Isolation of Bioactive Constituents from Kigelia Africana and Phlomis Stewartii; the Medicinal Plants of Pakistan

BISMILLAH

Isolation of Bioactive Constituents from Kigelia africana and Phlomis stewartii; The Medicinal Plants of Pakistan

A Dissertation Submitted for

The Fulfillment of The Requirement for The Award of Degree of Doctor of Philosophy in Chemistry

Bushra Jabeen Department of Chemistry The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan 2014

DECLARATION

I Bushra Jabeen, hereby declare that the research work, “Isolation of Bioactive

Constituents from Kigelia africana and Phlomis stewartii; The Medicinal Plants of

Pakistan” was carried out in the Department of Chemistry, The Islamia University of

Bahawalpur, Pakistan. All the work has been published by us in peer reviewed journals. I undertake that I did not submit the same work to any other institution or university for the degree of Doctor of Philosophy (Ph. D) and shall not, in future, be submitted for obtaining similar degree to any other university.

BUSHRA JABEEN

CERTIFICATE

It is certified that Bushra Jabeen D/O Muhammad Mushtaq has carried out the research work, embodied in this thesis, under my supervision in the Department of

Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Pakistan. The thesis entitled, “Isolation of Bioactive Constituents from Kigelia africana and Phlomis stewartii; The Medicinal Plants of Pakistan” submitted by her has been evaluated by us and is found appropriate to fulfill the requirement for the degree of Doctor of Philosophy

(Ph. D) in Chemistry.

Dr. Naheed Riaz Chairman (Research Supervisor) Department of Chemistry Associate Professor The Islamia University of Bahawalpur Department of Chemistry Pakistan The Islamia University of Bahawalpur Pakistan

DEDICATION

Dedicated to My Respected and Beloved Father and Mother Who Supported Me with Their Love Prayers and Guidance Also To My Ever Loving Sister and Brothers

CONTENTS

CONTENTS Acknowledgment i Summary iii Chapter # 1: Introduction (Part A) 1 1.1 Family Bignoniaceae 3 1.2 The Genus Kigelia 3 1.3 Kigelia africana 3 1.3.1 Pharmaceutical Importance of Kigelia africana 3 1.3.2 Literature Survey on Kigelia africana 4 1.4.1 Introduction of limonoids 13 1.4.2 Characteristics 13 1.4.3 Classification 13 1.4.3.1 Ring Intact Limonoids 15 1.4.3.1.1 Azadirone-Class 15 1.4.3.1.2 Cedrelone-Class 16 1.4.3.1.3 Havanensin-Class 16 1.4.3.1.4 Trichilin-Class 16 1.4.3.1.5 Vilasinin-Class 16 1.4.3.1.6 Others 16 1.4.3.2 Ring-seco Limonoids 17 1.4.3.2.1 Demolition of a Single Ring 18 1.4.3.2.2 Demolition of Two Rings 18 1.4.3.2.3 Demolition of Three Rings (Rings A,B,D-seco Group) 18 1.4.3.3 Rearranged Limonoids 18 1.4.3.3.1 1,n-Linkage Group 18 1.4.3.3.2 2,30-linkage Group 19 1.4.3.3.3 8,11-Linkage Limonoids (Trijugin-Class) 20 1.4.3.3.4 10,11-Linkage Limonoids (Cipadesin-Class) 20 1.4.3.3.5 Other Linkages Group 20 1.4.3.4 Limonoids Derivatives 20

CONTENTS

1.4.3.4.1 Pentanortriterpenoids, Hexanortriterpenoids, Eptanortriterpenoids, Octanortriterpenoids, and Enneanortriterpenoids Derivatives 20 1.4.3.4.2 Simple Degraded Derivatives 21 1.4.3.4.3 N-Containing Derivatives 21 1.4.4 Occurrence 23 1.4.5 Biological Activities 26 1.4.6 Biosynthesis of Limonoids 29 Chapter # 2: Results and Discussion 33 2.1 Structure Elucidation of New Compounds 35 2.1.1 1-O-Deacetyl-2α-methoxykhayanolide E (158) 35 2.1.2 Structure Elucidation of Kigelianolide (159) 40 2.2 Structure Elucidation of Known Compounds 42 2.2.1 Structure Elucidation of Deacetylkhayanolide E (160) 42 2.2.2 Structure Elucidation of 1-O-Deacetyl-2α-hyroxykhayanolide E (161) 44 2.2.3 Structure Elucidation of Khayanolide B (162) 46 2.2.4 Structure Elucidation of β-Sitosterol (5) 47 2.2.5 Structure Elucidation of β-Sitosterol 3-O-β-D-glucopyranoside (163) 49 2.2.6 Structure Elucidation of Oleanolic acid (164) 50 2.2.7 Structure Elucidation of Quercitrin (165) 52 2.2.8 Structure Elucidation of 1-O-methyl-D-chiro-inositol (166) 54 2.3 AChE, BChE and LOX inhibitory activities of compounds 158-162 55 Chapter # 3: Experimental 56 3.1 General Experimental Procedures 57 3.2 Plant Material 59 3.3 Extraction and Isolation 59 3.4.1 Characterization of 1-O-Deacetyl-2-α-methoxykhayanolide E (158) 61 3.4.2 Characterization of Kigelianolide (159) 62 3.4.3 Characterization of Deacetylkhayanolide E (160) 63 3.4.4 Characterization of 1-O-Deacetyl-2α-hyroxykhayanolide E (161) 64 3.4.5 Characterization of Khayanolide B (162) 65 3.4.6 Characterization of β-Sitosterol (5) 66

CONTENTS

3.4.7 Characterization of β-Sitosterol 3-O-β-D-glycopyranoside (163) 67 3.4.8 Characterization of Oleanolic Acid (164) 69 3.4.9 Characterization of Quercitrin (165) 70 3.4.10 Characterization of 1-O-Methyl-D-chiro-inositol (166) 71 Chapter # 4: Introduction (Part B) 72 4.1 The Family Labiatae 74 4.2 The Genus Phlomis 74 4.3 Phlomis stewartii 74 4.4 Ethnobotanical Importance of the Genus Phlomis 74 4.5 Pharmacological Importance of Genus Phlomis 75 4.6 Literature Survey on Genus Phlomis 75 4.7 Biosynthesis of 28-Nortriterpenoids 80 Chapter # 5: Results and Discussion 85 5.1 New Compounds Isolated from Phlomis stewartii 87 5.1.1 Structure Elucidation of Stewartiiside (186) 87 5.1.2 Structure Elucidation of Stewertiisin A (187) 91 5.1.3 Structure Elucidation of Stewertiisin B (188) 94 5.1.4 Structure Elucidation of Stewertiisin C (189) 97 5.2 Structure Elucidation of Known Compounds 99 5.2.1 Structure Elucidation of Notohamosin A (190) 99 5.2.2 Structure Elucidation of Phlomispentanol (191) 101 5.2.3 Structure Elucidation of Oleanolic acid (164) 102 5.2.4 Structure Elucidation of 2-Hydroxybenzoic acid (192) 104 5.2.5 Structure Elucidation of 4-Hydroxybenzoic acid (193) 105 5.2.6 Structure Elucidation of Caffeic acid (194) 106 5.2.7 Structure Elucidation of Tiliroside (195) 107 5.2.8 Structure Elucidation of Isorhamnetin 3-(6-p-coumaroyl)-β-D- glucopyranoside (196) 109 5.2.9 Structure Elucidation of Lunariifolioside (197) 111 5.3 α-Glucosidase Inhibition of Compounds 164, 186-197 113

