The Islamia University of Bahawalpur Pakistan

Home , Calligonum leucocladum, Calligonum polygonoides, Crateva adansonii, Crateva nurvala, Isoorientin, Isovitexin

Phytochemical and Biological Studies of Calligonum polygonoides and Crateva adansonii

A thesis submitted to the

Department of Pharmacy

In Partial Fulfillment of the Requirements for the Degree Of

DOCTOR OF PHILOSOPHY (PHARMACEUTICAL CHEMISTRY) BY IRFAN PERVAIZ (Pharm.D, MPhil)

Faculty of Pharmacy & Alternative Medicine

The Islamia University of Bahawalpur Pakistan

STUDENT’S DECLARATION

I, Irfan Pervaiz, PhD Scholar (Pharmaceutical chemistry) of the Department of Pharmacy, The

Islamia University of Bahawalpur, hereby declare that the research work entitled

“Phytochemical and Biological Studies of Calligonum polygonoides and Crateva adansonii” is done by me. I also certify that this thesis does not incorporate any material without acknowledgment; and to the best of my knowledge and belief that it does not contain any material previously published where due reference is not made in the text.

Irfan Pervaiz

SUPERVISOR’S DECLARATION

It is hereby certified that work presented by Irfan Pervaiz in the thesis titled, “Phytochemical and Biological Studies of Calligonum polygonoides and Crateva adansonii” is based on the research study conducted by candidate under my supervision. It is certified that no material has been used in this thesis which is not his own work, except where due acknowledgement has been made. Plagiarism as checked by Turnitin Software is 15% which is in the limit as described by Higher Education Commission. He has fulfilled all the requirements and is qualified to submit this thesis in partial fulfillment for the degree of Doctor of Philosophy

(PhD) in the Department of Pharmacy.

Prof. Dr. Saeed Ahmad Supervisor

List of contents

Acknowledgement i Abstract ii List of Tables v List of Figures vii Chapter 1 Introduction 1.1 General Introduction of Natural Products 1 1.1.1. Natural products as antimicrobial agents 2 1.1.2. Natural Products as Anticancer agents 8 Plant introduction/previous phytochemical Part A investigations on Calligonum Polygonoides 17 Chapter 2 Literature Review 17 2.1 Plant Introduction 18 2.1.1. Plant Classification 18 2.2. Family Polygonaceae 19 2.3. Genus Calligonum 19 2.3.1 Calligonum polygonoides 19 Bioactive constituents reported from the Family 2.4. Polygonaceae 20 2.4.1. Calligonum genus 20 2.4.1.1. Calligonum polygonoides 20 2.4.1.2. Calligonum leucocladum 20 2.4.2. Polygonum genus 22 2.4.2.1. Polygonum minus 22 2.4.2.2. Polygonum sachalinensis 22 2.4.2.3. Polygonum multiflorum 24 2.4.4.4. Polygonum chinense 24 2.4.4.5. Polygonum barbatum 26 2.4.3. Oxygonum genus 26 2.4.3.1. Oxygonum sinuata 26 2.4.4. Rumex genus 26 2.4.4.1. Rumex nepalensis 26 2.4.4.2. Rumex crispus 27 2.4.4.3. Rumex japonicus 27 2.4.4.4. Rumex aquaticus 27 2.4.4.5. Rumex patientia 28 2.4.4.6. Rumex dentatus 28 2.4.4.7. Rumex gmelini 29 2.4.4.8. Rumex bucephalophorus 30 Chapter 3 Phytochemical and Biological Profiling of aerial parts of Calligonum Polygonoides 32 Biological Evaluation of different fractions of 3.1. Calligonum polygonoides 33 3.1.1. DPPH Free Radical Scavenging Activity 33 3.1.2. α-glucosidase inhibition assay 34 3.1.3. Carbonic anhydrase inhibition assay 34 3.1.4. Urease Inhibition assay 35 3.1.5. Xanthine oxidase inhibition assay 35 3.1.6. Tyrosinase Inhibition Assay 36 GC-MS analysis of n-hexane fraction of Calligonum 3.2. Polygonoides 37 LC-MS Evaluation of Crude Methanolic Extract of 3.3. Calligonum Polygonoides 38 Structure Elucidation of Compounds Isolated from n- 3.4. hexane fraction (CpC-N) 48 3.4.1. Cycloartenol (145) 48 3.4.2. Beta-Amyrin (146) 49 3.4.3. Stigmasterol (147) 52 Structure Elucidation of Compounds Isolated from 3.5. Butanolic fraction (CpC-B) 54 3.5.1. Rhododendrin (148) 54 3.5.2. Glucogallin (149) 56 3.5.3. Hypolaetin-8-glucoside (150) 58 3.5.4. Isoetin-4’-O-glucuronide (151) 60 3.5.5. Medicarpin 3-O-glucoside-6'-O-malonate (152) 63 3.5.6. Melanoxetin (153) 65 3.5.7. Dihydrorobinetin (154) 66 3.6. Biological Evaluation of phytochemicals isolated from Calligonum polygonoides 68 3.6.1. Urease Inhibition assay 69 3.6.2. Xanthine oxidase inhibition assay 70 3.6.3. Carbonic Anhydrase Inhibition Assay 71 3.6.4. α-glucosidase inhibition assay 71 Chapter 4 Experimental 73 4.1. General Experimental Conditions 74 4.1.1. 1H-NMR, 13C-NMR & MS 74 4.1.2. LC-MS 74 4.1.2.1. HPLC System 74 4.1.2.2. Q-TOF high resolution mass spectrometry 75 4.1.3. GC-MS Analysis 75 4.1.3.1. GC Conditions 75 4.1.3.2. GC oven temperature programme 75 4.1.3.3. GC oven temperature programme 75 4.1.3.4. Full Scan Data acquisition 76 4.1.4. Chromatographic Separation 76 4.2. Materials and Methods 76 4.2.1. Plant collection 76 4.2.2. Extraction 76 4.2.3. Liquid-Liquid Extraction 77 LC-MS Profiling of crude methanolic fraction of 4.2.4. Calligonum Polygonoides 78 4.2.4.1. Positive Ionisation Mode 78 4.2.4.2. Negative Ionisation Mode 79 4.3. Isolation & purification of chemical constituents from n- hexane fraction CpC-N 80 4.4. Isolation & purification of chemical constituents from n- butanol fraction CpC-B 82 4.5. Characterization of constituents from Calligonum polygonoides 84 4.5.1. Cycloartenol (145) 84 4.5.2. Beta-Amyrin (146) 84 4.5.3. Stigmasterol (147) 85 4.5.4. Rhododendrin (148) 86 4.5.5. Glucogallin (149) 86 4.5.6. Hypolaetin-8-glucoside (150) 87 4.4.7. Isoetin-4’-O-glucuronide (151) 88 4.5.8. Medicarpin 3-O-glucoside-6'-O-malonate (152) 88 4.5.9. Melanoxetin (153) 89 4.5.10. Dihydrorobinetin (154) 90 4.6. Biological Evaluation of different fractions of Calligonum polygonoides and its phytochemicals 91 4.6.1. DPPH Free radical scavenging assay 91 4.6.2. α-glucosidase inhibition assay 91 4.6.3. Carbonic anhydrase inhibition assay 92 4.6.4. Urease Inhibiion assay 92 4.6.5. Xanthine oxidase inhibition assay 93 4.6.6. Tyrosinase Inhibition Assay 94 4.7. Statistical Analysis 95 Chapter 5 Conclusion 96 Plant introduction/previous phytochemical Part A investigations on Crateva adansonii 98 Chapter 5 Literature Review 99 5.1. Plant Introduction 99 6.1.1. Plant Classification 101 6.2. Family Capparaceae 101 6.3. Genus Crateva 101 6.3.1 Crateva adansonii 101 6.4. Bioactive constituents reported from Capparcaeae 101 6.4.1. Crateva genus 101 6.4.1.1. Crateva adansonii 101 6.4.1.2. Crateva nurvala 102 6.4.1.3. Crateva religiosa 102 6.4.1.4. Crateva tapia 103 6.4.2. Capparis genus 104 6.4.2.1. Capparis himalayensis 104 6.4.2.2. Capparis tenera 104 6.4.2.3. Capparis flavicans 104 6.4.2.4. Capparis spinosa 105 6.4.2.5. Capparis decidua 106 6.4.3. Cadaba Genus 107 6.4.3.1. Cadaba glandulosa/ Cadaba farinose 107 6.4.3.2. Cadaba rotundifolia 109 Phytochemical and Biological Profiling of aerial parts of Chapter 7 Crtaeva adnsonii 111 Biological Evaluation of different fractions of Crateva 7.1. adansonii 112 7.1.1. DPPH Free Radical Scavenging Activity 112 7.1.2. α-glucosidase inhibition assay 113 7.1.3. Tyrosinase inhibition assay 113 7.1.4. Carbonic anhydrase Inhibiion assay 114 7.1.5. Xanthine oxidase inhibition assay 114 7.1.6. Urease Inhibition Assay 115 7.2. GC-MS analysis of n-hexane fraction of Crateva adansonii 116 7.3. LC-MS Evaluation of Crude Methanolic Extract of Crateva adansonii 117 7.4. Structure Elucidation of Compounds Isolated from n- hexane fraction (CaC-N) 129 7.4.1. Lupeol (161) 129 7.4.2. Lupanol (201) 131 7.4.3. Lupenone (202) 133 7.5. Structure elucidation of Constituents isolated from chloroform fraction 135 7.5.1. Pheophorbide (203) 135 7.5.2. Pyropheophorbide (158) 138 7.5.3. Phytosphingosine (204) 140 7.5.4. Dehydrophytosphingosine (205) 141 7.6. Structure Elucidation of constituents isolated from butanolic fraction (CaC-B) 142 7.6.1. Cadabicine (177) 142 7.6.2. Isovitexin (206) 145 7.6.3. Isovitexin-7-O-rhamnoside (207) 147 7.7. Biological Evaluation of phytochemicals isolated from Calligonum polygonoides 149 7.7.1. Urease Inhibition assay 149 7.7.2. Tyrosinase inhibition assay 150 7.7.3. α-glucosidase Inhibition Assay 151 7.7.4. Carbonic anhydrase inhibition assay 152 7.7.5. Xanthine oxidase inhibition assay 153 Chapter 8 Experimental 154 8.1. General Experimental Conditions 155 8.1.1. 1H-NMR, 13C-NMR & MS 155 8.1.2. LC-MS 155 8.1.2.1. HPLC System 155 8.1.2.2. Q-TOF high resolution mass spectrometry 155 8.1.3. GC-MS Analysis 155 8.1.4. Chromatographic Separation 155 8.2. Materials and Methods 155 8.2.1. Plant collection 155 8.2.2. Extraction 155 8.2.3. Liquid-Liquid Extraction 156 8.2.4. LC-MS Profiling of crude methanolic fraction of Calligonum Polygonoides 156 8.2.4.1. Positive Ionisation Mode 156 8.2.4.2. Negative Ionisation Mode 157 8.3. Isolation & purification of chemical constituents from n- hexane fraction CaC-N 158 8.4. Isolation & purification of chemical constituents from chloroform fraction CaC-Cl 160 8.5. Isolation & purification of chemical constituents from chloroform fraction CaC-B 162 8.6. Characterization of constituents from Calligonum polygonoides 162 8.6.1. Lupeol (161) 164 8.6.2. Lupanol (201) 164 8.6.3. Lupenone (202) 165 8.6.4 Pheophorbide (203) 166 8.6.5. Pyropheophorbide (158) 167 8.6.6. Phytosphingosine (204) 167 8.6.7. Dehydrophytosphingosine (205) 168 8.6.8. Cadabicine (177) 168 8.6.9. Isovitexin (206) 169 8.6.10. Isovitexin-7-O-rhamnoside (206) 170 8.7. Biological Evaluation of phytochemicals isolated from Calligonum polygonoides 170 8.7.1. DPPH Free radical Scavenging assay 170 8.7.2. Urease Inhibition assay 170 8.7.3. Tyrosinase inhibition assay 170 8.7.4. α-glucosidase Inhibition Assay 170 8.7.5. Carbonic anhydrase inhibition assay 171 8.7.6. Xanthine oxidase inhibition assay 171 Chapter 9 Conclusion 172

Acknowledgment

In the name of Allah who is the Creator of the Universe who gave me the strength to accomplish this research work and the opportunity to explore the biological diversity amongst the flora of this world. I also pay homage to the Holy Prophet Hazrat Muhammad (PBUH) for helping us identify our true Creator and His blessings.

I feel elated for expressing imperceptible gratefulness to my encouraging and learnt supervisor Prof. Dr. Saeed Ahmad, Department of Pharmacy whose personal interest, guidance, advices, suggestions and discussions enfranchised me to complete my research work.

I convey my deepest gratitude and appreciation to ex-Dean Dr. Mahmood Ahmad and present Dean Dr. Naveed Akhtar for provision of research facilities. I also express gratitude to all faculty members of Pharmacy Department, IUB for their moral support. I am also grateful to Dr. Muhammad Imran Tousif (Assistant Professor, Chemistry Department, University of Education Lahore, D.G. Khan Campus) for his technical assistance in structure elucidation of isolated compounds.

I also acknowledge Department of Chemistry, F.C. College, Lahore for performing GC- MS analysis of my plant fractions.

I am highly thankful to Higher Education Commission of Pakistan for financial support under the “Access to Scientific Instrumentation Program” for spectroscopic techniques of isolated compounds at International Center for Chemical and Biological Sciences, University of Karachi.

The contribution of Dr. Ghulam Sarwar (Botany Department, the Islamia University Bahawalpur) in plant identification and collection is worth mentioning. I would also like to thank Islah Khan Saffi and Faisal Rehman for assisting me in plant collection.

I am thankful to all my lab fellows for providing a friendly environment for my research work in the lab. Other names worth mentioning in completion of my PhD include: Dr. Hamid Saeed, Dr. Mubashar Rehman, Dr.Arham Shabbir, Dr. Adeel Masood Butt, Dr. Zafar Iqbal and my friend, Ahmad Ijaz.

I highly appreciate and acknowledge the sacrifice of my wife and children who suffered a great deal during my strenuous journey of PhD. Last but not the least, I express my apology to those who ever had a soft corner for me but I missed to mention them personally.

Irfan Pervaiz

Abstract

The role of natural products in the treatment of diseases has inspired scientists in their search for new avenues in drug discovery. The use of medicinal plants is registered in most ancient archaeological sources, and, even today, plant-derived molecules occupy a significant portion of the pharmaceuticals. The relation of pharmaceutical chemistry and natural product is very old and it provides variety of leads for the development of drugs.

The aim of the present study is to collect the local medicinal plant and evaluate them for their biological activity. Therefore I selected two indigenous medicinal plants Calligonum polygonoides and Crateva adansonii and a comprehensive work has been done on these selected plants for their bioactive components. The whole dissertation has been divided into two parts A and B. The first chapter of this thesis gives a general overview on important compounds isolated from plant and now used as medicine in modern age. It also explains the role of major classes of natural products which serve as antimicrobial and anticancer agents.

Part A

In part A chapter 2 give description of plant family and various species of this family are discussed thoroughly. Brief literature review on bioactive constituents isolated from plants of this family are reported in this chapter.

The chapter 3 is composed of results obtained from present work on Calligonum polygonoides. The aerial portion of Calligonum polygonoides was extracted with methanol and fractionated into three fractions on the base of polarity (n-hexane, chloroform and n- butanol). The crude extract and polarity base fractions were evaluated for xanthine oxidase, carbonic anhydrase and α-glucosidase, tyrosinase and urease inhibitory activities. The

ii crude methanolic extract along with n-butanol showed high inhibition of xanthine oxidase with IC50 value of 43.68±0.4 and 37.74±0.56 µg/mL, carbonic anhydrase with IC50 value of 46.94±0.4 and 32.31± 0.6 µg/mL, α-glucosidase with IC50 value of 59 ± 0.64 and

27.61±0.18 µg/mL respectively. All other fractions failed to inhibit tyrosinase enzyme. n- hexane fraction displayed strong activity against urease (IC50 value of 12± 0.68 µg/mL).

The n-hexane fraction was further screened through GC-MS. The methanolic fraction was analysed via LC-MS. The two most bioactive fraction n-hexane and n-butanol were proceeded for isolation of bioactive compounds. The chromatographic separation result in isolation of total 10 compounds, three sterols from n-hexane fraction and seven flavonoids/glycosides from n-butanol fraction previously not reported from this source.

Structure elucidation of these compounds was carried out by using modern spectroscopic techniques (IR, NMR and mass spectrometry). Sterols displayed remarkable urease inhibitory activity and flavonoids isolated from n-butanol fraction showed strong inhibition of xanthine oxidase, carbonic anhydrase and α-glucosidase.

Part B

In part B Chapter 5 gives details of Capparaceae family and various species of this family are discussed thoroughly. A brief literature review on bioactive constituents isolated from plants of this family is reported in this chapter.

The chapter 6 is composed of results obtained from present work on Crateva adansonii.

The aerial parts of Crateva adansonii is extracted with methanol and fractionated into three fractions on the basis of polarity (n-hexane, chloform and n-butanol). The crude extract and different fractions were assessed for xanthine oxidase, carbonic anhydrase and α- glucosidase, tyrosinase and urease inibitory activities. The crude methanolic extract

iii showed high inhibition of xanthine oxidase with IC50 value of 42.2 ± 0.6µg/mL, carbonic anhydrase with IC50 value of 29.3 ± 0.6 µg/mL, α-glucosidase with IC50 value of 27.95 ±

0.19 µg/mL, tyrosinase with IC50 value of 28.96 ± 0.36µg/mL. Chloroform fraction exhibited high activity against α-glucosidase with IC50 value of 24.15 ± 0.45 and tyrosinase with IC50 value of 17.37 ± 0.47 µg/mL. n-hexane fraction displayed strong activity against urease enzyme (IC50 value of 10±0.5 µg/mL).

The n-hexane fraction was further screened through GC-MS. The methanolic fraction was analysed via LC-MS. The three most bioactive fractions (n- hexane, n-butanol and chloroform) were further selected for isolation of bioactive components. The chromatographic separation resulted in isolation of total 10 compounds, three lupane triterpenes from n-hexane fraction and two pheophorbides and two sphingolipids from chloroform fraction. One alkaloid and two glycosides were isolated from n-butanol fraction. All of above phytochemicals have been discovered for the first time from this source. Triterpenes displayed noteworthy urease inhibitory activity and pheophorbides isolated from chloroform fraction showed strong inhibition of tyrosinase and α- glucosidase. Compounds reported from n-butanol showed significant inhibition of only carbonic anhydrase. Structure elucidation of these compounds was carried out by modern spectroscopic methodologies i.e; IR, NMR and Mass spectrometry.

List of Tables

Table Title of Table Page No. no. 3.1. Antioxidant activity of Calligonum polygonoides 33 3.2. α-glucosidase inhibitory activity of all fractions of C. 34 polygonoides 3.3. Carbonic Anhydrase inhibitory activity of all fractions of C. 34 polygonoides 3.4. Urease inhibitory activity of all fractions of C. polygonoides 35 3.5. Xanthine oxidase inhibitory activity of all fractions of C. 35 polygonoides 3.6. Tyrosinase inhibitory activity of all fractions of C. polygonoides 36 3.7. Identification of the components of n-hexane extract of C. 36 polygonoides 3.8. Positive mode LC-MS Data of Calligonum polygonoides 37 3.9. Negative mode LC-MS Data of Calligonum polygonoides 42 3.10. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 145 in 48 CDCl3 3.11. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 146 in 50 CDCl3 3.12. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 147 in 52 CDCl3 3.13. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 148 in 54 DMSO 3.14. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 149 in 56 DMSO 3.15. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 150 in 58 DMSO 3.16. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 151 in 61 DMSO 3.17. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 152 in 63 DMSO 3.18. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 153 in 65 DMSO 3.19. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 154 in 66 DMSO 3.20. Urease Inhibitory activity of Phytochemicals isolated from 67 Calligonum polygonoides 3.21. Xanthine oxidase Inhibitory activity of Phytochemicals isolated 68 from Calligonum polygonoides 3.22. Carbonic Anhydrase inhibitory activity of phytochemicals 69 isolated from Calligonum polygonoides 3.23. α-glucosidase inhibitory activity of phytochemicals isolated from 71 Calligonum polygonoides

Table Title of Table Page No. no. 7.1. Antioxidant activity of Crateva adansonii 113 7.2. α-glucosidase inhibitory activity of all fractions of Crateva 113 adansonii 7.3. Tyrosinase inhibitory activity of all fractions of Crateva adansonii 114 7.4. Carbonic anhydrasee inhibitory activity of all fractions of Crateva 114 adansonii 7.5. Xanthine oxidase inhibitory activity of all fractions of Crateva 115 adansonii 7.6. Urease inhibitory activity of all fractions of Crateva adansonii 115 7.7. Identification of the components of n-hexane extract of Crateva 116 adansonii 7.8. Positive mode LC-MS Data of Crateva adansonii 117 7.9. Negative mode LC-MS Data of Crateva adansonii 122 7.10. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 161 in 128 CDCl3 7.11. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 201 in 130 CDCl3 7.12. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 202 in 132 CDCl3 7.13. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 203 in 134 DMSO 7.14. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 158 in 137 DMSO 7.15. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 204 in 139 DMSO 7.16. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 205 in 141 DMSO 7.17. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 177 in 144 DMSO 7.18. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 206 in 146 DMSO 7.19. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 207 in 148 DMSO 7.20. Urease Inhibitory activity of Phytochemicals isolated from 149 Crateva adansonii 7.21. Xanthine oxidase Inhibitory activity of Phytochemicals isolated 150 from Crateva adansonii 7.22. Carbonic Anhydrase inhibitory activity of phytochemicals isolated 151 from Crateva adansonii 7.23. α-glucosidase inhibitory activity of phytochemicals isolated from 152 Crateva adansonii

List of Figures

Figure Title of Figure Page No. no. 4.1. TCC and TIC scan (+ mode) of crude methanolic fraction of 78 Calligonum Polygonoides 4.2. LC-MS Ionogram of crude methanolic fraction of Calligonum 78 Polygonoides (+ mode) 4.3 TCC and TIC scan (- mode) of crude methanolic fraction of 79 Calligonum Polygonoides 4.4 LC-MS Ionogram of crude methanolic fraction of Calligonum 79 Polygonoides (- mode) 4.5. Scheme for isolation of pure compounds from n-hexane fraction 81 CpC-N 4.6. Scheme for isolation of pure compounds from butanol fraction 83 CpC-B 6.1. Crystal Structure of Crateva tapia bark lectin (CrataBL) 102 7.1. TCC and TIC scan (+ mode) of crude methanolic fraction of 156 Crateva adansonii 7.2. LC-MS Ionogram of crude methanolic fraction of Crateva 157 adansonii (+ mode) 7.3. TCC and TIC scan (- mode) of crude methanolic fraction of 157 Crateva adansonii 7.4. LC-MS Ionogram of crude methanolic fraction of Crateva 158 adansonii (- mode) 7.5. Scheme for isolation of pure compounds from hexane fraction 159 CaC-N 7.6. Scheme for isolation of pure compounds from chloroform fraction 161 CaC-Cl 7.7. Scheme for isolation of pure compounds from butanol fraction 163 CaC-B

vii

INTRODUCTION

1.1. General Introduction of Natural Products

Phytotherapy has been an imminent practice of medicine since time immemorial.

The Egyptian culture has provided a plethora of documentations regarding medicinal plants. The most famous of these documents is papyrus Ebers, named after the German

Egyptologist Georg Ebers. The documents contained at least 800 recipes and around 700 medicinal plants of both local and foreign origin which were also known among the

Babylonians. It includes names of many medicinal plants still used today like aloe, absinth, peppermint, colocynth, Indian hemp (cannabis) as well as garlic, opium poppy, juniper, cumin, ricinus seeds and Arabic gum (Griffith, 1893).

Li Shizhen, the Chinese pharmacologist of the 16th century, left a document named

Compendium of Materia Medica (Bencao Gangmu) that contained descriptions of plants such as opium poppy, liquorice, ergot, rhubarb, gentian and valerian, indicating that some of the same plants were used in different cultures during the same period, and several of these are still in use today (Zhao et al., 2018). Traditional Indian Ayurvedic system of medicine continues to provide a pertinent tool for the transition of complementary and alternative medicine into modern allopathic system of medicine (Aggarwal et al., 2006,

Chopra and Doiphode, 2002). De Materia Medica written by Pedanius Dioscorides describes 600 medicinal plants and about 1000 medicines obtained from these plants. This document served as a precursor of all of the modern pharmacopeias to date. Avicenna’s

Canon of Medicine established a systematic relationship between natural medicine and pharmacology. It describes the production of juices, tinctures, extracts of herbs, pills with coatings and distillation of alcohol alongwith systematic physiological experimentation and evidence based medicine.

Upto 20th century medicinal products were based on crude preparations like powders or extracts of medicinal plants. However, modern system of medicine relies on pure organic molecules that will activate or inhibit a target protein molecule, thus in turn, effecting a physiological process (Swinney and Anthony, 2011). For this purpose, the science of ethnopharmacology serves as a channel for translation of conventional traditional medicine systems to modern medicine. Ethnopharmacology helps in identification of bioactive phytochemicals from traditional herbal medicine (Leonti and

Casu, 2013, Reyes-Garcia, 2010). During the last 10 years, most of the new medicines that have been registered have their origin from the traditional medicinal plants.

Bioassay guided isolation is an interesting technique for the isolation of bioactive compounds from folk medicine (Balunas and Kinghorn, 2005, Bohni et al., 2013). This procedure includes successive extraction of plant fractions with solvents in order of ascending polarity. Subsequently all fractions are analysed for their activity on different targets (enzymes, tissues, organs, etc.). Following this, the most bioactive plant fraction is subjected to chromatographic techniques (column chromatography, flash chromatography, vaccum liquid chromatography, High Performance Liquid Chromatography, etc.) for isolation of bioactive phytochemicals which are again subjected to bioassay of concerned target for further validation.

Compounds derived from natural sources e.g., plants, animals and microorganisms and also possessing biological activities are defined as natural products. Natural products have been used by human societies for millennia. Historically pharmaceutical companies utilized plant extracts to produce relatively crude therapeutic formulations, but with the advancement of antibiotics in the mid-twentieth century, drug formulations of fairly

purified compounds have become more typical. Natural products have been the major sources of chemical diversity for starting materials for driving pharmaceutical discovery over the past century. Many natural products and synthetically modified natural product derivatives have been successfully developed for clinical use to treat human diseases in almost all therapeutic areas (Newman and Cragg, 2007). The phenomenon of biodiversity produces diverse complex chemical entities due to interactions among organisms and for the purpose of enhancing their survival and competitiveness (Mishra and Tiwari, 2011).

1.1.1. Natural products as antimicrobial agents

Microbial based natural products have proven to be most potent antimicrobials since the discovery of first microbial based antibiotic i.e; penicillin (Clardy et al., 2009).

But due to burgeoning use of antibiotics from mild to severe infections, antimicrobial resistance has been notified as the biggest global epidemic problem that could prove fatal to millions of population (Baker, 2015, Fair and Tor, 2014). Nevertheless, it has become pertinent to discover natural product based antimicrobials due to their structural diversity and added benefit of polypharmacology (Gu et al., 2013, Ho et al., 2018). Several alkaloids have been investigated as potential antimicrobials (Cushnie et al., 2014). Bontemps et al. has isolated pentacyclic pyridoacridine alkaloids from ascidian Cystodytes dellechiajei which include Shermilamine B (1), Kuanoniamine (2), N-deacetylshermilamine B (3), N- deacetyl Kuanoniamine D (4) (Bontemps et al., 2010)

These molecules showed MIC values for E. coli within range of 1.1-2.2 µM compared to gentamicin (MIC 0.08). For Mycobacterium luteus, they showed values between range of

17.4-4.5 µM compared to gentamicin (MIC 0.02). Benzophenanthridine alkaloids have been isolated from stem bark of Zanthoxylum rhoifolium, a brazilian herbal medicine which

were identified as Skimmianine (5), Chelerythrine (6), Magnoflorine (7). Antimicrobial activity was observed gainst seven Gram +ve bacteria and Gram –ve bacteria and seven yeasts which revealed Chelerythrine (6) to be the most active alkaloid (Tavares et al.,

2014). The marine fungus Curvularia produced alkaloid Curvulamine which exhibited

MIC value of 0.37 µM against five human pathogens i.e; Veillonella parvula, Actinomyces israelii, Streptococcus sp., Peptostreptococcus sp., and Bacteroides vulgatus (Han et al.,

2014). Axinellamine (9) isolated from Marine Sponge Axinella exhibited wide spectrum inhibitory activity against both Gram positive and negative organisms specifically inhibiting Candida albicans 32 times higher (Rodriguez et al., 2014).

