. Alpina; Lactuceae, Compositae. Five apomictic taxa from the Hieracium rohacsense group were studied for their phenolic constituent composition. Based on their characteristic profiles, H. rohacsense can be distinguished from a closely related and still undescribed taxon from Mt. Pip Ivan. The proportion of luteolin 7-O-glucoside to apigenin 4′-O-glucuronoside also clearly separates the individuals of two morphologically close species H. ratezaticum and H. pseudocaesium, which corresponds to a few slight but recognisable morphological and phenological characteristics.

Mitchell et al., (2001) analysed the flavonoid characters contributing to the taxonomic revision of the Hebe parviflora Complex. Flavonoid data and morphological characters were used to support the recognition of two species in this complex, Hebe stenophylla and Hebe parviflora, which were clearly distinguishable from each other and from the related Hebe traversii and Hebe strictissima. Six new

53 Chemotaxonomical Characterization of Solanum nigrum and its Varieties .

Chap.2: LITERATURE SURVEY compounds had been isolated in this study, including 6-hydroxyapigenin-7-O-β-[2- O-β-xyloxyloside] and-7-O-β-[2-O-β-xyloglucoside], 6-hydroxyluteolin-7-O-β-[2-O- β-xyloxyloside] and, luteolin-, 6-hydroxyluteolin- and 4′-O-methylluteolin-7-O-β-[6- O-β-xyloglucoside]. Other flavonoids included apigenin and luteolin 7- and 4′- mono-, di- and possibly tri-O-glycosides, 8-hydroxyluteolin 7- and 8-O-glucosides, and kaempferol and quercetin 3-O-mono- and di-glycosides. A detailed study of the flavonoid chemistry and the distributional data used to support these conclusions was presented.

Petrovic et al., (1999) reported the flavonoid and phenolic acid patterns in seven Hieracium species. Their methanolic extracts were analysed by HPLC and TLC in order to examine their flavonoids and phenolic acids. With the exception of H. rotundatum, which grow in shaded places, all species exhibited a similar pattern of phenolic substances with chlorogenic acid, 3,5-dicaffeoylquinic acid and luteolin 7-O-glucoside as the main compounds. The chemotaxonomic significance was discussed.

2.3.3. EPICUTICULAR WAXES: Urzua and Mendoza, (2008) investigated the epicuticular components from Psuedognaphalium robustum (Asteraceae) for its chemosystematic consideration with respect to other three taxa of this genus. Surface compounds were obtained by a methylene chloride extraction which was fractionated and the fractions were analyzed by GC-MS. Since flavonoids and terpenes were present only in small amounts in the epicuticular exudates of P. robustum, 80% of the surface compounds actually correspond to a complex mixture of saturated fatty acid esters, unsaturated fatty acid esters, alcohols, aldehydes, fatty acids, alkenes, triglycerides and monoglycerides. A minor hydrocarbon fraction of n-alkanes from C23 to C37 was also identified. On the contrary, in P. vira, P. cheiranthifolium, and P. heterotrichium, the epicuticular extracts contained from 70% to 80% of a mixture of diterpenoids and n-alkanes. These results showed a remarkable distance between P. robustum and other species of the genus, which share the same ecosystem. Also, these considerable differences in chemical composition were in agreement with authors that consider Pseudognaphalium as a heterogeneous taxonomic group.

54 Chemotaxonomical Characterization of Solanum nigrum and its Varieties .

Chap.2: LITERATURE SURVEY

Nguyen-Tu et al., (2007) reported the chemotaxonomical investigation of fossil and extant beeches by analysing their leaf lipids. The chemistry of lipid composition was suggested to be helpful in precising their taxonomic affinities as well as their taphonomic history.

Medina et al., (2006) studied the taxonomic significance of the epicuticular wax composition in species of the genus Clusia from Panama. Leaf surfaces were washed gently with hexane to extract epicuticular waxes, which were analyzed using gas chromatography and mass spectrometry. The predominant alkanes were C29, C31, and C33. In the extract the ratio C31/C29 was ≤1 in 6 of the 15 species analyzed. Concentration of linear alkanes in waxes from leaves of 15 species were used to group the as separate species or variety.

Tsydendambaev et al., (2004) identified unusual fatty acids of four alpine plant species from the Pamirs. Distribution of these fatty acids in 4 alpine plants were discussed with respect to their distribution in living organisms, pathways of biosynthesis and chemotaxonomic role.

Skorupa et al., (1998) discussed hydrocarbons of leaf epicuticular waxes of

Pilocarpus (Rutaceae) for taxonomic meaning. Most species presented either C29 or

C31 as main n-alkanes and they may be distinguished by the two most abundant alkanes of each distribution. Two varieties and three subspecies of P. spicatus were studied, which can be characterized by their alkane patterns. A cluster analysis using UPGMA split the 11 samples analyzed into three groups.

Mimura et al., (1997) analysed the alkanes from foliar epicuticular waxes of Hubaria species for chemotaxonomical implications.18 samples were grouped into different clusters and suggested the alkanes as good taxonomic characters at species level.

55 Chemotaxonomical Characterization of Solanum nigrum and its Varieties .

Chap.3: EXPERIMENTAL WORK

EXPERIMENTAL WORK

3.1. GENERAL EXPERIMENTAL CONDITIONS: 3.1.1. CHEMICALS AND STANDARDS Flavonoid standards Isoquercitrin, Quercetin-3-galactoside, Quercetin dehydrate and Naringin were purchased from MP. Biomedicals Inc. (Solon, Ohio). Alkaloid standards Solasonine, α-Solamargine, β-Solamargine, α-Solanine Solasodine and Solanidine were purchased from Sigma Co., St. Louis, MO. Chemicals were of analytical grade and purchased from E-Merck (Darmstadt, Germany), BDH, LABSCANE, FLUKA, RDH.

3.1.2. MELTING POINT Melting points were determined in glass capillaries using Gallenkemp melting point apparatus.

3.1.3. CHROMATOGRAPHIC TECHNIQUES Thin Layer Chromatography (TLC) was performed on precoated silica gel 60

F254 sheets (20×20 cm, 0.25 mm) (Merck, Darmstadt, Germany).

3.1.4. SPECTROSCOPIC TECHNIQUES Ultraviolet (UV) spectra were recorded on Shimadzu UV-1700 Pharma Spec. UV Spectrophotometer. Gas chromatography-Mass spectrometry (GC-MS) analysis was carried out on Shimadzu Model QP 2010A spectrophotometer in EI mode (70eV) equipped with a split/splitless injector using DB-5MS column (30 m×0.25 mm i.d., film thickness: 0.25 µm), J & W Scientific, Fulsom, CA, USA. Component identification was carried out using the internal standards and NIST 147 and NIST 27 libraries. The High Performance Liquid Chromatography (HPLC) was performed on Shimadzu LC-10A system equipped with a model LC-10AT pump, an SPD-10A variable wavelength detector, a CBM-10A interface module with class LC-10 HPLC software and a Rheodyne injection valve with a 20 µL loop was used. Chromatographic separation was performed using a Merck C-18 column (250×4.6, i.d., 5 µm particle size). Component identification was carried out using the internal standards. All analyses were carried out at GC University, Lahore, Pakistan.

56 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.1.5. DETECTION OF COMPOUNDS BY TLC TLC plates were viewed under UV light at 254nm for fluorescence quenching spots and at 366nm for fluorescent spots and with Iodine vapours for glycosides.

3.1.5.1. Spray Reagents Following spray reagents were applied to visualize different types of spots. They were prepared according to reported methods (Stahl, 1969; Harborne, 1974). 1. Ceric Sulphate Reagent 2. Dragendorff‘s Reagent 3.Marquis Reagent

3.2. PLANT MATERIAL: Plant samples of five morphologically different plant taxa (approx. 15 Kg each) of S. nigrum Complex were grown under controlled conditions in Botanic Garden of GC University Lahore, Pakistan, each in specified area. Third accession of each at flowering-seeding stage was collected for analysis. Voucher specimens were authenticated by Dr. Zaheer-ud-din Khan and deposited in Dr. Sultan Ahmad Herbarium of GC University Lahore, Pakistan (Table 2). The fresh plants were spread in clean stainless trays and stored under shade at room temperature (20-25oC) for 7 days. The dried plants (whole) were weighed, chopped and ground.

Table 2: List of plants investigated with voucher numbers

Species Voucher specimen S. americanum G.C. Herb., Bot.-146 S. chenopodioides G.C. Herb., Bot.-243 S. nigrum G.C. Herb., Bot.-241 S. retroflexum G.C. Herb., Bot.-244 S. villosum G.C. Herb., Bot.-242

3.2.1. Dewaxing with n-Hexane The dried plant powder of each sample was dipped in n-hexane for 45 sec. The n-hexane was then filtered and analysed. This process ensures the removal of surface fats and epicuticular wax so that they may not interfere in alkaloid and flavonoid analysis without disturbing the interior chemical make-up (Medina et al., 2006). 57 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

Plant Material n-Hexane

n -hexane Proximate & extract Mineral analyses Filter

n-hexane

extract Residual deffates plant material

Methanolic Alkaloid Extraction Extraction

Antibacterial & Concentration Flavonoids Steroidal glyce- Glycosides alkeloids antioxidant & acitivties Evaporation

Hydrolysis

TLC HPLC Alkaloid Hydrolysis aglycones

TLC HPLC Flavonoids Epicuticular aglycones Derivatization Wax GC-MS

Derivatization

TLC GC-MS GC-MS

Scheme 3: Analysis of the Plant Material

58 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.3. PHYTOCHEMICAL EXAMINATION OF PLANT MATERIAL: 3.3.1. DETECTING REAGENTS Following detecting reagents were prepared according to reported methods (Stahl, 1969; Harborne, 1974; Jeffery, 1989) to check the presence of different secondary metabolites: 1. Mayer‘s Reagent 2. Wagner‘s Reagent 3. Hagner‘s Reagent 4. Dragendorff‘s Reagent 5. Liebermann-Burchard Reagent

3.3.2. ANALYSIS Appropriate portion of the powdered plant material was subjected to phytochemical screening for the presence of alkaloids, flavonoids, tannins, saponins, steroids and anthraquinones using standard phytochemical procedures and reagents (Brain, 1975; Mayer, 1982; Jeffery, 1989; Feroz, 1993). The extracts were separately tested for alkaloids using Mayer‘s Reagent, Wagner‘s Reagent, Hagner‘s picric acid Reagent and Dragendorff‘s Reagent.

CHEMOTAXONOMIC INVESTIGATION The chemotaxonomic investigation of Solanum nigrum Complex was carried out by comparing their Alkaloids, Flavonoids and Epicuticular wax composition. An overview of this work is presented in Scheme 3.

3.4. ANALYSIS OF ALKALOIDS: The alkaloids were analysed as Steroidal Glyco-Alkaloids (SGA) as well as Aglycones (SGAA). Each of the plant samples was defatted by dipping in n-hexane for 45 sec before analysis of its alkaloid.

3.4.1. ESTIMATION OF TOTAL GLYCOALKALOID CONTENT Total Glyco-Alkaloid (TGA) contents of the defatted samples of five taxa of Solanum nigrum Complex were determined by titrimetric method of Fitzpatrick and Osman (1974).

59 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.4.1.1. Sample preparation for Titrimetric Analysis A sample (20 g) of each taxon was extracted with 100 mL of methanol- chloroform (2:1) by stirring with magnetic stirrer for 15 min, filtered through

Whatman filter paper No.1 and the extract was mixed with 100 mL of 0.8% Na2SO4. The mixture was shaken in a separating funnel and allowed to settle overnight. The upper chloroform layer was separated, dried to dryness. The residue was dissolved in

15 mL of 2 N H2SO4. The solution was then heated for 2 hrs on a steam bath and made basic with 10 mL of 4 N NaOH. The glycoalkaloids were extracted with three 15 mL portions of benzene. The benzene was then stripped off and the residue was taken up in 5 mL of methanol.

3.4.1.2. Titrimetric Analysis Samples were titrated with a solution of 0.067% bromophenol blue and 10% phenol in absolute methanol, against a blank of methanol. The TGA were calculated by using a standard curve prepared with known concentration of Solasodine and α- Solanine in methanol.

3.4.2. ALKALOID EXTRACTION AND DETECTION Individual alkaloids were detected and quantified by  TLC  HPLC  GC-MS Alkaloids for compositional analyses were extracted by a method based on a modification of the technique of Dao and Friedman (1996). Sample of 15 g of each powdered plant material with 200 mL of 5% aqueous acetic acid was placed in a 250 mL Erlenmeyer flask and stirred for 30 min. The sample was then vacuum filtered through a Whatman no. 42 filter paper, and the residue was reextracted three times with the same solution for 30 min each time. The four filtrates were combined and transferred to a 1 L separating funnel. The pH was adjusted to 11 with ammonium hydroxide, and the alkaline extract was partitioned four times with 50 mL of water saturated butanol. The combined butanol extracts were evaporated almost to dryness, and the residue was removed from the flash with three portions of 5 mL each of

60 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

methanol and placed in a vial with a pipette. The extract was evaporated to dryness, and the residue was weighed, redissolved in 10 mL of methanol and analyzed. Samples for HPLC and GC-MS analyses were filtered using 0.45µ polyamide filters (Sartorius, Germany).

3.4.3. TLC PROCEDURE FOR SGA The preliminary examination of the alkaloids extracted above was performed by TLC. The two solvent systems recommended for different glycoalkaloids of Solanum were used. a) Harborne (1974) used acetic acid:ethanol (1:3), spray reagent: Marquis reagent for TLC identification of α-Solanine. b) Boll‘s (1962) solvent whose composition was chloroform:methanol:1% aqueous ammonium hydroxide (2:2:1), the chromatogram was sprayed by Dragendorff‘s reagent. b) Boll and Anderson (1962) used ethyl acetate-pyridine-water (3:1:3) and the alkaloids were detected by colour reaction with antimony(III) chloride reagent. Experiments were repeated thrice and the results are given in Table 4.

3.4.4. HPLC ANALYSIS OF SGA The glycoalkaloids were analysed using HPLC apparatus consisting of Shimadzu LC-10A system as detailed previously. HPLC conditions were set as described by Sotelo and Serrano (2000) for S. tuberosum except that the buffer used was ammonium dihydrogen phosphate with pH 6.1 and the UV absorbance detector was set at 205 nm. The mobile phase was acetonitrile-0.05 M and ammonium dihydrogen phosphate buffer (30:70 v/v). Solvent flow rate was 1.5 mL/min. The injector loop was 20 μL. Mobile phase was prepared fresh, sonicated and filtered through a 0.45 µm polyamide filter. Solasonine, α-Solamargine, β-Solamargine and α-Solanine were used as internal standards. Results are presented in Table 5.

61 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.4.4.1. Validity study of the Method The validity was tested with glycoalkaloid standards and for a recovery study, different amounts (0.12-0.30 mg in triplicate) were added to 15 g of dried samples. The samples were thoroughly mixed, extracted as above, and analyzed by HPLC. The detector peak area response was linear over this range. The mean recoveries obtained from triplicate samples were 99.2 ± 0.47 to 99.6 ± 0.54%. The results of this validity study show that the method used is efficient and useful for glycoalkaloid analysis of S. nigrum Complex.

3.4.5. ACID HYDROLYSIS OF SGA Steroidal glycoalkaloid aglycones (SGAA) were obtained by the method reported by Laurila et al. (1999). Dried plant materials (10 g) and standards (20 μg each) were separately dissolved in 2 mL of 1 M HCl in methanol and heated for 3 h at 70 °C in a water bath. The free aglycones were liberated from the hydrolysate by adding 2 mL of 25% ammonia to the cooled tube and extracted with 2 mL of dichloromethane after a few minutes. After vigorous mixing and 5 min of centrifugation, the dichloromethane layer was removed with a pipette. This extraction procedure was repeated with two additional 2 mL aliquots of dichloromethane, and the extracts were combined. The aglycon extracts were then evaporated to dryness.