CONTENTS

Chapter # 6: Experimental 115 6.1 Plant Material 116 6.2 Extraction and Isolation 116 6.3 Acid Hydrolysis of Compounds 186 and 197 118 6.4 α-Glucosidase Inhibition Assay 118 6.5.1 Characterization of Stewartiiside (186) 119 6.5.2 Characterization of Stewertiisin A (187) 121 6.5.3 Characterization of Stewertiisin B (188) 122 6.5.4 Characterization of Stewertiisin C (189) 123 6.5.5 Characterization of Notohamosin A (190) 124 6.5.6 Characterization of Phlomispentanol (191) 125 6.5.7 Characterization of Oleanolic acid (164) 126 6.5.8 Characterization 2-Hydroxybenzoic acid (192) 127 6.5.9 Characterization 4-Hydroxybenzoic acid (193) 128 6.5.10 Characterization Caffeic acid (194) 129 6.5.11 Characterization of Tiliroside (195) 130 6.5.12 Characterization of Isorhamnetin 3-(6-p-coumaroyl)-β-D- glucopyranoside (196) 131 6.5.13 Characterization of Lunariifolioside (197) 132 Chapter # 7: References 133 List of Publications 148

ACKNOWLEDGEMENT

All praises to ALMIGHTY ALLAH, the most Beneficial, the most Merciful Who provided me not only this golden opportunity to enhance my skills, but also give me power and wisdom to work. I offer my humblest thanks from the core of my heart to the HOLY PROPHET (Peace be upon him) who is forever a model of guidance and knowledge for humanity.

Foremost, I would like to express my sincere gratitude to my research supervisor Dr. Naheed Riaz for his continuous support, patience, motivation, enthusiasm, and immense knowledge during the completion of this work. His guidance, keen interest and constructive criticism were the real source of inspiration.

I would like to thank the rest of all my teachers especially Prof. Dr. Abdul Jabbar, Dr. Muhammad Saleem, Prof. Dr. Faiz Ul Hassan Nasim, Dr. Muhammad Ashraf, Dr. Abdul Rauf, Dr. Shafquat Hussain and Dr. Muhammad Ashraf for their support, guidance, timely help, cooperation, encouragement and insightful comments during the completion of this work.

I would like to acknowledge the support of the Higher Education Commission (HEC), particularly in the award of Indigenous 5000 Ph. D Fellowship program and International Research Support Initiative Program that provided the necessary financial support for this research.

I also thankful to Dean of Science, Chairman, Department of Chemistry, Staff Library IUB, Technical and Non-technical staff of the Department of Chemistry for their support and assistance during the course of this study.

Page i ACKNOWLEDGEMENT

I would like to acknowledge of the best wishes to my friends especially Ansa Madeeha Zafar and special thanks are extended to my lab fellows Sara Mussadiq, Asia Tabussum, M. Akram Naveed, M. Imran Tousif, Nusrat Shafiq, Jallat Khan, Basharat Ali, Abdul Gaffar, my seniors and juniors for their valuable suggestions and encouragement during my stay at IUB.

Last but not the least I owe my deep gratitude to my loving Father and Mother who educated me and enabled me what I am today, May Allah bless them with His best in this world and life after.

I am also thankful to my Brothers and my Sister for their encouragement and being always there cheering me up and stood by me through the good and bad times.

BUSHRA JABEEN

Page ii SUMMARY

SUMMARY

The history of natural products is as ancient as human being and these are used to cure man since life started. Drugs isolated or derivative of natural products has played a great role in pharmaceutical industries and specially obtained from plants. Plants have provided us some of the very important life saving drugs used in the armamentarium of modern medicine. The exploration of the chemical constituent of the plants and pharmaceutical screening may provide us the basis for developing the lead for development of novel agents. Among the estimated 400,000 plant species, only 6% have been studied for biological activity and about 15% have been investigated phytochemically. This inadvertently shows a need for the in-depth dissertation of various chemical constituents, medicinal viability, biological and pharmacological evaluation of plants. The present Ph. D thesis deals with isolation of bioactive constituents from two medicinally important plants of Pakistan namely Kigelia africana and Phlomis stewartii. Therefore thesis is presented in following two parts Part A Isolation of Bioactive Constituents from Kigelia africana; The Medicinal Plant of Pakistan

Part B Isolation of Bioactive Constituents from Phlomis stewartii;

The Medicinal Plant of Pakistan

Page iii SUMMARY

Part A: Isolation of Bioactive Constituents from Kigelia africana; The Medicinal Plant of Pakistan

The name Kigelia africana is given due to its occurrence in Africa having synonym of Kigelia pinnata. It is used as folk medicine for its medicinal properties. Its crushed dried fruits are used as emollient, anti-eczema, anti-psoriasis and skin-firming properties and as dressing for ulcers and wounds. Its fresh fruits are purgative and cause blisters in the mouth and on the skin. The aqueous extract of the leaves and fruits of K. africana possess anti- diarrhoeal, anti-leprotic, anti-malarial and anti-implantation activities. The methanolic extract of Kigelia africana resulted in isolation and structural elucidation of two new (158,159) and eight known compounds (5, 160-166).

New Compounds from Kigelia africana 1. 1-O-Deacetyl-2α-methoxykhayanolide E (158)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 5 9 15 10 8 O O OH 29 30 1 4 2 O H3C 3 28

O OCH3 158

Z. Naturforsch. 2013, 68b, 1041-1048.

Page iv SUMMARY

2. Kigelianolide (159)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO 7 H3C 16 6 14 5 9 8 15 10 O O OH OH 29 30 1 4 2 O H3C 3 28

HO 159

Z. Naturforsch. 2013, 68b, 1041-1048.

Compounds Isolated for the First Time from Kigelia africana

1. Deacetylkhayanolide E (160) 2. 1-O-Deacetyl-2α-hyroxykhayanolide E (161) 3. Khayanolide B (162) 4. 1-O-Methyl-D-chiro-inositol (166)

Known Compounds from Kigelia africana

1. β-Sitosterol (5) 2. β-Sitosterol 3-O--D-glucopyranoside (163) 3. Oleanolic acid (164) 4. Quercitrin (165)

Page v SUMMARY

Part B: Isolation of Bioactive Constituents from Phlomis stewartii; The Medicinal Plant of Pakistan

The plants belong to the genus Phlomis are used pharmacologically in herbal medicine for the respiratory tract diseases and locally to cure wounds. Phlomis stewartii is found in Pakistan and Afghanistan. It is perennial shrub, stem is erect 30-40cm, leaves are elliptic, petiole, thick in texture and flowers are pink in color and is used in folk medicine as stimulant, tonic, analgesic, anti-diarrheatic, for the treatment of ulcers and hemorrhoids and active as anti-inflammatory, immunosuppressive, anti-mutagenic and anti-nociceptive. The methanolic extract of P. stewartii resulted in isolation and structural elucidation of four new (186-189) and nine known compounds (164, 190-197).

New Compounds from Phlomis stewartii

1. Stewartiiside (186)

6 CH3 OH 4 O 5 OH 2 OH 1 3

O O 2  OH 4 6 2 5 OH O O  1 3 1  O O  3 2 1 6 3 4 OH 6 4 OH 5 1 5 6 5 OH O CH3 3 O 2 4 OH 186 OH O 4 1 OH 5

2 3 OH OH

Phytochemistry 2013, 96, 443-448.

Page vi SUMMARY

2. Stewertiisin A [(17R)-19(18→17)-abeo-3α,18β,23,24-tetrahydroxy-28- norolean-12-ene] (187)

OH 22 21 12 30 18 20 17 25 11 26 19 29 1 9 14 16

10 H 27 3 5 7 HO H 24 23 OH OH 187

Phytochemistry 2013, 96, 443-448.