Several flavonoids have been reported to produce antimicrobial effects.

Phytochemical studies on Scutellaria oblonga yielded nine flavonoids which exhibited mild to moderate inhibitory activity aginst various food pathogens. Three of these flavonoids i.e; techtochrysin (10), negletein (11) and quercetin-3-glucoside (12) also proved to be potent anti-biofilm agents (Rajendran et al., 2016). Myricetin (13) was reported to show high MIC value (256 mg/mL) for Klebsiella pneumoniae expressing extended spectrum β-lactamase and also exhibited significant synergistic effect alongwith different antibiotics i.e; amoxicillin/clavulanate, ampicillin/sulbactam and cefoxitin (Lin et al., 2005)

Abreu et al. has shown that Quercetin (14) and Morin (15) decreased MIC values of ciprofloxacin, tetracycline, and erythromycin by 3-16 times more showing synergistic effect of these flavonoids (Abreu et al., 2015). Garcinia Travancorica, a plant species endemic to India produces a biflavonoid, Fukugiside (16) that has b een shown to inhibit biofilm formation of S. pyogenes at MIC value of 0.08 mg/mL (Nandu et al., 2018).

Triterpenes with various skeletons have been reported to show significant antibacterial effect aginst Staphyloccus aureus (Catteau et al., 2018). Mokoka et al. has studied inhibition potential of triterpenes isolated from Maytenus undata against

Staphyloccus aureus. This study revealed two triterpenes i.e; 3-oxo-11α hydroxyolean-12- ene-30-oic acid (17) and 3,11-dihydroxyolean-12-ene-30-oic acid (18) to possess

significant MIC value (0.024-0.063 mg/mL) (Mokoka et al., 2013). Rotundic Acid (19) has been reported to show broad spectrum antimicrobial activity with MIC values ranging between 25-100 (Haraguchi et al., 1999).

1.1.2. Natural Products as Anticancer agents

Cancer is one of primary causes of morbidity & mortality in humans which needs to be addressed through discovery of anticancer agents. Nature has provided us with several molecules that possess significant anticancer activity (Kinghorn et al., 2009, Kingston,

2011). Many natural products like Vincristine (20) and Vinblastine (21) have proven to be most effective anticancer agents (Martino et al., 2018).

Several Acridone alkaloids i.e. Citrusininine-I (22), Natsucitrine-I (23),

Glycofolinine (24), Citracridone-I (25), 5-hydroxynoracronycine (26), Citracridone-III

(27) isolated from Citrus aurantium demonstrated moderate to potent cytotoxicity (IC50 =

12.65 – 50.74 µM) against different cell lines but they have exhibited four times more cytotoxic activity as compared to PNT2 cell lines (Segun et al., 2018). Tylophorinine (28) and Tylophoriinidine (29) extracted from the roots of Tylophra atrofolliculata have

-8 exhibited significant cytotoxic effects against HCT-8 cell (IC50 8.3 -0.000018 mmol/mL)

and KB cells (IC50 0.00356-0.018 mmol/L) (Huang et al., 2004). Dimeric apomorphine alkaloids; Oviisocorydine (30), Ovihernangerine (31), Oxohernandaline (32) isolated from

Hernandia nymphaeifolia have shown significant cytotoxicity against P-388, KB16, A549, and HT-29 cell lines. (ED50 less than 1µg/ml) (Chen et al., 1996)

Likhitwitayawuid et al. has isolated many alkaloids from Crinum amabile.

Lycorine (33), Crinamine (34) and Augustine (35) showed significant cytotoxic effects against cell lines (Likhitwitayawuid et al., 1993). 2-N Methyltelobine (36), a bisbenzylisoquinoloine alkaloid isolated from Stephania erecta also displayed nonselective cytotoxicity against various human cell lines (Likhitwitayawuid et al., 1993). Three new pentacyclic aromatic alkaloids were reported from ascidian Amphicarpa meridiana and

Leptoclinides sp. All of the alkaloids showed significant cytotoxicity which include

Meridine (37), Meridin-12(13H)-one (38) (Schmitz et al., 1991).

Kim et al. has isolated seven alkaloids [Splendinine (39), Cepharadione B (40),

Norcepharadione B (41), Piperolactam A (42), Aristolactam A (43), Aristolactam B (44)] from Houttuynia cordata which exhibited moderate cytotoxic effects against five human tumor cell lines (A-549, SK-OV-3, SKMEL-2, XF-498 and HCT-15) in vitro (Kim et al.,

2001). Several anticarcinogenic mechanisms (carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of angiogenesis, antioxidation) have been noted for polyphenolic compounds i.e; flavonoids (Chahar et al.,

2011, Wenying et al., 2003). Miranda et al. has reported the cytotoxic effects of flavonoids;

Dehydrocycloxanthohumol (45), Xanthohumol (46), isoxanthohumol (47) isolated from

Humulus lupulus in human breast cancer (MCF-7), colon (HT-29) and ovarian cancer (A-

2780) cell lines (Miranda et al., 1999).

Ethyl acetate extract of flowers of Melastoma malabathricum produced two flavonoids namely; naringenin (48) and kaempferol 3-Sophorotrioside (49). MTT Assay of MCF-7 cell lines showed IC50 values in the range 0.28 and 1.3 µM for both flavonoids

(Susanti et al., 2007).

Baicalin (50) and its glycosidic derivative Baicalein (51) has been shown to produce cytotoxic effects in Leukemia, lymphoma and myeloma via various mechanisms

and acting on several targets (Chen et al., 2014). In leukemia these flavonoids have been noted to induce apoptosis, notch signal pathway, cleavage by caspases 3 & 9 cleavage by

Bid protein (Chen et al., 2013, Shieh et al., 2006, Wang et al., 2009). In Lymphoma they have been shown to induce apoptosis and suppress growth of CA46 cells (Huang et al.,

2012, Lin et al., 2013). In myeloma, Baicalein induces apoptosis, inhibits proliferation and migration (Li et al., 2006, Liu et al., 2010, Xu et al., 2012). Gardenia obtusifolia is a common herbal medicine used in Thailand for a variety of purposes like antibacterial, analgesic, diuretic and hypotensive agent (Hussain et al., 1991, Laurens et al., 1985). 5- hydroxy-2-(3-hydroxy-4-methoxyphenyl)-3,6,7,8-tetramethoxy-4H-chromen-4-one (52) reported from Gardenia obtusifolia has been shown to exhibit cytotoxicity in a variety of cell lines (Lichius et al., 1994, Shi et al., 1995, Zhang et al., 1999) via various mechanisms which include blocking of tumor cell proliferation, colony production, cell cycle at G2/M and sub G1 phases, expression of cell cycle regulated proteins, down regulation of cell survival proteins, apoptosis induction, BAX protein expression, activation of upstream and terminal caspases, suppression of AKT Pathway (Phromnoi et al., 2010).

Triterpenoids are another group of ubiquitous phytochemicals which express a range of biological activities including cytotoxic effects (Dzubak et al., 2006, Laszczyk,

2009, Salvador et al., 2012). Novel oleanane derivatives Leonuronin A (53) and B (54) isolated from Leonurus japonicus have exhibited weak cytotoxicity against A549 and Hela cancer cell lines (IC50 6.97 to 18.13 μM) (Peng et al., 2018).

Cao et al. has isolated three cytotoxic triterpenoids from woody portion of

Mitragyna diversifolia which include Hyptatic Acid B (55), 23-nor-24-exomethylene- 3,

6, 19-trihydroxy-urs-12-en-28-oic acid (56). These triterpenes exhibited significant

cytotoxicity against MCF-7 (breast) and HT-29 (colon) cell lines (Cao et al., 2014). Four novel tricullarane triterpenes ; 21,23-epoxy-21-methoxy-24, 25-dihydroxyapotirucall-7- en-3-one (57), 21,23-epoxy-21-methoxy-25-hydroxyapotirucall-7-en-3,24-dione (58),

21,23-epoxy-21,25-dimethoxyapotirucall-7-en3,24-dione (59), 21,23-epoxy-21a- methoxy-24,25-oxidoapotirucall-7-en-3-one (60) were isolated from Dysoxylum binectariferum which showed substantial toxicity for five tumor cell lines i.e; A-549 (IC50

20-25.6), HCT15 (IC50 22-24.4), HepG2 (IC50 7.5-8.4), SGC-7901 (IC50 21.7-25.8) and

SK-MEL-2 (IC50 23.7-25.4) (Hu et al., 2014)

Sesquiterpene lactones; Artemisinin (61), Artemisinic acid (62), Arteannuin B (63) and other triterpenes Friedelin (64) and Friedelanol (65) isolated from Artemisia annua have shown cytotoxic effects against five tumor cell lines. All compounds exhibited ED50 values greater than 10 (Zheng, 1994). Three new isopimarane diterpenes; Altavnol A-C

(66-68) and one new nor-triterpenes Altavolide D (69) were reported from Ephorbia alatavica and showed significant cytotoxic effects against MCF-8, HeLa and A549 cell lines in vitro by MTT assay (Rozimamat et al., 2018). Lanostane type triterpenes isolated from Tricholoma pardinum were evaluated for cytotoxic effects. Of all 9 isolated compounds Pardinol B (70) and Pardinols E- H (71-74) showed cytotoxic effects on different cell lines (HL-60 SMMC-7721 A-549 MCF-7 SW480). These compounds also inhibited nitric oxcide production within range of IC50 (5.3 ± 1.22- 14.7 ± 1.01 µM) (Zhang et al., 2018).

Ardisiacrispin B (75) is a triterpene saponin isolated from the fruit of Ardisia kivuensis. This compound was evaluated for its cytotoxic effects on leukemia and hepatocarcinoma cell lines. Its IC50 values were as: 0.0012 mmol/L (CCRF-CEM cells),

0.0067 mmol/L [HepG2 cells] and 0.12296 mmol/L (resistant CEM/ADR5000 leukemia cells). Its mechanism of action was noted as apoptosis induction by activating inititator caspases 8 and 9 and effector caspase 3/7, Matrix metalloproteinase alteration and increase in ROS production (Mbaveng et al., 2018).

Part-A

PLANT INTRODUCTION/PREVIOUS PHYTOCHEMICAL

INVESTIGATIONS ON

Calligonum Polygonoides

2.1. Plant Introduction

2.1.1. Plant classification

Kingdom: Plantae

Clade: Angiosperms

Clade: Eudicots

Order: Caryophyllales

Family: Polygonaceae

Subfamily: Polygonoideae

Genus: Calligonum

Species: Polygonoides

2.2. Family Polygonaceae

The Polygonaceae family is also known by the name of buck-wheat or knot-wheat or smartweed family consisting of 40 genera and 1000 species distributed mainly in north temperate regions, a few in tropical, arctic or Southern hemisphere. They are mostly annual or perennial herbs but some species are bushes and small trees (Bhandari, 1978).

2.3. Genus Calligonum

The genus Calligonum (family Polygonaceae) comprises about 80 species, has numerous therapeutic benefits already known and studied, and are distributed throughout northern Africa, southern Europe and western Asia. Most species are sand-fixing plants and are also used as animal feed and firewood. Plants of the genus Calligonum are shrubs, diffused with irregular branching and flexuous woody branches (Phondke G. P., 1992).

2.3.1. Calligonum polygonoides

Calligonum polygonoides Linnaeus, locally known as Phog or Phogra, is the only species of Calligonum found in Pakistan. It is a common woody shrub commonly found in

Thar desert region and adapt to extensively arid conditions. The plant is ecologically valuable as it stabilizes the sand dunes and prevents soil erosion, and is also an important source of food (Bhandari, 1978) . It’s branches are leafless and fleshy green. It produces small numerous flower buds and succulent flowers that are converted into hairy/spiny fruits. The flowers as well as buds have rich composition of different proteins henceforth they are locally used as food. However C. polygonoides also has potential use in folk medicine. Its juice is applied to the eye as an antidote to scorpion sting, the latex is used to treat eczema, dog bites and abortion, and decoction of the roots is used as gargle for sore gum alongwith catechu. Preliminary phytochemical screening of C. polygonoides shows

that flavonoids are present in buds, seeds, flowers and stems. Alkaloids are present in roots, buds, seeds and flowers. Proteins are present in flowers and seed. Tannins, steroids, phenols, carbohydrates and terpenoids are present in roots, stems, buds, flowers and seeds

(Samejo Mq et al., 2011).

2.4. Bioactive constituents reported from the Family Polygonaceae

2.4.1. Calligonum genus 2.4.1.1. Calligonum polygonoides

Fourteen flavonoids (76-85) have been isolated from aerial parts of Calligonum polygonoides which showed potential cytotoxicity against against liver HepG2 and breast

MCF-7 cancer cell lines (Ahmed H et al., 2016). Yawer et al. has isolated two butanolides

(86-87) from choloroform fraction Calligonum polygonoides with moderate inhibitory potential for lipoxygenase (Yawer et al., 2007). Samejo et al. has reported the presence of sterols (88-91) in chloroform fraction of roots of Calligonum polygonoides (Samejo et al.,

2013).

2.4.1.2. Calligonum leucocladum

Resveratrol (92) , Resveratrol 3-O-glucoside-6"-gallate (93), Resveratrol 3-O- xyloside -4"-acetate (94) and other glycosylated derivatives (95-97) have been reported from ethyl acetate and n-butanol fractions of Calligonum leucocladum. Pinosylvin displayed MIC of 125 µg/ml against MRSA whereas reseveratrol displayed synergistic inhibition of MRSA when used in combination with standard drug oxacillin (Okasaka et al., 2004). These compounds have already been reported to show remarkable anticarcinogenic activity (Savouret and Quesne, 2002).

2.4.2. Polygonum genus

2.4.2.1. Polygonum minus

Polygonum minus is traditionally used to treat microbial infections, inflammation

& for gastric protection (George et al., 2014, Hassim et al., 2015, Qader et al., 2012).

Polygonumins (98) are phenylpropanoid glycosides reported from polygonum minus that share structural similarity to vanicoside A (99) isolated from Polygonum sachalinense.

These compounds have shown significant cytotoxicity against several cell lines (HCT116,

C33A, HI 299, MCF7, A549, K 562, V79-4 cells). Polygonumin (98) also inhibited HIV-

1 Protease by 56% in comparison to standard Pepstatin. (Ahmad et al., 2018).

2.4.2.2. Polygonum sachalinensis

Polygonum sachalinensis is a Chinese folk medicine for the treatment of arthralgia, jaundice, amenorrhea, coughs, scalds and burns, traumatic injuries, carbuncles and sores

(Zhonghua Renmin Gongheguo Wei Sheng Bu Yao Dian Wei Yuan, 1997). Seven major compounds have been reported from this species which include Hyperoside(100),

Avicularin (101), and Hydropiperoside (103) and Vanicoside B (104) (Fan et al., 2010).

These compounds were evaluated for acetylcholine esterase and α/β glucosidase inhibitory activities. Hyperoside showed moderate inhibition of acetylcholineesterase. It also showed greater inhibition of α-glucosidase than acarbose. Avicularin failed to inhibit these enzymes (59.5±3.13%).

2.4.2.3. Polygonum multiflorum

P. multiflorum has been used in traditional chinese medicine for its anti-senescence potential (Xiao et al., 1993). Its main active constituent is Tetrahydroxystilbene glucoside

(105) which has been reported to show numerous pharmacological activities like inhibiting neurodegenerative disorders , anti-atherosclerotic, antioxidant and anti-inflammatory effect (Zhang and Chen, 2018).

Li et al. has isolated Tetrahydroxystilbene glucoside based oligomers from roots of

P. multiflorum which were named as multiflorumisides (106-110). These compounds displayed moderate inhibition of nitric oxide synthesis in lipopolysaccharide (LPS)- activated RAW264.7 cells in Griess assay (Li et al., 2019).

2.4.2.4. Polygonum chinense

Zheng et al. have reported five novel compounds; Maysedilactone A (111), maysedilactone D (112), 15-ethyl chebulate (113), 14-methyl, 15-ethyl, 11-desacetate chebulate (114), Lysciumamide D (115) from P. chinense with anti-complement activity

(Zheng et al., 2018).

2.4.2.5. Polygonum barbatum Three dihydrobenzofuran derivatives namely as methyl 2-(3,4-dimethoxyphenyl)-

4-((E)-3-ethoxy- 3-oxoprop-1-en-1-yl)-7-methoxy-2,3-dihydrobenzo-furan-3-carboxylate

(116), (E)-3,2-(3,4-dimethoxyphenyl)- 7-methoxy-3-(methoxy carbonyl)-2,3- dihydrobenzofuran-4-yl)acrylic acid (117) and 4-((E)-2-carboxyvinyl)- 2-(3,4- dimethoxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-3-carboxylic acid (118) were isolated from CH3CH2COOH fraction of P. barbatum. Compound (118) showed higher inhibition of oral cancer cells in comparison to other dihydrobenzofuran derivatives. All compounds suppressed angiogenesis.

2.4.3. Oxygonum genus 2.4.3.1. Oxygonum sinuata Oxygonum sinuata is a folk medicine that has been used in Kenya for variety of unrelated disorders (Kareru et al., 2007). Emodin (119) and Coleon A Lactone (120) have been isolated from Oxygonum sinuata which were evaluated for angiogenesis inhibition.

Both of compounds showed IC50 values similar to SU5416 in chick chorioallantoic membrane assay (CAM) (Crawford et al., 2011).

2.4.4. Rumex genus 2.4.4.1. Rumex nepalensis Rumex nepalensis is used for the treatment of nociception, inflammation, wounds, ringworm infection,carcinogenesis and bowel disorders in Traditional Chinese Medicine.

Two novel naphthalene acylglucosides, Rumexneposides A (121) and B (122) were evaluated for anti-tuberculosis activity. Only Rumexneposide A showed potent inhibition with MIC value of 20.7 µM.(Liang et al., 2010)

2.4.4.2 Rumex crispus

Nepodin/Musizin (123) is a 1,4 naphthoquinone that has been extracted from the underground parts of Rumex Crispus. Geun et al. has investigated antidiabetic effect of

Nepodin. It triggered uptake of glucose and phosphorylated 5’-AMP-activated PK enzyme in a dose dependent manner. Nepodin also suppressed fasting blood glucose levels in in vivo studies (Ha et al., 2014). This molecule has also shown significant inhibition against

Chloroquine sensitive Plasmodium falciparum (IC50=0.79 ± 0.06) and Chloroquine resistant Plasmodium falciparum (IC50= 0.74 ± 0.07) (Lee and Rhee, 2013). Nepodin has also inhibited Candida albicans in nematode infection model and also sufficiently suppressed biofilm formation (Lee et al., 2019).

2.4.4.3 Rumex japonicus

Nishina et al. has also reported Trachrysone (124) and 2-Methoxystypandrone

(125) from the root of R. japonicus. Both compounds exhibited greater antimicrobial activity in comparison to Musizin (123) (Nishina et al., 1993). Physicon 8-O-β- glucopyranoside (126) an active ingredeient of R. japonicus has shown potential cytotoxicity via apoptosis induction, cell cycle arrest (Xie and Yang, 2014) and anti- metastasis (Ding et al., 2016).

2.4.4.4 Rumex aquaticus

Ethyl acetate fraction of R. aquaticus yielded quercetin-3-O-galactoside (127) and quercetin-3-O-arabinoside (128). Both flavonoids greatly improved neuroprotection in rat pheochromocytoma (PC12) cell line which was also deprived of oxygen and glucose

(Orban-Gyapai et al., 2014).

2.4.4.5 Rumex patientia

Two Novel halogenated O-Glycosides Patientosides A (129) and B (130) were isolated from roots of R. patientia which inhibited secretion of IL-6 and overproduction of extracellular matrix production in high-glucose-induced mesangial cells at 10 µM

(Kuruuzum et al., 2001, Yang et al., 2013).

Several phenolics were reported from H2O and CH3OH extract of the roots of R. patientia including emodin-6-O-β-D-glucopyranoside (131), and a new simple halogenated flavan-3-ol, 6-chlorocatechin (132). These compounds showed high DPPH antioxidant activity and significant cytotoxicity against human breast and epidermoid carcinoma as well as melanoma cell lines (Demirezer et al., 2001).

2.4.4.6 Rumex dentatus

Two novel naphthalene glycosides were reported from C2H5OH fraction of roots of

Rumex dentatus; 6-methoxy-3-methyl-8-4,5,6-trihydroxy-2-(hydroxymethyl)tetrahydro-

2H-pyran-3-yl)oxy)-4a,5,6,7,8,8a-hexahydronaphthalene-2-carbaldehyde (133) and 3- methyl-8-4,5,6-trihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-

4a,5,6,7,8,8a-hexahydronaphthalene-2-carbaldehyde (134). These compounds exhibited reasonable antiproliferation activity agaiunst four cell lines namely (breast cancer MCF-7, gastric cancer 7901, melanoma A375 and oophoroma SKOV-3).

2.4.4.7 Rumex gmelini

1-O-caffeoyl glycoside (135), acteoside (136) and caffeic acid (137) isolated from

C2H5OH fractions of R. gmelini highly suppressed leukocyte migration and airway resistance in lungs of ovalbumin sensitized guinea pigs (Lee et al., 2011).

2.4.4.8 Rumex bucephalophorus

Glycosylated stilbenes; Piceid (138) and Rumexoid (139) from roots of Rumex bucephalophorus showed high antioxidant activity in trolox equivalent antioxidant capacity assay. These stibenoids inhibited α glucosidase two fold in comparison to acarbose (Kerem et al., 2006).

2.4.5. Triplaris genus

2.4.5.1. Triplaris cumingiana

Five cytotoxic flavonol glycosides were reported from Triplaris cumingiana which included 2-(3, 4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl-4,6-bis-O-α-

D-(3,4,5-trihydroxybenzoyl) glucopyranoside (140), 5,7-dihydroxy-2-(4-hydroxyphenyl)-

4-oxo-4H-chromen-3-yl-5-O-(3,4,5trihydroxybenzoyl)arabinofuranoside (141), and 2- hydroxy-4-O-(3,5,7-trihydroxy-4-oxo-4H chromen-2-yl) phenylarabinofuranoside (142), were isolated from the young leaves of Triplaris cumingiana, 5-O-galloyl quercetin 3-O- arabinofuranoside (143) and quercetin-3-O-α-D-(6”-O-galloyl)glucopyranoside (144).

These compounds showed potential anti-carcinogenic activity against MCF-7, H-460, and

SF-268 human cancer cell lines (Hussein et al., 2005).

PHYTOCHEMICAL AND BIOLOGICAL PROFILING OF

AERIAL PARTS OF

Calligonum

Polygonoides

3.1. Biological Evaluation of different fractions of Calligonum polygonoides Previous investigations on Calligonum polygonoides have revealed potential activity against lipoxygenase, carcinogenic cell lines and several microorganisms (Ahmad and Akram, 2019, Khan et al., 2015). Since different compounds have been reported from

Calligonum polygonoides (flavonoids, lactones, sterols), it was postulated to evaluate different fractions of Calligonum polygonoides and its phytochemicals against common drug targets; namely, α-glucosidase, carbonic anhydrase, xanthine oxidase, tyrosinase and urease because these activities of this plant species are unexplored. New drug development is a challenging process that requires high-risk, huge costs and long lead times. Therefore, drug repurposing is considered a strategic and economic way towards successful drug development (Gns et al., 2019, Lim et al., 2019). The aforementioned enzymes alongwith serving as potential targets in metabolic, skin and neurological disorders are also expressed by numerous bacteria and carcinogenic cell lines (Chang, 2009, Li et al., 2016, Monti et al., 2013, Nocentini and Supuran, 2018, Nocentini and Supuran, 2018, Pacher et al., 2006,

Supuran, 2008, Supuran, 2012, Supuran, 2018, Tan et al., 2018, Yuan et al., 2019). As

C.polygonoides has exhibited potential antibacterial and cytotoxic activity, significant inhibition of these enzymes will establish its role not only for treatment of bacterial infections and cancer but also as a multidimensional pharmacological agent in therapy of skin disorders, diabetes, glaucoma, epilepsy and other neurological disorders.

3.1.1. DPPH Free radical scavenging assay Following table represents the DPPH Free radical scavenging assay. Standard

Ascorbic acid showed the highest %age inhibition i.e. 92.67±0.33% with IC50 value 22.3

±0.57 followed by crude extract which showed 84±0.57 %age inhibition. Butanolic and n- hexane fractions also changed the color of DPPH and showed comparable %age inhibition

i.e. 89.63±0.65% with IC50 value 2.92±0.39 mg/ml and 50.6±0.31% with IC50 value

3.4±0.45 mg/ml. Chloroform fraction could not bring any change in color and their %age inhibition was less than 50%.

Table 3.1. Antioxidant activity of Calligonum polygonoides Samples Concentration %age IC50 (µg/ml) Inhibition Crude methanolic extract 5 mg/ml 84±0.57 2.7±0.24 n-butanol fraction 5 mg/ml 89.63±0.65 2.92±0.39 Chloroform fraction 5 mg/ml 30.7±0.18 --- n-hexane fraction 5 mg/ml 50.6±0.31 3.4±0.45 Ascorbic Acid 0.5 mmol/ml 92.67±0.33 22.3 ±0.57

3.1.2. α-glucosidase inhibition assay

Following table represents the α-glucosidase inhibition assay. Standard Acarbose showed the highest %age inhibition i.e. 92.67±0.33% with IC50 value. Methanolic

(78±0.4% with IC50 value of 59 ± 0.64) and butanolic fractions (91.68±0.5% with IC50 value of 27.61± 0.18) showed significantly higher % age inhibition. Chloroform fraction

(50.8±0.25%) and n-hexane fraction (20.8±0.11%) exhibited least activity.

Table 3.2. α-glucosidase inhibitory activity of all fractions of C. polygonoides

Samples tested Alpha glucosidase inhibition IC50 (µg/ml) Acarbose 97± 0.22 5.5 ± 0.2 Methanol fraction 78±0.4 59 ± 0.64 n-Butanol fraction 91.68±0.5 27.61± 0.18 Chloroform fraction 50.8±0.25 ---- n-hexane fraction 20.8±0.11 ----

3.1.3. Carbonic anhydrase inhibition assay

Major methanolic and other fractions were tested for inhibition of carbonic anhydrase in comparison to standard drug acetazolamide. Results revealed that butanolic fraction showed highest inhibitory activity at 88.3±0.5%, followed by crude methanolic

fraction at 75.5±0.36%, chloroform fraction at 45.8±0.25% and n-hexane fraction at 36.8±

0.22%.

Table 3.3. Carbonic Anhydrase inhibitory activity of all fractions of C. polygonoides

Samples tested Carbonic Anhydrase inhibition IC50 (µg/ml) Acetazolamide 98±0.11 0.26 ± 0.2 Methanol fraction 75.5±0.36 46.94±0.4 n-Butanol fraction 88.3±0.5 32.31± 0.6 Chloroform fraction 45.8±0.25 ----- n-hexane fraction 36.8± 0.22 -----

3.1.4. Urease inhibition assay

Table 3.4 demonstrates the %age inhibition of urease alongside IC50 values of different fractions of Calligonum polygonoides. The findings suggest that the n-hexane fraction exhibited highest inhibition of urease with inhibition %age of 93.25±0.16% and half maximal inhibitory concentration of 12 ± 0.67 µg/ml. This inhibition %age was very close to inhibition %age of standard inhibitor thiourea i.e: 97.5%.