3.4.6. DERIVATIZATION OF SGAA Trimethylsilylimidazole (20 μL) and dry acetonitrile (50 μL) were added via glass syringe to each sample and standard (Solasodine and Solanidine). The mixtures were placed in an oven at 60 oC for 15 min. After this, they were cooled to room temperature and 1 μL of each solution was injected into the chromatographic system.

3.4.7. GC-MS ANALYSIS OF SGAA DERIVATIVES The SGAA derivatives were determined by the method recommended by Laurila et al. (1999), for Solanum species using Shimadzu GC-MS QP2010A system operating at an ionization voltage of 70 eV (EI mode) with ion source temperature of 180 °C. Samples were analyzed on an NB-54 fused-silica capillary column (15 m, 0.20 mm i.d., Nordion, Finland) using split sampling mode and an oven temperature of 180−285 °C heated at 7.5 °C/min. A 1 μL sample was taken for GC/MS analysis. 62 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

Injector and detector temperatures were 285 °C. Helium was used as the carrier gas (flow rate = 0.5 mL/min). Identification of the aglycones in the plant materials was based on the GC-MS spectra of TMS derivatives of authentic standards and on reports of GC-MS glycoalkaloid aglycon data (Van Gelder et al., 1989; Laurila et al., 1996). Concentrations of SGAA calculated are given in Table 6.

3.4.7.1. Validity study of the Method The SGAA concentrations of the plant samples were calculated from peak areas (area normalization method) compared to those of standards. The validity of the quantitative analyses was determined from the same plant material (n = 3), which was extracted, hydrolyzed, derivatized and analyzed by GC-MS. The linearity of the method was determined for solasodine and solanidine by triplicate determinations at four concentration levels in the range between 20 and 400 μg, and the calibration curve was obtained by plotting the peak area ratio of standard against its amount. The calibration curves were linear in the range of 20−400 μg.

3.5. ANALYSIS OF FLAVONOIDS The flavonoid profile of S. nigrum Complex was examined before and after hydrolysis. Each of the plant samples was defatted by dipping in n-hexane before analysis of its flavonoids.

3.5.1. ESTIMATION OF TOTAL FLAVONOID CONTENT Total flavonoid contents of the five taxa of Solanum nigrum Complex were determined by colorimetric methods.

3.5.1.1. Sample preparation for Colorimetric analysis Flavonoids were extracted for colorimetric analysis as described by Chang et al. (2002). About 1 g (accurately weighed to 0.0001 g) of each of dewaxed powdered plant samples was extracted with 25 mL of 95% ethanol under 200 rpm shaking for 24 hr. After filtration, the filtrate was adjusted to 25 mL with 80% ethanol and stored in an amber bottle.

63 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.5.1.2. Colorimetric analysis

(I) Aluminum Chloride Colorimetric Method: The aluminum chloride colorimetric method was modified from the procedure reported by Woisky and Salatino (1998). Quercetin was used to make the calibration curve. Ten milligrams of quercetin was dissolved in 80% ethanol and then diluted to 25, 50 and 100 μg/mL. The diluted standard solutions (0.5 mL) were separately mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% AlCl3, 0.1 mL of 1M CH3COOK and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-1700 Pharma Spec. spectrophotometer. The amount of 10% AlCl3 was substituted by the same amount of distilled water in blank. Then 0.5 mL of each ethanolic extracts was reacted with

AlCl3 for determination of flavonoid content as described above.

(II) 2,4-Dinitrophenylhydrazine Colorimetric Method: The current method was modified from the procedure described by Nagy and Grancai (1996). Naringin was used as the reference standard. Twenty milligrams of naringin was dissolved in methanol and then diluted to 500, 1000 and 2000 μg/mL. One milliliter of each of the diluted standard solutions was separately reacted with 2 mL of 1% 2,4- DNPH reagent and 2 mL of methanol at 50°C for 50 min. After cooling to room temperature, the reaction mixture was mixed with 5 mL of 1% KOH in 70% methanol and incubated at room temperature for 2 min. Then, 1 mL of the mixture was taken, mixed with 5 mL of methanol and centrifuged at 1,000 x g for 10 min to remove the precipitate. The supernatant was collected and adjusted to 25 mL. The absorbance of the supernatant was measured at 495 nm. The ethanolic extracts were similarly reacted with 2,4-DNPH for determination of flavonoid content as described above. Results are given in Table 7.

64 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.5.2. FLAVONOIDS EXTRACTION AND DETECTION Individual flavonoids were detected and quantified by  TLC  HPLC  GC-MS

3.5.2.1. Sample preparation Samples for TLC, HPLC and GC-MS analyses were prepared in methanol. 10.0 g of dewaxed plant material was extracted in a go in a Soxhlet apparatus with methanol (50 mL). At 1 hour intervals, aliquots were removed and checked for the presence of flavonoids by TLC. After 5 hours, the extract showed absence of flavonoids. The extraction procedure was executed in triplicate, for 5.hours. Each extract was then filtered and the volume was completed to 100 mL with methanol. 10 mL from this extract was filtered through a small column of C18 silica (55-105 mm, 0.50 g) and the column eluted with 8 mL of methanol. The volume of the eluate was completed to 20 mL. More 5 mL of methanol was applied to the column and the eluate was checked for flavonoids, which were absent. Total volume of each extract was made 25 mL.

3.5.3. TLC PROCEDURE FOR FLAVONOIDS GLYCOSIDES Since quercetin, in its various glycosidic forms, has been the only flavonoid detected in S. nigrum Complex, the preliminary examination of the above extract by TLC was carried out according to the solvent systems recommended for quercetin glycosides (Harborne, 1974). Normal phase TLC was performed on precoated silica gel sheets. The plates were developed separately in a) BAW (n-butanol-acetic acid-water, 4:1:5, upper layer). b) 15% Acetic acid (AcOH) in water. The chromatograms were observed in UV light (254 nm) before and after exposing to ammonia vapors. Experiment was repeated thrice and the results are given in Table 8.

65 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.5.4. HPLC ANALYSIS OF FLAVONOIDS GLYCOSIDES Two gradients were applied as described in an optimized method (Wang and Li, 2007) for the analysis of flavonoid glycosides. The standards used were quercetin-3-glucoside (isoquercitrin) and quercetin-3-galactoside. HPLC apparatus consisting of Shimadzu LC-10A system equipped with a model LC-10AT pump, an SPD-10A variable wavelength detector, a CBM-10A interface module with class LC- 10 HPLC software and a Rheodyne injection valve with a 20 µL loop was used. Chromatographic separation was performed using a Merck C-18 column (250×4.6, i.d., 5 µm particle size). The mobile phase and gradient conditions were as follows:

Gradient I: Solvent A (methanol) and solvent B (1 M phosphoric acid adjusted at pH 2.0 with double deionized water). The gradient condition curve was set at G-five; A – B (30:70) used as the initial condition. Methanol concentration was increased from 30 to 52% A in 3.5 min, then to 61% A in 4 min, to 67% in 4 min, and finally to 40% in 4 min. after keeping at 40% for 6 min, the gradient was reversed to the initial condition in 35 min and then equiliberated for an additional 12 min before the next sample was injected. Gradient II: A – B (30:70) was used as initial condition. Methanol concentration was linearly (G-three) increased from 30% to 55% in 5 min, and finally to 68% in 4 min. The mobile phase flow rate was always 0.9 mL min-1. The absorption was measured either as a full spectrum (within 190-400 nm), at 350 nm for most constituents, or at 300 nm for flavones. Injection volume was 20 μL. Samples were run for 30-50 min. Results are presented in Table 9.

3.5.4.1. Validity study of the Method Validity of the extraction procedure was assessed by measuring the peak area variation for flavonoid standards peaks in three replicate analyses. The RSD values for the peaks were less than 1%, which is quite acceptable for quantification. By spiking the standard solutions of flavonoids, the recoveries of these components were measured. The amount of the spiked standard was calculated by subtracting the total amount of flavonoid standard after spiking from the amount found in the plant before spiking. The spiking experiments were repeated three times for two different concentrations to obtain the mean total amount of flavonoid after spiking. 66 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.5.5. ACID HYDROLYSIS OF FLAVONOIDS GLYCOSIDES The standard procedure used for the hydrolysis of quercetin glycosides in Solanum lycopersicum L. has been described by Hertog et al. (1992). HCl (1.2 M, 5 mL) was added in the methanolic extract (25 mL) of each sample and the mixture was stirred at 90oC under reflux for 2 hours to obtain the aglycones by hydrolysis of flavonoid glycosides. The extracts were cooled to room temperature and extracted with ethyl acetate (1:1, v/v). Fractionation with NaHCO3 was performed according to the method described by Sabatier et al. (1992). The ethyl acetate extract was treated with 0.5 M NaHCO3 (1:1, v/v) three times to eliminate the free phenolic acids. The ethyl acetate extract was evaporated to dryness under a flow of nitrogen and flavonols were re-dissolved in ethyl acetate. Each of the sample solution was filtered using 0.45µ polyamide filters (Sartorius, Germany) and was degassed by sonication for 3 min before injection.

3.5.6. GC-MS ANALYSIS OF FLAVONOIDS AGLYCONES GC-MS analysis was carried out using the conditions modified from the method of Tokusoglu et al. (2003) used for the characterization of quercetin aglycon from hydrolyzed extract of Solanum lycopersicum L. GC-MS spectra were recorded on Shimadzu GCMS-QP2010A system in EI mode (70eV) equipped with a split/splitless injector (280oC), at a split ratio of 30/70 using DB-5MS column. Helium was used as a carrier gas at the rate of 1mL/min. 1 µL of sample was injected keeping ion source temperature 200°C and interface temperature at 250°C. The column temperature was kept at 100°C for 1 min after injection and then increased at the rate of 10°C min-1 to 275oC which was held for 20 min. Standard stock solution 500 µg/mL of quercetin was prepared in methanol and the calibration curve was established using five dilutions of the standard solution in the concentration range of 0.1-2.0 µg/mL. R2 value was 0.99. Concentration of quercetin aglycone was calculated as given in Table 10.

67 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.6. ANALYSIS OF EPICUTICULAR WAXES The epicuticular was characterized physically and chemically. 3.6.1. EXTRACTION OF EPICUTICULAR WAX n-Hexane (GC-grade), purchased from Merck, Germany, was further distilled twice and microfiltered before use. Epicuticular waxes were extracted by immersing the plant samples in cold n-hexane for 90 seconds. This procedure extracts only surface n-hexane-soluble compounds without disturbing the inner chemical make-up (Medina et al., 2006). Extracts were filtered and concentrated by rotary evaporation. Complete dryness of residue was achieved by evaporation at room temperature in a ventilated fumehood. Dry extracts were weighed to calculate percentage yield. A measured amount of residue was redissolved in n-hexane and microfiltered to prepare diluted samples (10µg/250ml).

3.6.2. PHYSICOCHEMICAL ANALYSIS OF WAX Wax of the five taxa was characterized by applying different physical and chemical tests. Results are recorded in Table 11. 3.6.2.1. PHYSICAL TESTS (AOAC, 1993) Following physical tests were carried out on waxes of five taxa of Solanum nigrum Complex. i. Colour of Wax Colour of waxes was examined. ii. Melting Point(closed capillary tube method) Capillary tube was taken having one end closed. Filled 1/3rd of tube with wax and heated in the melting point apparatus till the wax melted. Melting point was determined as average of three concordant readings. iii. Refractive Index Abbe‘s refractometer was used for the determination of refractive index of wax. A drop of the melted wax was placed between the prisms of the refractometer. The telescope was rotated to bring the border line of the total refraction to the junction of cross wires in the telescope. The refractive index of the wax was observed up to the 4 decimal places from the scale present on the instrument and recorded the room temperature.

68 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.6.2.2. CHEMICAL TESTS Following chemical tests were applied to waxes

i. Saponification Value Apparatus: a) Erlenmeyer Flask –— 500 mL b) Air condenser –— minimum 650 mm long. c) Water bath or hot plate –— with variable heat control. d) Pipette –— 50 mL, volumetric.

Solutions: a) Hydrochloric acid, 0.5 N –— accurately standardized. b) Alcoholic potassium hydroxide –— placed a few grams (5-10) of KOH in a 2-L flask and added from 1-1.5 liters of 95% ethyl alcohol and boiled in a water bath under reflux condenser for 30-60 min distilled and collected the alcohol. Dissolved 27g of potassium hydroxide, low in carbonate, in 1 L of distilled alcohol while placing in a water bath. c) Phenolphthalein indicator solution –— 1% in 95% alcohol.

Procedure: About 1 g of sample wax was weighed. Added 50 mL of alcoholic KOH with pipette. Boiled under air condenser gently until the sample was completely saponified. Added 1 mL of Phenolphthalein indicator and titrated with 0.5N HCl until the pink color just disappeared. Noted the volume of acid used. Heated the flask to boiling on hot plate and again titrated to disappearance of pink color if developed. Added this titration volume to the previous volume. Took 3 concordant readings and calculated the average. Prepared and conducted blank determinations simultaneously with the sample, similar in all respects to the sample. Saponification value was calculated by the following formula:

Saponification value = 56.10 × N (B - S) Mass of sample in g

69 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

Where

B = Volume of HCl used for blank.

S = Volume of HCl used for sample. N = Normality of standardized HCl solution.

ii. Acid Value Apparatus: a) Erlenmeyer Flask –— 500 mL

Reagents:  Sodium hydroxide solution 0.5N –— accurately standardized.  Ethyl alcohol –— 95%. Added a few drops of phenolphthalein indicator solution. Boiled and neutralized with NaOH solution to a faint pink color just before using.  Phenolphthalein indicator solution –— 1% in 95% alcohol.

Procedure Weighed 1 g of wax into 500 mL Erlenmeyer flask and added 75-100 mL of neutral alcohol. Agitated and heated until the wax dissolved. Added 0.5 mL of phenolphthalein indicator and titrated immediately, while shaking, with 0.5N NaOH to the first pink color which persisted for 30 sec. calculated the Acid value by following formula:

Acid value = Vol. of titrant, mL ×N×56.10 Mass of sample in g Where ―N‖ is normality of NaOH solution.

iii. Ester Value Ester value was calculated for a wax sample, by difference, after determining Acid value and the saponification value.

Ester value = saponification value - acid value

70 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.6.3. DETECTION OF EPICUTICULAR WAX COMPONENTS Individual wax components were detected and quantified by  TLC  GC-MS

3.6.4. TLC PROCEDURE FOR EPICUTICULAR WAX TLC of the waxes obtained above was performed in different solvent systems suggested for epicuticular wax analysis in reported literature (Purdy and Truter, 1963; Holloway, 1977). For the detection of components, all the chromatograms were viewed in UV light at 254 and 366nm. a) Benzene (100%) b) n-Hexane (100%) c) n-Hexane and Diethylether (4:1) d) Chloroform and Ethanol (98:2)

3.6.5. GC-MS ANALYSIS OF EPICUTICULAR WAX The composition of the waxes was established using GC-MS technique with the method modified from that reported by Tulloch and Bergter (1981). GC-MS analyses were performed on a Shimadzu GCMS-QP2010A system given above in EI mode (70eV) equipped with injector at 250 oC, using DB-5MS column. Samples were injected at 250 oC with a split ratio of 50/50. Injection volume was 1 µl and electronic pressure programming was used to maintain a constant flow (0.67 ml/min) of the Helium carrier gas. The oven temperature was programmed from 150 oC (4 min) to 320 oC at a rate of 2 oC/min and held at this temperature for 2 min. The mass spectrometer was set to scan the mass range 40 amu to 600 amu with ion source temperature 200 oC and interface temperature 250 oC. Analyses were performed in triplicate with a blank run after every analysis. The resulting data was processed using Shimadzu Lab Solution GCMS Postrun Analysis software. The relative apparent percentage of each compound and of their classes was determined by area normalization method (Table 12 & 13). Compounds were identified by comparing the mass fragmentation pattern of the reported data and NIST 147 and NIST 27 libraries. Statistical analysis of the 34 compounds identified was carried out by Multivariate Cluster analysis using Minitab 3.2 Statistical software.