3. Stewertiisin B [(17R)-19(18→17)-abeo-2α,16β,18β,23,24-pentahydroxy-28- norolean-12-ene-3-one] (188)

OH 22 21 12 30 18 20 25 11 26 17 9 14 16 19 29 HO 1 10 OH H 27 3 5 7

O H 24 23 OH OH 188

Phytochemistry 2013, 96, 443-448.

Page vii SUMMARY

4. Stewertiisin C [(17R)-19(18→17)-abeo-3α,18β,23,24-tetrahydroxy-28- norolean-11,13-diene] (189)

22 21 12 18 30 11 20 25 26 17 19 29 9 14 16 HO 1 10 H 27 3 5 7 HO H 23 24 OH OH 189

Phytochemistry 2013, 96, 443-448.

Compounds Isolated for the First Time from Phlomis stewartii

1. Oleanolic acid (164) 2. Notohamosin A (190) 3. Phlomispentanol (191) 4. 2-Hydroxybenzoic acid (192) 5. 4-Hydroxybenzoic acid (193) 6. Caffeic acid (194) 7. Tiliroside (195) 8. Isorhamnetin 3-(6-p-coumaroyl)-β-D-glucopyranoside (196) 9. Lunariifolioside (197)

The structures of new compounds were elucidated by spectral studies including UV, IR, EI-MS, HR-EI-MS, FAB-MS, HR-FAB-MS, NMR techniques including 1D (1H, 13C) and 2D NMR (HMQC, HMBC, COSY, NOESY) and chemical transformations. The known compounds have been identified by comparison of their spectral data with those reported in literature. All the isolates were evaluated for their enzyme inhibitory activities and some of them showed promising results which were also reported.

Page viii CHAPTER # 01 INTRODUCTION

Part A: Isolation of Bioactive Constituents from Kigelia africana; The Medicinal Plant of Pakistan

Page 1 CHAPTER # 01 INTRODUCTION

INTRODUCTION

Page 2 CHAPTER # 01 INTRODUCTION

1.1 Family Bignoniaceae

Bignoniaceae, a member of order Lamiales is usually tree or shrub and rarely herbs. Its leaves are estipulate, opposite or rarely alternate, usually pinnately compound; terminal leaflet sometimes forming a tendril. Flowers often showy and their inflorescence usually a panicle, sometimes flowers solitary in axils. The stamens epipetalous and fruit is a capsule, often elongated (Gray, 2009). The family having 120 genera and 800 species is mostly found in tropical and neotropical areas of America, Africa and Asia. Some species are used as ornamental plants (Gentry, 1980). Most of species are rich in iridoids (Vonposer et al., 2000) showing insecticidal (Nieminen et al., 2003), anti-microbial (Gentry, 1992), anti-parasitic (Onegi et al., 2002), antidiabetic (Lozoya-Meckes and Mellado-Campos, 1985) and anti-leishmanial activities (Ventura Pinto and Lisboa de Castro, 2009).

1.2 The Genus Kigelia

The genus Kigelia have single specie known as Kigelia africana. It is widely distributed in tropical areas of Africa. It is evergreen tree up to 20m long and commonly known as sausage tree due to its cucumber like fruit.

1.3 Kigelia africana

The name Kigelia africana is given due to its occurrence in Africa having synonym of Kigelia pinnata. Balm kheera is its common name in India and its occurrence is in wet areas. Its flowers are bell shaped purplish green and leaves are pinnate with leaflets. The fruit is cucumber like woody berry up to 30-100 cm long, 18 cm broad and 5-10 kg weight hanging down from tree. Matured fruit is grey brown and seeds are obovoid enclosed in fibrous pulp (Gabriel and Olubunmi, 2009).

1.3.1 Pharmaceutical Importance of Kigelia africana

Kigelia africana is of great importance in Africa as herbal medicine and traditionally used for the cure of snakebites, evil spirits, rheumatism and many skin diseases. Its fresh fruit is used for preparation of beverages after drying, roasting or fermentation

Page 3 CHAPTER # 01 INTRODUCTION and to prepare red dye. Its fruit also used in dysentery, hemorrhage, malaria, diabetes, toothache and pneumonia. It is useful in treatment of ulcers, purgative and as cosmetics. Its seeds are edible and roots produce yellow dye and used for toothache and backache (Saini et al., 2009).

Biological investigations revealed its activities as anti-diarrheal (Akah, 1996), anti- leprotic (Lal and Yadar, 1983), anti-malarial (Carvalho et al., 1988), anti- inflammatory, analgesic (Carey et al., 2008), anti-microbial (Kela et al., 1989), anticancer (Graham et al., 2000), anti-implantation and used in cure of gynecological disorders (Prakash et al., 1985) and as stimulant for central nervous system (Owolabi and Omogbai, 2007).

1.3.2 Literature Survey on Kigelia africana

Kigelia africana is a unique plant embodied with variety of chemicals which plays important role in new drug discoveries. It is rich with naphthoquinones especially; kigelinol (1). Kigelinol (1), isokigelinol (2), isopinnatal (3) and 2-(1- hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (4) were isolated from K. africana which showed cytotoxicity and found active against Plasmodium falciparum so can be useful as anti-malarial drug (Claudia et al., 2000).

Stem bark and fruits of K. africana were extracted with dichloromethane which show cytotoxicity and the constituent responsible for this activity are isopinnatal (3), β- sitosterol (5), and norviburtinal (6) out of which β-sitosterol (5) showed no activity, isopinnatal (3) showed minor whereas norviburtinal (6) was found to be most active (Jackson et al., 2000).

The aqueous extract of the leaves of K. africana was evaluated for anti-diarrheal activity as it is locally used for this purpose and was proved by Peter A. Akah with experiments on mice and results indicates that it decreases anti-peristaltic movements (Akah, 1996).

Page 4 CHAPTER # 01 INTRODUCTION

The ethanolic extracts of stem bark exhibited antibacterial and antifungal activities which was comparable with standards amoxicillin (Owolabi et al., 2007).

O O O CHO OH OH HO O

HO HO

O O O 1 2 3

O O

O OH

OHC

H O H O O 4 HO 5 6 7

Verminoside (8) and verbascoside (9) were isolated from polar part of the fruits of K. africana evaluated for anti-inflammatory activity but only verminoside (8) showed positive results (Picerno et al., 2005). Carey et al., proved anti-inflammatory and analgesic activities of K. africana on rats and mice (Carey et al., 2010). Akunyili et al., isolated iridoids namely minecoside (10) and specicoside (11) from water extract of stem bark of K. africana which exhibited antimicrobial activity (Akunyili et al., 1991).

Page 5 CHAPTER # 01 INTRODUCTION

O HO O OH O OH O O HO O O O OH HO OH O OH O HO O HO HO O OH HO OH OH OH 8 9

O O H OCH3 O O OH O OH O O OH O OH HO O OH HO O O OH HO HO O OH 10 OH 11

Antimalarial constituents of K. africana were isolated by Zofou et al., are specicoside (11), atranorin (12), 2β,3β,19α-trihydroxy-urs-12,20-en-28-oic acid (13) and p- coumaric acid (14) from ethylacetate soluble fraction and 2β,3β,19α-trihydroxy-urs- 12-en-28-oic acid (15) and atranorin (12) from hexane soluble fraction of the stem bark of K. africana amongst which p-coumaric acid (14) found inactive whereas the remaining showed strong interaction in combination with quinine and each other (Zofou et al., 2011). These compounds were also tested for cytotoxicity on monkey kidney cells and 2β,3β,19α-trihydroxy-urs-12-en-28-oic acid (15) was found to be most toxic.