Table 3.4. Urease inhibitory activity of all fractions of C. polygonoides Samples tested Urease inhibition % IC50 (µg/ml) Thiourea 97.5±0.23 1 ± 0.1 Methanol fraction 58.1±0.26 75.96 ± 0.4 n-Butanol fraction 63.6±0.4 69.72± 0.23 Chloroform fraction 45.8±0.89 ---- n-hexane fraction 93.25±0.16 12± 0.67

3.1.5. Xanthine oxidase inhibition assay

Butanolic fraction showed highest %age inhibition (89.3± 0.7%) of xanthine oxidase with an IC50 value of 37.74±0.56 µg/mL which was followed by methanolic fraction (79.36± 0.5% with IC50 value of 43.68±0.4). n-hexane and chloroform fractions exhibited insignificant activity.

Table 3.5. Xanthine oxidase inhibitory activity of all fractions of C. polygonoides

Samples tested Xanthine oxidase inhibition IC50 (µg/ml) Allopurinol 91.7 ± 0.3 0.28±0.02 Methanol fraction 79.36±0.5 43.68±0.4 n-Butanol fraction 89.3± 0.7 37.74±0.56 n-Hexane fraction 48.2± 0.5 --- CHCl3 fraction 43.6± 0.4 ---

3.1.6. Tyrosinase inhibition Assay

Crude methanolic extract of Calligonum polygonoides and other three fractions were investigated for tyrosinase inhibitory property in comparison to standard drug i.e; kojic acid. Results disclosed that methanolic fraction inhibited tyrosinase at 18.5±0.05, n- butanol fraction at 33.8±0.45, CHCl3 fraction at 15.8±0.80, n-hexane fraction at

11.25±0.56. All fractions showed inhibition %age even less than 50%. Absence of tyrosinase inhibitory activity may be attributed to lack of secondary metabolites showing significant interactions with the active site of tyrosinase enzyme.

Table 3.6. Tyrosinase inhibitory activity of all fractions of C. polygonoides Samples tested Tyrosinase inhibition (5mg/mL) % IC50 (µg/ml) Kojic Acid 97.8±0.02 1.14±0.68 Methanol fraction 18.5±0.05 ------n-Butanol fraction 33.8±0.45 ------Chloroform fraction 15.8±0.80 ------n-hexane fraction 11.25±0.56 ------

3.2. GC-MS analysis of n-hexane fraction of Calligonum Polygonoides

The n-hexane fraction of Calligonum Polygonoides was subjected to GC-MS evaluation in a splitless mode thus revealing presence of 35 compounds.

Table 3.7. Identification of the components of n-hexane extract of C.polygonoides Relative area Peak No. Retention Time (min) Constituents Molecular Formula (%)

1 10.22 3-Hexen-1-ol C6H12O 1.5

2 10.66 1-Hexanol C6H14O 0.85

3 11.58 Benzaldehyde C7H6O 0.56

4 12.12 3-Pentanol, 2,4-dimethyl C7H16O 0.03

5 11.03 Hexanoic acid C6H12O2 1.54

6 12.36 2-Hexenal C6H10O 0.3

7 14.45 2-Octen-1-ol C8H16O 0.77

8 15.28 Benzeneacetaldehyde C8H8O 1.2

9 16.11 1-Octanol C8H18O 0.35

10 17.46 Octanal C8H16O 0.45

11 18.79 Nonanoic acid C9H18O2 1.89

12 19.55 Decanal C10H20O 3.5

13 21.1 Decanoic acid C10H20O2 2.5

14 22.2 Myristic acid C14H28O2 0.633

15 23.16 Palmitoleic acid C16H32O2 10.8

16 24.05 Palmitic acid C16H32O2 12.5

17 24.16 Linoleic Acid C18H32O2 6.2

18 24.25 Margaric acid C17H34O2 15.5

19 24.37 α-linolenic acid C18H32O2 9.3

20 24.56 Nonadecylic acid C19H38O2 1.5

21 24.85 Heneicosylic acid C21H42O2 4

22 28.24 Cycloartenol C30H50O 7.5

23 29.14 Cerotic acid C26H52O2 1.2

24 30.22 Campesterol C28H48O 1.8

25 30.65 Stigmasterol C29H48O 6.99

26 32.57 Nonacosane C29H60 3.7

27 32.89 Squalene C30H50 6.3

28 33.09 Sitostenone C29H48O 5.3

29 34.55 β-Sitosterol C29H50O 12.8

30 34.98 β-Amyrin C30H50O 9.7

31 35.12 Dotriacontane C32H66 20.65

32 36.11 Tritriacontane C33H68 4.55

33 37.58 Untriacontane C31H64 50.16

34 38.55 Tetratriacontane C34H70 12.66

35 39.99 Pentatriacontane C35H72 2.53

3.3. LC-MS Evaluation of Crude Methanolic Extract of Calligonum Polygonoides

Crude CH3OH fraction of Calligonum Polygonoides was submitted to LC-MS analysis (+ve &-ve mode) which thus revealed presence of 97 compounds in +ve mode and

93 compounds in –ve mode.

Table 3.8. +ve mode LC-MS Data of Calligonum Polygonoides Sr. No RT Molecular Mass Identification Formula 1 0.598 121.91752

2 0.6 217.03583 C7H11N3OS2

3 0.602 201.06126 C12H11NS

4 0.604 379.08867 C15H23Cl2N3O2S

5 0.604 161.06839 DL-2-Aminoadipic acid C6H11NO4

6 0.611 179.0788 D-Galactosamine C6H13NO5

7 0.622 341.13102 6-(alpha-D-Glucosaminyl)-1D- C12H23NO10 myo-inositol 8 0.629 103.09885 C5H13NO

9 0.634 220.03041 AG-123 C10H8N2O2S

10 0.637 180.06263 L-Galactose C6H12O6

11 0.645 197.08868 C6H15NO6

12 0.652 265.11451 D-1-[(3- C10H19NO7 Carboxypropyl)amino]-1- deoxyfructose

13 0.654 182.07775 C10H14OS

14 0.663 358.08986 Mebeverine metabolite C15H18O10 (Veratric acid glucuronide) 15 0.669 145.07507 C6H11NO3

16 0.671 144.04157 Methylitaconate C6H8O4

17 0.672 126.03077 C6H6O3

18 0.678 115.06327 D-Proline C5H9NO2

19 0.684 117.07826 C5H11NO2

20 0.693 192.0635 Quinic acid C7H12O6

21 0.693 162.05298 (2R,3S)-2,3-Dimethylmalate C6H10O5

22 0.695 277.11615 1-Deoxyprolyl-fructose C11H19NO7

23 0.712 135.05436 Adenine C5H5N5

24 0.713 304.12674 2'-Deoxymugineic acid C12H20N2O7

25 0.713 187.08388 2-Keto-6-acetamidocaproate C8H13NO4

26 0.727 279.13042 N-(1-Deoxy-1-fructosyl)valine C11H21NO7

27 0.728 129.07918 DL-pipecolic acid C6H11NO2

28 0.766 304.12719 2'-Deoxymugineic acid C12H20N2O7

29 0.788 299.13674 C14H21NO6

30 0.865 304.12649 2'-Deoxymugineic acid C12H20N2O7

31 0.891 129.07685 C6H11NO2

32 0.897 279.13039 N-(1-Deoxy-1-fructosyl)valine C11H21NO7

33 0.936 106.05894 C3H10N2S

34 0.954 299.13014 C13H21N3O3S

35 0.986 258.07606 C12H10N4O3

36 0.986 129.03389 Flucytosine C4H4FN3O

37 0.992 137.07173 C4H12ClN3

38 1.072 299.13056 C13H21N3O3S

39 1.156 120.05771 Phenylacetaldehyde C8H8O

40 1.349 299.13172 C9H21N3O8

41 1.542 99.10304 C6H13N

42 1.619 174.13594 C8H18N2O2

43 1.773 170.02104 Gallic acid C7H6O5

44 2.139 143.05784 Vinylacetylglycine C6H9NO3

45 6.777 300.11646 C9H20N2O9

46 7.339 171.12559 Gabapentin C9H17NO2

47 7.437 210.08924 3-(2,5- C11H14O4 Dimethoxyphenyl)propanoic acid 48 7.493 159.1252 8-Amino Caprylic acid C8H17NO2

49 7.542 446.17703 Crosatoside B C20H30O11

50 8.096 248.12602 N-4-glucosyl—methyl-2- C11H20O6 butenyl adenosine 51 8.416 248.10453 3-OH-4-methoxy-2-phenethyl- C14H16O4 2,3-dihydropyran-6-one 52 8.842 610.15403 Robinetin 3-rutinoside C27H30O16

53 8.967 190.08439 (R)-3-((R)-3- C8H14O5 Hydroxybutanoyloxy)butanoate 54 9.042 464.09584 5,6,7,3',4'-Pentahydroxy-8- C21H20O12 methoxyflavone 7-apioside 55 9.043 478.07541 Isoetin 4'-glucuronide C21H18O13

56 9.17 594.15713 Luteolin 7-rhamnosyl(1- C27H30O15 >6)galactoside 57 9.381 448.10082 6-Hydroxyluteolin 5- C21H20O11 rhamnoside 58 9.415 462.07983 5,6,7,2'-Tetrahydroxyflavone C21H18O12 7-glucuronide 59 9.539 492.09125 Tricetin 3'-methyl ether 7- C22H20O13 glucuronide

60 9.714 185.10562 Ecgonine C9H15NO3

61 9.972 476.09457 Scutellarein 4'-methyl ether 7- C22H20O12 glucuronide 62 10.47 204.09957 2-OH-3-methyl succinic acid C9H16O5 diethyl ether 63 10.719 531.13759 C25H25NO12

64 10.753 302.04263 Melanoxetin C15H10O7

65 11.05 263.22518 C17H29NO

66 11.15 518.14196 Medicarpin 3-O-glucoside-6'- C25H26O12 O-malonate 67 12.206 273.26595 C16 Sphinganine C16H35NO2

68 12.367 289.2619 C16H35NO3

69 12.615 287.28163 C17 Sphinganine C17H37NO2

70 13.392 317.29266 Phytosphingosine C18H39NO3

71 13.746 246.14709 3-Hydroxydodecanedioic acid C12H22O5

72 13.922 315.27786 Dehydrophytosphingosine C18H37NO3

73 13.932 352.2612 2 γ-linolenoyl glycerol C21H36O4

74 13.934 676.36647 (S)-Nerolidol 3-O-[α- C33H56O14 Rhamnose-(1->4)-α-rhamnose- (1->2)-β-glucose] 75 14.285 414.20329 Eplerenone C24H30O6

76 15.173 352.261 2 γ-linolenoyl glycerol C21H36O4

77 15.177 514.31333 C27H46O9

78 15.929 519.33293 C22H41N13S

79 16.239 290.2449 C16H34O4

80 16.294 246.21831 8,8-Diethoxy-2,6-dimethyl-2- C14H30O3 octanol 81 16.697 456.27242 C24H40O8

82 17.116 278.15198 Emmotin A C16H22O4

83 17.117 148.01587 C8H4O3

84 17.118 204.07873 3-Butylidene-7- C12H12O3 hydroxyphthalide 85 17.14 495.33234 PE(19:0/0:0) C24H50NO7P

86 17.173 556.29198 C25H48O11S

87 17.329 260.23414 C15H32O3

88 18.069 402.22475 Acetyl tributyl citrate C20H34O8

89 18.07 328.15163 Rhododendrin C16H24O7

90 18.081 335.30375 C19H37N5

91 18.327 274.25 C16H34O3

92 18.596 278.22499 α-linolenic acid C18H30O2

93 19.337 255.25597 Palmitic amide C16H33NO

94 19.578 281.27232 Oleamide C18H35NO

95 19.964 675.49361 C37H65N5O6

96 20.063 631.466 C34H65NO9

97 20.164 561.4507 C25H23NO14

Table 3.9. -ve mode LC-MS Data of Calligonum Polygonoides Compoun Molecular d No. RT Mass Name Formula

1 0.604 341.13345 His Ala Asp C13H19N5O6

2 0.615 226.07008 5-Acetylamino-6- C8H10N4O4 formylamino-3-methyluracil 3 0.644 216.04411 C6H14Cl2N2O 2 4 0.652 246.05177 C8H11ClN4O3

5 0.653 166.04861 9-Methylxanthine C6H6N4O2

6 0.665 378.09501 C13H19ClN4O 7 7 0.665 440.09276 C15H16N6O10

8 0.668 136.03825 Hypoxanthine C5H4N4O

9 0.67 111.99103

10 0.67 374.1399 C10H18N10O6

11 0.672 372.12781 C14H20N4O8

12 0.675 534.1793 C19H34O17

13 0.679 192.06458 C8H8N4O2

14 0.683 180.06432 Theobromine C7H8N4O2

15 0.698 126.00955 C9H2O

16 0.712 340.106 C13H17ClN6O 3 17 0.714 350.13401 C14H18N6O5

18 0.715 206.04371 Methylisocitric acid C7H10O7

19 0.751 134.02268 2,5-dimethoxy thiophene C5H10S2

20 0.923 291.09619 B181008 C12H13N5O4

21 0.977 192.02724 Citric acid C6H8O7

22 0.991 129.04298 N-Acryloylglycine C5H7NO3

23 1.087 345.14302 4,5-didemethylsimmondsin C15H23NO8

24 1.088 299.13797 C15H17N5O2

25 1.313 332.07541 beta-Glucogallin C13H16O10

26 1.345 148.03711 D-α-Hydroxyglutaric acid C5H8O5

27 1.385 299.13763 C14H21NO6

28 1.415 382.05796 C14H14N4O7S

29 1.588 154.03936 Diethylphosphate C4H11O4P

30 1.791 348.0304 C16H12O7S

31 1.793 170.02194 2,4,6-Trihydroxybenzoic acid C7H6O5

32 1.819 284.01505 C11H8O9

33 1.938 206.04263 Methylisocitric acid C7H10O7

34 3.065 316.07998 C13H16O9

35 3.193 154.02999 Diethyl sulfate C4H10O4S

36 3.832 154.02655 3,4-Dihydroxybenzoic acid C7H6O4

37 4.729 380.0786 4-Methoxybenzyl O-(2- C14H20O10S sulfoglucoside) 38 6.819 346.12687 1-(3-OH-4-Methoxyphenyl)- C15H22O9 1,2-ethanediol 3'-O-glucose 39 7.182 294.13177 Ethyl (S)-3-hydroxybutyrate C12H22O8 glucoside 40 7.208 426.08373 C15H22O12S

41 7.209 184.03722 4-O-Methyl-gallate C8H8O5

42 7.221 298.03064 1-Pyrenylsulfate C16H10O4S

43 7.458 290.04712 C12H10N4O3S

44 7.561 446.17985 Crosatoside B C20H30O11

45 7.596 240.06386 2-Succinyl 6-OH-2,4- C11H12O6 cyclohexadiene-1-carboxylate 46 7.63 290.06537 Isoprothiolane C12H18O4S2

47 7.776 282.13205 C11H22O8

48 7.834 320.05754 C13H12N4O4S

49 7.908 328.11647 Ethylvanillin glucoside C15H20O8

50 8.424 500.13745 Lyoniresinol 9'-sulfate C22H28O11S

51 8.433 372.10722 Dihydroferulic acid 4-O- C16H20O10 glucuronide 52 8.448 470.1265 C22H22N4O6S

53 8.55 440.11491 Hallactone B C20H24O9S

54 8.65 304.06177 C12H16O7S

55 8.711 198.0526 2-Hydroxy-3,4- C9H10O5 dimethoxybenzoic Acid 56 8.829 174.01568 Dehydroascorbic acid C6H6O6

57 8.859 610.15355 Robinetin 3-rutinoside C27H30O16

58 8.983 190.0838 (R)-3-((R)-3- C8H14O5 Hydroxybutanoyloxy)butanoa te 59 9.056 942.16909

60 9.059 464.09603 Hypolaetin-8-glucoside C21H20O12

61 9.06 478.07597 Isoetin 4'-glucuronide C21H18O13

62 9.128 190.08417 (R)-3-((R)-3- C8H14O5 Hydroxybutanoyloxy)butanoa te 63 9.184 594.15877 Luteolin 7-rhamnosyl(1- C27H30O15 >6)galactoside 64 9.253 248.08992 C10H16O7

65 9.293 304.05878 Dihydrorobinetin C15H12O7

66 9.315 274.05141 C11H14O6S

67 9.395 448.10191 6-Hydroxyluteolin 5- C21H20O11 rhamnoside 68 9.426 1386.243

69 9.426 1848.3207

70 9.431 462.08177 Scutellarein 5-glucuronide C21H18O12

71 9.553 492.09147 Tricetin 3'-methyl ether 7- C22H20O13 glucuronide

72 9.983 476.09596 Scutellarein 4'-methyl ether 7- C22H20O12 glucuronide 73 10.73 534.13944 6,8-Di-C-beta-D- C25H26O13 arabinopyranosylapigenin 74 10.77 302.04345 Melanoxetin C15H10O7 1 75 10.86 346.10919 C15H22O7S

76 10.96 376.11987 C16H24O8S 2 77 11.16 518.14349 Medicarpin 3-O-(6'- C25H26O12 1 malonylglucoside) 78 11.62 286.04835 5,7,2',3'-Tetrahydroxyflavone C15H10O6 5 79 12.53 222.12554 (6S)-dehydrovomifoliol C13H18O3 9 80 13.41 294.18381 Gingerol C17H26O4 8 81 13.93 722.3734 C35H54N4O12 6 82 14.61 724.38915 C35H56N4O12 3 83 15.13 578.27827 C28H42N4O7S 7 84 15.15 700.38915 C33H56N4O12

85 15.17 560.32087 C29H44N4O7 9 86 15.72 596.29705 Salannin C34H44O9 7 87 15.93 565.33921 C26H51N3O8S 4 88 17.04 312.17625 N-Undecylbenzenesulfonic C17H28O3S 1 acid 89 17.11 547.13931 C25H25NO13 3 90 18.17 326.19239 2-Dodecylbenzenesulfonic C18H30O3S 7 acid 91 18.60 278.2249 9Z,12Z,15E-octadecatrienoic C18H30O2 4 acid 92 20.33 356.16661 C19H24N4OS 6 93 20.36 135.90279 2

GC-MS Spectra displayed rich and diverse constituents of n-hexane fraction. LC-

MS analysis of crude methanolic fraction revealed the presence of a high amount of various secondary metabolites. Since methanolic fraction was further fractionated to n-hexane,

CHCl3 and Butanolic fractions, only bioactive n-hexane and butanol fractions were further subjected to chromatographic elution for isolation of bioactive natural products.

3.4. Structure Elucidation of Compounds Isolated from n-hexane fraction (CpC-N)

3.4.1. Cycloartenol (145)

Compound 145 was obtained as colorless crystals. The molecular formula was

+ determined as C30H50O by HR-ESI-MS [M+H] at m/z 426.3978 a.m.u. (calculated

426.7256 a.m.u.). The IR spectrum showed distinguishing bands at C–H broad band

-1 -1 -1 (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1380 cm ), sharp OH band (3600 cm ).

1H-NMR Spectrum (Table 3.10) indicated the presence of a doublet of doublet at δ

3.5 (J= 13.0, 4.5 Hz, 1H, H-3) which was attributed to an oxymethine. Six CH3 singlets at

δ 0.8, 0.99, 1.2, 1.4, 1.65, 1.75 and a methyl doublet at δ 1.0 (J=7.0 Hz, Me-21) displayed characteristic of tetracyclic triterpene skeleton of cycloartane series.

13C-NMR Spectrum (BB and DEPT) displayed seven methyl signals at δ 15.5, 18.2,

19.18, 20.1, 25.5, 26.0, 37.2, eleven methylene at δ 22.6, 24.9, 26.5, 27.1, 28.5, 30.0, 32.0,

33.0, 34.1, 36.0, 36.8, six methines at δ 19.1, 48.0, 48.1, 53.2, 79.1, 127.1 and six quarternary carbons at δ 20.2, 27.0, 40.8, 45.2, 50.2 and 131.0. 13C-NMR Spectrum also displayed a highly downfieleded shift in C-3 of ring A (δ79.1) which was postulated to be due to strong electronegative effect of –OH group on methine carbon.

ESI-MS Spectrum of compound 145 displayed characteristic fragments at m/z 69,

95, 109, 204, 340, 395, 410 (García Md et al., 1997). When all of spectra were equated with previous literature compound 145 was deciphered as Cycloartenol (Haba et al., 2007,

Zare et al., 2015).

1 13 Table 3.10. H (400 MHz) and C-NMR (200 MHz) of compound 145 in CDCl3

13C-NMR Multiplicity 1H-NMR (δH) coupling Position (δC) (DEPT) constants JHH (Hz) 1.68 (ddd, J = 13, 4.5,0.9),1.3 (ddd, J = 1 33 CH2 13, 4.5,0.9) 2 32.01 CH2 1.89, 1.9 (qd, J = 12.8, 4.5) 3 79.11 CH(OH) 3.5 (dd, J = 13,4.5) 4 40.88 C 5 48.05 CH 1.3 (dd, J = 13,4.5) 6 22.66 CH2 1.7 m 7 26.5 CH2 1.0, 1.4 (dddd, J = 12.5, 5.8, 4.2, 3.0) 8 48.15 CH 1.6 (dd, J = 13, 6) 9 20.23 C 10 27.01 C 11 27.09 CH2 2.0,1.2 (ddd, J = 15, 10, 8) 12 34.15 CH2 1.7 m 13 45.2 C 14 50.25 C 15 36.04 CH2 1.35 m 16 28.5 CH2 2.0 m 17 53.18 CH 1.65 m 18 19.18 CH3 0.99 (s) 19 30.09 CH2 0.62 (d , J = 5.0) 20 19.15 CH 1.5 m 21 37.19 CH3 1.01 (d, J = 7.0) 22 36.82 CH2 1.2, 1.6 (dddd, J = 13.8, 11.5, 6.9, 5.5) 23 24.95 CH2 2.0, 1.89 (ddt, J = 13.9, 11.5, 7.0) 24 127.11 CH 5.1 m 25 131.05 C 26 18.2 CH3 1.65 (s) 27 25.55 CH3 1.75 (s) 28 26.0 CH3 1.2 (s)

29 15.5 CH3 1.4(s) 30 20.1 CH3 0.89 (s)

3.4.2. Beta-Amyrin (146)

Compound 146 was purified in the form of colorless crystals. The molecular formula was ascertained as C30H50O by HR-ESI-MS [M+H]+ at m/z 426.40125 a.m.u.

(calcd. 426.6566 a.m.u.). IR spectrum showed characteristic bands at C–H broad band

-1 -1 -1 (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm ), sharp OH band (3615 nm ), C=C band (1650 cm-1).

The 1H-NMR spectrum (Table 3.11) exhibited eight methyl singlets at δ 0.75, 0.8,

0.85, 0.95, 0.98, 1.0, 1.01, 1.2. 1H-NMR spectrum shows a double doubelet at δ 3.18 (J =

4.6,11.0 Hz) which was attributed to an oxymethine proton and a multiplet at δ5.15 which was ascribed to an olefiniec proton.

13C-NMR spectrum of 146 revealed seven methyl reonances at δ 15.5, 17.2, 23.9,

26.3, 28.7, 29.06, 34.16, ten methylene resonances at δ 18.7, 23.5, 26.11, 26.15, 27.5, 32.6,

35.3, 36.82, 38.8, 47.2, five methine resonances at δ 33.0, 47.5, 48.2, 56.1, 79.8, and 120.9, and seven quarternary carbon resonances δ 30.8, 38.8, 37.2, 40.01, 41.2 and 144.9 13C-

NMR Spectrum depicted an olefinic –CH resonance at δ120.9 which was supported by an

olefinic triplet at highly downfielded shift of δ5.15 in electronegative 1H-NMR spectrum.

A highly downfielded methine resonance associated with ring A was found at δ79.8 in 13C-

NMR due to strong electronegative nature of -OH substitution. Both 1H-NMR & 13C-NMR elucidated structure of molecule to be a pentacyclic oleanane type triterpene. The mass spectrum exhibited characteristic peaks for β-amyrin at m/z 218, 204, 257, 69, 95, 120

(Ching et al., 2011). All the spectroscopic data was correlated with previous literature and compound 146 was reported as β-Amyrin. (Agrawal Pk and Jain Dc, 1992, Okoye et al.,

2014).

1 13 Table 3.11. H (400 MHz) and C-NMR (200 MHz) of compound 146 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 38.8 CH2 1.56, 1.52 2 27.5 CH2 1.53, 1.58 3 79.8 CH(OH) 3.18 (dd, J = 4.6; 11.0) 4 38.8 C 5 56.1 CH 0.71 (d, J = 10.8) 6 18.7 CH2 1.5, 1.28 7 32.6 CH2 8 40.01 C 9 47.5 CH 1.9 10 37.2 C 11 23.5 CH2 1.85 12 120.9 CH 5.15 m 13 144.9 C 14 41.2 C 15 26.11 CH2 1.9 (td, J = 4.1; 13.75) 16 26.15 CH2 1.8 (td, J = 4.0; 13.8) 17 33 CH 18 48.2 CH 1.98 19 47.2 CH2 1.95 (dd, J = 4.0; 14) 20 30.8 C 21 35.3 CH2 1.69 22 36.82 CH2 1.75 m 23 29.06 CH3 0.8 (s) 24 15.5 CH3 1.0 (s)

25 15.5 CH3 0.75 (s) 26 17.2 CH3 1.01 (s) 27 26.3 CH3 1.2 (s) 28 28.7 CH3 0.85 (s) 29 34.16 CH3 0.95 (s) 30 23.9 CH3 0.98(s)

3.4.3 Stigmasterol (147)

Compound 147 was obtained as amorphous solid. The molecular formula was determined as C29H48O by HR-ESI-MS [M+H]+ at m/z 426.40125 a.m.u. (calculated

426.6566 a.m.u.). The infra-red spectrum showed exclusive bands at C–H broad band

−1 -1 -1 -1 (2,970-2,850 cm ), CH3 (1375 cm ), sharp OH band (3600 cm ), C=C band (1630 cm ).

The mass spectrum of compound 147 exhibited characteristic fragment ions at 120,

125, 133, 160, 250, 270, 300 for stigmasterol according to fragmentation pattern of Δ5,22 sterol (Suttiarporn et al., 2015). The proton NMR Spectrum (Table 3.12) exhibited upfielded –CH3 singlets at δ 0.7 (Me-18) & 0.95 (Me-19) and methyl doublets at δ 0.8 (d,

J = 7.8, Me-29), 0.82 (d, J=7.0, Me-27) and 0.87 (d, J = 6.8, Me-26). Olefinic Methine protons resonated downfield at δ 5.56 (br s,H-6) and δ 5.15 (dd, J = 8.6,5.2, H-22,H-23) owing to sp2 hybridization. Electronegative hydroxyl substituent also downfielded methine resonance of cyclic carbon 3 to δ70.85. 13C-NMR Data showed 29 carbon atom signals

which were resolved via DEPT as quarternary at δ 40.25, 41.5, 44.1, eight methylene at δ

24.2, 29.8, 29.89, 37, 37.8, 38.12,70.03, 73.3, 124.4, six methyl at δ 12.5, 19.8, 20.1, 21.5,

23.0, 59.8 and ten methines at δ 29.3, 30.7, 31.1. 49.26, 61.0, 70.5, 70.85, 128.98, 143.0 and 151.0 All of the above data matched with reported literature and henceforth compound

147 was concluded to be stigmasterol (Forgo and Kover, 2004, Vassallo et al., 2013).