71 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.7. PROXIMATE ANALYSIS OF THE PLANT MATERIAL Moisture, ash, crude fibre, crude protein, fat, carbohydrate, nitrogen free extract (Table 14) and trace elements (Table 15) in the five taxa were determined according to the following procedures:

3.7.1. MOISTURE AND DRY MATTER (AOAC, 1995) Clean crucibles (25 mL) were dried in a muffle furnace at 600 oC for one hour and cooled to room temperature in desiccators. The crucibles were weighed by handling them with metal tong. Then 3 grams of the sample were taken in the dried and tarred crucible and it was placed in the oven at 105 oC for 24 hours. The crucible was removed from the oven and weighed after cooling to room temperature in desiccators. The percent dry matter and moisture were calculated as:

Dry Matter (%) = Weight of Dried Sample × 100 Weight of Sample before Drying

Moisture (%) = 100 - dry matter (%)

3.7.2. ASH (AOAC, 1995) Clean porcelain crucibles were heated in a muffle furnace at 600 oC for one hour and weighed after cooling it in desiccator to room temperature. The samples leaves, stems and roots (2 gram each) were accurately weighed and taken separately in the dried and tared porcelain crucibles. Each crucible was placed in a muffle furnace and its temperature maintained at 525 oC until carbon free ash was obtained. The crucibles were weighed after cooling to room temperature in desiccators and the ash contents were calculated as:

Ash (%) = Weight of ash × 100 Weight of Sample

72 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.7.3. CRUDE FIBRE (AOAC, 1984) 3.7.3.1. Reagents a. Sulphuric acid solution: 1.25% aqueous solution. b. Sodium hydroxide solution: 1.25% aqueous solution.

3.7.3.2. Procedure Two grams of the ground leaves, stems and roots were accurately weighed and transferred to a round bottom flask separately. Defatted the samples with n- hexane in soxhlet apparatus. The extracted sample was transferred to a 500 mL beaker. 200 mL of 1.25% sulphuric acid was added to the beaker. The beaker was placed on a preheated hot plate and solution was boiled for exactly 30 minutes, with periodic swirling of the contents. The level of the solution in the beaker was maintained with the addition of distilled water. The beaker was removed from the hot plate and the contents were filtered onto a linen cloth of fine mesh (about 200 mesh) in a fluted funnel and washed with boiling water until the washings were not acidic. The sample was then washed back into the beaker with 200 mL of NaOH (1.25%) solution. The beaker was placed on a hot plate. After the contents of beaker had boiled exactly for 30 minutes, it was removed and filtered onto a linen cloth as described above. The sample was washed with boiling water until free from alkali. The sample was filtered with suction into a Gooch crucible. Finally, the sample was washed in Gooch crucible with 10 mL of 95% ethanol. The contents of the crucible were dried at 105 oC in an oven for overnight, cooled to room temperature in a desiccator and weighed. The contents of the crucible were then ignited, in a muffle furnace at 600 oC to ash. After ignition the crucible was cooled to room temperature in a desiccator and weighed. The crude fiber was calculated as follows:

Crude Fiber (%) = W2-W3 × 100 W1

Where

W1 = Weight of the sample before drying and extracting with n-hexane.

W2 = Weight of dry material after extraction with acid and alkali.

W3 = Weight of ash.

73 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.7.4. NITROGEN AND CRUDE PROTEIN (AOAC, 1995) 3.7.4.1 Reagents A. Indicator Solution: a. Methyl red (0.1%) in ethanol b. Bromocresol green (0.1%) in ethanol The solutions ‗a‘ and ‗b‘ were mixed in ratio 1:5.

B. Standardized 0.01M H2SO4 and 0.01M HCl solutions.

C. Conc. H2SO4, 93-98% reagent grade. D. Catalyst Mixture, Premixed Selenium mixture of E.Merck for nitrogen estimation. E. Sodium hydroxide solution 40% nitrogen free prepared by dissolving 400 grams of Sodium hydroxide (A.R.grade) per dm3 solution. F. Boric acid solution, (4%), forty grams boric acid was dissolved per dm3 and then added to it 5 mL indicator solution.

3.7.4.2. Procedure The sample (leaves, stems and roots; 0.5 gram each) was accurately weighed and placed in a 100 mL long neck Kjeldahl flask. The catalyst mixture (0.5 gram) was added in to the flask followed by the addition of 10 mL conc. H2SO4. The flask was placed on the digestion heaters of the Kjeldahl apparatus. Then heater and exhaust fan were turn on. The digestion process was carefully watched until frothing ceased. The digestion was continued, with occasional turning of the flask for 30 minutes, till the solution had cleared and all carbon had been oxidized. After completion of digestion, the heater was turned off and the flask was allowed to cool. Then the volume was made 100 mL with distilled water and 5mL of this solution was taken in to the Markham distillation apparatus. About 5 mL of 40% sodium hydroxide solution was also added and the cup of the apparatus was rinsed down with distilled water. In a titration flask 10 mL of 4% boric acid solution was placed under the condenser, the end of which was dipped in the boric acid solution. The contents were heated by passing steam through the apparatus until the volume in the flask was about 20 mL. The indicator was added in the boric acid solution before distillation. The material in the flask was titrated against standard hydrochloric acid (N/70) taken in a burette. The end point was a sharp change of colour to pink.

74 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

The volume of N/70 hydrochloric acid was noted. The percentage of nitrogen and crude protein in the sample (leaves, stems and roots) was calculated as follows:

Nitrogen (%) = X × 100 × Y/10 Weight of Sample in mg

Where Volume of N/70 HCl used = X mL X/5 = Y g (Amount of Nitrogen)

Crude protein (%) = Nitrogen % × 6.25

3.7.5. FAT (AOAC, 1995) Leaf, stem and root samples were pulverized separately and placed in thimbers, which were extracted by soxhlet apparatus using n-hexane as solvent at 40 oC till the disappearance of colour. After that thimbers were opened, dried in oven and their weights were determined.

Fat (%) = Loss in weight × 100 Weight of Sample

3.7.6. CARBOHYDRATE (Harris, 1970) Carbohydrates of leaf, stem and root samples were determined separately by difference after the analysis had been completed for moisture, minerals, proteins, fats and fiber.

Carbohydrate (%) = 100-[Moisture (%) + Ash (%) + Crude Fibre (%) + n-Hexane Extract (%) + Crude Protein (%)]

75 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.7.7. NITROGEN FREE EXTRACT (Scales, 1920) Nitrogen free extract (N.F.E.) of leaf, stem and root samples water determined separately by difference after the analysis had been completed for ash, crude fiber, ether extract and crude protein.

N.F.E. (%) = 100-[Ash (%) + Crude Fibre (%) + n-Hexane Extract (%) + Crude Protein (%)]

3.7.8. METAL DETECTION (Gampton, 1969) 5 gram accurately weighted sample was taken in a porcelain crucible and ashed in a muffle furnace at 500-550 oC until free from carbon. The ash was cooled and dissolved in 5mL HCl-HNO3 mixture (9:1 V/V) for 15 minutes to complete solution. After cooling, the solution was diluted with distilled water and filtered into a 50mL volumetric flask. The filter paper was washed in hot water and the solution in the flask was diluted to volume. A blank solution was also prepared by diluting 5mL

HCl-HNO3 solution to 50mL. The standard solution of the element to be analyzed was prepared by diluting standard solutions of the metal (Fisher Chemicals) to the range of 2-10 ppm. The sample and standard solutions were stored in to polythene bottles. The sample was analyzed for metal ions by using a SOLAAR 969 Atomic Absorption spectrophotometer JUN AIR model 600. The air-acetylene gas mixture was used as a fuel for flame production. For each element, a specific hollow cathode lamp was used. The properly diluted sample solution and the standard solution were individually aspirated in the burner of the spectrophotometer and the atomic absorption of the sample and standard solutions was measured. Atomic absorption of blank was also determined for each element and corrections were made in the sample and standard absorption. The concentration of the element in the sample solution was determined by comparison of absorption with that of standards. The element concentration (ppm) of the samples were calculated and given in Table 15.

76 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.8. ANTIBACTERIAL STUDY A comparative antibacterial study of methanolic extracts of five taxa was carried out on seven microorganisms using the procedures of Wiesendanger et al., (1995) and Jain et al., (1996).

3.8.1. BACTERIAL STRAINS

3.8.1.1. Gram Positive  Bacillus subtilis (PCSIR-B-248)  Bacillus licheniformis (PCSIR-B-252)  Micrococus luteus (NRRL-B-287)  Nocardia asteroides (PCSIR-B-178)

3.8.1.2. Gram Negative  Escherichia coli (PCSIR-B-67)  Proteus mirabilis (ATCC-29245)  Salmonella typhimorium (ATCC-14028)

3.8.2. STANDARD ANTIBIOTICS The standard antibiotics, Benzyl penicillin, Ampicillin and Streptomycin were taken as positive control in the concentration of 1 µg/mL for bacterial strains. Distilled water was used as negative control against all the species.

3.8.3. SAMPLE PREPARATION Methanol from the concentrated dewaxed methanolic extracts of each taxa was completely removed and the residue was dissolved in distilled water to make concentration of 1000, 2000 and 3000 µg/mL.

3.8.4. PREPARATION OF NUTRIENT BROTH Dissolved 0.8 g nutrient broth in 100 mL distilled water by heating. Adjusted the pH to 7.4 and then sterilized it in an autoclave at 121 oC for 15 minutes.

77 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.8.5. PREPARATION OF NUTRIENT AGAR Dissolved 0.8g of nutrient broth in 100 mL distilled water by heating. Then added 1.3g of nutrient agar in it and heated till clear solution was prepared. Adjusted the pH to 7.4 and then sterilized it in anauto clave at 121 oC for 15 minutes.

3.8.6. PREPARATION OF INOCULUM Stock slants of bacterial culture were taken and a loop full of culture was added to the sterilized slants in the test tube. The cultures were incubated at 37 oC for 24 hours. After that a loop full from these cultures was transferred to conical flask of freshly prepared nutrient broth and incubated for 24 hours at 37 oC in a shaker. These cultures served as inoculum.

3.8.7. DETERMINATION OF ZONE OF INHIBITION The bacterial species were maintained on nutrient agar slants. Molten nutrient agar (20 mL) was poured into sterilized Petri dishes as a basal layer. Plates were inoculated with 0.5 mL inoculum of the respective organism. Covered the dishes with lids. Allowed them to cool and solidify. The agar core (4 mm) was then removed from the set agar at four peripheral points and each of these holes was filled with the standard and different concentration of extracts. After keeping the Petri dishes in the flat position for one hour, the incubation period was allowed to proceed for 24hours at 37 oC for bacteria. The diameters of the clear zones around the holes were observed and recorded. The test was carried out in triplicate. The results are given in Table 16.

3.8.8. MEASUREMENT OF MIC The Minimum Inhibitory Concentration (MIC) was evaluated on plant extracts that showed antimicrobial activity. The MIC, which inhibited the complete growth of the tested bacteria, was determined by regression analysis using the software STATGRAPHICS version 4 (Table 17).

3.8.9. STATISTICAL ANALYSIS SPSS 11.5 software was used for the statistical analysis. Analysis of variance (ANOVA) followed by the Duncan test was used to see the differences amongst the various groups. The significance level was set at p<0. 05. 78 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.9. ANTIOXIDANT STUDY A comparative antioxidant study was carried out on five taxa using six assays. All the chemicals and reagent used were of analytical grade purchased from Merck and Sigma.

3.9.1 PREPARATION OF REAGENTS

1. K2S2O8 Solution: 0.0270g of K2S2O8 was dissolved in 10ml of double distilled water to make its concentration to 10mM. 2. ABTS Solution: 0.0384g of ABTS was dissolved in 7.5 ml of double distilled water. 3. HCl Solution: Took 9.37 mL of HCl (36%) and dilute to 10 mL with distilled water. Now take 0.146 mL of above solution and again dilute it to10 mL.This is 40 mM HCl solution. 4. TPTZ Solution: Took 0.015 g of TPTZ and dissolve it in 5 mL of diluted HCl (which is diluted second time). If TPTZ does not dissolve, then heat it in water bath at 60Ċ till it dissolves. This is 10 mM TPTZ solution.

5. Ferric Chloride Solution: Took 0.054 g of FeCl3.6H2O and dissolve it in 10 mL of distilled water. This is 20 mM ferric chloride solution. 6. Sodium Acetate Buffer: Took 3.6 g of sodium acetate and add 950 mL of distilled water. Adjust its pH with acetic acid and make the volume up to one litre. This is 300 mM acetate buffer (pH 3.6).

7. 20% Na2CO3 Solution: 20 g of sodium carbonate was dissolved. The solution was boiled, cooled and then few crystals of fresh sodium carbonate was added in it. It was allowed to stay for twenty four hours at 4 oC.If sodium carbonate crystallizes, then filter it, otherwise use it. 8. 3.5% HCl Solution: 9.4594 cm3 of HCl solution was added to 100 cm3 of double distilled water. 3 9. FeCl2.4H2O Solution: 0.0397g of FeCl2.4H2O was dissolved in 10 cm of 3.5% of HCl solution. 3 10. FeSO4 Solution: 0.03 g of FeSO4 was dissolved in 100 cm of double distilled water. 11. Ferrozine Solution: 0.02 g of ferrozine was dissolved in 10 cm3 double distilled water.

79 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3 12. 5% NaNO2 solution: 5.0 g of NaNO2 was dissolved in 100 cm of distilled water. 3 13. 10 % AlCl3 Solution: 10.0 g of AlCl3 was dissolved in 100 cm of distilled water. 14. 1M NaOH Solution: 40.0 g of NaOH was dissolved in 1000 cm3 of distilled water.

15. Phosphate Buffer: 0.04 M KH2PO4 was prepared and pH was adjusted to 7.4 by adding concentrated solution of K2HPO4. 16. 30 % KSCN Solution: 30.0 g of KSCN was dissolved in 100 cm3 of distilled water. 17. Linoleic Acid Emulsion: 350 microgram of tween-20 was mixed with 310 microgram of linoleic acid and volume was made up to 100 cm3 with buffer. 18. NBT Solution: 0.00815 g of NBT was dissolved in 50 cm3 of buffer. 19. NADH Solution: 0.0221 g of NADH was dissolved in 50 cm3 of buffer. 20. PMS Solution: 0.00245 g of PMS was dissolved in 10 cm3 of buffer.

3.9.2. TOTAL PHENOLIC CONTENT ASSAY The total phenolic content of in the five methanolic extracts were determined by the method of Singleton and Rossi (1965). Stock solution of Gallic acid was made by dissolving 0.5 g gallic acid in 10 mL of ethanol in a 100 mL volumetric flask and diluting to volume with doubly distilled water. Sodium carbonate solution was prepared by dissolving 200 g of anhydrous sodium carbonate in 800 mL of double distilled water. After boiling and subsequent cooling of the solution, a few crystals of sodium carbonate were added. The solution was allowed to stand for 24 hours, filtered and volume was raised to 1L with double distilled water. To prepare a calibration curve, 0\1\2\3\5 and 10 mL of phenol stock solution were added into 100 mL volumetric flask separately and then diluted to volume with double distilled water. The resultant solutions contained concentrations of 0\50\100\150\250 and 500 mg/L gallic acid. From each calibration solution and sample or blank, 40 µL were pipetted into separate cuvettes, and to each 3.16 mL of double distilled water was added. Folin– Ciocalteu reagent (200 µl) was added, and mixed well. After 8 minutes, 600 µl of sodium carbonate solution was mixed thoroughly in the solution. The solution was 80 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK allowed to stand at 40 oC for 30 min and absorbance of each solution was determined at 765 nm against the blank (without phenolic solution). A concentration versus absorbance linear plot was thus obtained. The concentration of total phenolic compounds of each taxa of S. nigrum Complex (as milligram of gallic acid equivalent (GAE)), as determined by using the following equation was obtained from the standard gallic acid plot.