Page 6 CHAPTER # 01 INTRODUCTION

OH O OH

OH O OCH3 OHC O H COOH HO HO H 12 HO H 13

O H COOH HO OH H

HO HO H 15 14

Volatile components of leaves of K. africana were identified by using GC-MS are n- hentriacontane (16), 1-tricosene (17), 11-(2,2-dimethylpropyl)-heneicosane (18), 2,6,10-trimethyldodecane (19), pentafluoroheptadecyl ester (20), 2- ethylhexyloctadecyl sulfurous acid ester (21), heneicosane (22), 4,4-dimethylundecane (23), methyl-12-methyl tetradecanoate (24), 1-iododecane (25), 1-iodohexadecane (26) and hexyloctyl sulphurous acid ester (27). Both the crude wax and compounds showed moderate antimicrobial activity together with good anti-oxidant activity (Olubunmi et al., 2009).

Page 7 CHAPTER # 01 INTRODUCTION

16 17 18

F F F

F F 19 O 20 O

S O O

21 22 23

OCH3 I

24 25

O O S

I O

26 27

The constituents of K. africana roots and fruits were 3β,19α-dihydroxyurs-12-ene-28- oic acid (28), chlorogenic acid (29), caffeic acid (30), ferulic acid (31) and p-coumaric acid (14). The dietary contents of K. africana were evaluated by Chivandi et al., and the results showed that the seed and seed oil nutritional constituents as its organic matter is 7.98 g per kg, ash constituent 4.55 g per kg, oleic acid 17.6%, linoleic acid 12.9%, α-linoleic acid 54.3%, vitamin E 0.75 µg per g, protein 355.33 g per kg, arginine 6.1g per 100 g, hydroxy proline 0.13 g per 100 g, phosphorus 1123.2 mg per 100 g, calcium 56.2 mg per 100 g and gross energy was 29.5 MJ per kg. Hence it is proved as edible with good nutritional values (Chivandi et al., 2011).

Page 8 CHAPTER # 01 INTRODUCTION

O HO COOH OH HO O

OH HO O H 30 COOH OH OH O OH H 29 OH HO HO H 28 OH 31 OCH3

The methanolic extract of the leaves of K. africana have capability of wound healing and anti-bacterial activities along with toxicity (Hassan et al., 2011). Bharti et al., isolated and characterized verminoside (8), minecoside (10) and specioside (11) from butanolic extract of K. africana stem bark and evaluated their growth inhibitory effects against Entamoeba histolytica and in vitro anti-amoebic activity. These were tested against metronidazol (32); verminoside (8) showed double activity than it and specioside (11) showed equivalent to it (Bharti et al., 2006).

Gouda et al., isolated and characterized twelve compounds from methanolic extract of K. africana which are furanone 3-(2-hydroxyethyl)-5-(2-hydroxypropyl)- dihydrofuran-2(3H)-one (33) and eleven iridoids, namely 7-hydroxy viteoid II (34), 7- hydroxyeucommic acid (35), 7-hydroxy-10-deoxyeucommiol (36), 10- deoxyeucommiol (37), jiofuran (38), jioglutolide (39), 1-dehydroxy-3,4- dihydroaucubigenin (40), des-p-hydroxybenzoylkisasagenol B (41), ajugol (42) and verminoside (8) 6-transcaffeoyl ajugol (43) (Goudaa et al., 2003).

Page 9 CHAPTER # 01 INTRODUCTION

N OH HO HO OH CH O2N N 3 OH O HO HO O O O CH2CH2OH

33 HO O HO OH 32 34 35

OH HO HO HO HO OH OH HO OH HO O OH O O

HO HO 36 O 37 38 OH 39 40

HO OH HO HO OH HO HO HO OH O HO O OH OH HO O OH OH O OH OH O O 41 O

42 43

Isokigelinol (2), isopinnatal (3), pinnatal (44), kigeliquinol (45), 2-acetyl-naphtho[2,3- blfuran]-4,9-dione (46) and naphthoquinones (47) were isolated and characterized from the ethanolic extract of the root bark of K. africana (Akuigyili and Houghton, 1993). P. J. Houghton examined the stem bark and the fruits of K. africana by extracting its water, ethanol and dichloromethane for activity against melanoma and renal carcinoma cells. Only the dichloromethane extract and lapachol (48) one of its isolated constituent showed this activity. Stem bark showed less activity for renal carcinoma cell than melanoma cells (Houghton et al., 1994).

Page 10 CHAPTER # 01 INTRODUCTION

O CHO O O O HO HO O O

O O O 44 45 46

O O OH O OH

O O

47 48

Gouday et al. isolated and characterized darendoside A (49), 6-O-caffeoyl-β-D- fructofuranosyl-(2→1)-α-D-glucopyranoside (50), decaffeoylacteoside (51), acteoside (52), isoacteoside (53), jionoside D (54), echinacoside (55), 6-caffeoylglucose (56), 6- feruloylsucrose (57) and isoschaftoside (58) from the methanolic extract of the fruits of K. africana (Gouda et al., 2006).

Page 11 CHAPTER # 01 INTRODUCTION

HO OH HO O O O OH O O O O O O OH HO H CO HO OH OH 3 OH OH HO O OH OH O O O OH OH OH OCH3 OH OH 49 50 HO OH 51 HO

HO O O O O OH HO HO O O OH O O OH HO HO HO O OH O OH OH O O HO OH 52 OH HO OH 53

OH HO O HO OH O O OH HO HO O OH OH O OCH HO 3 O O OH HO HO O

O HO OH O O

OH O O 54 OH 55 HO OH

OCH3 HO OH

HO OH O OH

HO O OH OH OH OH O O O O HO OH O H OH HO O

OH OH O O OH OH O OH OH

56 OH O 57 HO OH

OH 58

Page 12 CHAPTER # 01 LIMONOIDS

As limonoids constitute main part of the thesis, so we included some details about limonoids and their biosynthesis also.

1.4.1 Introduction of Limonoids

The name ‘Limonoids’ is derived from the name of its first member limonin (59). Bernay isolated limonin (59) 1st time in 1841 from Citrus seeds. Higby isolated it from orange juice (Citrus) in 1938 and its structure was confirmed by using different techniques including X-ray crystallography in 1961. During this time period many other limonoids were also isolated from different sources and their structures were confirmed by using NMR spectroscopy. In 1965, applications of NMR to limonoids were defined along with confirmation of limonin (59) as major constituent of grapefruits. Till year 2000 thin layer chromatography, HPLC and liquid chromatography coupled with mass spectrometry used to identify and analyze limonoids. After such a great improvement in methodologies to purify limonoids, many more limonoids and their glycosides were isolated and evaluated biologically (Patil et al., 2009).

1.4.2 Characteristics

Limonoids have normal polarity but could be neutral and acidic, usually insoluble in water and nonpolar solvents and soluble in hydrocarbons, alcohol and ketones, mostly bitter in taste but its glycosides are tasteless (Roy and Saraf, 2006). They are commonly oxygenated triterpenes named as tetranorterpenoids, 4,4,8-trimethyl-17-furanyl terpenoid derivatives and stereochemically homogeneous. These are known as tetranortriterpenoids due to loss of four carbons in the side chain of triterpene apoeuphane during their biosynthesis. They contain furan ring at C-17 and are oxygenated at C-3, C-4, C-7 and C- 16 as ketonic, etherial, lactonic, caboxylic acid or alcoholic functional groups (Patil et al., 2009).