1 13 Table 3.12. H (400 MHz) and C-NMR (200 MHz) of compound 147 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 37.0 CH2 1.85, 1.1 m 2 29.8 CH2 1.85, 1.45 m 3 70.85 CH(OH) 3.50 m 4 29.89 CH2 2.31, 2.25 m 5 41.5 C 6 70.5 CH 5.56 br s 7 70.03 CH2 2.0 , 1.55 m 8 128.98 CH 1.48 m 9 143.0 CH 1.05 m 10 40.25 C 11 24.2 CH2 1.6 m 12 38.12 CH2 1.89, 1.2 m 13 44.1 C 14 49.26 CH 0.99 m 15 37.8 CH2 1.52, 0.98 m 16 73.3 CH2 1.75, 1.3 m 17 61.0 CH 1.28 (q, J= 10 ) 18 12.5 CH3 0.7 (s) 19 20.1 CH3 0.95 (s) 20 30.7 CH 2.1 m 21 19.8 CH3 1.0 (d J = 6.25) 22 37.09 CH 5.15 (dd, J = 8.6,5.2)) 23 29.3 CH 5.15 (dd, J = 8.6,5.2) 24 151.0 CH 1.55 m 25 31.1 CH 1.55 m 26 21.5 CH3 0.87 (d, J =6.8) 27 23.0 CH3 0.82 (d, J =7.0) 28 124.4 CH2 1.39 m 29 59.8 CH3 0.80 (d, J=7.8)

3.5 Structure Elucidation of Compounds Isolated from Butanolic fraction (CpC-B)

3.5.1 Rhododendrin (148)

Compound 148 was obtained as colorless needles with solubility in CH3OH. The

+ molecular formula was ascertained as C16H24O7 by HR-ESI-MS [M+H] at m/z 328.15163 a.m.u. (calcd. 331.16995 a.m.u.). The IR spectrum showed characteristic bands at 3355

(OH phenolic), 2200 cm-1 (C=C), 2880 (CH3 Aliphatic). This compound was identified in

LC-MS Spectrum (+ve mode) at retention time of 18.07 min (Compound 89).

1H-NMR Spectrum (Table 3.13) displayed doublets at 6.9 (d, J = 8.5 Hz, H-2’, H-

6’) alongside a doublet at 6.7 (d, J = 8.5, H-3’, H-9’) and a singlet at δ 8.2 (4’-OH).1H-

NMR spectrum also exhibited a multiplets at δ 3.9 ( H-2) for methine alongwith two methylene resonances at δ 1.73 (td , J = 7.1, 5.5 Hz, H-3) and δ 2.7 (t, J = 7.2 Hz, H-4). An upfielded methyl doublet at δ 1.27 (J=6.5 Hz, H-1) & downfielded hydroxymethine singlet at δ 8.2 (H-4’) in 1H-NMR Spectrum pointed towards presence of aliphatic pentanol side chain. A doublet at δ4.2 (J = 8.0 Hz, H-2”’) was attributed to anomeric proton of β-glucose.

13C-NMR Spectrum exhibited one methyl signal at δ 21, three methylene signals at

δ 32.2, 39.5, , 132, ten methine signals at δ 19.2, 72.5, 75.7, 78.8, 78.9, 79.8 100.11, 117.7,

117.8, 130.78 and 155.5, , and one quarternary carbon signal at 135.01. Carbon signals at

δ100.11, 75.7, 78.9, 72.5, 79.8 and 62.5 were attributed to a hexose moiety which was inferred as glucose by further elucidation of structural reporter region in the range of δ3.9-

4.2 within 1H-NMR Spectrum. All of the aforementioned data was matched with previously described literature & henceforth, compound 150 was deduced as

Rhododendrin (Kim et al., 2011, Smite E et al., 1993)

Table 3.13. 1H (400 MHz) and 13C-NMR (200 MHz) data of compound 148

Multiplicit 13C-NMR y 1H-NMR (δH) coupling Position (δC) (DEPT) constants JHH (Hz) 1 21 CH3 1.27(d, J=6.5) 2 78.8 CH 3.9 m 3 39.5 CH2 1.73 m 4 32.2 CH2 2.7 m 1' 135.01 C 2' 129.2 CH 6.9 (d, J = 8.5) 3' 117.7 CH 6.7 (d, J = 8.5) 4' 155.5 C-OH 8.2 (s) 5' 117.7 CH 6.7 (d, J = 8.5) 6' 130.78 CH 6.9 (d, J = 8.5) 2'" 100.11 CH 4.2 (d, J = 8.0) 3'" 75.7 CH 3.12 (dd, J = 7.8, 7.9) 4'" 78.9 CH 4.01 (dd, J = 11.8, 7.8) 5'" 72.5 CH 3.3 (d, J = 6.6) 6'" 79.8 CH 3.52 (dt, J = 10.3, 6.6) 3.9 (dd , J = 11.0, 2.0), 3.75 (dd , J = 7'" 62.5 CH2 11.0, 5.0)

3.5.2 Glucogallin (149)

Compound 149 was isolated in form of dark yellow amorphous powder with solubility in CH3OH. The molecular formula was ascertained as C13H16O10 by HR-ESI-MS

[M-H]- at m/z 332.07541 a.m.u. (calcd. 332.07143 a.m.u.). The infra-red spectrum

-1 exhibited characteristic bands at 3350 (OH phenolic), 1730 (C=O carboxyl) and 1520 cm

(C=C) and 1020 cm-1 (glycoside linkage).

This compound was identified in LC-MS Spectrum (-ve mode) at retention time of

1.313 min (Compound 25).

Aromatic region of 1H-NMR Spectrum (Table 3.14) revealed two methine doublets at δ 7.31 (J = 2.8, H-2) and δ 7.45 (J=2.8, H-6) and 3 hydroxymethine singlets at δ 5.02 ,

7.56, 7.67 (3-OH,4-OH,5-OH). This observation indicated the galloyl derived molecular nature of 149. Anomeric proton gave specific signals at δ 4.7 (J=2.9 Hz, H-2’) alongwith other glucose protons at δ 3.34, 3.39, 3.54, 3.80 and 4.02.

13C-NMR spectrum exhibited 5 -CH and two quarternary carbon resonances in aromatic region and five nethine and one methylene signal in glucose region (structural reporter region). Existence of gallic acid moiety was further verified on observation of upfielded δC 146.25, 140.01, 109.01 in the aromatic region due to strong shielding effect of polyhydroxylation. An additional downfielded carbonyl resonance at δC 167.07 highlighted presence of carboxylic group of gallic acid. 13C-NMR spectrum displayed showed additional two methines, three hydroxymethine, and one methylene resonance which averred the presence of glucose mediety as also confirmed via structural reporter region further upfield in the 1H-NMR Spectrum. All the data for compound 149 for was

correlated with previously reported work (Ayoub, 2003, Ling et al., 2002, Puppala et al.,

2012) and compound 149 was inferred as Glucogallin.

Table 3.14. 1H (400 MHz) and 13C-NMR (200 MHz)of compound 149 in DMSO Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 122.11 C 2 109.01 CH 7.31 (d, J = 2.8) 3 146.25 C-OH 7.67(s) 4 140.01 C-OH 5.02 (s) 5 146.25 C-OH 7.56 (s) 6 110 CH 7.31 (d, J = 2.8) 7 167.07 C=O 2' 98.62 CH 4.7 (d, J = 2.9) 3' 72.5 CH 3.34 m 4' 77.32 CH 3.39 m 5' 70.99 CH 3.54 m 6' 79.56 CH 3.8 m

7' 63.14 CH2 4.02, 4.03 m

3.5.3 Hypolaetin-8-glucoside (150) Compound 150 was isolated as dark yellow amorphous powder with solubility in

CH3OH. The molecular formula was elucidated as C21H20O12 by HR-ESI-MS [M-H]- at m/z 464.09603 a.m.u. (calculated 464.09201 a.m.u.). The infra-red spectrum showed

characteristic bands at 3350 (OH phenolic), 1710 (C=O carboxyl) and 1520 cm (C=C) and

1022 cm-1 (glycoside linkage).

This compound was identified in LC-MS Spectrum (-ve mode) at retention time of

8.86 min (Compound 60).

The proton NMR spectrum (Table 3.15) of 150 displayed typical ABX splitting pattern in aromatic region due to three B-ring protons at 훿H 7.0 (d, J = 1.8 Hz, H-2’), 7.2

(d, J = 8.4 Hz, H-5’) 7.32 (dd, J = 8.20, 1.9, H-6’). In this region, six methine singlets were noticed at δ 6.4, 6.5, 11.1, 12.25, 9.95, 8.02 . This data depicted the exitence of a flavone luteolin skeleton. The structural reporter region also exhibited a doublet due to resonance of anomeric proton δ5.7 (J=2.89 Hz, H-2”) alongwith other signals at δ 3.65 (dd, J= 2.5,

3.1 Hz), 3.32 (dd, J= 2.5, 4.1 Hz), 3.15 (dd, J= 4.1, 3.4 Hz) and 3.20 (dd, J= 10.2, 3.4 Hz) thus confirming presence of β-hexose moiety.

The 13C NMR spectrum displayed 21 signals, from which one carbonyl resonance and 14 aromatic carbons for a luteolin moiety were discerned. The remaining six carbon

NMR resonances, which were derived from the 1D carbon spectrum, comprised one quarternary carbon at δ 119.5, three hydroxymethine at δ 71.12, 76.1, 78.16, one oxymethine at δ 106.55 and one hydroxymethylene group at δ 62.54 suggested the presence

of β-D-Glucose. Downfield shift in a carbon of ring A (δC 130.12) corresponded to glycosylation in this portion. Anomeric methine resonated at δ 105.95 ppm suggesting the molecule to be an O-glycoside.

The existence of sugar was also substantiated by the peak at m/z 255.0712 a.m.u. in ESI spectrum due to loss of sugar portion at M+ 45 peak. The LC-MS, 1H-NMR and 13C-

NMR collectively established the structure of compound 150 to be a glycosidic derivative of Hypolaetin which is 8 hydroxylated derivative of luteolin. All the data described above corresponded to the data for Hypolaetin 8-glucoside (El-Garf I et al., 1999, Markham Kr and Porter Lj, 1975, Tomas F et al., 1985)

Table 3.15. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 150 in DMSO 13C-NMR Multiplicity 1H-NMR (δH) coupling Position (δC) (DEPT) constants JHH (Hz) 2 164.5 C 3 106.1 CH 6.5 (s) 4 183.6 C=O 5 158.2 C-OH 12.25 (s) 6 100.02 C-H 6.4 (s) 7 161.99 C-OH 11.1 (s) 8 130.12 C 9 151.22 C 10 104.21 C 1' 119.5 C 2' 114.3 CH 7.0 (d, J = 1.8) 3' 147.5 C-OH 9.95 (s) 4' 150.23 C-OH 8.02(s) 5' 115.42 CH 7.2 (d, J = 8.4) 6' 190.56 CH 7.32 (dd, J = 8.20, 1.9) 2" 62.54 CH 5.7 (d, J = 2.89) 3" 71.12 CH 3.65 (dd, J = 2.5, 3.1) 4'' 78.16 CH 3.32 (dd, J = 2.5,4.11) 5" 76.1 CH 3.15 (dd, J = 3.38,4.11) 6" 78.25 CH 3.7 (td, J = 6.5,3.2)

4.29 (d , J = 6.61), 3.20 (dd, J = 10.2, 7" 106.55 CH2 3.34)

3.5.4. Isoetin-4’-O-glucuronide (151)

Compound 151 was isolated as a yellow solid with solubility in CH3OH. The molecular formula was found out as C21H18O13 by HR-ESI-MS [M+H]+ at m/z 478.07541 a.m.u. (calcd. 464.09567a.m.u.). The infrared spectrum of compound 151 displayed characteristic bands for -OH, carboxylic C=O, and gamma-pyrone alongwith aromatic

C=C. The C=O band at 1742 cm–1 in the infrared spectrum proposed that the sugar portion was a uronic acid moiety.

This compound was identified in LC-MS Spectrum (+ve mode) at retention time of

9.043 min (Compound 55).

The proton NMR spectrum (Table 3.16) of 151 showed AX splitting in aromatic region with a doublet including two meta protons at δH 6.23 (1H, d, J = 2.01 Hz, H-6) and

δH 6.5 (1H, d, J = 2.2 Hz, H-8) as well as two doublets at δ 7.37 (1H, d, J = 0.5 Hz, H-2’) and δ 6.52 (1H, d, J = 0.61 Hz, H-5’). In addition to this, singlets were noticed at δ 6.52,

6.71, 7.37, 9.12, 11.5, 11.81, 10.67 (H-3,2’,5,6,3’,5’,6’). The structural reporter region

corroborated the sugar portion to be a 6-deoxysugar. A doublet of anomeric methine was observed at δ 45.01 (1H, J = 7.0, H-2”) and a single downfielded hydroxymethine singlet was observed at δ 11.71 (OH-7”).

The carbon NMR Spectrum (BB and DEPT) indicated the presence of 9 -CH resonances at δ 95.59, 99.89, 102.55, 105.1, 112.01, 141.1, 153.45 ,165.2, 165.6 and six quaternary carbons at δ 105.6, 115.5, 148.89, 160.12, 170.01, 180.55 and five methine signals at δ 71.45, 73.44, 74.0, 76.15 , 104.45, and a single quarternary carbon at δ 174.17 consistent with the molecular formula C21H18O13. Down field absorption of an additional quarternary carbon in 13C-NMR Spectrum at δC 174.17 was attributed carboxylic moiety of glucuronide portion. The existence of M+29 peak in the ESI-MS spectrum at m/z

175.0124 a.m.u. affirmed sugar portion to be glucuronide. Comparison with previous literature and above information indicated the structure of compound 151 as Isoetin-4’-O- glucuronide (Pauli Gf and Junior P, 1995).

Table 3.16. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 151 in DMSO Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 2 170.01 C 3 105.1 CH 6.71 (s) 4 180.55 C=O 5 165.2 C-OH 11.5 (s)

6 99.89 CH 6.23 (d, J = 2.01) 7 165.6 C-OH 11.81 (s) 8 95.59 CH 6.5 (d, J = 2.2) 9 160.12 C 10 105.6 C 1' 115.5 C 2' 112.01 CH 7.37 (s) 3' 141.1 C-OH 6.8 (s) 4' 148.89 C 5' 102.55 CH 6.52 (s) 6' 153.45 C-OH 10.67 (s) 2" 104.45 CH 5.01 (d, J = 7.0) 3" 73.44 CH 3.29 (dd, J= 3.5, 2.9) 4" 74 CH 3.4 (dd, J = 10.17, 3.5) 5" 71.45 CH 3.65 (dd, J = 10.16, 10.35) 6" 76.15 CH 4.2 (d, J = 10.44) 7" 174.17 C=O 7" OH 11.71 (s)

3.5.5 Medicarpin 3-O-glucoside-6'-O-malonate (152)

Compound 152 was obtained as white crystalline solid. The infrared spectrum displayed characteristic bands at 3350 (OH phenolic), two carbonyl absorptions at 1735 &

1695 cm-1, 1520 cm-1 (C=C), 1100 cm-1 (methoxy group). This compound was identified in LC-MS Spectrum (+ve mode) at retention time of 11.15 min (Compound 66).

Interpretation of the signals in the 1H NMR spectrum (Table 3.17) at δ 3.2 (ddd, J

= 10.06, 9.45, 1.98, H-6a), δ 4.3 (dd, J = 5.0,11.0 Hz, H-6), δ 3.65 (dd, J = 10.5,11.0 Hz,

H-6), 7.5 (d, J = 8.4, H-1) and 5.7 (d, J = 8.5, H-11a) with ABX splitting pattern indicated pterocarpananoid character of the molecule. The molecule has a penta substituted and tetra- substituted aromatic ring, as deduced by the presence of an isolated proton at δ 6.6 (d, J =

0.1) and two signals with ortho coupling at δ 7.3 (d, J = 8.6, H-7) and 6.71 (dd, J = 8.6,

2.5, H-8). The structural reporter region in the 1H-NMR spectrum exhibited multiplets at δ

3.25,3.3,3.35,3.7 due to presence of a protonated sugar moiety alongwith methylene doublet at δ 4.5 (J=3.8, Ha-7’,Hb-7’) and two singlets at δ 4.0, Ha-10’, Hb-10’) & δ2.03

(OH-12’).

The 13C-NMR (BB and DEPT) spectrum displayed total of thirteen methine at δ

40.12, 72.4, 75.0, 76.6, 77.11, 80.03, 97.05, 105.01, 111.07, 111.08, 112.0, 125.11, 130.0,

three methylene signals at δ 67.01, 63.45, and 42.12 in downfield region accompanied with two carbonyl signals further upfield at δ 167.5 and 169.13.

Presence of a mass fragment at 248.0142 a.m.u. reasserted the existence of a malonyl glucoside. In the HRESIMS spectrum of 152, a molecular ion peak [M]+ was observed at m/z 518.14196 coherent with the chemical formula C25H26O12. Complete data of compound 152 coincided with literature of Medicarpin 3-O-glucoside-6'-O-malonate

(Arman, 2011, Omar et al., 2016, Piccinelli et al., 2005, Weidemann et al., 1991).

Table 3.17. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 152 in DMSO 13C-NMR Multiplicity 1H-NMR (δH) coupling Position (δC) (DEPT) constants JHH (Hz) 130 CH 7.5 (d, J = 8.4) 1 2 112 CH 6.8 (dd, J = 7.6, 2.5)

3 155.05 C 4 105.01 CH 6.6 (d, J = 2.4) 4a 158.02 C 6 67.01 CH2 4.3 (dd, J=5.0, 11.0), 3.65 (dd, J=10.5, 11.0) 6a 40.12 CH 3.2 (ddd, J = 10.06, 9.45, 1.98) 6b 118.22 C 7 125.11 CH 7.3 (d, J =8.6) 8 111.08 CH 6.71 (dd, J=8.6, 2.5) 9 160.5 C 10 97.05 CH 6.4 (s) 10a 162 C 11a 80.03 CH 5.7 (d, J =8.5) 11b 115.38 C 13 56.01 CH3 3.75 (s) 2' 111.07 CH 4.78 (d, J = 6.5) 3' 75 CH 3.7 m 4' 77.11 CH 3.35 m 5' 72.4 CH 3.25 m 6' 76.6 CH 3.3 m 7' 63.45 CH2 4.5 (d, J = 3.8) 9' 167.5 C=O 10' 42.12 CH2 4.0 (s)

11' 169.13 C=O 12' OH 2.03 (s)

3.5.6 Melanoxetin (153)

Compound 153 was received as light yellow amorphous solid. The infrared spectrum exhibited characteristic bands at 3226 (phenolic OH), two absorptions at 1574 &

1517 cm-1 (C=C), 1640 cm-1(carbonyl group). This compound was identified in LC-MS

Spectrum (+ve/-ve mode) at retention time of 10.75 min (Compound 64).

ABX Splitting pattern was noted in the proton-NMR spectrum of 153 proposed the existence of a flavone skeleton, the two B-ring protons at 훿H 7.45 (dd, J = 8.6, 2.0, H-2’) and 7.6 (d, J = 1.5, H-3’) Pure singlet absorptions were verified at δ 9.5, 9.9, 8.9, 10.0, 8.89

(H-3,7,8,4’,5’)

BB and DEPT displayed 15 signals including five quarternary carbon signals at δ

147.5, 180.5, 145.12, 120.6, 123.5 and ten methine signals at 128.2, 107.8, 115.1, 115.5,

122.01, 131.0, 138.0, 147.8, 147.99, 148.6, The LC-MS, 1H-NMR and 13C-NMR spectrum collectively established the structure of compound to be flavone, melanoxetin (Tung et al.,

2010).

Table 3.18. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 153 in DMSO Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 2 147.5 C

3 138 C-OH 9.5 (s) 4 180.5 C=O 5 128.2 CH 6.9 (d, J= 8.85) 6 107.8 CH 6.8 (d, J = 8.85) 7 148.6 C-OH 9.9 (s) 8 131 C-OH 8.9 (s) 9 145.12 C 10 120.6 C 1' 123.5 C 2' 122.01 CH 7.45 (dd, J = 8.6,2.0) 3' 115.1 CH 7.6 (d, J = 1.5) 4' 147.89 C-OH 10.0 (s) 5' 147.99 C-OH 9.9(s) 6' 115.5 CH 7.0 m

3.5.7 Dihydrorobinetin (154)

Compound 154 was separated as yellowish crystalline solid. The infrared spectrum showed characteristic bands at 3226 (phenolic OH), 1515 cm-1 (C=C), 1640 cm-1 (carbonyl group). This compound was identified in LC-MS Spectrum (-ve mode) at retention time of

9.29 min (Compound 64).

1H-NMR spectrum (Table 3.19) displayed resonances for flavonoid nucleus with two additional –H doublets at H 5.0 (d, J = 11.0, H-2) and 4.8 (d, J = 11.0, H-3). These signals were accompanied with intense singlet absorption at  7.0, 7.1, 9.85, 10.1, 10.2,

11.0 (H-2’3’4’5’6’7).

13C-NMR Spectrum displayed eight methine signals at δ 72.5, 83.0, , 125.0, 117.8,

126.0, 127.0, 127.5, 128.0, 130.01, 134.0, and four quarternary carbon signals at δ 122.1,

123.5, 155.0, 190.0. Carbon atoms of ring B (C-3’,4’,5’) were upfielded 17-18 ppm. Strong upfield shift (approx. 70 ppm) was also noted for C-2 and C-3 in comparison to other flavonoids. Comparison with previous literature and above information indicated the structure of compound to be a flavanonol, dihydrorobinetin (Cerezo et al., 2009).

Table 3.19. 1H (400 MHz) and 13C-NMR (200 MHz) of compound 154 in DMSO 1H-NMR (δH) coupling Multiplicity constants 13 Position C-NMR (δC) (DEPT) JHH (Hz) 2 83 CH 5.0 (d, J = 11.0) 3 72.5 CH-OH 4.8 (d, J = 11.0) 4 190 C=O 5 125 CH 7.8 (d, J = 7.6) 6 126 CH 5.8 (d, J = 7.5) 7 134 C-OH 11 (s) 8 117.8 CH 6.0 (d, J = 1.5) 9 155 C 10 122.1 C 1' 123.5 C 2' 127 CH 7.0 (s) 3' 128 C-OH 10.1 (s) 4' 130.01 C-OH 10.2 (s) 5' 128 C-OH 9.85(s) 6' 127.5 CH 7.1 (s)

3.6. Biological Evaluation of phytochemicals isolated from Calligonum polygonoides 3.6.1. Urease inhibition assay Bioassay guided isolation led to isolation of 3 triterpenes from n-hexane fraction of Calligonum polygonoides. These molecules were further assessed for their urease inhibitory activity.

Table 3.20. Urease Inhibitory Activity of Phytochemicals isolated from Calligonum polygonoides

Compounds Urease inhibition % IC50 (µg/mL) Cycloartenol (145) 84.26±0.3 5.75±0.56 β-Amyrin (146) 88.1±0.8 4.29±0.27 Stigmasterol (147) 89.56±1.0 3.58±0.54 Thiourea 97.5 ± 0.5 1.6±0.01

It was revealed that stigmasterol showed highest inhibition with IC50 of 3.58±0.54, followed by β-amyrin with inhibitory concentration value of 4.29±0.27 and cycloartenol with IC50 of 5.75±0.56. Betulinic Acid and oleanolic acid have been isolated from extracts of Forsythia suspensa and reported to inhibit urease enzyme (Shin et al., 2009). Other oleanane triterpenes have also been reported to show significant inhibition of urease

(Golbabaei et al., 2013). Hydrophobic interactions between methyl groups of triterpenes and active site amino acids have been shown to play a important role in urease inhibition

(Golbabaei et al., 2013). Therefore this explains urease inhibitory action of n-hexane fraction and isolated triterpenoids.

3.6.2. Xanthine oxidase inhibition assay

Compounds isolated from n-butanol fraction were estimated for their xanthine oxidase inhibitory potential. The resulting inhibition values of each compound are listed in following table.

Table 3.21. Xanthine oxidase inhibitory activity of Phytochemicals isolated from Calligonum polygonoides Xanthine oxidase IC50 Compounds inhibition % (µg/mL) Rhododendrin (148) 20±1.25 ---- Glucogallin (149) 15±0.25 ---- Hypolaetin-8-glucoside (150) 91.3±0.75 3.25±0.6 Isoetin-4’-O-glucuronide (151) 87.1±0.3 4.14±0.5 Medicarpin 3-O-glucoside-6'-O-malonate (152) 30.56±1.52 ---- Melanoxetin (153) 84.74±0.47 5.17±0.4 Dihydrorobinetin (154) 30±0.45 ---- Allopurinol 95±0.5 0.3±0.05

Highest inhibition was exhibited by Hypolaetin-8-glucoside (150) (91.3±0.75% with IC50 3.25±0.6) followed by Isoetin-4’-O-glucuronide (151) (87.1±0.3 with IC50

4.14±0.5) and Melanoxetin (84.74±0.23 with IC50 5.17±0.4). Flavonoids have been known to show remarkable results regarding antioxidant/superoxide radical scavenging activity

(Chen et al., 1990). Previous studies have revealed that –OH substitution at C-5 or C-7 & conjugation between C-2 & C-3 are essential features required for xanthine oxidase inhibition (Cos et al., 1998).

3.6.3. Carbonic Anhydrase Inhibition Assay

Phytochemicals isolated from n-butanol fraction were assessed for their carbonic anhydrase inhibitory activity. The resulting inhibition values of each compound are listed in following table.

Table 3.22. Carbonic Anhydrase inhibitory activity of phytochemicals isolated from Calligonum polygonoides Carbonic anhydrase Compounds inhibition % IC50 (µg/mL) Rhododendrin (148) 20±1.25 ----- Glucogallin (149) 15±0.25 ----- Hypolaetin-8-glucoside (150) 85±0.2 5.44 ± 0.5 Isoetin-4’-O-glucuronide (151) 96.3±0.7 4.16 ± 0.25 Medicarpin 3-O-glucoside-6'-O-malonate (152) 30.56±1.52 ----- Melanoxetin (153) 98.45±0.2 3.21 ± 0.45 Dihydrorobinetin (154) 40.52±0.37 ----- Acetazolamide 99±0.5 0.3 ± 0.1

-2 Carbonic anhydrases catalyze the interconversion between CO2 and the HCO3 ion, and are thus needed for essential physiological processes. These enzymes contain a zinc anion in its active site which is essential for its functioning (Supuran and Scozzafava,

2007). Therefore, it was strongly believed carbonic anhydrase inhibitors must possess zinc binding groups (ZBG) like sulfonamide moiety (-SO2NH2), sulfamate (-O-SO2NH2) and sulfamide (-NH-SO2NH2) moieties. Since these functionalities rarely occur in natural products, it has been found that natural products inhibit carbonic anhydrase through bioisosteres or different mechanism of action. Coumarins are hydrolysed by Zn (OH)2/H2O molecule to cinnamic acid which in turn impedes any entrance to active site (Maresca et al., 2010). Phenolics inhibit carbonic anhydrase via interaction with H-bonding with –OH ion of H2O molecule in active site & -NH groups of active site amino acid residues (Karioti et al., 2016). Our investigations revealed molecules possessing the flavone nucleus to be most potent (Melanoxetin (153) with an IC50 of 3.21±0.45 followed by Isoetin-4’-O- glucuronide (151) with an IC50 4.16±0.25 and Hypolaetin-8-glucoside (150) with an IC50

5.44±0.5) compared to Acetazolamide (IC50 10.3±2.5). Previous investigations on

flavonoids revealed that flavones inhibited carbonic anhydrase at a higher rate in comparison to flavanols, flavonols, isoflavones, flavanonols etc. Essential features necessary for inhibition were interaction of ring A with active site, double bond b/w C-2

& C-3. Glycosylated flavonoids showed higher inhibition than non-glycosylated ones which was possibly due to H-bonding of glycone moiety with external amino acid residues at entrance of binding site, thereby stabilizing aglycone portion at the catalytic site (Karioti et al., 2016). Compounds 152 and 154 failed to inhibit Carbonic anhydrase possibly due to lack of flavone nucleus.

3.6.4. α-glucosidase inhibition assay

Compounds separated from n-butanol fraction were also assessed for their α- glucosidase inhibitory potential. The resulting inhibition values of each compound are listed in following table 3.13.