Absorbance = 0.0128 × Gallic acid (mg/L)

3.9.3. LIPID PEROXIDATION ASSAY Total antioxidant activity of aqueous and organic extracts of all the plants was determined according to the lipid peroxidation assay employed by Mitsuda et al. (1996). The solution, which contained 100 L each of neat or diluted plant extract of all the plants in 2.5 mL of potassium phosphate buffer (0.04 M, pH 7.0) was added to 2.5 mL of linoleic acid emulsion in potassium phosphate buffer (0.04 M, pH 7.0). Each solution was then incubated at 37 oC in sealed bottles, in dark. The solution without added extract was used as blank, while the solutions containing 100 L (50 g/20L) of Trolox was used as positive control. At intervals of 24 hours during incubation, 0.1 mL of each solution was transferred to a beaker containing 3.7 mL of ethanol. After addition of 0.1 mL each of FeCl2 (20mM in 3.5% HCl) and thiocyanate solution (30%) to ethanolic sample, the solution was stirred for one minute. The absorption values of the solutions measured at 500 nm were taken as lipid peroxidation values.

3.9.4. FRAP ASSAY The reducing capacity of plant extract was measured according to the method of Benzie and Strain (1996). Freshly prepared FRAP solution contained; 25 mL of 300 mM acetate buffer (pH 3.6), 2.5 mL of 10 mM TPTZ solution in 40 mM HCl solution and 2.5mL of 20 mM ferric chloride solution. The mixture was incubated at 37°C throughout the monitoring period. 3 mL of FRAP reagent was mixed with 100µl of sample and 300µL of distilled water. Absorbance reading was taken at 593 nm after every minute for 6 minutes. Results were compared with standard curve of ferrous sulphate.

81 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

3.9.5. ABTS•+ ASSAY PROTOCOL ABTS•+ Assay protocol, as developed by Re et al., (1999) was followed for analysis of the methanolic extracts. ABTS was dissolved in double distilled water to a 7 mM concentration. ABTS radical cation (ABTS•+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand, in the dark, at room temperature for 12-16 hrs before use. For study of antioxidant activity of standard antioxidant and plant samples, the ABTS stock solution was diluted with PBS buffer (pH 7.4) to an absorbance of 0.70 (+0.02) at 745 nm and equilibrated at 30 oC. For plant extracts, dilutions were made in the respective organic solvents with which they had been extracted from aqueous solutions. After addition of 10 μL of neat or diluted stock solution (as necessary) to 2.99 mL of diluted ABTS•+ solution (A= 0.700 + 0.020), the absorbance was taken at 30 °C, with exactly 1 minute intervals for 8 minutes. Solvent blanks were run in each assay for accurate readings. All determinations were carried out at least three times in succession, and in triplicate at each separate concentration level of the standards. The percentage inhibition of absorbance was calculated by the following formula.

% inhibition (at 734 nm) = (1- Af /Ao) x 100

Where Ao is the absorbance of radical cation solution before addition of sample/standard antioxidants and Af is the absorbance after addition of the sample/standard antioxidants. The resultant data was plotted between concentration of antioxidants and that of Trolox for the standard reference curve.

3.9.6. DPPH RADICAL SCAVENGING CAPACITY ASSAY The DPPH radical scavenging effect was determined according the method described by Sanchez-Moreno et al., 1998. DPPH (Diphenyl-1-picrylhydrazyl) solution (3 mL, 25 mg/L) in methanol was mixed with appropriate volumes of neat or diluted sample solutions. The reaction progress of the mixture was monitored at

517 nm over a time period until TEC50 was obtained. Upon appropriate reduction, the color of the solution faded. The percentage of the DPPH remaining was calculated as

%DPPHrem = [DPPH] t=t/[DPPH]t=0 x 100 82 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.3: EXPERIMENTAL WORK

Where [DPPH]t=0 is the concentration of DPPH radical before reaction with antioxidant samples and [DPPH]t=t is the concentration of DPPH radical after reaction with antioxidant sample at time t. A kinetic curve showing the scavenging of DPPH radical in terms of decrease in absorbance at 517 nm as a function of time (min) was plotted for each fraction of the samples. The concentration that causes a decrease in the initial DPPH concentration by 50% (EC50), and the time needed to reach the steady state with EC50 concentration (TEC50) were measured from the kinetic curve as given in Table 18.

3.9.7. METAL CHELATING ACTIVITY Ferrous ion (Fe++) chelation by plant extracts was estimated by the Ferrozine assay (Dinis et al., 1994), 100 µL of plant extract was added to a solution of 2 mM

FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and total volume was adjusted to 4 mL ethanol. Then, the mixture was shaken vigorously and left standing at room temperature for ten minutes. After the mixture had reached equilibrium, the absorbance of the solution was measured spectrophotometrically at 562 nm. The results were expressed as percentage of inhibition of ferrozine-Fe2+complex formation. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the formula given below:

2+ Bound Fe (%) = [(AControl-ASample)/AControl] x 100

Where AControl is the absorbance of the control, and ASample is the absorbance in the presence of the sample of plants (Elmastas et al., 2005).

83 Chemotaxonomical Characterization of Solanum nigrum and its Varieties

Chap.4: RESULTS AND DISCUSSION

RESULTS & DISCUSSION

4.1. OVERVIEW OF THE WORK The current study involves the chemotaxonomic studies on the locally available taxa of S. nigrum Complex. The comparative work on these taxa covers the following areas:

1. Phytochemical examination. 2. Alkaloids. 3. Flavonoids. 4. Epicuticular waxes. 5. Proximate and mineral analyses. 6. Antibacterial study. 7. Antioxidant study.

4.2. SPECIES DELIMITATION The plant family Solanaceae - the nightshade or potato family is one of the most familiar flowering plant families which are commercially, medicinally and economically important. It shows incredible morphological and chemical diversity and worldwide distribution. The ancient classification of the plants was predominantly based on comparative morphological and anatomical characters. However with the progress in taxonomy; structure/configuration and chemical constituents of the plants became keys to classify different plant varieties. Taxonomy of some morphologically different taxa of S. nigrum (now known as S. nigrum Complex) had been controversial among the international renowned taxonomists while considering their morphological characters. The difficulty of distinguishing genetically-controlled characteristics from phenotypic plasticity had long been known to impede species level taxonomy in Solanum (Edmonds and Chweya, 1997).

84 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Chemical analysis of the work carried out on different morphological variants of S. nigrum in past was either lacking the true identification or was controversial among the various available reports. Classification of the taxa with black and that with orange berries was particularly uncertain. Linnaeus suggested them as varieties of the species S. nigrum whereas Lamarck and Miller differentiated them as distinct species of genus Solanum. Similar controversy existed among these famous taxonomists for S. americanum, S. Chenopodioides and S. retroflexum. (Yasin, 1985; Edmonds and Chweya, 1997). That was why some researchers reported them as separate species (e.g. Xu et al., 2001; Al Chami et al., 2003; Dehmer and Hammer, 2004) while other regarded them as varieties (Campos et al., 2002).

As described in literature review, secondary metabolites like Alkaloids, Flavonoids and Epicuticular waxes are primarily useful in assessing relationship among closely related species and they can be used for solving taxonomic problems. Thus chemotaxonomic studies on following five locally available taxa of S. nigrum Complex were carried out.

 Solanum americanum  Solanum chenopodioides  Solanum nigrum  Solanum retroflexum  Solanum villosum

Morphological characters of these five taxa are compared in Table 3.

85 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 3: Morphological comparison of five taxa of Solanum nigrum Complex

Species (Code)a Character SA SC SN SR SV Leaf shape Ovate- Lanceolate Ovate- Rhomboidal Rhombic to lanceolate to lanceolate, to ovate- ovate- lanceolate ovate- lanceolate lanceolate rhombic Leaf margin Entire to Entire to Sinuate or Deeply Entire to sinuate sinuate irregularly lobed, 3- sinuate- rarely dentate 5(7)b dentate sinuate- dentate Leaf lengthc 2.5-3.2(4.0) 2.7-5.4 2.5-7 4-4.8 2.0-7.0(10) Leaf breadth 1.1-4.0(4.6) (1.8)2.2-5.4 2.0-4.5 3-4.2 1.5-4.0(6.0) lnflorescence Umbellate Umbellate Extended Simple to Umbellate to type cymes cymes cymes Umbellate solitary cymes cymes Calyx lengthd 1.1-2.0(2.4) 2.4-3.5 1.2-2.5 2-3 1.2-2.2 Corolla color White, White White White with White occasionally purple purple stripes on each petal Style lengthd 1.2-3.5(4.5) 4.0-5.5(6.5) 2.8-3.5(4.5) 2-4.5 2.9-5.0(6.0) Stylar exsertion Usually Exserted up Not exserted Exserted up Rarely exserted to 2 mm beyond to 2 mm exserted beyond beyond anthers beyond beyond anthers anthers anthers anthers Berry diameterd 4-7(8) 6.3-8.5 6-10 7-9 6-10 Berry shape Globose Globose to Broadly Spherical Occasionally broadly ovoid globose ovoid Berry color Black, rarely Purple Dull purple Purple Red, orange dark green Cuticle Shiny Dull opaque Shiny Dull opaque Translucent opaque opaque Seed lengthd 0.8-l.5 1.0-1.8 1.7-2.4 1.5-2.3 1.6-2.2 a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum. b Figures in parentheses refer to infrequent values below or above the regular range. c Values are given in cm d Values are given in mm

86 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.3. PHYTOCHEMICAL EXAMINATION OF PLANT MATERIAL: The preliminary phytochemical screening for the secondary metabolites was carried out. The presence of alkaloids, flavonoids, steroids, terpenoids, tannins and saponins was investigated. All these secondary metabolites were indicated in the five taxa which was confirmatory of previous literature.

4.4. ANALYSIS OF ALKALOIDS The Solanum glycoalkaloids have been intensively studied during recent decades and as a result of substantial research efforts, thousands of articles concerning various aspects of glycoalkaloids have been published. S. nigrum is especially known for its toxicity because it contains solanine, a neurotoxic glycoalkaloid (Abbas, 1998). The development of new glycoalkaloid analysis techniques is still continuing although hundreds of research articles, based on analytical methods, have already been published. HPLC is the most commonly used application and its advantage is that both entire glycosides and also aglycones can be analysed. However, if the glycoalkaloid profile of material is unknown or novel compounds are expected, the techniques coupled with MS are relevant, such as LC- MS or GC-MS. The most important advantage of GC analysis is its sensitivity and good separation of aglycone mixtures. Alkaloids are said to be excellent taxonomic markers by a number of researchers (e.g. Izaddoost, 1975; Tetenyi, 1987; Greinwald et al., 1995; Jamil et al., 2007; Dinchev et al., 2008; Loaiza et al., 2008). Actually, as suggested by Tetenyi (1987), alkaloids were helpful for the classification of the family Solanaceae on the basis of the metabolism of alkaloids. GC-MS technique is cited (Suau et al., 2002) for the easy determination and identification of alkaloids and the application of the technique to chemotaxonomic studies. In the present study, variation was investigated in qualitative and quantitative glycoalkaloid aglycone contents in interspecific Solanum hybrids of various genome constitutions.

4.4.1. ESTIMATION OF TOTAL GLYCOALKALOID (TGA) CONTENT The TGA after extraction and purification can be determined by various methods like titrimetric, gravimetric, colorimetric and precipitation. The titrimetric method was selected for present study because it has been successfully used for S. nigrum L. by several researchers. From the results, it is evident that the percentage

87 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

yield of TGA was highest in S. villosum (68.9±0.6%) but with a little difference with S. chenopodioides (68.6±0.3%). Also good yields of TGA were observed for S. nigrum (55.7±0.3%) and S. retroflexum (53.3±0.6%). This indicated that these plants, particularly their leaves and fruits, can be an important raw material for the commercial preparation of steroidal drugs. But their unchecked use by the local community as medicine or food can produce adverse effects as the steroidal alkaloids are proved to be toxic if taken in more than a specified concentration. On the contrary the yield of TGA in S. americanum was comparatively much lower (25±0.8%).

4.4.2. TLC Analysis There had been many reports on the compositional analysis of alkaloids of S. nigrum by TLC. Three of these reported methods were selected (Boll, 1962; Boll and Anderson, 1962; Harborne 1974) and a preliminary comparison of the five taxa was carried out. Various steroidal glycoalkaloids (SGA) were detected by TLC of the crude alkaloids (Table 4) with some minor differences in their occurrence. β- Solamargine and α-Solamargine were not detected in S. americanum and S. chenopodioides, respectively. The 5-α-solasodan3-β-ol was found to be accumulated in S. nigrum and S. retroflexum only who showed similar type of SGA profile.

Table 4: TLC of alkaloids of five taxa of S. nigrum Complex

a R Species (Code) No Compound f Colour value SA SC SN SR SV 1 α-Solanine 0.46b Purple + + + + + 2 Solanigridine 0.1c Brown + + + + + 3 Solasonine (-Solanigrine) 0.24c Brown + + + + + 4 α-Solamargine 0.39c Brown + – + + + 5 β-Solamargine (-Solanigrine) 0.55c Brown – + + + + 6 Solasodine 0.23d Red + + + + + 7 5-α-solasodan3-β-ol 0.21d Grey red – – + + – a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum. b Rf in acetic acid:ethanol (1:3), spray reagent: Marquis reagent c Rf in chloroform:methanol:1% aq. NH4OH (2:2:1), spray reagent: Dragendorff reagent d Rf in chloroform:methanol (95:5), spray reagent: SbCl3 reagent

88 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

The SGA detected fall into two aglycon groups due to their different basic structures (Fig. 10):

1. Solanidine 2. Solasodine

Fig. 10: The structures of solanidine- and solasodine-based glycoalkaloids.

89 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.4.3. HPLC ANALYSIS All the five taxa showed much similar SGA profile on HPLC. This is due to the fact that these taxa belong to the genus Solanum which is very well-known for the presence of SGA. So to make a chemotaxonomic comparison the quantitative analysis was required. The glycoalkaloids are particularly difficult to separate due to their similarity in structure. α- and β-Solamargine have identical sugar constituents, but different attachment pattern with aglycones, i.e. solasodine and solanidine, respectively. Similarly, solasonine and solanine contain the same sugar moieties, but have the solasodine and solanidine aglycone backbones, respectively. There had been many reports on the SGA of different species of genus Solanum but not a single one on S. nigrum. Therefore, different reported conditions that can affect selectivity of SGA of Solanum appreciably were applied. The best results were obtained by the method of Sotelo and Serrano (2000) so it is discussed here. Based on the relative areas obtained in the chromatograms, greater signal intensities were seen for standard analytes at 205 nm so it was selected for analysis. Solasonine, α-Solamargine, β-Solamargine and α-Solanine are the SGA of interest to our studies. The SGA were, therefore, further analysed qualitatively and quantitatively by the HPLC of the alkaloids extracted using the standard compounds. Four of the peaks were tentatively identified on the basis of retention time and peak response when standards were added (one by one) in each sample. The Solasonine, α- Solamargine, β-Solamargine and α-Solanine contents of each taxon is given in Table 5.

Table 5: Concentration of SGA in five taxa of S. nigrum Complex by HPLC Concentration in species (Code)a No Compound (mg/g) SA SC SN SR SV 1 β-Solamargine ndc 2.53 1.69 4.78 9.8 2 α-Solamargine 1.96 nd 5.03 1.45 1.17 3 Solasonine 3.5 4.4 5.8 2.9 2.01 4 α-Solanine 3.29 3.08 4.7 5.7 1.5 a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum. c nd: not detected.

90 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

According to our study β-Solamargine levels varied among different taxa of S. nigrum Complex (1.69-9.8 mg g–1). Its concentration in S. villosum was distinctly higher than other taxa specially in contrast to S. americanum in which it was not detected. The α-Solamargine was detected at lower levels than the β-Solamargine. Infact it had not been detected in S. chenopodioides but its level in S. nigrum was slightly higher than other samples. Solasonine and α-Solanine were detected in all taxa with less concentration variations. The chemical profile for each subfamily, as expressed by occurrence of the major categories of secondary metabolites (indole alkaloids, iridoids, triterpenes and anthraquinones) is remarkably distinctive (Young et al., 1996). So far, secondary metabolites profile can contribute to the taxonomic position of some tribes, which remain with a morphological controversy (Cardoso et al., 2008). SGA determined in this study corroborated the evolutionary taxonomic distribution made by Miller, Dunal and Lamarck, who recommended these taxa as distinct species contrary to that proposed by Linnaeus , who classified these as the varieties of S. nigrum. Statistical comparison (Fig. 11) of the taxa by Minitab 3.2 Statistical Software segregated S. americanum and S. chenopodioides more early than others but with a less similarity index. Then S. nigrum and S. retroflexum join this group at almost similar position. But S. villosum was unique and depicted very low similarity with rest of the taxa.