1.4.3 Classification

Hasegawa and Miyake classified Citrus limonoids into four groups on the basis of their biosynthetic origins. First one is limonin group which is present in all plants of the genus

Page 13 CHAPTER # 01 LIMONOIDS

Citrus and Rutaceae family and contain limonin (59), nomilin (60) and obacunone (61) like limonoids. Only tissues of Fortunella and its hybrid contain second group named as calamin group having calamin (62), retrocalamin (63) like limonoids. Third group of limonoids occur usually in tissues of Citrus ichangensis and its hybrids having ichangensin (64). Fourth group 7-acetate limonoid group found in tissues of Poncirus and its hybrids having limonyl acetate (65) and 7α-obacunyl acetate (66) like limonoides (Hasegawa and Miyake, 1996).

O O O O

O OH O OAc O O O O MeOOC O O O H O O H H O H O O O O O HO OH O O O H O H O H O 61 62 59 60

O O O

O OH O O O O

MeOOC O O O HO O H O O O HO OH O OAc O H O 63 64 65

Champane et al divided the limonoids into ten groups on the basis of oxidation and modification of limonoids rings during their biosynthetic conversions. Group one named as protolimonoides like maliantriol (66), group two as apo-euphol limonoids contain sendanal (67) like compounds, group three named D-seco-limonoids e.g gedunin (68), group four and five contain methyl angolensate (69), group six also named as A-seco- limonoids contain tecleanin (70), group seven called A,B-seco-limonoids having rohitiukin (71), group eight, C-seco-limonoids having nimbinen (72), group nine, A,D-

Page 14 CHAPTER # 01 LIMONOIDS seco-limonoids having limonin (59) and group ten named as B-seco-limonoids having toonacilin (73) (Champagne et al., 1992).

Kim et al also classified the limonoids into four groups on the basis of their biosynthetic origin and structural variability. These are named as their major component like limonin group having limonin (59) and its similar structural compounds in a same way calamin group having calamin (62), ichangensin group and 7α-acetate limonoids group (Jinhee et al., 2012).

OH O O O O HO OH OAc

OH O O O

H O H O H AcO OH O O HO H H OAc H OH CHO COOMe 66 67 68 69

O O O O O O H3CO OAc O AcO O O OAc O

H O H OH O O O O O O OH H H OAc COOCH3 O O 72 73 70 71

Chinese Scientists Qin-Gang Tan and Xiao-Dong Luo classified the limonoids into four main classes according to their skeletal rearrangements which are given bellow.

1.4.3.1 Ring Intact Limonoids

1.4.3.1.1 Azadirone-Class

This class of limonoid exhibits the characteristics of oxidized C-3 and C-7 also having conjugated ketone at C-3 with double at C-1 and C-2. Its basic skeleton is present in azadiron (74) isolated from the oil of Azadirachta indica.

Page 15 CHAPTER # 01 LIMONOIDS

1.4.3.1.2 Cedrelone-Class

These have characteristic 5,6-enol-7-one in basic skeleton of cedrelone (75) and is its basic component isolated from Cedrela toona. Anthothecol (76) is examples of this class.

1.4.3.1.3 Havanensin-Class

C-1, C-3 and C-7 are oxygenated in this class and C-28 is also oxidized in some compounds. Trichilia havanensin contain havanensin (77), a member of this class.

1.4.3.1.4 Trichilin-Class

These have characteristic 14,15-epoxide and C-19/29 lactone ring. Usually occur in genus Trichilin, Melia and trichilin A (78) was isolated from Trichilia roka.

1.4.3.1.5 Vilasinin-Class

Ether linkage between C-6 and C-28 is its identity and nimbidinin (79) is its examples occurring in Melia azedarach and Azadirachta indica.

1.4.3.1.6 Others

1α,2α-epoxy-17β-hydroxyazadiradione (80) and 1α,2α-epoxynimolicinol (81) isolated from Azadirachta indica oil are also ring intact limonoids.

Page 16 CHAPTER # 01 LIMONOIDS

O O O O

H H O H H O

O OAc O O O O HO OH H H OH OH 74 75 76 77

O O O O

O O OH OH OH OH O O AcO O O O H O O H H H HO OH H HO OH O OAc O OAc H H H O HO 78 79 80 81

1.4.3.2 Ring-seco-Limonoids

1.4.3.2.1 Demolition of a Single Ring

 Ring A-seco-limonoid group contain lactone linkage in C-3 and C-4 as in carapolide I (82).

 Ring B-seco-limonoid have C-7/8 lactone linkage like in turraflorins A (83) and found only in genus Turraea and Toona.

 Ring C-seco-limonoids are further classified as

Azadirachtin/Meliacarpin class having azadirachtol (84).

Azadirachtinin/Meliacarpinin class having C-7,13 ether linkage e.g meliacarpins A (85).

Salannin-Class having salannin (86).

Nimbolinin-Class having nimbolinin A (87).

Nimbin-Class e.g nimbin (88).

Nimbolidin-Class e.g Walsogyne A (89).

Page 17 CHAPTER # 01 LIMONOIDS

Ring D-seco-limonoids have δ-lactone in D-ring and gedunin (68) is its member.

1.4.3.2.2 Demolition of Two Rings

 Rings A,B-seco Group

Prieurianin-Class having 3/4 or 7/4 lactone and C-11 acetoxy group in limonoid nucleus. e.g nymania 4 (90).

Others have 3/4 lactone linkage along with 8/14 or 14/15 epoxide e.g surenolactone (91).

 Rings A,D-seco Group

This group have ketone at C-11 along with δ-lactone in ring D e.g 11-oxo-7α- obacunol (92).

 Rings B,D-seco Group

Andirobin-Class. These have 8/30 exocyclic double bond and δ-lactonic D ring and occurred in genus khaya. e.g Methyl angolensate (69).

Others having a rare five membered γ-lactone fused to the C-ring at C-8 and C-14 e.g Secomahoganin (93).

1.4.3.2.3 Demolition of Three Rings (Rings A,B,D-seco Group)

Meliaceae family contain this class of limonoids and Methyl ivorensate (94) is its member.

1.4.3.3 Rearranged Limonoids

1.4.3.3.1 1,n-Linkage Group

This group include carapolide class e.g carapolide G (95) of genus Carapa, dukunolide class e.g dukunolides A (96) of genus Lansium, neotecleanin-class e.g neotecleanins (97) of genus Turraea.

Page 18 CHAPTER # 01 LIMONOIDS

O O

H3COOC OAc H3COOC OCH O 3 HO O OH O AcO CinO OAc HO O O O O O H H HO H OH HO H O O O H COOC O OH OAc 3 O H COOCH3 83 84 85 82 OH O O O HO H COOC O OH 3 H3COOC HO O O TigO O OAc O

CHO AcO O O H AcO Bz H H OTig H O O H3COOC OAc O 86 88 89 87 O O O O OAc

AcO AcO O O O O O H O O H O O O O O O COOCH3 O O O H O OH O H OAc 90 91 92 93

O O O O

OAc O HO O O O O HO O H O OH O H O O O H H O O O OH O OAc H COOCH3 O O O 94 95 97 96

1.4.3.3.2 2,30-linkage Group

The genus Khaya is rich with this group of limonoids.

 Mexicanolide-Class

Page 19 CHAPTER # 01 LIMONOIDS

Mexicanolide (98) is major constituent Carapa procera and many other compounds are reported from this class.

 Phragmalin-Class

This class of limonoids have rings of A and B as tricyclo[3.3.1.1]decane or tricyclo[4.2.1.1]decane.

 Phragmalin-ortho Esters

Phragmalin class mostly posses ester group and have four subtypes due to acetate group substitution which are 1,8,9-, 8,9,11-, 8,9,14-, and 8,9,30-phragmalin ortho-esters (99- 102).