Table 3.23. α-glucosidase inhibitory activity of phytochemicals isolated from Calligonum polygonoides α-glucosidase inhibition IC50 Compounds % (µg/mL) Rhododendrin (148) 32±1.28 ----- Glucogallin (149) 23±0.27 ----- Hypolaetin-8-glucoside (150) 87.5±0.5 6.27±0.46 Isoetin-4’-O-glucuronide (151) 78.2±0.7 5.77±0.32 Medicarpin 3-O-glucoside-6'-O-malonate (152) 25.76±0.23 ----- Melanoxetin (153) 89.9±0.2 5.91±0.25 Dihydrorobinetin (154) 35.5±0.52 ----- Acarbose 97.56±0.41 4.2±0.25 α-glucosidase is an enzyme bound inside membrane that occurs in the small intestinal epithelium & catalyzes the hydrolytic cleavage of oligosaccharides into absorbable monosaccharides by targeting α (1→4) linkages. Henceforth alpha glucosidase inhibition decreases postprandial hyperglycemia via reduction in hydrolysis of

oligosaccaharides subsequently leading to delayed carbohydrate digestion (Lebovitz He,

1997). Different phytochemicals such as alkaloids & terpenoids have been explored as potential sources for lead and development of novel anti-diabetics (Gaikwad Sb et al.,

2014, Nazaruk J and Borzym-Kluczyk M, 2015, Sharma B et al., 2010, Tiong Sh et al.,

2013) as well as α-glucosidase inhibitors (Kumar S et al., 2011, Telagari M and Hullatti

K, 2015, Xie J-T et al., Yin Z et al., 2014, Zafar M et al., 2016). Intriguingly, several flavonoids have also been reported to show significant inhibition of intestinal α- glucosidase (Ota A and Ulrich Np, 2017). α-glucosidase inhibition was noted for 4 compounds in the following order: Isoetin-4’-O-glucuronide (154) 78.2±0.7 (IC50

5.77±0.32) > Melanoxetin (153) 89.9±0.2 (IC50 5.91±0.25) > Hypolaetin-8-glucoside (150)

87.5±0.5 (IC50 6.27±0.46). SAR studies have revealed that –OH substitution at C-3, C-4’,

C-6 and C-7 positions greatly enhance glucosidase inhibitory activity. The inhibitory property is further augmented by glycosidation of the flavone nucleus (Li et al., 2018,

Sohretoglu et al., 2018). Jong-Sang Kim et al. have exhibited that flavone luteolin inhibits

α-glucosidase at a five fold higher rate in comparison to standard acarbose (Kim et al.,

2000). Dihydrorobinetin (154) and Medicarpin 3-O-glucoside-6'-O-malonate (152) failed to show inhibition of α-glucosidase possibly due to lack of an essential pharmacophore i.e; flavone nucleus (Proença C. et al., 2017, Şöhretoğlu and Sari, 2019).

EXPERIMENTAL

4.1. General Experimental Conditions 4.1.1. 1H-NMR, 13C-NMR & MS Mass spectrum was recorded at Finnigan-MAT-311 and Varian MAT 312 Mass spectrometer (250 oC and 70 eV). HR-ESI-MS was observed on JEOL JMS-600H Mass spectrometer. IR spectra was recorded on JASCO A-302 spectrophotometer. 1H-NMR

Spectra were recorded on Bruker-Avance Spectrometers at 400 MHz and 13C-NMR

Spectra were recorded on Bruker Avance on spectrometer at 200 MHz using TMS as an internal standard.

4.1.2. LC-MS 4.1.2.1. HPLC System The liquid chromatography analyses were performed using an Agilent 1290 infinity

LC System coupled to an AGILENT 6200 Accurate-Mass Q-TOF mass spectrometer with dual ESI source. The high-pressure liquid chromatography (HPLC) system consisted of a binary pump, a cooling auto-sampler set at 4°C with an injection loop of 10 µL. The column heater was set at 25°C and a Agilent ZORBAX Eclipse XDB column-C18 (150 mm x 2.1 mm; 3.5 µ) was used for the separation of the metabolites. The mobile phase consisted of

A: 0.1 % formic acid in water and B: 0.1 % formic acid in acetonitrile. Chromatographic separation was achieved using a linear gradient: 0-7.0 min, 25-98% B; 7-9.5 min, 98% B;

9.5-10 min, 98-25% B; 10-15 min, 25% B; at a flow rate of 0.4 mL min-1. Weak wash solvent was 10 % acetonitrile. The strong and needle wash solvents were a mixture of acetonitrile, propan-2-01, methanol and water (30:30:30:10 v/v/v/v).

4.1.2.2. Q-TOF high resolution mass spectrometry

This was carried out on a Agilent 6200 series TOF/6500 UHR-Q-TOF mass spectrometer under the following conditions: ionisation mode: ESI (-) and ESI (+); MS

Scan range: 50-2500 m/z; end plate offset: -500 V; capillary: -3000 V. Nebulizer gas (N2):

0.4 bar; dry gas (N2): 4 L min-1; dry temperature: 180°C. Ion transfer conditions: funnel

RF: 200 Vpp; multiple RF: 200 Vpp; Quadruple Low Mass: 55 m/z; Collision Energy: 0 ev; Collision RF: 600 Vpp; Ion Cooler RF: 50-250 Vpp ramping. Transfer time: 121 µs;

Pre-Pulse Storage time: 1 µs. Calibration was achieved before each run through a loop injector 20 µL of sodium formate (10 mM).

4.1.3. GC-MS Analysis

GC-MS investigations were undertaken using GC Q-TOF MS (Quantitative time of flight mass spectrometry) using an Agilent 7693A ALS (Automatic Liquid Sampler), a GC

7890A gas chromatograph, and an Agilent 5975C quadrupole mass spectrometer (Agilent,

Santa Clara, CA 95051, USA)

4.1.3.1. GC conditions

Manual injection of 1ul, with splitless mode, into GC injector at 230°C, onto 30-m capillary column HP-5 MS of 0.25 mm internal diameter with an integrated guard column and a 0.25 μm film (Agilent, Santa Clara, CA 95051, USA)

4.1.3.2. GC oven temperature programme

2 min isothermal heating at 60°C, followed by a 5°C/min oven temperature ramp to

80°C and a final 5-min heating at 10 – 310°C. The system was then temperature equilibrated for 6 min at 70°C prior to injection of the next sample

4.1.3.3. MS conditions

Ion source temperature 200°C. EI+ ionisation mode, using 125 V energy.

4.1.3.4. Full scan data acquisition

(m/z 50-650)

4.1.4. Chromatographic separation

Column Chromatography was executed over Merck silica gel (200 μm) and

Sephadex G-15 (Sigma-Aldrich). For preparative TLC, silica gel plates (60 G F254 20x20) with thickness of 0.2 mm were used. Compound Purity was evaluated on these plates under

UV lamp (245 and 365 nm). Ce(SO4)2, Vanillin, Iodine and Dragendorff’s Reagent were utilized as spraying reagents.

4.2. Materials and Methods 4.2.1. Plant collection Aerial parts of Calligonum polygonoides was collected from from different places of Cholistan desert region and identified by Mr. Ghulam Sarwar (Lecturer at Department of Botany, Islamai University of Bahawalpur). A voucher specimen was deposited at the

Herbarium of the Department of Life Sciences, Islamia University, Bahawalpur with the voucher number of 471/LS.

4.2.2. Extraction

Plant material was collected and disesased, deteriorated, foreign parts were removed. It was shade dried with frequent agitation after 6 hours. Grinding mill was utilized for pulverization. 10 kilos of pulverized material was taken and macerated in 30 llitres of solvent (H2O:CH3OH 20:80) for 3 days with occasional shaking. Solvent was filtered after 3 days. Filtrate was evaporated in rotary evaporator (Buchi, Switzerland) and fume hood. Marc was extracted for second time using same solvent and procedure.

Semisolid material obtained after drying of crude extract was designated as CpC. The weight of CpC was 900 g and stored for further processing.

4.2.3. Liquid-Liquid Extraction

For fractionation, liquid-liquid extraction of CpC was accomplished. Briefly, 900 g of CpC was suspended in 2500 mL of hot H2O. Separating funnel was utilized to extract this water suspension 3 times with n-hexane. Organic layer was separated and dried in

o rotary evaporator at 35 C to obtain n-hexane fraction (CpC-N). H2O layer was agin extracted 3 times first with chloroform then with methanol. These fractions were subsequently subjected to various biological assays.

4.2.4. LC-MS Profiling of crude methanolic fraction of Calligonum Polygonoides 4.2.4.1. +ve Ionisation Mode

Fig 4.1. TCC and TIC scan (+ mode) of crude methanolic fraction of Calligonum Polygonoides

Fig 4.2. LC-MS Ionogram of crude methanolic fraction of Calligonum Polygonoides (+ mode)

4.2.4.2. -ve Ionisation Mode

Fig 4.3. TCC and TIC scan (- mode) of crude methanolic fraction of Calligonum Polygonoides

Fig 4.4. LC-MS Ionogram of crude methanolic fraction of Calligonum Polygonoides (- mode)

4.3. Isolation & purification of chemical constituents from n-hexane fraction CpC-N

Owing to high biological activity of CpC-N (57 g) against urease; it was further submitted to chromatographic elution to isolate its different bio-active constituents. 57 grams of n-hexane fraction of Calligonum polygonoides was obtained which was then subjected to column chromatography using silica gel as stationary phase and mobile phase of n-hexane:CHCl3 starting from the ratio of 85:25 upto 25:85. This resulted into many subfractions which were collected and mixed on basis of TLC evaluation leading to major subfractions. Fraction A (12.6 g) and Fraction B (20.66 g). ratio was subsequently purified on column via silica gel which lead to isolation of compound (145) i.e; Cycloartenol. Silica gel column chromatography was also applied for purification of fraction B which yielded

β-amyrin (146) & stigmasterol (147).

Fig 4.5. Scheme for isolation of pure compounds from n-hexane fraction CpC-N

4.4. Isolation & purification of chemical constituents from n-butanol fraction CpC-B:

Owing to high biological activity of CpC-B against α-glucosidase, carbonic anhydrase, urease; it was further subjected to chromatographic separation to isolate its different bio-active constituents. Furthermore it was also obtained in highest amount (400 g).

CpC-B was subjected initially to normal phase chromatography starting from 100%

CHCl3 leading upto 100% methanol. This yielded four major subfractions i.e; fraction A

(12.5 g), fraction B (25.5 g), fraction C (200 g), fraction D (40 g). Fraction A was purified on silica gel column chromatography which afforded 4 subfractions (A1,A2,A3,A4). TLC analysis lead to the combination of A2 & A3. Combined fraction A2+A3 was purified using size exclusion chromatography with 2 % decrease in polarity using solvent system

H2O: Methanol which yielded Rhododendrin (148). Glucogallin (149) was also obtained through normal phase column chromatography of fraction B followed by preparative TLC.

Fraction C was purified on silica gel using CHCl3: MeOH system which afforded 8 subfractions (C1→C8). Fractions (C1→C3) and fractions C5& C6 were combined via TLC profile. Combined fractions (C1→C3) & C5+C6 were purified via reverse phase chromatography using solvent system H2O: MeOH with 0.5% decrease in polarity resulting in isolation of Hypolaetin 8-glucoside (150) & Isoetin-4’-glucuronide (151). Medicarpin-

3-O-6’- malonyl glucoside (152) was obtained from subfraction C8 via preparative TLC using solvent system CHCl3: MeOH. Normal phase chromatography of fraction D yielded

5 subfractions (D1→D5). Combined fraction D3+D4 was eluted with H2O: MeOH solvent system using Sephadex G-15 (GE Healthcare, 17002001) with 1% decrease in polarity which furnished Melanoxetin (155) & Dihydrorobinetin (154).

Fig 4.6. Scheme for isolation of pure compounds from butanol fraction CpC-B

4.5. Characterization of constituents from Calligonum polygonoides

4.5.1. Cycloartenol (145)

Physical State: Colorless crystals

Yield: 2.9 mg

ESI-MS (m/z): 464 a.m.u.

Molecular Formula: C30H50O

HR-ESIMS: 426.3978 a.m.u. (calculated 426.7256 a.m.u.)

−1 -1 - IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm ), CH2 (1450 cm ), CH3 (1380 cm

1), sharp OH band (3600 cm-1).

4.5.2 β-Amyrin (146)

Physical state: Colorless crystals

Yield: 3.1 mg

ESI-MS (m/z): 426 a.m.u.

Molecular formula: C30H50O

HR-ESIMS: 426.40125 a.m.u. (calcd. 426.6566 a.m.u.).

-1 - IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm

1), sharp OH band (3615 cm-1), C=C band (1650 cm-1).

4.5.3 Stigmasterol (147)

Physical state: amorphous solid

Yield: 2.5 mg

ESI-MS (m/z): 426 a.m.u.

Molecular Formula: C30H50O

HR-ESI-MS: 426.40125 a.m.u. (calculated 426.6566 a.m.u.)

−1 -1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm ), CH3 (1375 cm ), sharp OH band

(3600 cm-1), C=C band (1630 cm-1).

4.5.4 Rhododendrin (148)

Physical State: Colorless needles

Yield: 3 mg

ESI-MS (m/z): 328 a.m.u.

Molecular Formulaa: C16H24O7

HR-ESI-MS: 328.15163 a.m.u.(calculated 331.16995 a.m.u.)

−1 -1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm ), CH3 (1375 cm ), sharp OH band

(3600 cm-1), C=C band (1630 cm-1).

4.5.5. Glucogallin (149)

Physical state: Dark Yelllow Amorphous Powder

Yield: 5.6 mg

ESI-MS (m/z): 328 a.m.u.

Molecular Formula: C13H16O10

HR-ESI-MS: 328.15163 a.m.u. (calculated 332.07143 a.m.u.)

−1 -1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm ), CH3 (1375 cm ), sharp OH

band (3600 cm-1), C=C band (1630 cm-1).

4.5.6. Hypolaetin-8-glucoside (150)

Physical State: Dark Yellow Amorphous powder

Yield: 6.8 mg

ESI-MS (m/z): 464 a.m.u.

Molecular Formula: C21H20O12

HR-ESI-MS: 464.09603 a.m.u. (calculated 464.09201 a.m.u.)

-1 IRmax cm (CHCl3): 3350 (OH phenolic), 1710 (C=O carboxyl) and 1520 cm-1 (C=C) and 1022 cm-1 (glycoside linkage).

4.5.7. Isoetin-4’-O-glucuronide (151)

Physical State: Yellow solid

Yield: 8.9 mg

ESI-MS (m/z): 478 a.m.u.

Molecular Formula: C21H18O13

HR-ESI-MS: 478.07541 a.m.u. (calculated 464.09567 a.m.u.)

-1 IRmax cm (CHCl3): 3350 (OH phenolic), 1700 (C=O carboxyl)

4.5.8. Medicarpin 3-O-glucoside-6'-O-malonate (152)

Physical State: White crystalline solid

Yield: 4.6 mg

ESI-MS (m/z): 561 a.m.u.

Molecular Formula: C25H23NO14

HR-ESI-MS: 561.4507 a.m.u. (calculated 561. 5617 a.m.u.)

-1 -1 -1 IRmax cm (CHCl3): 3535 cm (phenolic hydroxyl group), 1741 cm (ketonic C=O group)

1580 cm-1 (aromatic C=C stretching) and 3400 cm-1 (–NH group)

4.5.9. Melanoxetin (153)

Physical State: Light Yellow Amorphous solid Yield: 4.6 mg ESI-MS (m/z): 303 a.m.u.

Molecular Formula: C15H10O7

HR-ESI-MS: 303.0497 a.m.u. (calculated 302.04263 a.m.u.)

-1 IRmax cm (CHCl3): 3226 (OH), 1640 (C=O), 1574 (C=C), 1517 (C=C)

4.5.10. Dihydrorobinetin (154)

Physical State: Light Yellow amorphous solid Yield: 2.2 mg ESI-MS (m/z): 303 a.m.u.

Molecular Formula: C15H12O7

Hr-esi-ms: 303.0515 a.m.u. (calculated 304.05878 a.m.u.)

-1 IRmax cm (CHCl3): 3225 (OH), 1640 (C=O), 1515 (C=C)

4.6. Biological Evaluation of different fractions of Calligonum polygonoides and its phytochemicals

4.6.1. DPPH Free radical scavenging assay

1 mL of 0.000135 moles of DPPH was prepared in CH3CH2COOH and mixed with

1 mL of extract (0.2-0.8 mg/mL) and pure compound ranging from 20, 40, 60, 80, 100

μM/mL. The reaction mixture was mixed thoroughly and left in dark at 20-30 oC for 1800 seconds. The absorbance was calculated at 517 nm. The scavenging power of the plant extract and pure compounds were calculated using the following equation;

[ ] Percentage of DPPH Scavenging = 퐴푏푠 푐표푛푡푟표푙−퐴푏푠 푠푎푚푝푙푒 푥 100 100

Abs control: Where Abs control is the absorbance of DPPH + methanol, Abs sample is the absorbance of DPPH radical + sample i.e. plant extract(Dorman Hj et al.,

2004, Kwon Y.-I. et al., 2008). The inhibitory concentration (50%) were estimated from nonlinear regression analysis via Microsoft Excel and GraphPad Prism v. 5.0

4.6.2. α-glucosidase inhibition assay

α-glucosidase inhibition was performed in accordance to the method reported by

Y.I Kwon et al. (Kwon Y.-I. et al., 2008). A volume of 50 µl of sample solution and 100

µl of 0.1 M phosphate buffer (pH 6.8) containing crude α-glucosidase solution (25 mg/ml) were incubated in 96 well plates at 25oC for 10 min. Following pre-incubation, 50 µl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at an interval of 5 seconds. The reaction mixtures were incubated at

30oC for 15 min, absorbance readings were recorded at 405 nm by UV Microplate reader

(Biotek, Synergy HT) in both conditions before and after incubation and compared to a control which had 50 µl of buffer solution in place of the sample. The α-glucosidase inhibitory activity was expressed as inhibition percentage and was calculated as follows:

control extract control Percentage Inhibtion (%) = [λ 405- λ 405/λ 405] x 100

4.6.3. Carbonic anhydrase inhibition assay

Carbonic anhydrase inhibition assay for the test samples was performed according to previous methods with a slight modification(Rauf et al., 2017, Sahin et al., 2012) .

Briefly, total mixture volume was 200 μL in a well contained 140 μL (20 mM HEPES,

Bioworld - cat# 40820000-1) Tris (Invitrogen - cat# 15504-020) buffer (pH 7.4), 20 μL of the enzyme (from bovine, Sigma-Aldrich, C2624, PCode: 1001584424), 20 μL (0.5mg ̸ mL in dimethylsulfoxide) of the test compounds, which were mixed and incubated at 25

°C for 15 minutes. After incubation, pre-read was taken at 400 nm and, then, 20 μL of substrate (4-nitrophenyl acetate, Sigma-Aldrich, N-8130, lot# BCBK4587V) (0.7 mM in

MeOH) was added. The reaction was run at the same conditions for 30 minutes and final read was taken at 400 nm. Acetazolamide was used as the reference (positive control).

Results obtained after the reactions done in triplicate were measured by the equation given below.

Inhibition % = 100 - (absorbance of test compound /absorbance of control) × 100

4.6.4. Urease inhibition assay

The assay mixture, incorporating 100 μL (2 mg/mL) of Jack-bean urease and 100

μL of the plant fraction with 0.2 mL of 100 mM phosphate buffer (pH 6.8) containing 25 mM urea was pre-incubated for 30 min in water bath at 37°C. The urease reaction was stopped after 30 min incubation with 600 μL of 4% H2SO4 acid. Ammonia released was determined by the phenol– hypochlorite method (Weatherburn, 1967). NH3 was approximated via employing 500 μL of solution I (5.0 g phenol and 25 mg of sodium nitroprusside) and 500 μL of solution II (2.5 g sodium hydroxide and 4.2 mL of sodium

hypochlorite in 500 mL of distilled water) at 37°C for 30 min and the absorbance was measured at 625 nm against the control. All assays were performed in triplicate in a final volume of 1 mL. The urease inhibitory activity was expressed as inhibition percentage and was calculated as follows:

control extract control Percentage Inhibition (%) = [λ 625- λ 625/λ 625] x 100

4.6.5. Xanthine Oxidase inhibition assay

The enzyme xanthine oxidase catalyzes the oxidation of xanthine to uric acid.

During this reaction, molecular oxygen acts as an electron acceptor, producing superoxide radicals according to the following equation:

Xanthine oxidase activity was evaluated under aerobic condition by the spectrophotometric measurement of the production of uric acid from xanthine. The inhibition of xanthine oxidase activity was followed by measuring the increase of uric acid absorbance at 290 nm in accordance to the proposed methodology(Chaabane et al., 2012,

Hudaib Mm et al., 2011) The assay mixture consisted of 100 μl of sample test solution,

200 μl xanthine (final concentration 0.1 mM) as the substrate, hydroxylamine (final concentration 0.2 mM), 200 μl EDTA (0.1 mM) and 300 μl distilled water. The reaction was initiated by adding 200 μl xanthine oxidase (11 mU) dissolved in phosphate buffer

(KH2PO4, 0.2 M, pH 7.5). The assay mixture was incubated at 37°C for 30 min. Before measuring the uric acid production at 290 nm, the reaction was stopped by adding 100 μl of 0.58 mM HCl. The absorbance was measured spectrophotometrically against a blank solution prepared as described above, but replacing xanthine oxidase with buffer solution

(no production of uric acid). A control solution without test compound was prepared in the same manner as the assay mixture to measure the total uric acid production. The uric acid production was calculated from the differential absorbance.

4.6.6. Tyrosinase inhibition assay

Tyrosinase inhibition assay was performed using kojic acid as standard inhibitor for tyrosinase as previously described (Kishore et al., 2018, Momtaz et al., 2008, Ngankeu

Pagning et al., 2016) with slight modifications. Assays were conducted in a 96-well microplate, and ELISA plate reader was used to determine the absorbance at 490 nm (BIOTEK).

The test sample was dissolved in aqueous DMSO, and incubated with L-tyrosine (2.5 mg/ml) in 50 mM phosphate buffer (pH 6.8). All samples were dissolved in DMSO. Then,

25 U/ml of mushroom tyrosinase in the same buffer was added, and the mixture was incubated at 37°C for 30 min.

The percentage tyrosinase inhibition was calculated as follows:

% inhibition = [(A control-A sample)/ Acontrol] X 100 where

Acontrol is the absorbance of DMSO and Asample is the absorbance of the test reaction mixture containing sample or kojic acid. The IC50 values of samples and kojic acid were calculated.

4.7. Statistical Analysis

All the measurements were carried out in triplicate and results were conveyed as mean ± Standard error of mean. One way ANOVA and LSD Post-Hoc Analysis were applied for statistical analysis. One-way ANOVA followed by LSD post Hoc analysis was used for statistics. A p-value of less than or equal to 0.05 was considered significant.

CONCLUSION

5.1. Conclusion

o GC-MS analysis of n-hexane fraction was performed and triterpenes, esters, fatty acids and

waxes were identified.

o The methanol and n-butanol extracts of Calligonum polygonoides revealed strong activity

against xanthine oxidase, alpha glucosidase and carbonic anhydrase. These extracts can

be further utilized as herbal medicine.

o LC-MS screening of methanolic fraction revealed presence of huge amount of flavonoids

and glycosides.

o For further studies, different parts (stem, roots, leaves, fruits, flowers) of Calligonum

polygonoides could be evaluated separately for their biological potential.

o Rhododendrin (148), Glucogallin (149), Hypolaetin 8-glucoside (150) Isoetin-4’-O-

glucuronide (151) Medicarpin-3-O-6’- malonyl glucoside (152), Melanoxetin (155) and

Dihydrorobinetin (154) have been reported for first time from Calligonum polygonoides.

o Compounds isolated from n-butanol fraction also inhibited xanthine oxidase, alpha

glucosidase and carbonic anhydrase significantly. These findings prompt further

investigation for development/lead optimization of these phytochemicals as potential

therapeutic agents against these targets.

Part-B

PLANT

INTRODUCTION/PREVIOUS

PHYTOCHEMICAL INVESTIGATIONS ON Crateva Adansonii

6.1. Plant Classification

Plant description Kingdom:Plantae

Clade:Angiosperms

Clade:Eudicots

Order:Brassicales

Family: Capparaceae

Genus:Crateva

Species:adansonii

6.2. Family Capparaceae

The Capparaceae family is also known by the name of Caper family. It consists of

29 genus and 700 species that are disseminated worldwide mostly in tropical, subtropical

& mediterranean regions. They are mostly annual or perennial herbs, subshrubs, shrubs or trees. (Kers, 2003)

6.3. Genus Crateva

The genus Crateva (family Capparaceae) comprises about 8 species four of which are found in the subcontinent. It consists of medium sized deciduous trees. All plants of this genus are used to in folk medicine (Kher et al., 2016)

6.3.1. Crateva adansonii

Crateva adansonii is the least explored Crateva species. Its leaves are used as an ethnomedicinal treatment of syphilis, jaundice and yellow fever (Burkill H.M., 1985)

6.4. Bioactive constituents reported from the Family Capparaceae:

6.4.1. Crateva genus: 6.4.1.1. Crateva adansonii Ngozichukwuka Peace Igoli et al. evaluated leaf extracts both phytochemically as well as biologically. Hexane extract and ethyl acetate extract of C. adansonii DC leaves possessed a minimum inhibitory concentration value of 12.5 μg/ml, corresponding to that of standard Suramin when assessed invitro against African trypanosome blood forms like

T. brucei Further chromatographic analysis of these extracts resulted in isolation of aurantiamide acetate (155), Ethyl pyropheophorbide A (156), purpurin-18 ethyl ester (157) and pyropheophorbide A (158). Insilico analysis revealed strong binding interactions of these components with validated T. brucei protein targets (Igoli et al., 2014).

6.4.1.2. Crateva nurvala

100

The most widely investigated amongst Crateva species is Crateva nurvala.

Pharmacological investigations have revealed its multifarious biological activities such as cardioprotective (Sudharsan et al., 2006), anti-nociceptive (Alam et al., 2008,

Moniruzzaman and Imam, 2014) antidiabetic activity (Sikarwar and Patil, 2010), anti- fertility (Bhaskar et al., 2009). Ethnobotanically its most common use is as an anti- inflammatory (Cho et al., 2015), anti-arthritic (Geetha and Varalakshmi, 1999), anti- urolithiatic agent (Agarwal et al., 2010, Anand et al., 2012). A cholestane type pentacyclic steroidal glycoside crataenoside (159) and quinolone alkaloid crateamine (160) has been isolated from the barks of Crateva nurvala. These compounds showed cytotoxic activity against HeLa, PC-3 and MCF-7 cells (Sinha et al., 2013).

6.4.1.3. Crateva religiosa

Ethnopharmacological uses of Crateva religiosa include diuretic, laxative, lithotriptic, antirheumatic, antiperiodic, bitter, tonic, rubifacient and counterirritant

(Bhatachagee S.K., 2001). Lupeol (161) isolated from Crateva religiosa has been shown to inhibit IL-2 (interleukin-2) production by CD4+ T cells in a dose dependant manner.

(Bani et al., 2006).

101

6.4.1.4. Crateva tapia

Crateva tapia is an edible plant that has also been used as folk medicine in Brazil for treatment of diabetes mellitus (Nunes et al., 2018). Lectins are proteins derived from plants which provide defense to plants from bacteria, fungi and viruses. They bind to polysaccharides composing the cell membrane resulting in cell agglutination. These molecules have exhibited a variety of pharmacological activities namely; antitumor, immunomodulatory, antifungal, HIV-1 reverse transcriptase inhibition, and insecticidal activity (Bah et al., 2013, Coelho et al., 2017, Lam and Ng, 2011). Silva et al. has purified a lectin from the bark of C. tapia named as CrataBL (Fig 5.1) (Ferreira et al., 2013). This lectin has been shown to enhance wound healing via promotion of angiogenesis through differenbt mechanisms (Batista et al., 2019). Alloxan induced diabetes mice models were administered CrataBL intraperitoneally at dose of 10 mg/kg and 20 mg/kg daily which reduced glucose levels (14.9-55.9%) alongwith serum urea, creatinine, aspartate

102

aminotransferase and alanine aminotransferase (Da Rocha et al., 2013). It has also exhibited potential cytotoxicity against human prostate cancer cell lines (Ferreira et al.,

2013). Additionally it has also exhibited insecticidal and larvicidal activity (Araujo et al.,

2012, Nunes et al., 2015).