S. S S. retroflexum S. nigrum S. villosum

. chenopodioides

americanum

Fig. 11: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of SGA analysed by HPLC and determined by similarity and Multivarial cluster analysis. 91 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.4.4. GC-MS ANALYSIS Volatility and thermal stability of the compounds are desirable in gas chromatographic analysis. Luckily, by modifying the functionality of a molecule (e.g., -OH, COOH, =NH, -NH2, -SH, and other functional groups) with derivatizing reagents, it is possible to analyze compounds that otherwise are not readily monitored using GC. In some cases the mass spectrum of the underivatized molecule exhibits poor diagnostic ions. After derivatization, the fragmentation pattern may change so that structural elucidation is more clear. A large number of reagents are used to prepare derivatives for gas chromatography, but most of the derivatization reactions belong to one of three categories: acylation, alkylation, or silylation. Of these, silylation is the most widely used for GC-MS. Silyl derivatives are formed when active proton displacement (in –OH, -SH or –NH groups) by an alkylsilylgroup occurs. Trimethysilylation is the most common silylation procedure. Gas chromatography has been applied for the determination of the aglycones of steroidal glycoalkaloids in potato materials. Using GC with a nitrogen-specific detector (Holstege et al., 1995) or FID detector (Herb et al., 1975; Lawson et al. 1992), several aglycones can be separated and quantified in a single run. The aglycones can be analyzed without derivatization (Van Gelder et al., 1989), but it has been shown that high temperatures (>280 oC) can lead to aglycon decomposition and shorten the column life (Lawson et al., 1992). The steroidal glycoalkaloid aglycones (SGAA) important to our work were solanidine and solasodine. The lack of GC-MS reports, on the alkaloids of S. nigrum Complex prompted to quantify the aglycones by this useful technique. Solanidine produced mono-TMS derivatives with molecular ion peaks at m/z 469. In its MS spectrum, solasodine showed the di-TMS derivative after silylation with base peak at m/z 125 and at m/z 559 [M++2H+]. According to the literature the tetrahydrofuran ring opens, after which the formed hydroxyl group had been attached to the TMS group. Moreover, it has been stated that such a phenomenon can be related to the presence of the nitrogen ring, for example the silylation of diosgenin containing oxygen instead of nitrogen gave a mono-TMS derivative only (Laurila et al., 1999). Quantification of aglycones was carried out using an external standard calibration method. The principal glycoalkaloid present in all taxa was solasodine with a percentage range of 66.94-85.67%. Solanidine concentration was much lower ranging from 8.85-20.31%. Solanidine is reported to be toxic so care must be taken 92 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION in using S. americanum in herbal medicine and as food. Calibration was performed by injecting standard mixtures of solasodine and solanidine at levels ranging from 4 to 200 mg/L. Good linearity of response was found for solanidine and solasodine in this concentration range belonging to cited interval, with correlation coefficients greater than 0.995.

Table 6: SGAA concentration in five taxa of S. nigrum Complex by GC-MS Retention timea Percentage of aglyconesb Sample (min) (%) Solanidine Solasodine Solanidine Solasodine S. americanum 21.613 26.645 8.85 85.67 S. chenopodioides 25.773 26.645 11.08 66.94 S. nigrum 25.779 29.843 16.03 75.90 S. retroflexum 24.918 26.662 20.31 71.76 S. villosum 25.337 28.672 10.15 74.70 a Results were presented as mean (n=3). b Determined by area normalization method.

Cluster analysis (Fig. 12) separated the taxa into three main groups. S. nigrum and S. retroflexum formed a much closely related group. S. chenopodioides and S. villosum constitute another group but with slight less similarity index. However S. americanum showed a characteristic behavior of its own with highest percentage of solasodine and lowest of solanidine. So it aligned distantly with the above two groups.

Fig. 12: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of SGAA analysed by GC-MS and determined by similarity and Multivarial cluster analysis. 93 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.5. ANALYSIS OF FLAVONOIDS Flavonoids are widely occurring polyphenolic compounds and are extremely important because of their medicinal effects (Bernardi et al., 2007). Flavonoids from different species of Solanum have been reported and reviewed (da Silva et al., 2003). Quercetin and its derivative glycosides make up most of the flavonoid content in S. nigrum (Nawwar et al., 1989). But the reports mostly do not take into account the morphological taxonomic complication associated with S. nigrum Complex and hence cause uncertainty. Flavonoids are useful secondary metabolites in assessing the relationship among closely related species or in studies of intraspecific variation, and they are also occasionally useful in assessing phylogenetic relationships at higher levels (Bate-Smith, 1968; Gornall et al., 1979; Harborne and Turner, 1984). The flavonoids have systematic significance and can be used for solving taxonomic problems (Mimura et al., 2004; Citoglu et al., 2005). So to search out the boundaries between these five taxa using the chemotaxonomy to help resolve the International morphological taxonomic controversy on S. nigrum Complex we aimed to study their flavonoid profiles by comparing the total flavonoid contents, quantitative comparison of glycosides by HPLC and quercetin aglycon by GC-MS along with qualitative study of their glycosides using TLC.

4.5.1. COLORIMETRIC ANALYSIS It is old conventional method but is still used because it is easy to work. The flavonoid content was determined by two independent colorimetric methods.

(I) AlCl3 Colorimetric method: Aluminium chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition it also forms acid labile complexes with the ortho-dihydroxyl groups in the A- or B-ring of flavonoids. Complexes were scanned at different wavelengths Ortho-dihydroxyl groups had maximum absorbance at 415- 440nm, C-4 keto group at 385nm, C-5 hydroxyl group and Ortho-dihydroxyl groups in B ring at 415nm. In compromise, therefore, the wavelength 415nm was chosen for absorbance measurement. However absorbance of complexes formed by flavanones such as naringin at 415 nm was too low to make the meaningful contribution to total absorbance. So these were estimated by 2,4-dinitrophenylhydrazine method. 94 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Quercetin is reported to be suitable for building the calibration curve (Chang et al., 2002; Woisky and Salatino, 1998). Therefore standard Quercetin solutions ranging from 50 to 150 µg/ml concentrations were used to build up the calibration curve. The coefficient of determination (R2) was 0.953. Total amount of flavones, flavonols and isoflavones was calculated from the curve (Table 7). (II) 2,4-dinitrophenylhydrazine for determination of flavanones: The principle of this method is that 2,4-dinitrophenylhydrazine reacts with ketones and aldehydes to form 2,4-dinitrophenylhydrazones. It was found that flavones, flavonols and isoflavones with the C2-C3 double bond could not react with 2,4- dinitrophenylhydrazine while the hydrazones of flavanones showed maximum absorbance at 495 nm, and so this wavelength was selected for all measurements in the 2,4-dinitrophenylhydrazine reaction. Flavanone naringin, which was reported to show maximum absorbance at the above selected wavelength (Chang et al., 2002), was used to make the calibration curve. R2 value was 0.96. Total flavonoid content: Total flavonoid contents were represented as sum of two individual colorimetric methods. In fact, the amounts of total flavonoid content determined by HPLC can be greatly influenced by the selected authentic standards. Sometimes, limited by the availability of authentic standards, the identification of flavonoid peaks in chromatograms may be incomplete. Therefore to stay away from preconception, we conducted the quantitative determination of flavonoid contents in S. nigrum Complex by colorimetric analysis. Results showed that flavonoid contents of five taxa as determined by aluminum chloride method was much higher than those determined by 2,4-dinitrophenylhydrazine method (Table 7). The former ranged from 0.766 ± 0.012% to 1.616 ± 0.031% while the later ranged from 0.062 ± 0.005% to 0.5 ± 0.001%. As suggested by Chang et al. (2002), the flavones, flavonols and isoflavones formed complexes only with aluminum chloride, while flavanones strongly reacted only with 2,4- dinitrophenylhydrazine, the contents determined by the two methods were added up to evaluate the total content of flavonoids. Results indicated that, among the five taxa investigated, S. chenopodioides contained the lowest level of total flavonoids (0.883 ± 0.020%), while S. nigrum showed the highest level of total flavonoids (2.116 ± 0.032%) (Table 7). Overall, there was great variation in total flavonoid contents of the investigated taxa of S. nigrum Complex indicating that the quality of its medicinal use does require specification of the taxa taken. 95 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 7: Flavonoid contents of S. nigrum Complex by Colorimetric methods Flavonoid content (%)a Sample b c AlCl3 2,4-DNPH Total S. americanum 1.229 ± 0.024 0.438 ± 0.010 1.667 ± 0.034 S. chenopodioides 0.821 ± 0.015 0.062 ± 0.005 0.883 ± 0.020 S. nigrum 1.616 ± 0.031 0.500 ± 0.001 2.116 ± 0.032 S. retroflexum 0.954 ± 0.014 0.250 ± 0.001 1.204 ± 0.015 S. villosum 0.766 ± 0.012 0.125 ± 0.002 0.891 ± 0.014 a Results were presented as mean (n=3). b Flavonoid content (%) = quercetin equivalent (μg/mL) × total volume of ethanol extract (mL) ÷ sample weight (g) × 10-6 (g/μg) × 100. c Flavonoid content (%) = naringin equivalent (μg/mL) × total volume of ethanol extract (mL) ÷ sample weight (g) × 10-6 (g/μg) × 100.

4.5.2. TLC ANALYSIS TLC is a simple and reliable technique to compare the flavonoid profiles of different taxa and hence can be used to as an aid of their chemotaxonomy (Kharazian and Rahiminejad, 2008). Different solvent systems according to the reported literature were tried. However the best systems are discussed here. Various simple and complex glycosides of quercetin had been reported from S. nigrum Complex (Schilling, 1984; Nawwar et al., 1989). TLC of the extracts of the five taxa under study displayed variability in their flavonoid pattern (Table 8).

Two spots were identified on the basis of their Rf values, color in UV light (254 nm) and UV light/ + ammonia (Harborne, 1974). Quercetin-3-glucoside

(isoquercitrin) with Rf values 0.58 (BAW) and 0.37 (15% acetic acid) was detected in all the five taxa. But quercetin-3-galactoside having Rf values 0.55 (BAW) and 0.35 (15% acetic acid) was detected only in S. nigrum and S. retroflexum. Color of both spots was yellowish brown in UV light (254 nm) but turned to bright yellow when examined in the same light after treating with vapors of ammonia. Since these glycosides had been previously identified in different taxa of S. nigrum Complex (Nawwar et al., 1984) and they are easy to detect by TLC, therefore, their presence in the samples can be indicated by comparing the observed properties with those reported in literature but other spots could not be characterized.

96 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 8: TLC of flavonoids of five taxa of S. nigrum Complex R value Plant Taxa No. of spot f BAWa 15% AcOH 1 0.93 0.80 S. americanum 2 0.58 0.37

3 0.15 0.05 1 0.93 0.90 2 0.83 0.62 S. chenopodioides 3 0.58 0.37

4 0.36 0.12 5 0.13 0.02 1 0.93 0.80 2 0.83 0.62 S. nigrum 3 0.58 0.37 4 0.55 0.35 5 0.36 0.12 6 0.15 0.05 1 0.93 0.80 2 0.83 0.62 S. retroflexum 3 0.58 0.37

4 0.55 0.35 5 0.15 0.05 1 0.93 0.80 S. villosum 2 0.83 0.62 3 0.58 0.37 4 0.13 0.02 a BAW: n-butanol-acetic acid-water, 4:1:5, upper layer.

4.5.3. HPLC ANALYSIS HPLC of the flavonoid glycosides was performed using two different gradient systems. Gradient I: Detection of quercetin glycosides was carried out but the response of two standards was not linear. Therefore this gradient could not be used for quantification of these glycosides. Gradient II: This gradient system was used for the quantification of flavonoids from the plant extracts. A calibration curve was plotted using peak areas against five different concentration levels (2.0-20 mg/20µL) of standards (quercetin-3-glucoside and quercetin-3-galactoside) and the concentration of flavonoid glycosides was determined (Table 9). The highest concentration of Quercetin-3-glucoside was

97 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION observed in S. americanum and the lowest in S. retroflexum. Level of Quercetin-3- galactoside was highest in S. americanum and lowest in S. chenopodioides.

Table 9: Concentration of Flavonoid Glycosides in S. nigrum Complex by HPLC Retention timea Concentrationb (min) (mg/100mL) Sample Quercetin-3- Quercetin-3- Quercetin-3- Quercetin-3- glucoside galactoside glucoside galactoside S. americanum 15.347 16.535 0.03520 0.00750 S. chenopodioides 15.089 16.992 0.00725 0.00051 S. nigrum 15.346 16.524 0.00550 0.00252 S. retroflexum 15.265 16.817 0.00450 0.00256 S. villosum 15.270 16.480 0.00957 0.00063 a Results were presented as mean (n=3). b Determined from the calibration curves of standards.

Concentrations of these two quercetin glycosides helped comparing the taxa statistically. S. nigrum and S. retroflexum resembled much closely and were first to segregate as a cluster. This is because of resemblance in their glycosides concentrations. But S. villosum and S. chenopodioides formed another cluster having slightly lower similarity index as of the previously discussed cluster. These two clusters were distantly related to S. americanum which showed relatively higher concentration of the quercetin glycosides under study (Fig. 13).

Fig. 13: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of flavonoid glycosides analysed by HPLC and determined by multivariate cluster analysis. 98 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.5.4. GC-MS ANALYSIS GC-MS has been used in a number of occasions for the analysis of flavonoids in plant. For example, in one study (Tokusoglu et al., 2003), it was used to characterize the flavonol aglycones in tomatoes (Solanum lycopersicum L.). In another study, the flavonoid aglycones isolated from Propolis were identified by GC-MS (Maciejewicz et al., 2001). Also GC-MS was claimed to be useful in chemosystematics helping, for example, to characterize species on the basis of their cuticular wax (Szafranek and Synak, 2006; Urzua and Mendoza, 2008). We could not find any report on the analysis of flavonoids of S. nigrum Complex by GC-MS and use of flavonoids in its chemotaxonomy. Quercetin in its different glycosidic forms has been the only flavonol reported from S. nigrum Complex (Nawwar et al., 1984). The presence of quercetin aglycone in the hydrolyzed extracts of the five taxa under study was confirmed by GC-MS. Retention time of quercetin standard was 13.012 ± 0.001 min. It showed molecular ion peak of m/z (relative intensity in %) at 304 (22.8), base peak at 153 (100) with other characteristics peaks at 286 (4.0), 275 (27.6), 195 (2.8), 165 (12.4), 152 (24.4), 150 (21.2) and 123 (42.4). Quercetin in the samples was detected by spiking the samples with 0.5 µg/mL of standard quercetin solution and identified on the basis of comparison with the retention time and mass fragmentation pattern of the standard, and of the mass spectrum with the data from NIST 147 and 27 Libraries linked to the mass detector. The extreme variation in its percentage composition in the extracts from the taxa grown, harvested and analyzed under similar conditions decides its part in their chemotaxonomy (Table 10). Comparing the area percentage, quercetin made up only 7.28% of the hydrolyzed flavonoid extract of S. nigrum and this continued to increase irregularly while moving to S. retroflexum, S. villosum and S. chenopodioides. But S. americanum stands alone in the group with such a high percentage (92.92%). The calibration curve of standard was linear with R2 value of 0.99 in the concentration range of 0.1-2.0 µg/mL. Amount of quercetin varied from 3.06 ± 0.01 to 6.46 ± 0.01 mg/100g of plant (Table 10). Yang et al. (2008), reported the quercetin content of S. nigrum and S. villosum. Our results were in agreement with this study for S. nigrum (3.7 mg/100g) but there was contradiction for S. villosum (18.1 mg/100g). However, no reports could be found for quantitative studies of quercetin on other three taxa under study.