 Polyoxyphragmalins

Xylocarpins A (103) isolated from Xylocarpus granatum is its example.

1.4.3.3.3 8,11-Linkage Limonoids (Trijugin-Class)

This class of limonoids contain contract ring C as in E.P 5 (104)

1.4.3.3.4 10,11-Linkage Limonoids (Cipadesin-Class)

Limonoids in this class have linkage between rings A and C through C-10 and C-11 as in cipadesin C (105).

1.4.3.3.5 Other Linkages Group

Some other linkages of limonoids also reported e.g 3,4-peroxide bridge in A-seco- skeleton and C-3/19 linkage in walsuronoid A (106).

1.4.3.4 Limonoids Derivatives

1.4.3.4.1 Pentanortriterpenoids, Hexanortriterpenoids, Heptanortriterpenoids, Octanortriterpenoids and Enneanortriterpenoids Derivatives

Pentanortriterpenoids e.g voamatin C (107), hexanortriterpenoids e.g carapolide A (108), hepanortriterpenoids e.g entilin A (109), octanortriterpenoids e.g azadironol (110), and

Page 20 CHAPTER # 01 LIMONOIDS enneanortriterpenoids e.g 7α-acetoxy-4,4,8-trimethyl-5α-(13α-Me)-17-oxa-androsta- 1,14-dien-3,16-dione (13α-nimolactone) (111).

1.4.3.4.2 Simple Degraded Derivatives

Limonoids of this category have degradation in ring-C which contract to five member ring as in trichiconnarins A (112).

1.4.3.4.3 N-Containing Derivatives

These limonoids have pyridine ring in the skeleton which is due to involvement of microbes during their biosynthesis as in turrapubesin B (113) (Tan and Luo, 2011).

Page 21 CHAPTER # 01 LIMONOIDS

O OAc OAc OAc O HO O O O O AcO O MeOOC O MeOOC O O H OAc O COOCH3 OH H O AcO O O O MeOOC OBu

99 OH 98 OAc 100 O OAc OAc

O O O

PivO O O O O O O H MeOOC O OAc O OAc O OH OH O MeOOC O H COOC OBu 3 O OAc OH OH O OAc O OAc OTig 103 101 102 O

O OH O

O AcO O OAc AcO AcO H COOC O 3 O O HO

O H AcO O O OAc O COOCH3 OAc OAc OAc 105 104 106

AcO O O

O PalmitylO O O O O O O OAc CHO O 107 108

Page 22 CHAPTER # 01 LIMONOIDS

OH O O O HO H O H H O O O OAc O OFer H OH H 111 OH 110 O 109 HN OiBu O O AcO

H O O O O

112 COOCH3 113

1.4.4 Occurrence

Upto 1992 more than 300 limonoids were isolated which belongs only to order rutales of plant kingdom. Families Maliaceae, Rutaceae and Cneoraceae are rich with versatile limonoids but in family Simaroubaceae only the plant Harrisonia abbysinica contains few limonoids. Neem tree Azadirachta indica and Melia azadirachtin have most abundant variety of limonoides (Champagne et al., 1992).

Cipadessa cinerascens, family Maliaceae contain six limonoids of B,D-seco-type named as cipadesin-type. These are cipadonoids B-G (114-119), which were cytotoxic and weak antifeedant (Fang et al., 2009). Xylocarpus granatum member of family Meliaceae contain a limonoids xyloccensin K (120) isolated from its dichloromethane extract of its dried fruits (Kokpol et al., 1996). Ang and Alex have isolated 7α-acetoxydihydronomili (121), mexicanolide (98), gedunin (68) and xylocarpin (122) from same species.

Page 23 CHAPTER # 01 LIMONOIDS

MeOOC O MeOOC O

OAc O O AcO AcO O O H O O O O O

COOMe 114 115 116

O MeOOC O O

OAc HO OAc OAc MeOOC MeOOC OH AcO O O O O O

O O O O O O AcO AcO

117 118 119

O O O

O AcO O O MeOOC COOMe O O O O OH H H O O O O O O H OAc

OAc 120 121 122

Citrus plants are rich with limonoids which causes a bitter taste of Citrus fruits juices. Rutaceae and Maliacea families are rich with limonoids and limonin (59) is the most abundant constituent which is basic cause of bitterness in taste. But some fruits have delayed bitterness which is due to absence of lactones in D-ring which undergo acidic hydrolysis and form limonin (59).

Page 24 CHAPTER # 01 LIMONOIDS

O O

O O O O O OH Acidic O Hydrolase H O O O O O H O O H H O

Bitter limonin Nonbitter Limonin

Hasegawa and Miyake reported that 36 limonoids have been isolated from Citrus among them limonin (59), nomilin (60), obacunoic acid (123), ichangin (124) and deoxylimonic acid (125) are bitter and calamin (62), retrocalamin (63), ichangensin (64), limonyl acetate (65), cyclocalamin (126), deacetylnomilic acid (127) etc. are not bitter (Hasegawa and Miyake, 1996).

Kim et al isolated 62 limonoids from genus Citrus till 2012 among them 44 are aglycones and 18 are limonoid glycosides but their number is increasing day by day. They also reported same causes of bitterness, delayed bitterness and non bitter components (Jinhee et al., 2012).

Page 25 CHAPTER # 01 LIMONOIDS

O O O

O O O O O O O

O HO O O H O H O H HOOC O HO HO H O H O COOH

123 124 125

O O

O MeOOC O O O

O O O O H O OH COOH O

126 127

1.4.5 Biological Activities

Limonoids are rich in biological potential as they have various biological activities as Khaya anthotheca is locally used for cure of malaria. Lee et al isolated anthothecol (76), a limonoid which showed a remarkable anti-malarial activity against Plasmodium falciparum as compare to other Citrus limonoids (Lee et al., 2008). Methyl 3β-n- butyryloxy-1-oxomeliac-8(14)-enate (128) isolated from methanolic extract of seeds of Khaya ivoresis show strong anti-feeding activity against larvae of the lepidopteran Agrotis segetum (Vanucci et al., 1992). Poulose et al work on aglycone parts of Citrus limonoids and there glycosides to evaluate the anticancer activity and confirmed that glycosidic limonoids are responsible for the said activity. They isolated limonin (59), nomilin (60), obacunone (61) and deacetylnomilin (129) and their glycosides from seeds and molasses of Citrus fruits. These were checked against two human cancer cell lines, SH-SY5Y neuroblastoma and CaCo-2 colonic adenocarcinoma, and a noncancerous mammalian epithelial Chinese hamster ovary (CHO) cells. They showed no effect on

Page 26 CHAPTER # 01 LIMONOIDS

Chinese hamster ovary (CHO) cells but very toxic for cancerous cells (Poulose et al., 2006).

Citrus limonoids specially limonin glycoside (130) showed versatile biological activities and is important nutrition content of Citrus fruit juices. Manners et al evaluated the bioavailability of Citrus limonoids i.e limonin (59) glycoside from molasses. They injected the limonin glycoside (130) doses to human plasma and observed the bioavailability of this constituent in human plasma by using liquid chromatography along with mass spectrometry. By this experiment they concluded that the increase of dose increases the concentration of limonin (59) in plasma and this increase was not harmful to human body so it was nontoxic. They also proposed that limonin glycoside (130) is converted into limonin (59) or epilimonin (131) by hydrolysis followed by lactonization in plasma. Hence limonoid glycosides have good nutritional values and can be used pharmacologically (Manners et al., 2003).