Fig 6.1 Crystal Structure of Crateva tapia bark lectin (CrataBL) 6.4.2. Capparis genus 6.4.2.1 Capparis himalayensis Yun-Qiu Li et al. has reported two novel alkaloids Capparin A (162) and B (163) from Capparis himalayensis (Li et al., 2008). Xin-xin Lei et al. has isolated a novel anthraquinone Cappariquinone A (164) from Capparis himalayensis (Lei et al., 2015)

6.4.2.2. Capparis tenera

Two novel glucosidic lignans; 3,5-dimethoxy-4-(((3R,4S,6R)-3,4,5-trihydroxy-6-

(((4-hydroxy-3,5-dimethoxybenzoyl)peroxy)methyl)tetrahydro-2H-pyran-2- yl)oxy)benzoic acid (165) and Leoneruside A (166) were reported from butanolic fraction of roots of Capparis tenera (Su et al., 2007).

103

6.4.2.3. Capparis flavicans

It is traditionally used in Thailand to promote lactation(Brun, 2003). The antiestrogenic effect of this species was attributed to glycosides isolated from it, of which

Capparoside A (167) was a novel & important constituent.

6.4.2.4. Capparis spinosa

Capparis spinosa has been traditionally used to treat paralysis, diabetes, liver and kidney diseases (Tlili et al., 2011). It is used in China as a therapy for Gout, rheumatoid arthritis and haemorrhoids (Mahboubi and Mahboubi, 2014). Yang et al. has isolated a novel antioxidant molecule (168) from Capparis spinosa (Yang et al., 2010). Quarternary

Ammonium compounds such as Choline (169), Stachydrine (170), Betaine (171) have been reported from roots and leaves of Capparis spinosa which play an important role in protection against different environmental stresses (Al-Tamimi et al., 2018). Carageenen induced paw edema in rats was alleviated by hydroalcoholic extracts of Capparis spinosa

(Al-Said et al., 1988). 2-thiophenecarboxaldehyde (172) and methyl isothiocyanate (173) isolated from methanolic fraction prepared from stem of C. spinosa have shown strong nematocidal activity against Meloidogyne incognita EC50 = 7900 ± 1.6, and 14100 ± 1.9

µg/L (Caboni et al., 2012).

Zhou et al. has isolated two novel bioflavonoids; isoginkgetin (173) and sakuranin

(174) alongside a known molecule, i.e; ginkgetin (175). Secreted Placental Alkaline phosphatase Reporter Assay revealed only isoginkgetin and ginkgetin to inhibit NF-κB activation, an important inflammatory mediator (Zhou et al., 2011).

104

6.4.2.5.Capparis decidua

Traditionally Capparis decidua is used as analgesic, anti-rheumatic, anti- inflammatory and anthelmintic (Singh et al., 2011). Codonocarpine type alkaloids have been isolated from Capparis decidua which include Cadabicine (177), Isocadabicine (178),

N-Acetylisocodonocarpine (179), Isocodonocarpine (180), Capparisine (181), N-

Acetylcapparisine (182), Capparidisine (183), Capparisinine (184) (Forster et al., 2016).

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6.4.3. Cadaba Genus 6.4.3.1. Cadaba glandulosa/ Cadaba farinose Cadaba glandulosa leaves have been used as a therapy for haemorrhoids and urinary tract infections (Al-Fatimi et al., 2007). Several flavonoids have been isolated from

Cadaba glandulosa. AA Gohar has isolated a novel flavonol glucoside 7- methylkaempferol-3-O-neohesperoside-4-O-β-D-glucopyranoside (185) alongwith two known glycosides 7-methylkaempferol-3-O-neohesperoside (186) and 7- methoxyquercetin-3-neohesperoside (187) (Gohar, 2002).

Antinflammatory flavonoids were discovered from Cadaba glandulosa which exhibited activity close to indomethacin. New flavonoids were identified as 4-methoxy- benzaldehyde (188), kaempferol-3-methylether (189), 2-(3,5-dimethoxy-4- phenoxyphenyl)-5,7-dihydroxy-3-methoxy-4H-chromen-4-one (190). Other flavonoids were elucidated as 7-methylkaempferol -4'-(4-hydroxy-3-methoxy)phenoxy-3-methyl ether (191), and 7-methylkaempferol-3-O-neohesperoside-4'-O-rhamnoside (192)

(Mohamed et al., 2014).

106

107

Al-Musayeib et al. has isolated different compounds from leaves of Cadaba farinosa which included Lupeol (161), a novel ester of Lupeol; Lupeol-3-O-Decanoate

(193) Ursolic Acid (194), Quercetin-3-O-Glucose (195), Dillenetin-3-O-Glucose (196)

(Al-Musayeib et al., 2013).

6.4.3.2.Cadaba rotundifolia

This plant has been traditionally used in Sudan for treatment of tumors and abscesses (Teklehaymanot, 2017). Four novel acylated glycosides of kaempferol were yielded from n-butanol fraction of upper parts of Cadaba rotundifolia. These molecules were named as kaempferol 3-O-[2-O-(trans-feruloyl)-3-O-glucopyranosyl]- glucopyranoside (197), kaempferol 3-O-neohesperidoside-7-O-[2-O-(cis-p-coumaroyl)-3-

O-glucopyranosyl]-glucopyranoside (198), kaempferol 3-O-[2,6-di-O-l- rhamnopyranosyl]-glucopyranoside-7-O-[6-O-(trans-feruloyl)]-glucopyranoside (199) and kaempferol 3-O-[2,6-di-O-l-rhamnopyranosyl]-glucopyranoside (200). These compounds displayed potential antioxidant activity in DPPH Assay. Compound 200 showed high inhibition of production of advanced glycation end products with inhibitory concentration value of 0.0855 ±3.5 mM (Abdulaziz Al-Hamoud et al., 2019).

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109

PHYTOCHEMICAL AND BIOLOGICAL PROFILING OF AERIAL PARTS OF Crateva adansonii

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7.1. Biological Evaluation of different fractions of Crateva adansonii:

Previous investigations on Crateva adansonii have revealed potential activity against xanthine oxidase, carcinogenic cell lines, Trypanosma brucei and inflammation (Abdullahi et al., 2012, Ahama-Esseh et al., 2017, Igoli et al., 2014, Thirumalaisamy et al., 2018,

Zingue et al., 2016). Xanthine oxidase, Carbonic anhydrase, Urease and Tyrosinase have been implicated as potential factors involved in inflammation and carcinogenic cell proliferation (Büttner et al., 2019, Henry et al., 2016, Hudalla et al., 2019, Konieczna et al., 2012, Shimoda, 2019, Supuran, 2016, Uberti et al., 2013, Winum, 2018). Additionally, carbonic anhydrase and α-glucosidase are also expressed by Trypanosoma brucei (Jones et al., 2004, Vermelho et al., 2017). Henceforth, these drug targets were evaluated for their inhibition by Crateva adansonii for development of natural produsct based antitrypanosomal, anti-inflammatory and anticancer agents.

7.1.1. DPPH free radical scavenging assay Following table represents the DPPH Free radical scavenging assay. Standard

Ascorbic acid showed the highest %age inhibition i.e. 95.55±0.15% with IC50 value 20.15

±0.75 μmol followed by crude extract which showed 90.99±0.88 %age inhibition.

Chloroform & Butanolic fractions also showed considerably higher %age inhibition i.e.

88.6±0.11% with an IC50 value of 2.32±0.45 mg/ml and 90.7±0.56% with IC50 value

1.56±0.39 mg/ml. n-hexane fraction could not bring any change in color and their %age inhibition was less than 50%.

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Table 7.1. Antioxidant activity of Crateva adansonii

Samples Concentration %age IC50 (µg/ml) Inhibition Crude methanolic 5 mg/ml 84±0.57 2.7±0.24 extract Chloroform fraction 5 mg/ml 88.6±0.11 2.32±0.45 n-butanol fraction 5 mg/ml 90.7±0.56 1.56±0.39 n-hexane fraction 5 mg/ml 30.7±0.18 ---- Ascorbic Acid 0.5 mmol/ml 92.67±0.33 22.3 ±0.57

7.1.2. α-glucosidase inhibition assay

Following table represents the α-glucosidase inhibition assay. Standard Acarbose showed the highest %age inhibition i.e. 97.5±0.5% with IC50 value of 20.5 ± 0.72.

Chloroform fraction exhibited highest inhibition %age 94.6±0.43 with IC50 of 24.15 ± 0.45 followed by Methanolic fraction (85.7±0.3% with IC50 value of 27.95 ± 0.19) and butanolic fractions (60±0.5% with IC50 value of 70.95 ± 0.6). n-hexane fraction failed to show a significant inhibition.

Table 7.2. α-glucosidase inhibitory activity of all fractions of C. adansonii Alpha glucosidase inhibition Samples tested % IC50 (µg/mL) Acarbose 97.5±0.5 5.45 ± 0.22 Methanol fraction 85.7±0.3 27.95 ± 0.19 n-Butanol fraction 60 ±0.5 70.95± 0.6 Chloroform fraction 94.6±0.43 24.15 ± 0.45 n-hexane fraction 40.5±0.96 ----

7.1.3. Tyrosinase inhibition assay

Chloroform and n-hexane fractions showed noteworthy tyrosinase inhibition i.e;

94.6±0.5% (IC50 17.37 ± 0.47) & 88.6±0.4% (IC50 30.42 ± 0.29). Methanolic extract also showed significant inhibition 91.5±0.7 (IC50 28.96 ± 0.36) but butanolic fraction showed moderate inhibition as is evident in Table 6.3.

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Table 7.3 Tyrosinase inhibitory activity of all fractions of C. adansonii

Samples tested Tyrosinase inhibition % IC50 (µg/mL) Kojic Acid 97.9±0.52 1.2 ± 0.02 Methanol fraction 91.5±0.7 28.96 ± 0.36 n-Butanol fraction 61.46±0.7 65.9± 0.53 Chloroform fraction 94.6±0.5 17.37 ± 0.47 n-hexane fraction 88.6±0.4 30.42 ± 0.29

7.1.4. Carbonic anhydrase inhibition assay

Crude methanolic extract and other fractions were evaluated for inhibition of carbonic anhydrase in comparison to standard drug acetazolamide. Results revealed that methanol fraction showed highest inhibition rate at 91.6±0.5%, followed by crude butanol fraction at 85.2±0.7%, chloroform fraction at 37.68±0.14% and n-hexane fraction at

25.46±0.85%.

Table 7.4. Carbonic Anhydrase inhibitory activity of all fractions of C. adansonii

Samples tested Carbonic Anhydrase inhibition % IC50 (µg/mL) Acetazolamide 97.7±0.25 0.3 ± 0.12 Methanol fraction 91.6±0.5 29.3 ± 0.6 n-Butanol fraction 85.2±0.7 33.99± 0.5 Chloroform fraction 37.68±0.14 ----- n-hexane fraction 25.46±0.85 -----

7.1.5. Xanthine oxidase inhibition assay

Table 6.5 shows inhibition %age alongside IC50 values of all fractions of C. adansonii in comparison to standard drug Allopurinol. Only methanolic fraction showed significant inhibition at 80.7± 0.9% with IC50 value of 42.2 ± 0.6 followed by moderate inhibition by butanol (68.2± 0.7% with IC50 value of 57.86 ± 0.56). Chloroform fraction and n-hexane fraction was inactive.

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Table 7.5. Xanthine oxidase inhibitory activity of all fractions of C. adansonii

Samples tested Xanthine oxidase inhibition % IC50 (µg/mL) Allopurinol 92 ± 0.3 0.26±0.05 Methanol fraction 80.7± 0.9 42.2 ± 0.6 n-Butanol fraction 68.2± 0.7 57.86 ± 0.56 Chloroform fraction 46.3± 0.4 ----- n-hexane fraction 38.2± 0.5 -----

7.1.6. Urease Inhibition Assay

Table 6.6 demonstrates the %age inhibition of urease alongside IC50 values of different fractions of Crataeva adansonii. The findings suggest that hexane fraction exhibited most strong inhibition of urease with inhibition %age of 93.25% and IC50 value of 10±0.5 µg/ml. This inhibition %age was very close to inhibition %age of standard inhibitor thiourea i.e: 98±0.85%. Remaining fractions displayed insignificant inhibition lower than 50%.

Table 7.6. Urease inhibitory activity of all fractions of C. adansonii

Samples tested Urease inhibition % IC50 (µg/mL) Thiourea 98±0.85 1.2 ± 0.08 Methanol fraction 39.1±0.6 ---- n-Butanol fraction 41.26±0.55 ---- Chloroform fraction 32.6±1.4 ---- n-hexane fraction 95.65±0.23 10±0.5

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7.2. GC-MS Analysis of n-hexane fraction of Crateva adansonii:

n-hexane fraction of Crateva adansonii was subjected to GC-MS evaluation in a splitless mode thus revealing presence of 25 compounds.

Table 7.7. Identification of the components of n-hexane extract of C.adansonii

Relat ive Peak Retention Molecular area No. Time (min) Constituents Formula (%) 1 14.55 Pentadecane C15H32 0.6 2 15.757 2,6,10-Trimethyl Tetradecane C17H36 0.785 6,10,14-trimethyl-2- 3 18.479 Pentadecanone C18H36O 0.66 Hexadecanoic Acid, Methyl 13.58 4 19.348 Ester C17H34O2 9 5 19.959 Hexadecanoic Acid, Ethyl Ester C18H36O2 2.801 11.46 6 20.97 Methyl Linoleate C19H34O2 6 10.76 7 21.045 Methyl Linolenate C19H32O2 8 8 21.154 Phytol C20H40O 3.379 9 21.222 Octadecanoic Acid,Methyl Ester C19H38O2 2.092 9,12-Octadecadienoic Acid 10 21.534 (Z,Z)-,Ethyl ester C20H36O2 1.464 11 21.785 Octadecanoic Acid,Ethyl Ester C20H40O2 0.688 12 22.688 Erucic Acid C22H42O2 2.286 13 24.534 Docosanoic Acid, Methyl Ester C23H46O2 0.66 2-(ethylhexoxycarbonyl)- 14 24.758 benzoic acid C16H22O4 3.944 15 25.777 Heptacosane C27H56 1.288 16 27.182 Nonacosane C29H60 2.941 20.65 17 28.58 Tetratriacontane C34H70 2 18 29.205 17-Pentatriacontene C35H70 0.549 19 29.836 Hexatriacontane C36H74 8.216 20 30.366 γ-Sitosterol C29H50O 4.206 21 31.125 Lupenone C30H48O 4.65 22 31.106 Lupeol C30H50O 5.036 23 33.32 ψ-Taraxasterol C30H50O 6.25 24 29.65 Oleanolic acid C30H48O3 7.55

115

25 28.56 Lupanol C30H52O 10.52

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7.3. LC-MS Evaluation of Crude Methanolic Extract of Crateva adansonii:

Crude CH3OH fraction of Crateva adansonii was submitted for LC-MS analysis

(+ve &-ve mode) which thus revealed presence of 115 compounds in +ve mode and 132 compounds in –ve mode.

Table 7.8. +ve mode LC-MS Data of Crateva adansonii

Compoun Molecular d No. RT Mass Name Formula 1 0.561 127.01293

2 0.561 103.997

3 0.561 109.00248

4 0.561 150.02859 C4H10N2S2

5 0.562 199.96654 C11HClO2

6 0.606 174.11074 C6H14N4O2

7 0.624 202.04567 C4H6N6O4

8 0.626 242.17557

9 0.629 218.0188 C7H10N2O2S2

10 0.629 103.09714 C5H13NO

11 0.636 119.05181 MEG (sulfate) C3H9N3S

12 0.642 180.05697 2S,5S-Methionine C5H12N2O3S sulfoximine 13 0.642 131.04959 C7H5N3

14 0.656 146.06094 C6H11ClN2

15 0.662 112.05558

16 0.678 137.04319

17 0.691 174.09741 C3H10N8O

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18 0.712 334.179 10-isovaleryloxy-8,9- C19H26O5 epoxythymol-3- isovalerate 19 0.725 129.08086 C6H11NO2

20 0.731 100.05276 β-Penteic acid C5H8O2

21 0.734 115.06376 D-Proline C5H9NO2

22 0.737 172.13214 Acetylagmatine C7H16N4O

23 0.743 143.09385

24 0.881 100.05161 C5H8O2

25 0.883 129.07961 C6H11NO2

26 0.888 334.1836 Thr Ser Lys C13H26N4O6

27 0.902 123.02902

28 0.933 143.09404 1- C7H13NO2 Aminocyclohexanecarb oxylic acid 29 0.934 172.13187 Acetylagmatine C7H16N4O

30 0.936 106.06345 Diethylene glycol C4H10O3

31 0.987 129.0424 N-Acryloylglycine C5H7NO3

32 1.069 131.09453 L-Leucine C6H13NO2

33 1.171 131.09334 C6H13NO2

34 1.319 159.10056 delta-Guanidinovaleric C6H13N3O2 acid 35 1.435 158.06918 4-Methylene-L- C6H10N2O3 glutamine 36 1.543 99.10488 Cyclohexylammonium C6H13N

37 2.121 165.07821 4-(3-Pyridyl)-butanoic C9H11NO2 acid 38 2.145 143.05739 C6H9NO3

39 7.554 343.12654 N-(1-Deoxy-1- C15H21NO8 fructosyl)tyrosine 40 7.846 287.12701 SR95531 C15H17N3O3

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41 8.4 561.18495 C27H31NO12

42 8.421 164.04753 m-Coumaric acid C9H8O3

43 8.423 146.0369 Phenylpropiolic acid C9H6O2

44 8.439 341.11102 Diethylpropion(metabo C15H19NO8 lite VIII-glucuronide) 45 8.45 435.21521 Cadabicine C25H29N3O4

46 8.592 566.16341 Butein 4'-arabinosyl- C26H30O14 (1->4)-galactoside 47 8.633 594.15915 Luteolin 7- C27H30O15 rhamnosyl(1- >6)galactoside 48 8.658 434.12059 Coreopsin C21H22O10

49 8.659 432.10521 Apigenin 5-galactoside C21H20O10

50 8.665 902.26759 Mauritianin-7-O-β- C39H50O24 glucoside 51 8.694 435.2154 Cadabicine C25H29N3O4

52 8.769 432.10929 1-Methyl-4-nitro-5-(S- C14H20N6O8S Gluctathionyl) Imidazole 53 8.774 740.22049 C31H40N4O15S

54 8.861 435.2146 Cadabicine C25H29N3O4

55 8.889 886.25133 Kaempferol 3-(4''-(E)- C42H46O21 p- coumarylrobinobioside )-7-rhamnoside 56 8.889 1048.30396 Capilliposide II C48H56O26

57 8.943 902.24689 Saponaretin 2''-O-(6'''- C42H46O22 (E)-p- coumaroyl)glucoside 4'-O-glucose 58 8.974 432.10625 Apigenin 5-glucoside C21H20O10

59 8.979 578.16431 Isovitexin 7-O- C27H30O14 rhamnoside 60 9.186 248.12646 2-hydroxymethyl-6-[2- C11H20O6 methylbut-2- enoxy]oxane-3,4,5-triol

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61 9.202 886.25637 Kaempferol 3-(4''-(E)- C42H46O21 p- coumarylrobinobioside )-7-rhamnoside 62 9.332 196.10968 4-(2-OH-propoxy)-3,5- C11H16O3 dimethyl carbolic acid 63 9.366 1194.33907

64 9.372 397.36594 C23H47N3O2

65 9.409 886.25213 Kaempferol 3-(4''-(E)- C42H46O21 p- coumarylrobinobioside )-7-rhamnoside 66 9.428 395.35198 C23H45N3O2

67 9.993 338.09986 4-p-Coumaroylquinic C16H18O8 acid 68 10.18 320.09009 1-Naphthyl β-D- C16H16O7 3 glucuronide 69 10.18 338.10063 4-p-Coumaroylquinic C16H18O8 3 acid 70 10.18 164.04701 m-Coumaric acid C9H8O3 4 71 10.18 146.03714 Phenylpropiolic acid C9H6O2 4 72 10.38 277.09568 C14H15NO5 8 73 10.43 142.06298 7-OH-5-Heptynoic C7H10O3 3 Acid 74 10.43 174.08949 Suberic acid C8H14O4 5 75 10.59 306.07266 Robinetinidol-4alpha- C15H14O7 7 ol 76 11.05 263.22507 C17H29NO 3 77 11.34 464.22513 Linalool oxide D 3- C21H36O11 6 [apiofuranosyl-(1>6)- glucoside] 78 12.21 273.26642 C16 Sphinganine C16H35NO2

79 12.40 180.11442 3-tert-Butyl-5- C11H16O2 9 methylcatechol 80 12.82 315.27673 Dehydrophytosphingos C18H37NO3 5 ine

120

81 12.86 390.18858 LMFA02020206 C18H30O9

82 13.4 317.29267 Phytosphingosine C18H39NO3

83 13.75 307.28665 C20H37NO 9 84 13.85 216.13594 Undecanedioic acid C11H20O4 2 85 14.38 309.30274 Oleoyl Ethyl Amide C20H39NO 6 86 15.88 539.50123 C32H65N3O3 1 87 16.03 256.24125 2-hexyl-decanoic acid C16H32O2 4 88 16.24 278.22445 9,12,15- C18H30O2 3 octadecatrienoic acid 89 16.49 294.21871 9-epoxy-12,15- C18H30O3 4 octadecadienoic acid 90 16.61 294.21939 9-Epoxy-12,15- C18H30O3 5 octadecadienoic acid 91 16.80 294.21863 9-epoxy-12,15- C18H30O3 4 octadecadienoic acid 92 17.12 278.15398 C17H18N4

93 17.12 148.01736 C8H4O3

94 17.12 204.08025 C12H12O3 2 95 18.07 402.22486 Acetyl tributyl citrate C20H34O8 1 96 18.16 270.25685 12-Methyltetradecyl C17H34O2 8 acetate 97 18.59 278.22427 9,12,15- C18H30O2 1 octadecatrienoic acid 98 18.66 308.23459 Methyl (9,12)-14-(3- C19H32O3 7 ethyloxiran-2-yl)- tetradeca-9,12-dienoate 99 18.83 308.2345 Methyl (9,12)-14-(3- C19H32O3 4 ethyloxiran-2-yl)- tetradeca-9,12-dienoate 100 19.24 624.29393 C35H44O10 3 101 19.33 255.25525 Palmitic amide C16H33NO 3 102 19.42 608.26217 Harderoporphyrin C35H36N4O6 1

121

103 19.47 280.23982 Octadeca-6,9-dienoic C18H32O2 6 acid 104 19.50 624.25868 C35H36N4O7 2 105 19.54 330.2759 1-Monopalmitin C19H38O4 4 106 19.57 281.27177 Oleamide C18H35NO 3 107 19.73 608.26471 Harderoporphyrin C35H36N4O6 1 108 19.99 638.27284 C35H42O11 8 109 20.01 620.26276 C39H41ClN2OS 2 110 20.01 592.26801 Pheophorbide a C35H36N4O5 7 111 20.05 620.26411 C36H36N4O6 3 112 20.37 592.26886 Pheophorbide a C35H36N4O5 8 113 20.59 638.30005 C38H42N2O7 9 114 20.67 534.26332 Pyropheophorbide a C33H34N4O3 7 115 20.70 622.27986 C36H38N4O6 1

Table 7.9. -ve mode LC-MS Data of Crateva adansonii

Compoun d No. RT Mass Name Formula 1 0.56 387.94661

2 0.561 273.96579 C17H3ClS

3 0.561 159.98585 C3H4N4S2

4 0.612 226.06958 Glucoheptonic acid C7H14O8

5 0.638 180.0647 L-Galactose C6H12O6

6 0.652 166.04794 L-Arabinonic acid C5H10O6

7 0.654 196.05893 L-Gulonate C6H12O7

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8 0.663 243.05978 C6H13NO9

9 0.666 378.09399 C13H19ClN4O7

10 0.669 538.17485 C16H22N14O8

11 0.67 136.03743 Erythronic acid C4H8O5

12 0.674 405.11305 C13H19N5O10

13 0.683 192.06375 Quinic acid C7H12O6

14 0.694 552.19192 C20H32N4O14

15 0.698 210.07549 1,3,7-Trimethyluric C8H10N4O3 acid 16 0.706 180.06455 Theobromine C7H8N4O2

17 0.711 397.1834 C15H19N13O

18 0.758 134.02271 2,5-dimethoxy C5H10S2 thiophene 19 0.897 160.03797 2,3-diketo-4-deoxy- C6H8O5 epi-inositol 20 0.975 192.02852 C7H4N4O3

21 0.99 129.043 N-Acryloylglycine C5H7NO3

22 0.993 178.04822 5-Deoxy glucuronic C6H10O6 acid 23 1.132 160.0377 3D-(3,5/4)- C6H8O5 Trihydroxycyclohexane -1,2-dione 24 1.217 118.02748 C4H6O4

25 1.353 148.03763 2-Dehydro-3-deoxy-D- C5H8O5 xylonate 26 1.896 160.03733 3D-(3,5/4)- C6H8O5 Trihydroxycyclohexane -1,2-dione 27 2.172 160.03707 3D-(3,5/4)- C6H8O5 Trihydroxycyclohexane -1,2-dione 28 7.561 326.10112 Acetylaminodantrolene C16H14N4O4

29 7.63 316.11682 Vanilloloside C14H20O8

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30 7.833 340.08 Sinapoyl malate C15H16O9

31 7.985 296.14756 C12H24O8

32 7.99 432.20041 Val Trp Glu C21H28N4O6

33 8.114 566.25742 C25H42O14

34 8.137 450.11704 8-C- C21H22O11 Glucopyranosyleriodict ylol 35 8.264 450.11721 8-C- C21H22O11 Glucopyranosyleriodict ylol 36 8.36 610.15422 5-deoxymyricetin-3- C27H30O16 rutinoside 37 8.426 160.0373 3D-(3,5/4)- C6H8O5 Trihydroxycyclohexane -1,2-dione 38 8.435 324.08582 Mahaleboside C15H16O8

39 8.457 178.04786 2,4,6/3,5- C6H10O6 Pentahydroxycyclohex anone 40 8.533 756.21392 Kaempferol 3-(6””- C33H40O20 rhamnosyl-2’”-glucosyl rutinoside) 41 8.606 566.16536 Butein 4'-arabinosyl- C26H30O14 (1->4)-galactoside 42 8.636 594.16042 Luteolin 7- C27H30O15 rhamnosyl(1- >6)galactoside 43 8.649 1028.2808 1 44 8.659 657.15565 C28H27N5O14

45 8.668 902.2712 Mauritianin-7-O- C39H50O24 Glucose 46 8.671 434.12284 Coreopsin C21H22O10

47 8.771 2960.8917 3 48 8.774 854.20916

49 8.775 740.21519 robinin C33H40O19

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50 8.781 803.21111

51 8.889 1048.3086 Capilliposide II C48H56O26 3 52 8.942 902.25017 LMPK12110329 C42H46O22

53 8.964 578.16126 Isovitexin 7-O- C27H30O14 rhamnoside 54 8.969 550.25813 C18H30N16O5

55 8.971 944.28034

56 8.977 2312.6621 8 57 8.979 692.15352 C23H24N12O14

58 8.985 641.15508 C22H31N3O19

59 8.994 578.1593 C24H30N6O7S2

60 9.022 164.03699 C11H4N2

61 9.061 548.24271 C18H28N16O5

62 9.14 1210.3397 7 63 9.199 2658.7609

64 9.201 1000.2463 6 65 9.205 949.24961

66 9.205 886.25804 LMPK12111706 C42H46O21

67 9.259 566.16657 Butein 4'-arabinosyl- C26H30O14 (1->4)-galactoside 68 9.327 868.24377

69 9.367 1194.3482

70 9.375 1048.3064 Capilliposide II C48H56O26 7 71 9.401 448.10134 6-Hydroxyluteolin 5- C21H20O11 rhamnoside 72 9.413 886.25313 LMPK12111706 C42H46O21

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73 9.471 594.1602 Luteolin 7- C27H30O15 rhamnosyl(1- >6)galactoside 74 9.522 263.07993 C13H13NO5

75 9.532 1194.3460 5 76 9.644 852.24875

77 9.689 188.10484 Nonic Acid C9H16O4

78 9.762 1194.3471

79 9.879 870.2573

80 9.994 338.09899 4-p-Coumaroylquinic C16H18O8 acid 81 9.998 306.07245 Robinetinidol-4alpha- C15H14O7 ol 82 10.184 338.10012 4-p-Coumaroylquinic C16H18O8 acid 83 10.189 306.07312 Robinetinidol-4alpha- C15H14O7 ol 84 10.19 164.04563 C9H8O3

85 10.392 277.09561 C14H15NO5

86 10.467 432.10667 Isovitexin C21H20O10

87 10.601 164.04726 m-Coumaric acid C9H8O3

88 10.601 306.07474 Robinetinidol-4alpha- C15H14O7 ol 89 10.944 306.07452 Robinetinidol-4alpha- C15H14O7 ol 90 11.069 178.06328 8-OH-2-Decene-4,6- C10H10O3 diynoic acid 91 11.554 330.24126 5,8,12-OH-octa-9- C18H34O5 Decenoic acid 92 12.534 222.12568 (6S)-dehydrovomifoliol C13H18O3

93 13.411 294.18365 Gingerol C17H26O4

94 13.591 312.23051 9(S)-HpODE C18H32O4

95 14.251 314.246 9,10-Epoxy-18- C18H34O4 hydroxystearate

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96 14.42 314.24617 9,10-Epoxy-18- C18H34O4 hydroxystearate 97 14.604 724.38791 C34H60O16

98 15.205 316.26219 10,11-dihydroxy stearic C18H36O4 acid 99 15.22 294.21986 α-9(10)-EpODE C18H30O3

100 15.349 294.21992 α-9(10)-EpODE C18H30O3

101 15.882 330.19738 C18H31ClO3

102 16.038 296.23624 12-oxo-10Z- C18H32O3 octadecenoic acid 103 16.179 580.29238 C27H48O11S

104 16.5 294.21992 α-9(10)-EpODE C18H30O3

105 16.615 294.21994 α-9(10)-EpODE C18H30O3

106 16.681 332.21212 C18H33ClO3

107 16.806 294.21991 α-9(10)-EpODE C18H30O3

108 16.82 332.21258 C18H33ClO3

109 16.859 298.25122 cis-9,10-Epoxystearic C18H34O3 acid 110 16.925 312.17666 N- C17H28O3S Undecylbenzenesulfoni c acid 111 17.064 312.17644 N- C1H28O3S Undecylbenzenesulfoni c acid 112 17.086 556.29322 C26H44N4O7S

113 17.617 334.22875 9-hydroxy-10-chloro- C18H35ClO3 octadecanoic acid 114 17.723 582.30871 C28H46N4O7S

115 17.73 334.22796 9-hydroxy-10-chloro- C18H35ClO3 octadecanoic acid 116 18.169 326.19269 2- C18H30O3S Dodecylbenzenesulfoni c acid 117 18.603 278.22555 Octadeca-9,12,15- C18H30O2 trienoic acid

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118 18.684 272.23554 2- C16H32O3 Hydroxyhexadecanoic acid 119 19.497 380.16711 Ecabet C20H28O5S

120 19.498 280.24181 C18H32O2

121 19.547 366.25537 C20H35ClN4

122 20.195 300.2684 C18H36O3

123 20.2 328.29956 C20H40O3

124 20.333 356.16753 C19H24N4OS

125 20.382 133.93164 3H-1,2-Dithiole-3- C3H2S3 thione 126 20.388 101.94267

127 20.392 145.93219 C4H2S3

128 21.185 650.34938 C38H51ClN2O5

129 21.607 650.34595 C38H50O9

130 21.784 652.36297 C39H48N4O5

131 22.312 664.36243

132 22.434 227.98708 C4H8N2O5S2

GC-MS Spectra of n-hexane fraction revealed high content of sterols, triterpenes and esters. LC-MS Analysis of crude methanolic fraction disclosed the presence of a high amount of diverse natural products (flavonoids, glycosides, pheophorbides). Since methanolic fraction was further fractionated to n-hexane, CHCl3 and butanolic fractions, it was decided to isolate bioactive phytochemicals from these fractions rather than crude methanolic fraction.