99 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 10: Quercetin concentration in five taxa of S. nigrum Complex by GC-MS Rta Quercetin concentration Sample (min) %age in extractb mg/100g of plantc S. americanum 13.019 92.92 ± 0.45 6.46 ± 0.01 S. chenopodioides 13.008 38.31 ± 0.23 3.81 ± 0.02 S. nigrum 13.011 7.28 ± 0.33 3.06 ± 0.01 S. retroflexum 13.013 13.03 ± 0.38 3.18 ± 0.01 S. villosum 13.012 23.94 ± 0.28 4.91 ± 0.01 a Results were presented as mean (n=3). b Determined by area normalization method. c Calculated from the calibration curve.

The percentage variation of the flavonoids observed in the five taxa by colorimetric and GC-MS analyses were used to group them statistically. Here again S. nigrum and S. retroflexum formed a closely related cluster with a high similarity index showing much similarity in their flavonoid profiles. S. chenopodioides and S. villosum, although not so closely related to one another as compared to the previously discussed cluster, made another cluster. S. americanum aligned more distantly with above mentioned clusters. This is due to its very high quercetin concentration (shown by GC-MS analysis) and very low flavanone percentage (in 2,4-dinitrophenylhydrazine method) as compared to other taxa (Fig. 14).

Fig. 14: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of flavonoid aglycones analysed by GC-MS and determined by multivariate cluster analysis. 100 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.6. ANALYSIS OF EPICUTICULAR WAXES Epicuticular wax from each of the five taxa was extracted using n-hexane. The procedure used extracts only epicuticular wax without disturbing the internal chemical makeup (Medina et al., 2006). The wax yield of the plant samples varied from 0.022% to 0.97% (Table 11). The epicuticular waxes had often been considered as a potential character for chemotaxonomy of the plants in general (Evans et al., 1975; Griffiths et al., 1999; Tsydendambaev et al., 2004; Ercisli et al., 2009). Hanna et al. (1996), although, had reported the presence of some fatty acids such as Palmitic, Stearic, Linolenic acids and Squalene without specifying the taxon of S. nigrum Complex, yet no detailed chemotaxonomic study based upon chemical constituents of epicuticular waxes of the complex has been undertaken. The objectives of the present study were: a) to study the composition of epicuticular wax of five locally available taxa of S. nigrum Complex; and b) to mark the boundaries among these five taxa using the chemotaxonomic data to help resolve the International taxonomic controversy regarding S. nigrum Complex.

4.6.1. PHYSICOCHEMICAL ANALYSIS OF WAX The physicochemical analysis was carried out and the results had been reported in Table 11. The melting point of wax was not sharp and taken as the average of three readings. The saponification value of S. nigrum was higher than other taxa which indicated high concentration of saturated fatty acids, however it was close to that of S. retroflexum. Saponification value is related to molecular weight of wax and, therefore, provides information of the mean molecular weight of combined fatty acids. The shorter the carbon chain length of an acid molecule, the lesser will be its molecular weight and hence there will be greater number of molecules in one gram of the sample. Thus it will require greater number of KOH molecules, as saponification value is the number of milligram of KOH required to saponify one gram of wax. Acid value determines the amount of free fatty acids present in wax. Low acid values of waxes indicated low concentration of free fatty acid with the exception of S. villosum. High ester value indicates that wax is natural because waxes are the esters of fatty acids with monohydric fatty alcohols. Wax of S. nigrum showed high ester value than other plant waxes analysed.

101 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 11: Physicochemical analysis of the waxes of S. nigrum Complex Species (Code)a Parameter SA SC SN SR SV Wax Yield (%) 0.022 0.42 0.076 0.29 0.97 Dark Dark Light Yellow Colour Green Green Green Green Green Melting point (oC) 71 78 82 80 75 Refractive index 1.131 1.038 1.522 1.513 1.046 Saponification value 712.47 742.19 830.28 823.58 985.4 Acid value 67.32 72.1 100.98 103.22 613.18 Ester value 718.08 493.18 729.3 724.34 499.29 a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum.

4.6.2. TLC ANALYSIS TLC of wax was performed in four solvent systems recommended for preliminary examination of epicuticular waxes (Purdy and Truter, 1963; Holloway and Brown, 1977). In benzene (100%), four spots were given by extracts of S villosum, three spots were given by S. chenopodioides. Whereas S. americanum, S. retroflexum and S. nigrum all showed two spots each. In each variety separation of components was good. Rf value (0.25) indicated presence of Ketol in S. americanum.

Presence of ester (Rf: 0.53) and primary alcohol (Rf: 0.10) in all taxa, excluding S. americanum for alcohols, were indicated. Five spots were given by S. americanum when n-Hexane (100%) was used. Whereas, each of S. villosum and S. nigrum showed four spots. Both S. chenopodioides and S. retroflexum showed two spots.

Presence of ester (Rf: 0.53) was indicated in all taxa. With n-Hexane and Diethyl ether (4:1), five spots were given by S. americanum, while S. villosum and S. nigrum showed three spots. S. chenopodioides and S. retroflexum each showed two spots Rf values shows presence of terpinol (Rf: 0.14) in four taxa (S. americanum was exception). Separation of components was good in these three systems and spots of different colours were observed.

102 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

However separation of components was not good when the mixture of Chloroform and Ethanol (98:2) was used as solvent, so this system was not used for concluding any result.

4.6.3. GC-MS ANALYSIS: Different classes of compounds were found by applying GC-MS analysis (Table 12). Waxes had consistently been found to contain hydrocarbons, alcohols, fatty acids, esters, aldehydes and ketones (Tulloch and Hoffman, 1971; Bianchi et al., 1979; Reynhardt and Riederer, 1994; Koch et al., 2005).

Table 12: Wax yield and the distributiona of different classes of compounds Percentage in species (Code)b Parameter SA SC SN SR SV Alkanes - 42.19 15.48 23.58 15.06 Chloroalkanes 1.45 - - - - Aromatic hydrocarbons 2.07 - - - - Alkenes 24.86 1.76 13.52 12.1 0.83 Alcohols - 19.24 7.04 12.85 2.72 Free fatty acids 1.18 6.1 4.24 5.15 67.37 Esters 53.91 30.7 59.72 46.34 11.25 Chloroesters 2.45 - - - - Aldehydes - - - - 2.78 Ketones 14.07 - - - - a Expressed as relative apparent percentages: combined areas of peaks for compounds of given class ÷ combined areas of all identified peaks for all classes ×100. b Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum.

In addition to the four compounds which had been reported previously (Hanna et al., 1996), 30 more compounds were detected in quantifiable amounts in the five plant species analysed (Table 13). To identify possible chemotaxonomic features, the results obtained were compared with respect to morphology and taxonomy of the taxa.

103 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Table 13: Composition of Epicuticular wax extracted from S. nigrum Complex Sr. M+, Base Percentage in species (Code)a Compound No peak SA SC SN SR SV 1 n-Tetradecane [14C]b 198, 57 - 2.45 trc - 0.6 2 n-Pentadecane [15C] 212, 57 - 3.43 tr tr 1.03 3 n-Hexadecane [16C] 226, 57 - 4.13 tr 1.14 1.19 4 n-Heptadecane [17C] 240, 57 - 4.45 - 2.41 1.43 5 n-Octadecane [18C] 254, 57 - 3.89 tr 2.96 1.44 6 n-Nonadecane [19C] 268, 57 - 3.51 0.95 2.83 1.41 7 n-Eicosane [20C] 282, 57 - 3.24 1.73 3.13 1.23 8 n-Heneicosane [21C] 296, 57 - 2.93 1.63 2.46 1.27 9 n-Tricosane [23C] 324, 57 - 4.05 1.09 1.4 0.63 10 n-Tetracosane [24C] 338, 57 - 2.48 1.96 2.55 0.9 11 n-Pentacosane [25C] 352, 57 - 1.77 1.99 1.13 0.89 12 n-Heptacosane [27C] 380, 57 - 3.81 4.29 1.58 2.22 13 n-Octacosane [28C] 394, 57 - 2.05 1.84 1.99 0.82 14 1-Chloroheneicosane 340, 57 tr - - - - 15 1-Chloropentacosane 386, 57 tr - - - - 16 1-Chloroheptacosane 414, 57 1.45 - - - - 17 Octadecyl chloroacetate 346, 57 2.45 - - - - 18 2,6-Diisopropylnaphthalene 212, 197 2.07 - - - - 19 10-methyl-1-octadecene 266, 57 - - - 1.23 - 20 Squalene 410, 69 24.86 1.76 13.52 10.87 0.83 21 n-Cetyl alcohol 242, 55 - - - 1.01 - 22 9-Eicosen-1-ol 296, 55 - - - 1.08 1.02 23 3,13-Octadecadiene-1-ol 308, 43 - - - - 0.75 24 Phytol 296, 71 - 19.24 7.04 10.76 0.95 25 7,10,13-Hexadecatrienal 234, 79 - - - - 2.78 26 3-Hydroxyspirost-8-en-11-one 428,314 14.03 - - - - 27 Palmitic acid 256, 43 1.18 4.68 2.94 4.2 24.04 28 Stearic acid 284, 43 - 1.42 1.3 0.95 3.84 29 α-Linolenic acid 278, 41 - - tr - 19.81 30 -Linolenic acid r 306, 41 - - - - 19.68 31 Palmitoleic acid methyl ester 236, 55 53.91 30.7 59.72 46.34 9.15 32 Palmitic acid, methyl ester 270, 74 - - - - 0.63 33 Palmitic acid, ethyl ester 284, 88 - - - - 0.84 34 Linolenic acid,methyl ester 292, 79 - - - - 0.63 Total ------99.9 99.9 100 100 100 a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum. b Alkane Carbon number. c tr: trace (0.01-0.1% detectable). 104 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.6.3.1. Alkanes and Alkenes The epicuticular wax of many Solanaceous plants like tobacco leaves, tomato leaves/fruit, bell peppers, aubergines, potato leaves have been reported to contain alkanes (Severson et al., 1984; Smith et al., 1996; Bauer et al., 2004; Bauer et al., 2005; Szafranek and Synak, 2006). In the present study, n-alkanes ranged from 15.06-42.19% of the total wax samples except S. americanum in which free alkanes were not detected (Table 12). Unlike the other plant species analysed, 1- chloroalkanes of C21, C25, C27 and aromatic hydrocarbons were also found in S. americanum (Table 13). The individual n-alkanes were composed of C14 to C28. The alkanes C15, C16, C18, C19, C20, C21, C23, C24 C25, C27 and C28 were found common in all the samples in varying amounts. But the alkanes C14 and C17 showed selective occurrence. The taxa can be distinguished from one another by the distribution of the n-alkanes, taking into account the two main alkanes (Skorupa, et al., 1998), e.g. by quoting one main alkane outside and the second main alkane inside the parenthesis as: S. chenopodioides — C17 (C16); S. nigrum — C27 (C25); S. retroflexum — C20 (C18); S. villosum — C27(C18). The occurrence of the individual alkane was almost the same in S. chenopodioides and S. villosum qualitatively and matched closely with the alkanes of S. nigrum and S. retroflexum, but there were marked quantitative variations. The highest percentage of alkanes was observed in S. chenopodioides (42.19%) followed by S. retroflexum (23.58%), S. nigrum (15.48%) and S. villosum (15.06%). The absence of n-alkanes and the presence of 1-Chloroalkanes and 2,6- Diisopropylnaphthalene positioned S. americanum unique among the Solanum species under study. Grossi and Raphel (2003) reported 1-Chloro alkanes in the leaf waxes of Chenopodioides plants and showed that these compounds had potential value as chemotaxonomic and/or environmental markers. Squalene was detected in all the five taxa in variable quantity and could be regarded as a characteristic compound of genus Solanum. The variation in quantity could be attributed to differentiation at species level. It was the major component in S. americanum (24.86%). A small quantity of another alkene, 10-Methyl-1- octadecene, was also detected only in S. retroflexum (Table 13).

105 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.6.3.2. Alcohols, Aldehydes and ketones Free alcohols are widespread components of plant waxes (Bianchi, 1995). Homologous series of primary and secondary alcohols were found in S. tuberosum (Szafranek and Synak, 2006). In the present study, variable amounts of alcohols ranging from 2.72-19.24% were found in all the taxa except S. americanum (Table 12). There was not much variety in long chain alcohol types. Phytol was detected in the four taxa and it accounted for 19.24% of the total wax in S. chenopodioides. Two more unsaturated alcohols, 3,13-Octadecadiene-1-ol and 9-Eicosen-1-ol, were found in S. villosum in minute quantities. In addition to 9-Eicosen-1-ol, S. retroflexum also showed the presence of n-Cetyl alcohol and an aldehyde that were not found in other taxa. Alcohols were not detected in S. americanum, instead 3-hydroxyspirost-8-en- 11-one (14.07%) was detected (Table 13).

4.6.3.3. Free fatty acids and esters Free aliphatic fatty acids are common components of leaf waxes, but are usually present in low concentrations (Bianchi, 1995). Small amounts of free fatty acids were found in waxes of all the taxa except S. villosum where they constituted 67.37% of wax (Table 12). Stearic acid, detected in small quantity in the four taxa, matched in quantity in S. chenopodioides, S. nigrum and S. retroflexum but was more than double in S. villosum. α-Linolenic acid was also detected in reasonable amount (9.81%) in S. villosum and in a trace amount in S. nigrum but not found in detectable amount in other three taxa. Its methyl ester (0.63%) was also detected in S. villosum. -Linolenic acid (19.68%) was present in S. villosum only. Palmitic acid was previously reported in S. nigrum (Hanna et al., 1996) and S. tuberosum (Szafranek and Synak, 2006) and seems to be a characteristic acid of genus Solanum. Our findings were also in line with the already reported data. Concentration of Palmitic acid was relatively high in S. villosum (24.04%) and here its methyl and ethyl esters were also detected though in small amounts. S. americanum showed the unique presence of chlorinated ester of acetic acid in small quantity (Table 13). Palmitoleic acid methyl ester was detected as the major constituent (except for S. villosum) of the waxes, S. americanum (53.91%), S. chenopodioides (30.70%), S. nigrum (59.72%) and S. retroflexum (46.34%). Palmitoleic acid had been reported from S. nigrum (Hanna et al., 1996).

106 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Statistical analysis of the data presented in Table 13 was carried out using Minitab 3.2 Statistical Software. By cluster analysis (Fig. 15), the two samples S. nigrum and S. retroflexum were first to segregate. They showed many similarities in the distribution of epicuticular wax constituents except some minor differences like presence of 10-Methyl-1-octadecene, n-Cetyl alcohol and 9-Eicosen-1-ol in S. retroflexum that were not detected in S. nigrum. Also Tetradecane and Palmitic acid were present in trace amounts in S. nigrum but not detected in S. retroflexum. S. americanum deviated from the average distribution pattern of wax components due to unique occurrence of a ketone, aromatic hydrocarbon, chlorinated hydrocarbon and chlorinated ester and the absence of free hydrocarbons, some fatty acids and some esters occurring in other taxa. These features aligned S. americanum more distantly with above mentioned cluster as shown in Fig. 15. S. chenopodioides and S. villosum, although not much closely related to one another as compared to the previously discussed cluster, made another cluster. Their epicuticular wax showed all the hydrocarbons, Squalene, Phytol, Palmitic acid, Stearic acid and ester of Palmitoleic acid with different amounts. However, eight such compounds were present in S. villosum that were not detected in S. chenopodioides.

S. chenopodioides S. villosum S. americanum S. nigrum S. retroflexum

Fig. 15: Affinity relationships among different taxa of S. nigrum Complex based on the distribution of epicuticular wax components analysed by GC-MS and determined by similarity and Multivarial cluster analysis.