O O

O OH O H MeOOC O O O H H O O O O

OCOCH2CH2CH3 129 128

O O O O

O Glucose O O O O O OH O O O O O O O H O O O O H O H O H O O H O O O H O O H O H H O 130 59 131

Limonoids induce glutathione 5-transferase activity, anticancer and anti-feedant activity (Hasegawa and Miyake, 1996). Jinhee et al summarized that limonoids posses many

Page 27 CHAPTER # 01 LIMONOIDS nutritional and pharmaceutical values as it exhibit antibacterial, antifungal, antiviral, antioxidant, anti-HIV, moderate radical scavenging, inhibit bacterial cell-cell signaling and biofilm formation and anticancer activity. They also purposed that the presence of furan and an intact A ring structure are responsible for chemo preventive activity in cancer cells and anti-proliferative activity (Jinhee et al., 2012).

The unique skeleton of limonoids give a large rang of biological activities in every field of research. In case of agriculture these posses anti-feedant, growth regulator, insecticidal, anti-phytopathogen and inhibited seed germination activities. On the other hand, limonoids have pharmacological importance as these show anti-neoplastic activity as these are cytotoxic with the IC50 values to result as anticancer potential drug, anti- protozoal activity, especially as anti-malarial along with antimicrobial, antiviral, anti- hermitic and anti-kinase activities. These are also anti-inflammatory, anti-edematogenic, possess analgesic effects, spermicidal, anti-secretary and anti-gastric ulcer activities (Tan and Luo, 2011).

Page 28 CHAPTER # 01 BIOSYNTHESIS OF LIMONOIDS

1.4.6 Biosynthesis of Limonoids

Biosynthetically, limonoid can be synthesized through a variety of reactions like oxidation and rearrangement in the basic skeleton. Basically, they are formed by biosynthetic degradation of terpenoids. The basic precursor of limonoids is 4,4,8- trimethyl-17-furanylsteroid which is derived from squalene (132) (Champagne et al., 1992). Roy et al purposed that squalene (132) undergo cyclisation through protosterylcation (133) formation which on series of 1,2-methyl and 1,2-hydride shift forms euphane (134) than butyrospermol (135). It is supposed to be biosynthetic precursor of limonoids by Endo, Bagge, Wasterman and Suarez (Roy and Saraf, 2006).

series of 1,2-methyl and 1,2 hydride shift H O HO 133 132 H

HO HO H 135 H 134

Oxidation of butyrospermol (135) forms epoxide (136) at C-6/7 which after apo-euphol rearrangement shifts Me-14 to C-8, -OH at C-7 and double bond between C-14/15 (137). It further undergo oxidation to form aldehyde of side chain methyl C-20 and two -OH at C-22 and C-23 (138) which on cyclization with removal of water forms ether linkage between C-21 and C-23 (139). On oxidation 139 loss the four carbons from the side chain (140) and after removal of two water molecules a tetranortriterpenoid formed having furan ring at C-17 (141).

Page 29 CHAPTER # 01 BIOSYNTHESIS OF LIMONOIDS

apo-euphol epoxidation H H H O H rearrangement HO HO HO OH H H H 135 136 137 [O]

O O O HO HO HO O O

H OH CH-

[O] H - 2H2O H H H HO OH HO OH HO H OH H H HO H OH 141 140 139 138

Basic skeleton of all limonoids is derived from this precursor whose stereochemistry is homogeneous. Tetranortriterpenoid (141) undergo olefination, oxidative cleavage to form 17-OH and 16-COOH (142) which on cyclisation form lactone linkage in ring D (143). A large range of limonoids have this lactone in ring D e.g 11-oxo-7α-obacunol (92).

Epoxide (144) at C-14/15 and C-7 ketone are formed by oxidation and C-1, C-3 acetylation form 7-oxo-7-deacetoxykhivorin (146). It undergo oxidative cleavage and than cyclisation to form another lactone linkage in ring B. Mild basic condition break this seven member lactone ring and than its methylation form ester of C-16 caboxylic acid. Thionyl chloride replace -OH at C-8 and Jones reagent generates double bond at C-8 along with ketones at C-1 and C-3 (147). Epoxidation at C-14/15 change into double bond in the presence of chromylchloride and product undergo cyclisation between C-2 to C-7 to form maxicanoloid (149) (Connolly et al., 1971).

Page 30 CHAPTER # 01 BIOSYNTHESIS OF LIMONOIDS

O O O

OH O

COOH H Oxidative cleavage O H Esterification H HO H OH HO OH HO OH H H 141 142 143

[O] O O O

OAc O OAc O OAc O

Oxidative cleavage O O MeCO O H H O 3 H O O O HO AcO O AcO AcO COOH H O 144 146 145 1. mild base 2. methylation

3. SOCl2 4. Jones O O O

O O O O O MeOOC Aq. NaHCO3 O O CHCl3 H H CrCl2 O O O

O O H COOMe COOMe O 147 148 149

Maxicanoloids a large range of limonoids including khayanolides. Tylor has proposed the biosynthethesis of pharagmalin type limonoids which help out Abdelgaleil et al to propose a biosynthtic pathway of khyanoloids from maxicanoloids.

Maxicanoloide (149) first undergo hydroxylation and cyclisation to generate ketal of xyrocarpus type (151). Free radical method was supposed to be used for formation of oxygen radical (152). It oxidises the methyl at C-29 and forms its ketal (153) which may form another oxygen radical by its transfer to oxidise C-30 (154). C-30 radical (156) attacks 1,2-bond and form ketonic compound (157) which on reduction forms

Page 31 CHAPTER # 01 BIOSYNTHESIS OF LIMONOIDS khayanoloid and its methylation forms its methoxy derivative (158-162) (Abdelgaleil et al., 2001).

O O O O

O O O O COOMe COOMe COOMe COOMe O H O O H O H H O O OH O O HO HO HO O OH 149 OH 150 151 152 O O OH OH OH OH

O O O

O O O OH OH OH O O O H O O O O H MeOOC MeOOC MeOOC O HO H H OH OH OH H2C H2C H-O-H OH OH 153 OH 155 154

O O O

CH3 O O OH H O H CO H OH 3 H C OH O OH 3 OH O O OH O O MeOOC O OH MeOOC O H3C

OH OH 162 OH O HO 156 157

O O

CH3 CH3 OH O H CO H OH 3 H C H O 3 H3CO OH H3C O O OH OH O O OH O H3C 158 R = OCH H C O 3 3 160 R = H HO 159 O R 161 R = OH

Page 32 CHAPTER # 02 RESULTS AND DISCUSSION

RESULTS & DISCUSSION

Page 33 CHAPTER # 02 RESULTS AND DISCUSSION

2 Results and Discussion

The shade-dried powdered plant material of Kigelia africana was extracted thrice in methanol at room temperature which further divided into n-hexane, ethyl acetate and water soluble fractions, respectively. The ethyl acetate soluble fraction (95 g) was subjected to column chromatography over silica gel using n-hexane/EtOAc, EtOAc,

EtOAc/MeOH and MeOH as eluent resulted into six fractions E1 up to 20% EtOAc in n- hexane, E2 up to 50% EtOAc in n-hexane, E3 up to 75% EtOAc in n-hexane, E4 up to

100% EtOAc, E5 up to 5% MeOH in EtOAc and E6 up to 30% MeOH in EtOAc. After repeated column chromatography the fraction E1 on gradient elusion using 10% EtOAc in n-hexane provided β-sitosterol (5). The fractions E2 on gradient elusion using 40% EtOAc in n-hexane gave deacetylkhayanolide E (160) and 45% EtOAc in n-hexane resulted to purify 1-O-deacetyl-2α-methoxykhayanolide (158). The fraction E3 on gradient elusion using 50% EtOAc in n-hexane to get 1-O-deacetyl-2α-hydroxykhanolide E (161), 55% EtOAc in n-hexane to get kigelianolide (159) and khayanolide B (162), respectively. The fraction E4 on gradient elusion using 80% EtOAc in n-hexane provided oleanolic acid (164).The fraction E5 on gradient elusion using 2% MeOH in EtOAc purified β-sitosterol 3-O-β-D-glucopyranoside (163) and 5% MeOH in EtOAc to get quercitrin (165).The fraction E6 on gradient elusion using 10% MeOH in EtOAc to get 1- O-methyl-D-chiro-inositol (166).