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7.4. Structure elucidation of constituents isolated from n-hexane fraction (CaC-N)

7.4.1. Lupeol (161)

Compound 161 was obtained as white amorphous powder. The molecular formula

+ was estimated as as C30H50O by HR-ESI-MS [M+H ] at m/z 426.38125 a.m.u. (calculated

426.5566 a.m.u.). The infrared spectrum exhibited exclusive bands at C–H broad band

−1 -1 -1 -1 (2,970-2,850 cm ), CH2 (1450 cm ), CH3 (1370 cm ), sharp OH band (3720cm ), C=C

-1 band (1650 cm ), 950 (C=CH2)

The 1H-NMR Spectrum (Table 6.10) revealed 7 methyl singlets (δ0.79, 0.81, 0.85,

0.98, 1.01, 1.71), one methylene singlet at δ4.6 (H-29), a doublet of methine at δ 0.69 (J =

9.6, H-5) alongside a double doublet of methine at δ 1.29 ( J = 10.4, 2.9, H-9) and δ 1.7

(J=10.15, 2.87, H-18) and a methine triple doublet at δ1.4 (J = 9.95, 3.1, H-13). A characteristic resonance was a downfielded olefinic methylene singlet at δ4.6 ppm (H-29) and oxymethine double doublet at δ3.3 (J= 5.5, 10.9 Hz, H-3). These resonances were reminiscent of a presence of pentacyclic triterpene with lupane skeleton. Further evidence was supported by 13C-NMR spectrum which displayed 30 carbon signals consisting of ten methylene at δ 18.56, 21.1, 25.5, 27.5, 28.0, 31.1, 34.0, 35.5, 38.0, 40.8, 110.0, six methine at δ 40.01, 48.1, 48.2, 50.8, 55.15, 79.0, six methyl at δ 14.8, 15.8, 16.0, 18.8, 20.1, 29.5

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and five quarternary carbon signals at δ 37.2, 39.0, 41.0, 43.1, and 151.0. The mass spectrum displayed mass fragment peaks at 425, 410, 395, 380, 370, 330 which displayed specific fragmentation pattern of lupeol (Suttiarporn et al., 2015). The above mentioned data was correlated with previously reported literature which revealed compound 161 to be pentacyclic triterpene lupeol (Mahato and Kundu, 1994, Sholichin et al., 1980, Silva et al., 2017, Wang et al., 2017).

1 13 Table 7.10. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 161 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 38.0 CH2 1.7 m 2 25.5 CH2 1.5, 1.82 (dddd, J = 13.0, 10.3, 10.5, 3.0) 3 79.0 CH(OH) 3.3 (dd, J = 5.5, 10.8) 4 39.0 C 5 55.15 CH 0.69 (d, J = 9.6) 6 18.56 CH2 1.6, 1.75 m 7 34.0 CH2 1.5, 1.45 m 8 41.0 C 9 50.8 CH 1.29 (dd, J = 10.4, 2.9) 10 37.2 C 11 21.1 CH2 1.42, 1.7 m 12 27.5 CH2 1.39, 1.65 m 13 40.01 CH 1.4 (td, J = 9.95, 3.1) 14 43.1 C 15 28.0 CH2 1.5, 1.7 m 16 35.5 CH2 1.52, 1.4 m 17 43.1 C 18 48.1 CH 1.7 (dd, J = 10.15, 2.87) 19 48.2 CH 2.45 m 20 151.0 C 21 31.1 CH2 1.76 m 22 40.8 CH2 1.54 m 23 29.5 CH3 1.04 (s) 24 14.8 CH3 1.01 (s) 25 15.8 CH3 0.98 (s) 26 16.0 CH3 0.85 (s) 27 14.8 CH3 0.81 (s) 28 18.8 CH3 0.79 (s)

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29 110.0 CH2 4.6 (s) 30 20.1 CH3 1.71(s)

7.4.2. Lupanol (201)

Both carbon and proton NMR resonances indicated the presence of a pentacyclic triterpene with lupane skeleton similar to 161. The 1H-NMR Spectrum (Table 6.11) revealed 5 methyl singlets (δ1.0, 0.98, 0.85, 0.81, 0.79), a doublet of methine at δ 0.69 (J

= 9.6 Hz, H-5) alongside a double doubletof methine at δ 1.29 (J = 10.4, 2.9 Hz, H-9) and

1.7 (J=10.15, 2.87, H-18) and a methine triple doublet at δ 1.4 (J = 9.95, 3.1 Hz, H-13). A distinguishing feature was appearance of an additional methyl doublet at δ 1.04 (J = 1.67

Hz, H-29,30). This was accompanied by disappearance of methylene singlet at δ4.6 (H-

29).

13 C-NMR Spectrum displayed the presence of five –CH3 signals at δ 14.8, 15.8,

16.0, 18.8, 20, 20.1, 29.5, ten methylene signals at δ 25.5, 18.56, 21.1, 27.5, 28.5, 31.1,

35.0, 35.8, 39.1, 40.8, seven methine signals at δ 40.01, 48.25, 48.2, 51.0, 56.0, 80.2, 151.0 and five quarternary signals at δ 37.2, 40.0, 40.89, 43.1, 43.2. The C-NMR was in complete

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correspondence with mass and 1H-NMR Spectrum. Further comparison with published literature hence proved 201 to be lupanol (Suttiarporn et al., 2015).

1 13 Table 7.11. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 201 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 39.1 CH2 1.69 m 2 25.5 CH2 1.5, 1.82 (dddd, J = 13, 10.3, 10.5, 3.0) 3 80.2 CH(OH) 3.3 (dd, J = 5.5, 10.8) 4 40.0 C 5 56.0 CH 0.69 (d, J = 9.6) 6 18.56 CH2 1.6, 1.75 m 7 35.0 CH2 1.5, 1.45 m 8 40.89 C 9 51.0 CH 1.31 (dd, J = 10.4, 2.9) 10 37.2 C 11 21.1 CH2 1.42, 1.7 m 12 27.5 CH2 1.39, 1.65 m 13 40.01 CH 1.4 (td, J = 9.95, 3.1) 14 43.1 C 15 28.5 CH2 1.5, 1.7 m 16 35.8 CH2 1.52, 1.4 m 17 43.1 C 18 48.25 CH 1.8 (dd, J = 10.15, 2.87) 19 48.2 CH 1.86 (dddd, J = 9.0, 8.1, 5.2, 4.6) 20 151.0 CH 1.5 m 21 31.1 CH2 1.76 m 22 40.8 CH2 1.54 m 23 29.5 CH3 1.02 (s) 24 14.8 CH3 1.0 (s) 25 15.8 CH3 0.98 (s) 26 16.0 CH3 0.85 (s) 27 14.8 CH3 0.81 (s) 28 18.8 CH3 0.79 (s) 29 20.0 CH3 1.04 (d, J = 6.7) 30 20.1 CH3 1.04 (d, J = 6.7)

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7.4.3. Lupenone (202)

Compound 202 was obtained as white amorphous powder. The molecular formula

+ was estimated as C30H48O by HR-ESI-MS [M+H ] at m/z 424.37125 a.m.u. (calculated.

424.7025 a.m.u.). The infrared spectrum exhibited characteristic bands at C–H broad band

-1 -1 -1 (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm ), sharp OH band (3720cm ).

Both carbon and proton NMR resonances indicated the presence of a pentacyclic triterpene with lupane skeleton similar to 161. The 1H-NMR Spectrum (Table 6.12) exhibited loss of hydroxymethine resonance at δ3.3 ppm. 13C-NMR spectrum also demonstrated an extra strongly downfielded C=O resonance at δ218.02 ppm. Another distinguishing feature was an upfield shift methylene protons by 1 ppm as compared to lupeol at δ 2.5, 2.8 (ddd, J= 14.1, 10, 3.3 Hz, 2H, C-2) which was due to increase in electron density owing to substitution of C=O functionality in place of –CH(OH) moiety causing deshielding of methylene protons. This was accompanied by a downfield shift in methine proton by 1 ppm compared to lupeol 1.7 (dd, J = 9.8,2.0 Hz, H-5) which was due to pronounced shielding effect of 2 methylene groups at C-4.

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The mass spectrum displayed mass fragment peaks at 425, 410, 380, 368, 343 which displayed specific fragmentation pattern of lupenone (Suttiarporn et al., 2015). The above mentioned data was correlated with previously reported literature which revealed compound 202 to be pentacyclic triterpene lupenone (Mahato and Kundu, 1994, Sholichin et al., 1980, Silva et al., 2017, Wang et al., 2017).

1 13 Table 7.12. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 202 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 C/H. No. C-NMR (δC) (DEPT) constants JHH (Hz) 1 38 CH2 1.7 m 2 25.5 CH2 2.5, 2.8 (ddd, J = 14.1, 10,3.3) 3 218.02 C=O 4 39 C 5 55.15 CH 1.7 (dd, J = 9.8,2) 6 18.56 CH2 1.6, 1.75 m 7 34 CH2 1.5, 1.45 m 8 41 C 9 50.8 CH 1.29 (dd, J = 10.4, 2.9) 10 37.2 C 11 21.1 CH2 1.42, 1.7 m 12 27.5 CH2 1.39, 1.65 m 13 40.01 CH 1.4 (td, J = 9.95, 3.1) 14 43.1 C 15 28 CH2 1.5, 1.7 m 16 35.5 CH2 1.52, 1.4 m 17 43.1 C 18 48.1 CH 1.7 (dd, J = 10.15, 2.87) 19 48.2 CH 2.45 m 20 151 C 21 31.1 CH2 1.76 m 22 40.8 CH2 1.54 m 23 29.5 CH3 1.12 (s) 24 14.8 CH3 1.13 (s) 25 15.8 CH3 0.98 (s) 26 16 CH3 0.85 (s) 27 14.8 CH3 0.81 (s) 28 18.8 CH3 0.79 (s) 29 110 CH2 4.6 (s) 30 20.1 CH3 1.71(s)

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7.5. Structure elucidation of Constituents isolated from chloroform fraction

(CaC-Cl)

7.5.1. Pheophorbide A (203)

Compound 203 was obtained as dark brown amorphous solid. The molecular

+ formula was ascertained as C35H36N4O5 by HR-ESI-MS [M+H ] at m/z 592.26801a.m.u.

- (calcd. 593.27555 a.m.u.). The IR spectrum showed characteristic bands at CH2 (1450 cm

1 -1 -1 -1 ), CH3 (1370 cm ), broad OH band (2400 and 2800 cm ), C=C band (1622 cm ), 1702

-1 -1 cm (C=O, broad band) and 1100 cm (OCH3 group). This compound was identified in

LC-MS Spectrum (+ve mode) at retention time of 20 min (Compound 110).

Both 1H-NMR and 13C-NMR (Table 6.13) displayed a broad range of chemical shifts indicative of the presence of a cyclic tetrapyrrole nucleus. These effects were encountered owing to diamagnetic ring current. (Gomes and Mallion, 2001, Levin and

Roberts, 1973).

In the 1H-NMR spectrum, the methine bridge protons at δ 9.4, 9.5, 8.5 were extremely downfielded owing to strong deshielding effects of pyrrole & pyrroline rings.

Protons at C-5 & C-10 are more deshielded (δ9.4, 9.5) because they experience a higher

135

electron density environment due to proximity to a ring I methylene (C-31 and C-32) & ring

V keto carbonyl group (C-131) compared to methine proton at C-20 (δ8.5). A spin-spin splitting of AMX pattern was observed within range of 6-8 ppm which was assigned to vinyl functionality of ring I at δ 8.0 (dd, J= 11, 17.5, H-31), 6.10 (d, J=11.2, H-32 a), 6.25

(d, J =17.5, H-32 b). In the region of δ3.00~4.00, some sharp singlets were detected for methyl or methoxy groups, and some methyl singlets were also noticed between δ1.60 and

δ1.90.

The 13C NMR spectra displayed four olefinic methines between δ90.0 and δ130.0, one olefinic methylene at around δ123.0, three methines and one methoxy in the region of

δ50.0~65.0, and four methyl singlets between δ10.0 and δ25.0. The spectra also exhibited three methylenes between δ20.0 and δ31.0, and three carbonyls were observed in the region of δ170.0~190.0.

On the basis of the above information and comparing with previous literature, the data obtained for compound 203 was identified as pheophorbide A. The spectra results were in good agreement with published data for this compound.(Bafor et al., 2013, Chen et al., 2015, Holt and Jacobs, 1955, Juin et al., 2015, Kamarulzaman et al., 2011, Katz Jj and Brown Ce, 1983, Zhao et al., 2014).

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1 13 Table 7.13. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 203 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 142 C 2 132.01 C 1 2 11.89 CH3 3.4 (s) 3 137 C 3 1 130.1 CH 8.0 (dd, J = 11, 17.5) 2 3 123.03 CH2 6.10 (d, J = 11.2), 6.25 (d, J = 17.5) 4 137.2 C 5 97.8 CH 9.4 (s) 6 156.2 C 7 135.78 C 1 7 10.8 CH3 3.2 (s) 8 146 C 1 8 20.1 CH2 3.65 m 2 8 17.6 CH3 1.71( t, J = 3.5) 9 151.06 C 10 103.56 CH 9.5 (s) 11 138 C 12 129 C 1 12 12.2 CH3 3.75 (s) 13 128.67 C 1 13 190.05 C 13 2 64.98 CH 6.4 (s) 3 13 170.12 C 4 13 53.3 CH3 4.01 (s) 14 150.4 C 15 105 C 16 160.6 C 17 51.5 CH 4.35 (d, J = 9) 17 1 30.1 CH2 2.3 m, 2.7 m 2 17 31 CH2 2.4 m, 2.6 m 3 17 178 C 18 50.1 CH 4.56 (d, J =7.5) 1 18 23.2 CH3 2.01 (d, J = 7.8) 19 173 C 20 92.5 CH 8.5 (s)

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7.5.2 Pyropheophorbide A (158)

Compound 158 was obtained as greenish black amorphous solid. The molecular

+ formula was ascertained as C33H34N4O3 by HR-ESI-MS [M+H ] at m/z 534.26332 a.m.u.

(ccalculated. 535.27073 a.m.u.) suggesting 19 degrees of unstauration. The infrared

-1 spectrum exhibited exclusive bands at -CH=CH2 (920 cm ), broad OH band (2400 and

2800 cm-1), C=C band (1621 cm-1), 1705 cm-1 (C=O, broad band). This compound was identified in LC-MS Spectrum (+ve mode) at retention time of 20.67 min (Compound 114).

Both 1H-NMR & 13C-NMR showed high similarity to data obtained for compound

203. A significant change in both of these spectra was loss signals for carbomethoxy chain at C-132. 1H-NMR Spectrum exhibited a doublet due to magnetically equivalent protons at

δ4.07 (2H, d, J = 17.1, Ha-172, Hb-172).

The 13C NMR spectra displayed three olefinic methines at δ20.1, 29.87, 31 93.6,

98, 104, 130, two methines at δ50.1 and δ51.5. Methylene carbons were recognized at

δ123.03, alongside an additional upfielded methylene signal at δ44.5. Two carbonyl resonances were noticed at δ178 and δ189.8. Three methlyl singlets associated with pyrrole

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rings of corrin nucleus were identified at δ3.2, 3.4, 3.75 alongwith a methyl doublet at δ

1.8 (d , J = 8.1).

The spectra of 158 (Table 6.14) was compared with the chlorophyll degradation product pyropheophorbide A which confirmed the structure of 158 as pyropheophorbide A

(Lai et al., 2010, Holt and Jacobs, 1955, Katz JJ and Brown CE, 1983, Juin et al., 2015, Bafor et al., 2013) (Lai et al., 2010).

1 13 Table 7.14. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 158 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 142 C 2 132.01 C 1 2 11.89 CH3 3.4 (s) 3 137 C 3 1 130 CH 7.95 (dd, J = 11.4, 17.9) 2 3 123.03 CH2 6.10 (d, J = 11.2), 6.25 (d, J = 18) 4 137.2 C 5 98 CH 9.35(s) 6 156.2 C 7 135.78 C 1 7 10.8 CH3 3.2 (s) 8 146 C 1 8 20.1 CH2 3.65 (q, J = 7.9) 2 8 17.6 CH3 1.65 (t, J = 7.9) 9 151.06 C 10 104 CH 9.5 (s) 11 138 C 12 129 C 1 12 12.2 CH3 3.75 (s) 13 128.67 C 1 13 189.8 C 2 13 44.5 CH2 5.10 (d, J = 20) 5.3 (d, J = 20) 14 150.4 C 15 118 C 16 160.6 C 17 51.5 CH 4.4 (d, J = 8.4) 1 17 29.87 CH2 2.2 m, 2.3 m

139

2 17 31 CH2 2.4 m, 2.56 m 3 17 178 C 18 50.1 CH 4.56 (dq, J =7.5,1.5) 1 18 23.2 CH3 1.8 (d, J =8.1) 19 171.5 C 20 93.6 CH 8.5 (s)

7.5.3 Phytosphingosine (204)

Compound 204 was obtained as dark coloured waxy semi-solid. This compound was UV inactive. The molecular formula was estimated as C20H37NO by HR-ESI-MS

[M+H+] at m/z 308.2940 a.m.u. (calculated 309.297 a.m.u.). The infrared spectrum showed characteristic bands at 3315 (hydroxyl), 1650, and 1550 cm−1 (amide). The 1H NMR spectrum (Table 6.15) showed highly overlapped signals at δ 1.29-1.31, indicatαive of long aliphatic carbon chain.

13C NMR spectrum hinted the presence of one C=O carbon, four carbons attached to heteroatoms, and six olefinic carbons on the basis of their characteristic chemical shifts.

All of this information of 1 allowed us to deduce a ceramide nature. Spectral data confirmed the structure of 204 as Phytosphingosine upon comparison (Tan and Chen,

2003).

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1 13 Table 7.15. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 204 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 Position C-NMR (δC) (DEPT) constants JHH (Hz) 1 63 CH2 3.5, 3.2 (d, J = 5.5) 2 55 CH 2.78 (td, J = 6.0,5.1) 3 81 CH 3.3 (dd, J = 5.0,4.21) 4 71.5 CH 3.6 m

5 32.8 CH2 1.4 m 6 26 CH2 1.25 m 7 30.5 CH2 1.25 m 8 30.5 CH2 1.3 m 9 30.5 CH2 1.27m 10 30.5 CH2 1.27m 11 30.5 CH2 1.27m 12 30.5 CH2 1.27m

13 30.5 CH2 1.27m 14 29.9 CH2 1.27m 15 30 CH2 1.31m 16 32 CH2 1.31m 17 23 CH2 1.31m 18 15 CH3 0.91 (t, J = 7)

7.5.4. Dehydrophytosphingosine (205)

Compound 205 had wax like semi solid appearance. It exhibited no signals in UV spectrum. The molecular formula was estimated as C18H37NO3 by HR-ESI-MS [M+H+]

HR-ESI-MS [M+H]+ at m/z 316.2841 a.m.u. (calculated 317.2871 a.m.u.). The infrared spectrum exhibited absorption bands at 3315 (hydroxyl), 1650, and 1550 cm−1 (amide).

Both 1H NMR and 13C NMR (Table 6.16) spectrum showed absorptions similar to compound. However there was loss of 2 methylene proton signals in overlapping region at

δ 1.29-1.31, accompanied by highly downfielded methine proton signal at  5.5. Downfield shift was also observed in 13C NMR spectrum at 130 which confirmed presence of a C=C bond. Compound 205 was henceforth confirmed as dehydrogenated derivative of

Phytosphingosine i.e; Dehydrophytosphingosine (Tan and Chen, 2003).

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1 13 Table 7.16. H- (400 MHz) and C-NMR (200 MHz) chemical shifts of 205 in CDCl3 Multiplicity 1H-NMR (δH) coupling 13 C/H. No. C-NMR (δc) (DEPT) constants JHH (Hz) 1 63 CH2 3.5, 3.2 (d, J = 6.01) 2 55 CH 2.78 (td, J = 5.89,4.99) 3 81 CH 3.33 (dd, J = 5.1,4.21) 4 71.5 CH 3.6 m 5 32.8 CH2 1.4 m

6 26 CH2 1.25 m 7 30.5 CH2 1.25 m 8 130.5 CH 1.3 m 9 130.5 CH 1.27 m 10 30.5 CH2 1.27 m 11 30.5 CH2 1.27 m 12 30.5 CH2 1.27 m 13 30.5 CH2 1.27 m 14 29.9 CH2 1.27 m 15 30 CH2 1.31 m 16 32 CH2 1.31 m 17 23 CH2 1.31 m 18 15 CH3 0.91 (t , J = 6.9)

7.6. Structure Elucidation of constituents isolated from butanolic fraction (CaC-B)

7.6.1 Cadabicine (177)

Compound 177 was obtained as greenish black amorphous solid. The molecular

+ formula was ascertained as C25H29N3O4 by HR-ESI-MS [M+H ] at m/z 436.2225 a.m.u.

(calculated 437.22554 a.m.u.). Absorption bands of IR Spectrum appeared at 3410 (NH),

1643 and 1635 (C=O), 1537 and 1505 cm-1 for aromatic rings. This compound was identified in LC-MS Spectrum (+ve mode) at different retention times (8.45, 8.694, 8.861 min)

(Compound 45, 51, 54).

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The 1H NMR spectrum (Table 6.17) of 177 revealed readily the ABX splitting patterns of the ring A which was inferred from a double doublet at δ 6.93 (J =8.5, 2.01, H-

24) a doublet at δ 7.01 (J =8.23, H-25) and a doublet at δ 6.35 (J=2.0, H-27). AA'XX' splitting was noted due to 2 doublets at δ 7.72 and 7.23 (J =8.9, H-2, H-29) for ring B due to the para-substitution of the benzene moiety. Three downfielded broad –NH singlets were observed at δ8.0, 8.3, 9.89. Moreover, mass spectrum revealed a fragment ion at m/z 203, which confirmed presence of spermidine backbone.

13C NMR spectra displayed two singlets in the carboxylic region (δ165, 165.1) and

14 signals in the aromatic/alkene region (at approx. δ 111-160) which indicated presence of 2 cinnamic acid units. The distinctive signals for the aliphatic carbons of a spermidine moiety were found within range of δ25-50.

Comparison of all spectroscopic data of compound 177 with previously reported literature confirmed it to be codonocarpine type alkaloid, cadabicine (Ahmad et al., 1985,

Forster et al., 2016, Fu et al., 2008, Khanfar et al., 2003).

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Table 7.17. 1H- (400 MHz) and 13C-NMR (200 MHz) chemical shifts of 177 in DMSO

Multiplicity 1H-NMR (δH) coupling 13 C/H. No. C-NMR (δC) (DEPT) constants JHH (Hz) 1 149 C 3 156.2 C 4 122.5 CH 7.23 (d, J = 8.7) 5 130 CH 7.8 (d, J = 8.9) 6 132.9 C 7 141.03 CH 7.55 (d, J = 15.9) 8 124.6 C 6.63 (d, J = 16.1) 9 165 C=O 10 NH 8.3 br (s) 11 36.05 CH2 3.35 m 12 25.5 CH2 1.87, 1.89 m 13 44.2 CH2 2.8, 2.9 m 14 NH 9.01 br (s) 15 46.5 CH2 2.8, 2.9 m 16 23.2 CH2 1.61, 1.69 m 17 25.2 CH2 1.38,1.42 m 18 39 CH2 3.15, 3.19 m 19 NH 8.0 br (s) 20 165.1 C=O 21 119 CH 6.01 (d, J = 16.1) 22 138.5 CH 7.22 (d, J = 15.8) 23 126 C 24 125.5 CH 6.93 (dd, J = 8.5, 2.01) 25 117.1 CH 7.02 (d, J = 8.23) 26 148.5 C-OH 9.89 br (s) 27 111.1 CH 6.35 (d, J = 2.0) 28 122.5 CH 7.23 (d, J = 8.9) 29 130 CH 7.77 (d, J = 8.9)

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7.6.2 Isovitexin (206)

Compound 206 was obtained as yellow solid. The molecular formula of this

- compound was estimated as C21H20O10 by HR-ESI-MS [M-H ] at m/z 432.10667 a.m.u.