107 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.7 PROXIMATE ANALYSIS AND MINERAL COMPOSITION The proximate analysis includes the determination of dry matter, ash, protein, carbohydrate and fat. The nutritional potentials of the medicinal plant parts could be evaluated through their proximate compositions as well as percentage mineral elements composition (Abolaji et al., 2007). Because of the high contents of total carbohydrate, total fat, total ash and crude fibre contents of Xylopia aethiopica, it is particularly recommended as a tonic in Ivory Coast (Burkill, 1985). Moisture contents of the herbs investigated were high and closely related. The ash of a foodstuff is the inorganic residue remaining after the organic matter has been burnt away. The ashes of the dried husks and the seeds are used in the preparation of a kind of soap (Irvin, 1961). However all the taxa yielded very small amounts of ash. Further analysis of macro and micro-minerals using atomic absorption spectrophotometer (AAS) of the ash revealed that Ca, Mg, Fe, Na, K, Cu, Zn and Mn present at the highest concentration in water extract corn silk as compared to other samples (Wan Rosli et al. 2007).

Dietary fibre consists mainly of cellulose, hemicellulose and lignin, which exert different physiological effects on human health (Zia-ur-Rehman et al. 2003). Our data suggested that the plants of S. nigrum Complex are a good source of dietary fibre. Nutritionally, this is of beneficial effect since it had been reported that food fibre aids absorption of trace elements in the gut (Kelsay, 1981) and reduce absorption of cholesterol (Le Veille and Sanberlich, 1966). Proteins contain amino acids utilized by the cells of the body to synthesize all the numerous proteins required for the function of the cell and also to furnish energy (Robinson, 1978). Nutritionally, S. nigrum Complex is beneficial because of having a good amount of crude protein content. Proteins and amino acids of S. nigrum had been investigated (Khanna and Sharma, 1981) but the taxon investigated was not taxonomically specified. Taxa of S. nigrum Complex were low in fat and carbohydrate percentage so could be used as low calorie high protein diet. Our results for fats and carbohydrate concentrations were in good agreement with those of Kareav et al. (1955), who worked on one plant of this group. S. nigrum Complex do contain fatty acids and vitamins but in small amount.

108 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Also it could not be a good source of carbohydrate which constitutes a major class of naturally occurring organic compounds that are essential for the maintenance of plant and animal life and also provide raw materials for many industries (Ebun- Oluwa and Alade, 2007). Overall, the plants have nutritional qualities which when used in the right proportions could be of tremendous benefit to the body. Comparatively, because of the relatively high contents of total protein, total ash and crude fibre, S. nigrum is particularly recommended as food.

Table 14: Proximate analysis of the five taxa of S. nigrum Complex Percentage in species (Code)a Parameters (%) SA SC SN SR SV Moisture 55.15 59.11 56.41 56.61 58.84 Ash 4.81 1.2 2.70 2.82 1.6 Crude fibre 18.26 14.62 15.83 15.1 14.91 Crude protein 18.14 20.08 21.59 20.92 20.22 Fat 1.85 2.61 2.64 2.6 2.56 Carbohydrate 1.79 2.38 1.6 1.18 1.87 Nitrogen free extract 56.94 61.49 58.01 57.79 60.71 a Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum.

Minerals are the nutrients that exist in the body, and are as essential as our need for oxygen to sustain life. Minerals are also found in organic and inorganic combinations in food. They are most important factors in maintaining all physiological processes, are constituents of the teeth, bones, tissues, blood, muscle, and nerve cells. Acting as catalysts for many biological reactions within the human body, they are necessary for transmission of messages through the nervous system, digestion, & metabolism or utilization of all nutrients in foods. Vitamins cannot be properly assimilated without the correct balance of minerals. For example; calcium is needed for vitamin "C" utilization, zinc for vitamin "A", magnesium for "B" complex vitamins, selenium for vitamin "E" absorption, etc.

109 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

The mineral elements which are needed by the body in substantial amounts are calcium, phosphorus, iron, sulfur, magnesium, sodium, potassium and chlorine. In addition, the body needs minute (trace) amounts of iodine, copper, cobalt, manganese, zinc, selenium, and some others. If the body requires more than 100 milligrams (i.e., more than 1150th of a teaspoon) of mineral each day, the substance is labeled a mineral. If the cellular body requires less than this, it is labeled a trace mineral. Ten mineral and trace mineral elements were analysed in the ashes of five taxa of S. nigrum Complex (Table 15). Calcium, Potassium and Magnesium were found in significant amounts. Lead, cadmium and chromium were least detected, this is beneficial to consumers, since it has been reported that some of these minerals like lead, cobalt and cadmium are highly toxic (Asaolu et al., 1997). Overall these taxa are good supplements of minerals for human diet.

Table 15: Mineral element concentrationa of the five taxa of S. nigrum Complex Composition in species (Code)b (ppm) Element SA SC SN SR SV Cadmium 0.044 0.049 0.048 0.049 0.054 Calcium 193.21 145.20 348.15 340.76 162.24 Chromium 0.091 0.041 0.075 0.062 0.034 Iron 0.018 0.002 0.051 0.062 0.007 Lead 0.041 0.031 0.039 0.042 0.021 Magnesium 223.61 400.25 117.14 143.73 430.40 Manganese 0.003 0.003 0.004 0.005 0.002 Potassium 510.04 217.33 400.23 370.80 200.17 Sodium 53.11 40.47 26.02 29.15 32.14 Zinc 3.38 2.634 3.018 3.112 2.801 a n = 3 b Abbreviation of species; SA: S. americanum, SC: S. chenopodioides, SN: S. nigrum, SR: S. retroflexum, SV: S. villosum.

110 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.8. ANTIBACTERIAL STUDY Five morphologically different taxa of S. nigrum Complex were taken for antibacterial study to evaluate their individual characteristic medicinal value. The antibacterial activity of the methanolic extracts from five taxa were determined against four Gram-positive and three Gram-negative bacteria by agar diffusion method. All the plants showed significant antimicrobial activity in regards to the seven microorganisms tested (Table 16). S. americanum was most active among the taxa studied and showed highest activities against Bacillus licheniformis, Micrococus luteus (Gram-positive) and Escherichia coli (Gram-negative) at all concentrations. S. villosum was much active against Gram-positive bacteria Bacillus subtilis [food contaminant but non-pathogenic (Kunst et al., 1997) and Nocardia asteroides [causing cutaneous and pulmonary Nocardiosis (Jodlowski et al., 2007)]. However this taxa was least effective against Micrococus luteus which had been a reported cause of meningitis in humans (Greenblat et al., 2004) and septic shock (Albertson et al., 1978). S. chenopodioides showed moderate activity against all bacteria that was slightly related to S. villosum. S. chenopodioides and S. nigrum had parallel activities against Proteus mirabilis. Proteus mirabilis causes 90% of all 'Proteus' infections in humans including wound infections, urinary tract infections, septicemia and pneumonias (Feigin, 2004). For Salmonella typhimorium, which leads to the development of typhoid or enteric fever (Giannella, 1996), S. nigrum and S. retroflexum were more active than others. These two taxa showed much similar results against all other bacteria as well. All these results were concluded on the basis of zones of inhibition produced by the extracts at different concentrations. One-way analysis of variance (ANOVA) and Duncan t-test were carried out to validate the experimental results. All the plants have different kinds of secondary metabolites. The Solanum plants are particularly famous for their alkaloid contents and are considered as a good source of alkaloidal drugs (Khan and Ikram, 1983). Former studies associated the alkaloid with its biological activity (Oyedeji et al., 2005). Quercetin is also a highly biologically active flavonoid (So et al., 1981) which is present in its various glycosidic forms in S. nigrum Complex (da Silva et al., 2003). These compounds could be responsible for the antimicrobial activity of the plants investigated. Former studies on the glycoproteins of S. nigrum revealed antimicrobial activity against 111 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Escherichia coli and Listeria monocytogenes (Lim et al., 2002). Results suggested that S. americanum have higher concentration of the biologically active components. The activities of S. nigrum and S. retroflexum were much similar confirming our idea of S. retroflexum being the variety of S. nigrum. S. chenopodioides and S. villosum also gave comparable results but were less coordinated depicting them as separate species (Table 16).

Table 16: Zones of inhibition of different concentrations of methanolic extracts of S. nigrum Complex and standards against different bacterial strains Plant taxa/ Concentration Zone of Inhibition against bacterial strainsa drug (µg/mL) BS BL ML NA EC PM ST 1000 5.1 11.2 10.1 5.9 7.2 5.1 4.8 S. americanum 2000 10.2 12.1 11.9 9.1 11.0 6.0 6.2 3000 11.0 20.0 13.1 7.9 17.0 7.1 8.0 1000 10.8 8.1 9.2 7.0 6.2 6.0 5.1 S. chenopodioides 2000 13.0 10.0 10.0 9.0 7.9 8.2 7.0 3000 15.0 11.1 11.0 10.1 10.2 10.1 8.2 1000 10.1 6.0 8.0 7.0 6.1 6.2 10.1 S. nigrum 2000 11.0 8.1 10.0 8.9 8.1 8.0 12.1 3000 13.2 12.0 12.2 10.1 12.0 10.1 14.0 1000 9.1 6.2 8.2 7.0 5.0 6.0 10.0 S. retroflexum 2000 9.9 8.0 9.8 9.2 6.0 7.1 12.0 3000 12.0 11.8 12.0 9.8 11.1 9.9 14.1 1000 11.9 9.1 8.1 8.5 6.1 5.9 5.1 S. villosum 2000 15.0 10.0 8.9 9.0 7.1 6.9 8.1 3000 16.0 11.9 10.0 10.0 9.8 10.2 8.8 Ampicillin 1000 17.7 19.7 16.7 24.7 20.0 18.3 24.0 Benzyl penicillin 1000 18.0 20.0 18.3 20.3 20.0 21.0 25.7 Streptomycin 1000 25.7 25.7 28.3 30.0 30.0 28.3 30.0 Negative control 0 0 0 0 0 0 0 0 a Abbreviation of bacterial strains; BS: B. subtilus; BL: B. licheniformis; ML: M. luteus; NA: N. asteroides; EC: E. coli; PM: P. mirabilis; ST: S. typhimorium.

112 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Except for S. americanum, all the taxa presented lowest MICs against Escherichia coli among the seven bacterial strains studied, so they could be a part of herbal medicines treating gastroenteritis, urinary tract infections, haemolytic-uremic syndrome, peritonitis, mastitis, septicemia, Gram-negative pneumonia and neonatal meningitis that are caused by Escherichia coli (Todar, 2007). Excluding S. chenopodioides, the plant extracts also showed good MIC values against Bacillus licheniformis which is the cause of toxinogenic and food poisoning in humans characterised by diarrhoea, although vomiting occurs in half of reported cases (Veith et al., 2004; Voigt et al., 2004). Table 17 show that S. nigrum and S. retroflexum presented similar MICs. Moreover, these two plants manifested a better MIC against Bacillus subtilis, Micrococus luteus and Escherichia coli than other taxa. S. retroflexum presented the lowest MIC against Escherichia coli (30 μg/mL) compared to Benzyl penicillin (32 μg/mL). Likewise, S. villosum manifested a comparable value of MIC against Proteus mirabilis (45 μg/mL) compared to Benzyl penicillin (32 μg/mL).

Table 17: Minimum Inhibitory Concentration (MIC) of methanolic extracts from S. nigrum Complex against different bacterial strains MIC for Bacterial strainsa (µg/mL) Plant taxa/ drug BS BL ML NA EC PM ST S. americanum 114 34 225 85 135 118 77 S. chenopodioides 560 184 178 170 78 382 185 S. nigrum 78 49 105 195 49 66 96 S. retroflexum 74 49 95 127 30 53 100 S. villosum 137 84 141 116 53 45 111 Ampicillin 8 1 28 32 8 2 7 Benzyl penicillin 8 4 40 18 32 32 16 Streptomycin 4 2 16 8 2 8 2 a Abbreviation of bacterial strains; BS: B. subtilus; BL: B. licheniformis; ML: M. luteus; NA: N. asteroides; EC: E. coli; PM: P. mirabilis; ST: S. typhimorium.

113 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.9. ANTIOXIDANT STUDY Food and beverage companies are touting the presence of antioxidants in their products in response to consumer interest in the potential health benefits of antioxidants in the diet. For example, Lipton teas carry a logo, ―AOX, Naturally Protective Antioxidants,‖ POM Wonderful pomegranate juice says it‘s ―the real Antioxidant Superpower™,‖ and Hershey’s Nuggets Special Dark Mildly Sweet Chocolate bears a logo stating ―Natural Source of Flavonol Antioxidants‖.

4.9.1. ANTIOXIDANT ACTIVITY OF S. NIGRUM COMPLEX The extract of fruits of S. nigrum L. has anti-tumor and neuropharmacological properties and it can be used as an anti-oxidant and cancer chemo-preventive matter (Perez et al., 1998; Son et al., 2003). S. nigrum L. has been reported to show antioxidant activity (Surveswaran et al., 2007), so a comparison of the other taxa was performed with respect to it. Methanol is usually recommended for the extraction of antioxidant compounds (Iqbal et al., 2005). All determinations of antioxidant capacity were conducted in triplicate. The reported value for each sample was calculated as the mean of three measurements. In the present work six different antioxidant assays were employed to evaluate the antioxidant activity of five taxa of S. nigrum Complex.

4.9.2. TOTAL PHENOLIC CONTENT ASSAY This assay measures the change in color when metal oxides are reduced by polyphenolic antioxidants such as gallic acid, resulting in a blue solution. Fig. 16 shows the comparison of total phenolic content (TPC) as determined by the Folin- Ciocalteu reagent method. It was found that TPC of the five samples showed slight variation, ranging from 20.31-26.58 mg of gallic acid equivalents (GAE)/100 g dry weight (DW). The highly significant correlations obtained in this study support the hypothesis that phenolic compounds contribute significantly to the total antioxidant capacity of medicinal plants. The contents of total phenolics of S. americanum and S. nigrum were equal (26.58 mg/100g) and higher than the values determined for other taxa though much closer to S. retroflexum (26.4 mg/100g). The GAE of S.

114 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION chenopodioides (20.31 mg/100g) and S. villosum (22.695 mg/100g) were matching and lower among other extracts. Iqbal et al., (2005) reported the TPC of four varieties of rice bran indigenous to Pakistan ranging from 251-359 mg/100g. The TPC of ―Akron‖ wheat bran (Zhou and Yu, 2004) was reported as a potent source of antioxidants, thus suggesting the exploitation of rice bran as a viable source of antioxidant for neutraceuticals and functional foods.

The plant phenols are regarded as those substances derived from the shikimate pathway and phenylpropanoid metabolism, following the phosphoenolpyruvate → phenylalanine → cinnamate → 4-coumarate course, leading to chalcone, flavanone, dihydroflavonol and anthocyanin (Robards and Antolovich, 1997). Many of these phenolic compounds are essential to plant life, e.g., by providing defense against microbial attacks and by making food unpalatable to herbivorous predators (Bennick, 2002). Previous studies have found that phenolic compounds are major antioxidant constituents in selected herbs, vegetables and fruits, and there are direct relationships between their antioxidant activity and total phenolic content (Velioglu et al., 1998; Dorman et al., 2004).

TPC(mg/L GAE)

Plant Species

Fig. 16: Total phenolic contents (TPC) of S. nigrum Complex

115 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.9.3. LIPID PEROXIDATION ASSAY (TOTAL ANTIOXIDANT ACTIVITY) The results of Fig. 17 depict that methanolic extracts of five taxa exhibited moderate antioxidant activity in the Linoleic acid peroxidation system in terms of the measurement of inhibition of peroxidation. Trolox was used as a positive control. Higher absorbance values show lower concentration of antioxidant components in sample. All the five samples exhibited a good inhibition to peroxidation comparable to that of standard Trolox. So they can be utilized as an alternative and effective source of natural antoxidants. Methanolic extract of S. retroflexum was found to show significantly higher inhibition of peroxidation in Linoleic acid system and thus reflected the highest antioxidant activity among the extracts. Whereas methanolic extract of S. nigrum exhibited lowest antioxidant activity as evident by its least inhibition of peroxidation. Oryeneho and Hettiarachchy (1992) reported that the phenolic contents of various agriculture wastes contained potential antioxidant activity against the Linoleic acid peroxidation system. Velioglu et al., (1998) also described that the total phenolic contents of fruits and vegetables contained potential antioxidant activities against the Linoleic acid peroxidation system. The inhibition of peroxidation as exhibited by these different methanolic extracts might be attributed to the presence of various polyphenolics in S. nigrum Complex.

S. americanum S. chenopodioides S. nigrum

S. retroflexum S. villosum Trolox

Blank Lipidperoxidation value

Time (Hours)

Fig. 17: Total Antioxidant capacity of five taxa of S. nigrum Complex evaluated by the Lipid peroxidation method

116 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.9.4. FRAP ASSAY This method measures the ability of antioxidants to reduce ferric iron. It is based on the reduction of the complex of ferric iron and 2,3,5-triphenyl-1,3,4-triaza- 2-azoniacyclopenta-1,4-diene chloride (TPTZ) to the ferrous form at acidic pH. The FRAP assay is different from DPPH assay only in the fact that it is carried out at acidic pH while former at neutral pH. The FRAP values of the five samples varied from 5.89 to 8.5 mM/mL of extracts (Fig. 18). The FRAP method also showed a higher antioxidant capacity for the three taxa (S. americanum, S. nigrum and S. villosum) with respect to other two (S. chenopodioides and S. retroflexum). Apparently, the biosynthesis of the natural products is profoundly influenced by a number of factors, such as locations, weather conditions, and harvest periods, etc. Therefore, it is expected that the FRAP values vary accordingly. Another recent study has also shown that the antioxidant capacities of apples are dependent on cultivar (van der Sluis et al., 2001). The FRAP assay measures the reducing capability by increased sample absorbance based on the formed ferrous ions, and the assay may not be complete even several hours after the reaction starts, such that a single end-point of the reaction cannot be determined (Prior et al., 2005). Ou et al. (2002) also noted that the FRAP assay has some drawbacks, such as interference, reaction kinetics, and quantitation methods. Considering all these factors several researchers (e.g., Arnao, 2000; Cai et al., 2004; Shan et al., 2005) favour the improved ABTS assay, which was rapid, robust and accurate for systematically assessing total antioxidant capacity of crude extracts from plant materials on a large scale. However, it had been recommended that at least two methods be used due to

the differences between the test systems investigated (Schlesier et al., 2002).

FRAP FRAP Value (mM FeSO4.7H2O FeSO4.7H2O equivalent)

Plant Species

Fig. 18: Antioxidant capacity of five taxa of S. nigrum Complex evaluated by the FRAP method 117 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.9.5. ABTS•+ ASSAY PROTOCOL This method uses a diode-array spectrophotometer to measure the loss of color when an antioxidant is added to the blue-green chromophore ABTS·+, 2,2'- azino-bis(3-ethylbenzthiazoline- 6-sulfonic acid). The antioxidant reduces ABTS·+ to ABTS, decolorizing it. ABTS·+ is a stable radical not found in the human body. Trolox equivalent antioxidant capacity (TEAC) is the concentration of Trolox required to give the same antioxidant capacity as 1mM test substance (Rice-Evans and Miller, 1994). The analyzed taxa of S. nigrum Complex showed a free-radical scavenging activity equivalent to that of a Trolox solution 6 mM with the ABTS method. The TEAC values of ABTS assay exhibited slight variation from 25.69 to 33.88 mM Trolox equivalents per 100 g dry weight (mM TEAC/100 g DW). The comparison of different TEAC values of the tested plants by the ABTS assay is shown in Fig. 19. Of the five samples studied, S. retroflexum had highest antioxidant capacity (33.88 mM/100 g DW) that was comparable to S. villosum (31.065 mM/100 g DW) Rest of the samples i.e. S. americanum, S. chenopodioides and S. nigrum had matching TEAC values 25.69, 27.2 and 26.82 mM/100 g DW respectively. ABTS assay is reported to be rapid, robust and accurate for systematically assessing total antioxidant capacity of crude extracts from plant materials particularly on a large scale (Surveswaran et al., 2007).

TEAC TEAC (mM)

Plant Species

Fig. 19: Antioxidant capacity of S. nigrum Complex evaluated by the ABTS method. The values represent the Trolox equivalent antioxidant capacity

118 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.9.6. DPPH RADICAL SCAVENGING CAPACITY ASSAY DPPH is a method based on the scavenging of the 1,1-diphenyl-2- picrylhydrazyl radical. The reaction of DPPH with numerous antioxidants has been published and the stoichiometry characterized (Hogg et al., 1961; Cuvelier et al., 1992). The odd electron in the DPPH free radical determines the decrease in sample absorbance at 517 nm, and the coloured compounds such as anthocyanins and carotenoids present in the test sample may have the spectra that overlap with DPPH at 517 nm and thus interfere with the OD measurements (Arnao, 2000; Prior et al., 2005). The resulting decolorization is stoichiometric with respect to number of electrons captured The sample size that can lower the initial absorbance of DPPH solution by 50% (EC50) has been chosen as the endpoint for measuring the antioxidant activity and TEC50 was calculated. Different concentrations of samples were applied and dose response was observed (Table 18).

Table 18: EC50 and TEC50 values of methanolic extracts of S. nigrum Complex Antioxidant activity a b Sample EC50 TEC50 (µg) (min) S. americanum 76.6 15 S. chenopodioides 48.7 7 S. nigrum 88.2 18 S. retroflexum 67.5 10 S. villosum 125 25 a Concentration that causes a decrease in the initial DPPH concentration by 50% b Time needed to reach the steady state with EC50 concentration

119 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

Fig. 20 demonstrate the decrease in DPPH radical due to the scavenging activity of five taxa of S. nigrum Complex and standard trolox and BHA solutions. The curves showed a sharp decrease in first five minutes but they remain steadier (except for S. retroflexum) in next 35 min. S. chenopodioides gave a better response as compare to other taxa however the difference was not large. The kinetic curves depicted that S. villosum had a slow response and took more time for exhibiting the complete antioxidant activity. Results suggested that all taxa have moderate effects on scavenging DPPH free radical so they could be considered suitable as antioxidants in living organisms particularly human beings. Antioxidants in food may be water soluble, fat soluble, insoluble, or bound to cell walls and thus not necessarily freely available to react with DPPH, hence they react at different rates i.e. differing kinetics, and the reaction will often not go to completion in a reasonable assay time.

S. americanum S. nigrum S. chenopodiodes S. retroflexum S. villosum BLank

Trolox % % [DPPH] Remaining

Time (minutes)

Fig. 20: Antioxidant capacity of S. nigrum Complex evaluated by DPPH method

4.9.7. METAL CHELATING ACTIVITY Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating agents, the complex formation is disrupted, resulting in decrease of the red colored complex. Measurement of color reduction is the measure of the metal chelating activity. The transition metal, iron, is capable of generating free radicals from peroxides by Fenton reactions and may be implicated in human cardiovascular disease (Halliwell and Gutteridge, 1990). Because Fe2+also has been shown to cause 120 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION the production of oxyradicals and lipid peroxidation, minimizing Fe2+ concentration in Fenton reactions affords protection against oxidative damage. In this assay, crude extracts and standard compounds interfered with the formation of ferrous and ferrozine complex, suggesting that they have chelating activity and are able to capture ferrous ion before the formation of ferrozine. Fig. 21 describes the effect of the S. chenopodioides (70.37%) which was found to be more than all the other taxa. While the chelating potentials of S. nigrum, S. retroflexum and S. villosum were almost the same (55.78%, 52.08% and 53.24% respectively). In this case S. americanum showed lowest chelating potential (42.361%) than other samples. Iron is known to generate free radicals through the Fenton and Haber–Weiss reaction. Metal ion chelating activity of an antioxidant molecule prevents oxyradical generation and the consequent oxidative damage. Metal ion chelating capacity plays a significant role in antioxidant mechanism since it reduces the concentration of the catalysing transition metal in lipid peroxidation (Duh et al., 1999). It is reported that chelating agents, which form s-bonds with a metal, are effective as secondary antioxidants since they reduce the redox potential thereby stabilizing the oxidized form of the metal ion (Gordon, 1990).

% % BoundIron

Plant Species

Fig. 21: Metal chelating ability of methanolic extracts of S. nigrum Complex

121 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4.10. CONCLUSION This research work attempts to identify the taxa related to the S. nigrum/ black nightshade more accurately, by providing their chemical composition as identification key. Documented uses of the ‗black nightshades‘ suggest that the taxa do exhibit many potentially valuable characteristics which deserve conservation and utilization. Chemical profile of a plant can be regarded as its fingerprints as it is highly genus-specific and can be used as chemotaxonomic indicators to elucidate phylogenetic relationships. The results are described as a model for the origin of the chemical races. Analyses of alkaloids from five taxa of S. nigrum Complex as glycones and aglycones demonstrated that each of the five taxa is unique regarding the chemical composition. But still there was a close relationship among all with respect to occurrence of compounds like Solasonine and α-Solanine. The reason behind this may be ascribed as belonging to a common genus Solanum. S. americanum was found to be slightly related to S. chenopodioides in its SGA profile however the SGAA marked it unique to other taxa. Similarly S. villosum was also found different in SGA concentration for showing presence of all the compounds analysed but it had matching in SGAA contents with S. chenopodioides. S. retroflexum showed many similarities in qualitative and quantitative chemical composition of alkaloids (glycones and aglycones) with S. nigrum. With various biological activities, flavonoids are the principal components in evaluating the quality as well as taxonomy of various taxa of S. nigrum Complex. The use of one colorimetric methods utilizing aluminum chloride reaction to determine flavonoid contents was proved to be specific only for flavones and flavonols, while another colorimetric method utilizing 2,4-dinitrophenylhydrazine reaction was specific for flavanones. Therefore, we used both analyses so that the sum of the results may better represent the real content of total flavonoids. TLC was helpful in the preliminary examination of flavonoid glycosides. HPLC and GC-MS analysis enabled the quantitative comparison of quercetin glycones and aglycones respectively. Concentrations of two glycosides of quercetin were found to be different in the five taxa. Though Quercetin was detected in all five taxa however it showed marked variations quantitatively.

122 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

TLC helped identifying the classes of compounds present in epicuticular waxes. GC-MS analyses demonstrated that each of the five taxa had its own specific set of chemical constituents. But still there was a close relationship among all with respect to some compounds like Squalene, Palmitic acid and ester of Palmitoleic acid. The reason behind this may be ascribed to having a common genus Solanum. S. americanum was found to contain very small number of compounds. Most of these compounds (e.g. chlorinated compounds and one each of aromatic hydrocarbons and ketones) were found unique to S. americanum. S. villosum was also found different for having greatest variety of compounds detected (26 out of 34). Some of them such as some alcohols, esters, an aldehyde and an acid were not detected in other taxa. The qualitative chemical composition of epicuticular waxes of S. americanum, S. chenopodioides, S. nigrum and S. villosum suggested that they had significant differences. In case of S. retroflexum, many similarities in chemical constituents particularly in hydrocarbon composition were observed. All the extracts showed varying degrees of antimicrobial activity on the microorganisms tested. Some of these plants were more effective than traditional antibiotics to combat the pathogenic microorganisms studied. Two taxa (S. retroflexum and S. villosum presented the low MIC comparable to the antibiotic standard. These plants could be a source of new antibiotic compounds. Proximate and mineral analyses revealed that all the plants have nutritional qualities which when used in the right proportions could be of tremendous benefit to the body. The millenarian use of these plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases. Systematic evaluation of a large number of medicinal plants is useful for understanding their functionality and chemical constituents, and also supports them as potential sources of potent natural antioxidants. Five taxa of S. nigrum Complex were analysed by six reported antioxidant assays. Total antioxidant activity was calculated by lipid peroxidation assay. There was a variable response of each taxon towards different assays applied. In comparing the five taxa, S. chenopodioides was found to be highly active in DPPH, Lipid peroxidation and Metal chelating assays but least active in FRAP and Total phenolic content assays. On contrary, S. nigrum showed highest activity in FRAP and Total phenolic content assays but least in Lipid peroxidation assay. Total phenolic contents of S. americanum were also highest as of S. nigrum but it was 123 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION least active in ABTS and Metal chelating activities. S. retroflexum was most effective in ABTS assay. S. villosum was least active against DPPH radical. Overall S. nigrum and S. retroflexum had comparable activities in almost all assays. The results suggested that S. americanum, S. chenopodioides, S. nigrum and S. villosum had significant differences and might be treated as separate species and not the varieties/subspecies of S. nigrum. In case of S. retroflexum, many similarities with S. nigrum were observed. So it could be regarded as the variety/ subspecies of S. nigrum. Some minor differences could be attributed to differentiation at variety/ subspecies level. Moreover S. retroflexum had many morphological variations as compared to S. villosum and showed no relationship with later in this study also thereby rejecting the Burbank‘s claim to be its hybrid. Because of the taxonomic misunderstanding surrounding the component species and the tendency to refer to all members as ‘S. nigrum’, it is advisable that the information found in literature should be reinterpreted and any medicinal/commercial use of the taxa should be carried out in the light of above chemotaxonomic suggestion. While the taxonomic account compiled for this document covered a revision of the species concerned, it is hoped that it will stimulate continued biosystematic work on material of known origin so that these ‘Solanums’ can be understood, controlled and utilized appropriately throughout the world.

124 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

1. HPLC CHROMATOGRAMS OF STEROIDAL GLYCO-

ALKALOID (SGAA) OF FIVE TAXA OF S. NIGRUM COMPLEX

1. HPLC chromatogram of SGAA of S. americanum

125 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

2. HPLC chromatogram of SGAA of S. chenopodioides

126 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

3. HPLC chromatogram of SGAA of S. nigrum

127 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4. HPLC chromatogram of SGAA of S. retroflexum

128 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

5. HPLC chromatogram of SGAA of S. villosum

129 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

2. GC-MS CHROMATOGRAMS OF STEROIDAL GLYCO- ALKALOID AGLYCONES (SGAA) OF S. NIGRUM COMPLEX

6. GC-MS chromatogram of SGAA of S. americanum

130 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

7. GC-MS chromatogram of SGAA of S. chenopodioides

131 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

8. GC-MS chromatogram of SGAA of S. nigrum

132 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

9. GC-MS chromatogram of SGAA S. retroflexum

133 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

10. GC-MS chromatogram of SGAA of S. villosum

134 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

3. HPLC CHROMATOGRAMS OF FLAVONOID GLYCOSIDES

FIVE TAXA OF S. NIGRUM COMPLEX

11. HPLC chromatogram of flavonoid glycosides of S. americanum

135 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

12. HPLC chromatogram of flavonoid glycosides of S. chenopodioides

136 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

13. HPLC chromatogram of flavonoid glycosides of S. nigrum

137 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

14. HPLC chromatogram of flavonoid glycosides of S. retroflexum

138 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

15. HPLC chromatogram of flavonoid glycosides of S. villosum

139 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

4. GC-MS CHROMATOGRAMS OF FLAVONOID AGLYCONES

OF FIVE TAXA OF S. NIGRUM COMPLEX

16: GC-MS chromatogram of flavonoid aglycones of S. americanum

140 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

17: GC-MS chromatogram of flavonoid aglycones of S. chenopodioides

141 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

18: GC-MS chromatogram of flavonoid aglycones of S. nigrum

142 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

19: GC-MS chromatogram of flavonoid aglycones of S. retroflexum

143 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

20: GC-MS chromatogram of flavonoid aglycones of S. villosum

144 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

5. GC-MS CHROMATOGRAMS OF EPICUTICULAR WAXES OF FIVE TAXA OF S. NIGRUM COMPLEX

21: GC-MS chromatogram of epicuticular wax of S. americanum

145 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

22: GC-MS chromatogram of epicuticular wax of S. chenopodioides

146 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

23: GC-MS chromatogram of epicuticular wax of S. nigrum

147 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

24: GC-MS chromatogram of epicuticular wax of S. retroflexum

148 Chemotaxonomical Characterization of Solanum nigrum and its Varieties Chap.4: RESULTS AND DISCUSSION

25: GC-MS chromatogram of epicuticular wax of S. villosum

149 Chemotaxonomical Characterization of Solanum nigrum and its Varieties

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