(calculated 431.09913 a.m.u.). Bands of infra red spectrum were observed at 3350 (OH phenolic), 1710 (C=O carboxyl) and 1520 (C=C) and 1022 cm-1(glycoside linkage). This compound was identified in LC-MS Spectrum (-ve mode) at a retention time of 10.46 min

(Compound 86).

The 1H NMR spectrum (Table 7.9) of the compound 206 showed typical resonances for a glycosylated flavone. Six aromatic proton resonances were identified within range of δ 6-

8. A set of four AA’BB’ proton signals at δ 7.88 (d, J=8.5 Hz, H-2' and H-6') and 7.01 (d,

J=8.88 Hz, H-3', H-5') located in ring B alongside a highly downfielded hydroxymethine singlet at δ 12.25 indicated presence of flavone moiety. Anomeric –CH doublet was observed at δ 4.56 (J = 9.8 Hz) alongwith other –CH double doublets at δ 3.81 (J = 2.5,

3.1), 3.4 (dd, J=2.5, 4.1), 3.2 (dd, J=3.4, 4.1) indicating the sugar moiety to be β-glucose.

The 13C NMR spectrum (Table 6.18) supported above data by showing fifteen signals in aromatic region at δ 165.89, 104.02, 184.0, 161.99, 110.0, 164.3, 95.3, 157.7,

104.88, 121, 130.01, 116.5, 163.0, 116.5, 130.0 The remaining six carbon NMR

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resonances, which were derived from the 1D carbon spectrum, five methine at δ 76.12,

71.12, 78.16, 73.0, 80.88 and one hydroxymethylene group at δ 62.54 suggested the presence of β-D-Glucose. Anomeric methine resonated at δ 76.12 ppm suggesting the molecule to be a C-glycoside. Loss of proton resonance at C-6 of ring A of apigenin flavone portion alongwith a downfield shift of δ110 indicated glycosidic linkage at C-6 of apigenin flavone.

The existence of glucose moiety was also substantiated by the presence of peak at m/z

255.0712 a.m.u. in the esi spectrum due to loss of sugar peak from M+55 peak. The LC-MS,

1H-NMR & 13C-NMR collectively established the structure of compound 206 to be a C- glucosylated flavonoid Isovitexin (Ganbaatar C et al., 2015, Gattuso et al., 2006, Peng et al.,

2005, Yang et al., 2015).

Table 7.18. 1H-(400 MHz) and 13C-NMR (200 MHz) chemical shifts of 206 in DMSO C/H. 13C-NMR Multiplicity 1H-NMR (δH) coupling No. (δC) (DEPT) constants JHH (Hz) 2 165.89 C 3 104.02 CH 6.6 (s) 4 184.0 C=O 5 161.99 C-OH 12.25 (s) 6 110.0 C-Gluc 7 164.3 C-OH 10.5 (s) 8 95.3 C 9 157.7 C 10 104.88 C 1' 121.0 C 2' 130.01 CH 7.88 (d, J = 8.5) 3' 116.5 CH 7.01 (d, J = 8.8) 4' 163.0 C-OH 8.05 (s) 5' 116.5 CH 7.01 (d, J = 8.8) 6' 130.0 CH 7.88 (d, J = 8.5) 2" 76.12 CH 4.56 (d, J = 9.8) 3" 71.12 CH 3.81 (dd, J = 2.5, 3.1) 4'' 78.16 CH 3.4 (dd, J = 2.5,4.1)

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5" 73.0 CH 3.2 (dd, J = 3.4,4.1) 6" 80.88 CH 3.5 m 4.0 (dd, J = 12.2, 1.9), 3.89 (dd, J = 7" 62.54 CH2 12.2,5.6)

7.6.3 Isovitexin 7-O-Rhamnoside (207)

Compound 207 was obtained as yellowish white crystals. The molecular formula

- was ascertained as C27H30O15 by HR-ESI-MS [M-H ] at m/z 577.15536 a.m.u. (calculated

578.15463 a.m.u.) The infrared spectrum demonstrated cabsorption bands at 3355 (OH phenolic), 1710 (C=O carboxyl) and 1520 (C=C) and 1022 (glycoside linkage), 1450 cm-1

(aromatic group). This compound was identified in LC-MS Spectrum (+ve mode) at a retention time of 8.98 min (Compound 53) and –ve mode at retention time of 8.96 min.

All spectroscopic data for compound 207 showed high similarity to compound 206 indicating the presence of isovitexin. (Yang et al., 2015, Peng et al., 2005, Ganbaatar C et al., 2015, Gattuso et al., 2006).

Additional proton resonances densely populated the structural reporter region which confirmed presence of an extra sugar moiety and upfielded methyl doublet δ1.26 (J

= 6.4, 3H, H-6’”) confirmed the new glycone portion to be a deoxysugar. Since the anomeric methine proton resonated at δ5.2 (J=11.02, 1H, H-1’”), this confirmed the

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additional glycine moiety to be Rhamnose. Loss of hydroxyl singlet at δ10.5 associated with carbon 7 of ring A of flavone portion indicated that rhamnose sugar was attached to carbon 7.

13C-NMR (Table 6.19) also displayed additional 6 signals in comparison to 206.

Anomeric carbon in these additional resonances resonated at δ95.01 further confirming presence of rhamnose. By comparison of the spectral data (1H- and 13C NMR) of compound

207 (Table 7.10) with a known compound isovitexin 7-O-rhamnoside from literature it was confirmed that 207 is isovitexin 7-O-rhamnoside (Blunder et al., 2017, Mabry et al., 1970).

Table 7.19. 1H- (400 MHz) and 13C-NMR (200 MHz) chemical shifts of 207 in DMSO C/H. 13C-NMR Multiplicity 1H-NMR (δH) coupling No. (δC) (DEPT) constants JHH (Hz) 2 165.89 C 3 103.99 CH 6.6 (s) 4 185.2 C=O 5 162 C-OH 12.25 (s) 6 110 C-Gluc 7 153.8 C-Rham 8 95.3 C 9 157.7 C 10 104.88 C 1' 121 C 2' 130.01 CH 7.89 (d, J = 8.5) 3' 116.5 CH 6.87 (d, J = 9) 4' 163 C-OH 7.98 (s) 5' 116.5 CH 6.87 (d, J = 9) 6' 130 CH 7.89 (d, J = 8.5) 2" 80.88 CH 3" 71.12 CH 4.02 (dd, J = 2.5, 3.1) 4'' 78.16 CH 3.4 (dd, J = 2.5,4.11) 5" 73 CH 3.2 (dd, J = 3.38,4.11) 6" 76.12 CH 4.56 (d, J = 9.8) 4.0 (dd, J = 12.2, 1.89), 3.89 (dd, J = 7" 62.54 CH2 12.2,5.6) 1''' 95.01 CH 5.2 (d, J = 11.02) 2''' 70.4 CH 3.6 m 3''' 73.2 CH 3.1 (dd, J = 3.5,2.7)

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4''' 72.4 CH 3.6 (t, J = 3.5) 5''' 74.2 CH 3.2 (dd, J = 3.4,2.7) 6''' 18.5 CH3 1.26(d, J = 6.4)

7.7. Biological Evaluation of phytochemicals isolated from Crateva adansonii: 7.7.1. Urease inhibition assay Bioassay guided isolation led to isolation of 3 Lupane type pentacyclic triterpenes from n-hexane fraction of Crataeva adansonii. These compounds were further evaluated for their urease inhibitory profile.

Table 7.20. Urease inhibitory activity of phytochemicals isolated from Crateva adansonii

Compounds Urease inhibition % IC50 (µg/mL)

Lupeol (161) 89.3±0.6 4.32±0.56 Lupanol (201) 94.4±0.8 3.66±0.54 Lupenone (202) 90.2±0.6 3.87±0.45 Thiourea 98 ± 0.5 1.0±0.08

Lupane type pentacyclic triterpenes have been reported to show urease inhibition

(Kazmi et al., 2011). Inhibition for urease was observed in following pattern: Lupanol

(201) [94.4±0.8%-IC50 3.66±0.54] > Lupenone (202) [90.2±0.6%-IC503.87±0.45] >Lupeol

(161) [89.3±0.6%-IC50 4.32±0.56]. Relatively higher activity of lupanol could be attributed to an additional methyl group in place of methylene moiety.

7.7.2. Tyrosinase Inhibition Assay

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Bioassay guided isolation of chloroform fraxction led to isolation of 4 compounds which were further evaluated for their tyrosinase inhibition potential.

Table 7.21. Tyrosinase inhibitory activity of phytochemicals isolated from Crateva adansonii

Compounds Tyrosinase Inhibition Assay % IC50 (µg/mL) Pheophorbide A (214) 85.7±0.7 5.52±0.34 Pyropheophorbide (160) 24±0.22 ---- Phytosphingosine (215) 25.6±0.86 ---- Dehydrophytosphingosine (216) 15.5±0.5 ---- Kojic acid 99±0.8 1.5±0.14

Tyrosinase catalyzes two essential reactions in the melanogenesis, oxidation of monophenols to diphenols, and diphenols to o-quinone that ultimately transforms to melanin (Kanteev et al., 2015). Pheophorbide A (214) showed IC50 values [5.52±0.34].

- Mushroom Tyrosinase consists of Cu (II) ions at its active site ligated via –OH/O2 ion when the enzyme is activated. An important inhibitory mechanism shared amongst different molecules is presence of ketonic or hydroxyl moiety that chelates copper ions by

- displacing –OH/O2 ion. Metal chelation studies on chlorophyll have concluded that ß- ketoester system at the periphery of the chlorin ring plays important role in chelating Zn

(II) or Cu (II) ions (Bechaieb et al., 2016). Since pheophorbides have not been evaluated for their anti-tyrosinase potential before, it can be postulated that β-ketoester chain (C-131 and C-132) of ring V chelates Cu(II) ions of tyrosinase active site. Pyropheophorbide (17) totally lacks β-ketoester chain (C-131 and C-132) and formyl butyrate chain at C-17 of ring

IV, so it fails to show more than 50% inhibition. Sphingolipids (215 & 216) failed to inhibit tyrosinase.

7.7.3. α-glucosidase inhibition assay

150

Since chloroform fraction showed remarkably higher inhibition of alpha glucosidase (94.8±0.77 with IC50 value of 24±0.45) which was close to standard acarbose, pheophorbides isolated from this fraction were also evaluated for α-glucosidase inhibition.

Table 6.22. α-glucosidase inhibitory activity of phytochemicals isolated from Crateva adansonii

Compounds α-glucosidase Inhibition Assay % IC50 (µg/mL) Pheophorbide A (203) 81±0.5 6.84±0.83 Pyropheophorbide (158) 85.5±0.5 6.3±0.28 Phytosphingosine (204) 30.5±0.86 ---- Dehydrophytosphingosine (205) 26.5±0.98 ---- Acarbose 96.9±0.48 4.5±0.14

Pheophorbides have never been evaluated before for inhibitory potential of glucosidase. Chlorophyll rich ethanolic extracts of Camellia sinensis and Eugenia uniflora showed IC50 values 5 times lower than standard acrabose when used in combination

(Vinholes et al., 2017). D.G. Semaan et al. has shown that pheophytin rich crude extract of antidiabetic plant Allophylus cominia inhibited α-glucosidase albeit at a lesser rate than flavonoid rich fraction (Semaan et al., 2017). Nonetheless, several pyrrole based inhibitors have been evaluated for their α-glucosidase inhibitory potential (Bekircan et al., 2015,

Chaudhry et al., 2017). Molecular modeling studies have revealed that pyrrole nucleus shows interactions with key amino acid residues of glucosidase active site in a manner similar to acarbose (Jadhav et al., 2017, Kaur et al., 2018, Taha et al., 2015). But pyropheophorbide (160) showed relatively higher IC50 value of 6.3±0.28 than pheophorbide which may be attributed to the loss of β-ketoester chain (C-131 and C-132) and formyl butyrate chain at C-17 of ring IV. Sphingosines (215 & 216) failed to show significant inhibition.

151

7.7.4. Carbonic Anhydrase inhibition assay

Since butanolic fraction exhibited highest inhibition of carbonic anhydrase

92.35±0.25% with IC50 value of 13±0.5, the compounds isolated from n-butanol fraction were further evaluated for Carbonic Anhydrase inhibition.

Table 6.23. Carbonic anhydrase inhibitory activity of phytochemicals isolated from Crateva adansonii Carbonic Anhydrase Inhibition IC Compounds 50 %age (µg/mL) Cadabicine (177) 97.52±0.51 2.04±0.45 Isovitexin (206) 95.25±0.6 4.71±0.28 Isovitexin 7-O-rhamnoside 64.46±0.5 7.54±0.3 (207) Acetazolamide 98.65±0.22 0.24±0.06

Several natural & synthetic polyamine alkaloids containing spermine/spermidine have been reported to inhibit carbonic anhydrase (Davis et al., 2014, Karioti et al., 2016,

Poulsen and Davis, 2014). As Cadabicine shows lowest IC50 value of 2.04±0.45 it could be attributed to presence of spermidine backbone. Crystallographic studies of spermidine- carbonic anhydrase complex revealed that necessary factors required to inhibit carbonic anhydrase are H-bonding between –NH. Group and Zn(OH)2 moiety, a network of hydrophobic/H-bond interactions between aliphatic chain and active site amino acids, presence of bulky terminal groups (Carta et al., 2010). C-Glucosylated flavonoids, in specific, synthetic C-cinnamoyl glycosides have shown to be excellent inhibitors of carbonic anhydrase (Riafrecha et al., 2015, Zaro et al., 2016) Isovitexin, a natural C- glycoside inhibited at %age of 95.25±0.6 with IC50 value of 4.71±0.28 whereas it’s rhamnosidyl derivative (218) showed relatively low inhibition. This could be attributed to additional rhamnosidic sugar moiety which might have obscured key interactions between flavone portion and active site amino acid residues.

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7.7.5. Xanthine oxidase inhibition Assay

Butanolic fraction exhibited moderate inhibition of xanthine oxidase. So compounds isolated for analyzed for inhibition of xanthine oxidase.

Table 6.24. Xanthine oxidase inhibitory activity of phytochemicals isolated from Crateva adansonii Xanthine Oxidase Inhibition IC Compounds 50 %age (µg/mL) Cadabicine (183) 50.2±0.3 ---- Isovitexin (206) 33.12±0.52 ---- Isovitexin 7-O-rhamnoside 46.2±1.4 ----- (207) Allopurinol 90.5±0.5 0.24±0.04

None of compounds isolated from n-butanol fraction of Crateva adansonii showed significant inhibition of xanthine oxidase. Cos et al. has reported that 6/8 C-glycosidic derivatives showed extremely reduced inhibition of xanthine oxidase (Cos et al., 1998).

153

EXPERIMENTAL

154

8.1. General Experimental Conditions

8.1.1. 1H-NMR, 13C-NMR & MS Same conditions were applied for LC-MS as discussed in the previous section 4.1.1.

8.1.2. LC-MS

Same conditions were applied for LC-MS as discussed in the previous section 4.1.2.

8.1.3. GC-MS Analysis

Same conditions were applied for LC-MS as discussed in the previous section 4.1.3.

8.1.4. Chromatographic separation

Same conditions were applied for LC-MS as discussed in the previous section 4.1.4.

8.2. Materials and Methods

8.2.1. Plant collection

Aerial portion of Crateva adansonii were gathered from Bahawalpur zoo and identified by Mr. Ghulam Sarwar (Lecturer at Department of Botany, Islamia University of Bahawalpur). A voucher specimen was deposited at Herbarium of the Department of

Life Sciences, Islamia University, Bahawalpur with the voucher number of 472/LS.

8.2.2. Extraction

Plant material was accumulated and foreign and diseased parts were removed. This material was shade dried for 6 hours with continuous agitation and pulverized via grinding mill. A total of 7 kg of shade dried and crushed aerial parts were macerated in 30 litres of solvent (H2O:CH3OH-20:80) for 3 days with occasional shaking. Filteration was carried out after days. Filtrate was evaporated under vacuum rotary evaporator (Buchi,

155

Switzerland) and fume hood.The marc was extracted for 2nd time with same solvent. The semisolid material obtained after drying of filterate was called as CaC. Weight of CaC was

700 grams.

8.2.3. Liquid-Liquid Extraction

For fractionation, solvent-solvent extraction of CaC was performed. 700 grams of

CaC was suspended in 2.5L of hot water. Furthermore, extraction was performed with n- hexane (2.5Lx3) via a separating funnel. Organic layer, thus obtained was evaporated under vacuum (35oC). Residue thus obtained was named as CaC-N. Aqueos layer was again extracted with chloroform (2.5Lx3) and n-butanol (2.5Lx3). Residue obtained after fractionation with chloroform was named as CaC-Cl and after fractionation with butanol was named as CaC-B. These fractions were subsequently subjected to various bioassays.

8.2.4. LC-MS Profiling of crude methanolic fraction of Crateva adansonii 8.2.4.1. +ve Ionisation Mode

Fig 8.1. TCC and TIC scan (+ mode) of crude methanolic fraction of Crateva adansonii

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Fig 8.2. LC-MS Ionogram of crude methanolic fraction of Crateva adansonii (+ mode) 8.2.4.2. -ve Ionisation Mode

Fig 8.3. TCC and TIC scan (- mode) of crude methanolic fraction of Crateva adansonii

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Fig 8.4. LC-MS Ionogram of crude methanolic fraction of Crateva adansonii (- mode) 8.3. Isolation & purification of chemical constituents from n-hexane fraction CaC-N

Due to high biological activity of CaC-N (55g) against urease; it was further subjected to isolation via normal phase chromatography to isolate its different bio-active constituents. CaC-N was eluted via silica gel column chromatography using the solvent system n-hexane:CHCl3 which afforded various fractions which were then merged to give

2 major fractions A (10.5g) & B (25g). Fraction B was subsequently purified via normal phase column chromatography with mobile phase of chloroform:methanol to afford Lupeol

(161). Fraction A was further puriﬁed by preparative TLC over silica gel 60 G using hexane-ethyl acetate (1:1) as developing solvent which yielded two compounds Lupenone

(201) and Lupanol (202).

158

Fig 8.5. Scheme for isolation of pure compounds from hexane fraction CaC-

159

8.4. Isolation & purification of chemical constituents from chloroform fraction CaC-

Bioassay guided isolation of chloroform fraction led to isolation of 4 constituents.

CHCl3 fraction (60 g) of aerial parts were separated via column chromatography (n- hexane-chloroform in ascending order of polarity) yielded four major fractions (A,B,C,D).

Fraction A was purified on silica gel using solvent system of n-hexane-CHCl3 which afforded Pheophorbide A (203) at solvent ratio of 35:65. The fraction B was further processed by column chromatography yielding 5 sub-fractions i.e; B1-B5. Subfraction B2 was resolved via preparative TLC which afforded compound Phytosphingosine (204).

Fraction B4 and B5 were combined and subjected to column chromatography followed by preparative TLC resulting in purification of Dehydrophytosphingosine (205). Three subfractions C1,C2,C3 were obtained from fraction C. Fractions C1 and C2 were combined and submitted to column chromatography using solvent system n-hexane-chloroform in ascending order of polarity, which furnished pyropheophorbide (158).

160

Fig 8.6. Scheme for isolation of pure compounds from chloroform fraction CaC-Cl

161

8.5. Isolation and Purification of Chemical Constituents butanolic fraction CaC-B

CaC-B was subjected to chromatographic isolation which afforded 4 major fractions (A,B,C,D). Fraction A was further subjected to normal phase chromatography using solvent system CHCl3: MeOH which yielded four subfractions (A1,A2,A3,A4).

Fraction A1 and A2 were merged on the basis of TLC Profile. Combined sub-fraction

A1+A2 was purified by size exclusion column chromatography using Sephadex G-15 (GE

Healthcare, 17002001) which afforded cadabicine (177). Fraction B afforded isovitexin

(206) through size exclusion column chromatography using Sephadex G-15 and mobile phase of H2O: CH3OH in descending order of polarity. Fraction C was further subjected to normal phase column chromatography (silica gel) using solvent system CHCl3: MeOH which yielded two subfractions C1 & C2. C2 was not further proceeded due to insufficient quantity. C1 was again processed via reverse phase column chromatography which yielded isovitexin-7-O-rhamnoside (207) on solvent ratio of 80:20 (H2O: MeOH).

162

Fig 8.7. Scheme for isolation of pure compounds from CHCl3 fraction CaC-B

163

8.6.Characterization of constituents from Crateva adansonii

8.6.1 Lupeol (161)

Physical State: White amorphous powder

Yield:1.8 mg

ESI-MS (m/z): 426 a.m.u.

Molecular formula: C30H50O

HR-ESIMS: 426.38125 a.m.u.(calculated 426.5566 a.m.u.)

-1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm-1), sharp OH band (3720cm-1), C=C band (1650 cm-1), 950 (C=CH2)

8.6.2 Lupanol (201)

164

Physical State: White amorphous powder

Yield: 2.4 mg

ESI-MS (m/z): 428 a.m.u.

Molecular Formula: C30H52O

HR-ESIMS: 428.40125 a.m.u. (calculated 428.41562 a.m.u.)

-1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm-1), sharp OH band (3720cm-1), C=C band (1650 cm-1), 950 (C=CH2)

8.6.3 Lupenone (202)

Physical State : White Amorphous powder

Yield: 3 mg

ESI-MS (m/z): 424 a.m.u.

Molecular formula: C30H48O

HR-ESIMS: 424.37125 a.m.u. (calculated 424.7025 a.m.u.)

-1 IRmax cm-1 (CHCl3): C–H broad band (2,970-2,850 cm−1), CH2 (1450 cm ), CH3 (1370 cm-1), sharp OH band (3720cm-1).

165

8.6.4. Pheophorbide A (203)

Physical state: dark brown amorphous solid

Yield:2.5 mg

ESI-MS (m/z): 592 a.m.u.

Molecular Formula: C35H36N4O5

HR-ESIMS: 592.26801 a.m.u. (calculated 593.27555 a.m.u.)

-1 -1 - IRmax cm-1 (CHCl3): CH2 (1450 cm ), CH3 (1370 cm ), broad OH band (2400 and 2800cm

1 -1 -1 -1 ), C=C (1622 cm ), 1702 cm (C=O, broad band), 1100 cm (OCH3 group).

166

8.6.5. Pyropheophorbide A (158)

Physical state: greenish brown amorphous solid

Yield : 4.25 mg

ESI-MS (m/z): 534 a.m.u.

Molecular Formula: C33H34N4O3

HR-ESIMS: 534.26332 a.m.u. (calculated 535.27073 a.m.u.)

-1 -1 IRmax cm-1 (CHCl3): -CH=CH2 (920 cm ), broad OH band (2400 and 2800 cm ), C=C

(1621 cm-1), 1705 cm-1 (C=O, broad band).

8.6.6. Phytosphingosine (204)

Physical state: waxy semi-solid

Yield: 2.5 mg

ESI-MS (m/z): 308 a.m.u.

167

Molecular Formula: C20H37NO

HR-ESIMS: 308.2940 aa.m.u. (calculated 309.297 a.m.u.)

−1 IRmax cm-1 (CHCl3): 3315 (-OH), 1650, and 1550 cm (-NH).

8.6.7 Dehydrophytosphingosine (205)

Physical state: waxy semi-solid

Yield: 2.2 mg

ESI-MS (m/z): 316 a.m.u.

Molecular Formula: C18H37NO3

HR-ESIMS: 316.2841 a.m.u. (calculated 317.2871 a.m.u.)

−1 IRmax cm-1 (CHCl3): 3315 (-OH), 1650, and 1550 cm (-NH).

8.6.8. Cadabicine (177)

168

Physical state: greenish black amorphous solid

Yield: 4.2 mg

ESI-MS (m/z): 436 a.m.u.

Molecular Formula: C25H29N3O4

HR-ESIMS: 436.2225 a.m.u. (calculated 437.22554 a.m.u.)

−1 -1 -1 IRmax cm-1 (DMSO): 3410 cm (NH), 1643 and 1635 cm (C=O), 1537 and 1505 cm

(aromatic).

8.6.9. Isovitexin (206)

Physical state: Yellow powder

Yield: 2.7 mg

ESI-MS (m/z): 432 a.m.u.

Molecualr Formula: C21H20O10

HR-ESIMS: 432.10667 a.m.u. (calculated 431.09913 a,.m.u.)

IRmax cm-1 (DMSO): 3350 (OH phenolic), 1710 (carboxyl) and 1520 cm-1 (alkene) 1022 cm-1 (glycoside linkage).

169

8.6.10. Isovitexin 7-O-Rhamnoside (207)

Physical State: Light yellow powder

Yield: 3.67 mg

ESI-MS (m/z): 577 a.m.u.

Molecular Formula: C27H30O15.

HR-ESIMS: 577.15536 a.m.u. (calcd. 578.15463 a.m.u.)

IRmax cm-1 (DMSO): 3355 (OH phenolic), 1710 (carboxyl) and 1520 (alkene) and 1022 cm-1 (glycoside linkage), 1450 cm-1 (aromatic group).

8.7. Biological Evaluation of different fractions of Crateva adansonii and it’s phytochemicals 8.7.1. DPPH free radical scavenging assay It was performed as described in previous section 4.6.1

8.7.2. α-glucosidase inhibition assay:

It was performed as described in previous section 4.6.2.

8.7.3. Carbonic Anhydrase Inhibition Assay:

It was performed as described in previous section 4.6.3.

8.7.4. Urease inhibition assay:

It was performed as described in previous section 4.6.4.

170

8.7.5. Xanthine Oxidase inhibition assay:

It was performed as described in previous section 4.6.5.

8.7.6. Tyrsosinase inhibition assay:

It was performed as described in previous section 4.6.6.

171

CONCLUSION

172

9.1. Conclusion

o GC-MS analysis of n-hexane fraction was performed and triterpenes, esters, fatty acids and

pheophorbides were identified.

o The methanol and n-butanol fractions of Crateva adansonii revealed strong activity against

xanthine oxidase and carbonic anhydrase. Chloroform fraction showed high activity

against tyrosinase and α-glucosidase whilst n-hexane fraction exhibited significant

inhibition of urease. These fractions can be further utilized as herbal medicine.

o LC-MS screening of methanolic fraction revealed presence of huge amount of flavonoids,

glycosides and pheophorbides.

o For further studies, different parts (stem, roots, leaves, fruits, flowers) of Crateva adansonii

could be evaluated separately for their biological potential.

o Isovitexin (206) and Isovitexin-7-O-rhamnoside (207) have been isolated from Crateva

adansonii for first time.

o Bioassay guided isolation led afforded three compounds from n-hexane fraction namely;

Lupeol (161), Lupenone (201), Lupanol (202) which showed high inhibition of urease as

well. Pheophorbide A (203), Phytosphingosine (204), Dehydrophytosphingosine (205) and

pyropheophorbide (158) were isolated from Chloroform fraction of Crateva

adansonii.Pheophorbides significantly inhibited tyrosinase and α-glucosidase

o Theis study expedites further investigation for development/lead optimization of these

phytochemicals as potential therapeutic agents against these targets.

173

REFERENCES

174

9.1. References

Abdulaziz Al-Hamoud, G., Saud Orfali, R., Sugimoto, S., Yamano, Y., Alothyqi, N., Mohammed Alzahrani, A. and Matsunami, K. (2019). Four new flavonoids isolated from the aerial parts of cadaba rotundifolia forssk. (qadab). Molecules. 24.

Abdullahi, A., Hamzah, R. U., Jigam, A. A., Yahya, A., Kabiru, A. Y., Muhammad, H., Sakpe, S., Adefolalu, F. S., Isah, M. C. and Kolo, M. Z. (2012). Inhibitory activity of xanthine oxidase by fractions crateva adansonii. Journal of Acute Disease. 1: 126-129.

Abreu, A. C., Serra, S. C., Borges, A., Saavedra, M. J., Mcbain, A. J., Salgado, A. J. and Simoes, M. (2015). Combinatorial activity of flavonoids with antibiotics against drug- resistant staphylococcus aureus. Microb Drug Resist. 21: 600-9.

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