Page 34 CHAPTER # 02 RESULTS AND DISCUSSION

2.1 STRUCTURE ELUCIDATION OF NEW COMPOUNDS 2.1.1 Structure Elucidation of 1-O-Deacetyl-2α-methoxykhayanolide E (158)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 5 9 15 10 8 O O OH 29 30 1 4 2 O H3C 3 28

O OCH3 158

1-O-deacetyl-2α-methoxykhayanolide E (158) was obtained as crystalline solid. The IR spectrum showed the presence of O-H (3435 cm-1) and C=O (1736, 1718 cm-1) and C=C -1 (1615 cm ). Its molecular formula C28H34O11 was deduced by HR-EI-MS through molecular ion peak at m/z 546.2110 with 12 double bond equivalence (DBE). The 1H NMR spectrum of 158 (Table 1) showed three tertiary methyls at δ 1.32, 1.07 and 1.01, two oxygenated methyls at δ 3.71 and 3.48, (3H each, s) oxygenated methines δ 5.51 (1H, s), 4.22 (1H, d, J = 8.4 Hz), various signals between δ 0.94-2.89 for cyclic methylenes and methines and a furan moiety at δ 7.54, 7.49, 6.46 (1H each, s).

The 13C NMR spectra of 158 (Table 1) supported the above data as it displayed total 28 carbon resonances for five methyl (δ 53.1, 52.7, 18.7, 15.7, 15.6), four methylene (δ 43.8, 37.5, 28.8,17.2), eight methine (δ 144.2, 142.7, 111.1, 82.5, 73.7, 71.5, 56.8, 44.1) and eleven quaternary carbons (δ 204.7, 175.5, 173.9, 122.1, 101.9, 88.3, 86.4, 84.9, 61.0, 50.2, 38.7). The above spectroscopic data are very similar to those of 1-O-deacetyl-2α- hyroxykhayanolide E (161) (Zhang et al., 2009) indicating both compounds have the same carbon framework. The only difference lies in the increased molecular weight of 158 by 14 units that was attributed to the presence of a methoxyl group in 158 instead of

Page 35 CHAPTER # 02 RESULTS AND DISCUSSION hydroxyl group in the reference compound 1-O-deacetyl-2α-hydroxykhayanolide E (161) with molecular formula C27H32O11. The position of methoxyl group was confirmed at C-2 through HMBC spectrum, in which methoxy protons (δ 3.48) showed 3J correlation with C-2 (δ 101.9). The remaining substitution pattern was confirmed by the combination of HSQC, HMBC and COSY correlations.

The structure was also confirmed by X-ray crystallography (Figure 1). The absolute configuration of 158 could be established by means of the so-called solid-state ECD/TDDFT method (Pescitelli et al., 2009). It consists in comparing the electronic circular dichroism (ECD) spectrum measured on a microcrystalline sample with that calculated with time-dependent density functional theory (TDDFT) (Autschbach et al., 2011) using the X-ray coordinates as input structure. This approach renders a full conformational analysis unnecessary (Pescitelli et al., 2011) and is especially useful in assigning the absolute configuration of flexible natural products. In the case of compound 158, for instance, the rotation of the furan chromophore around the C17-C20 bond, which is expected to affect ECD spectra, is frozen in the crystals. In Figure 2 (left) the experimental absorption and ECD spectra of (+)-158 measured in acetonitrile solution and in the solid state, as KCl pellet, are displayed. The solid state spectrum has a long- wavelength cutoff because of the strong furan absorption, however, the positive ECD band around 305 nm is consistently found both in solution and in the solid state. In Figure 2 (right) the calculated spectra are shown, employing the TDDFT method at CAM- B3LYP/TZVP level (other functional/basis set combinations gave consistent results). The X-ray geometry was used as input structure with initially arbitrary (1S,2R,4R,5R,6S,8S,9R,10S,13S,14R,17S,30S) configuration, after optimization of hydrogen atoms only (see Computational Section). The calculated ECD spectrum nicely reproduces the experimental ones; in particular, the band calculated at 302 nm has positive sign for the above configuration. This band is associated with an n-* transition mainly localized on the C-3 carbonyl group, which is quite distant from the furan, and depends only on the rigid ring system. Based on the above discussion, the compound was

Page 36 CHAPTER # 02 RESULTS AND DISCUSSION assigned the structure (1S,2R,4R,5R,6S,8S,9R,10S,13S,14R,17S,30S)-158 and named as 1- O-deacetyl-2α-methoxykhayanolide E. The assigned absolute configuration corresponds to that established for related khayanolides such as khayanolide A (Nakatani et al., 2000) and 1-O-acetylkhayanolide B (162) (Zhang et al., 2009).

Figure 1. X-ray drawing of compound 158

Page 37 CHAPTER # 02 RESULTS AND DISCUSSION

Figure 2. Experimental (a, left) and calculated (b, right) absorption (top) and ECD spectra (bottom) of (+)-(1S,2R,4R,5R,6S,8S,9R,10S,13S,14R,17S,30S)-1-O-deacetyl-2-α- methoxykhayanolide E (158). Solution spectra measured on a 3.0 mM sample using 0.05 cm and 1 cm (expansions) cells. Calculated spectra obtained by CAM-B3LYP/TZVP calculations on the X-ray input geometries after application of Gaussian band-shape with 0.4 eV exponential half-width, red-shifted by 10 nm.

Page 38 CHAPTER # 02 RESULTS AND DISCUSSION

1 13 Table 1. H and C NMR data, HMBC and COSY correlations of 158 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) 1 - 84.9 - - 2 - 101.9 - - 3 - 204.7 - - 4 - 50.2 - - 5 2.89 (d, 8.4) 44.1 1,4,6,7,10,19,28,29 H-5/H-6 6 4.22 (d, 8.4) 71.5 4,5,7,10 H-6/H-5 7 - 175.5 - - 8 - 88.3 - - 9 2.28 (d, 9.0) 56.8 1,5,8,11,12,14,19,30 H-9/H-11 10 - 61.0 - - 11 1.97 (d, 13.8) 17.2 8,9,10,12,13 H-11/H-9,12 1.81 (dd, 14.1, 5.4) 12 1.73 (dt, 13.8, 3.0) 28.8 9,11,13,14,18 H-12/H-11 0.94 (d,12.0) 13 - 38.7 - - 14 - 86.4 - - 15 3.19 (s) 37.5 8,13,14,16 - 16 - 173.9 - - 17 5.51 (s) 82.5 12,13,14,18,20,21,22 - 18 1.07 (s) 15.7 12,13,14,17 - 19 1.32 (s) 18.7 1,5,9,10 - 20 - 122.1 - - 21 7.54 (s) 142.7 17,20,22,23 - 22 6.46 (s) 111.1 17,20,21,23 - 23 7.49 (s) 144.2 20,21,22 - 28 1.01 (s) 15.6 3,4,5,29 - 29 2.10 (d, 12.6) 43.8 1,3,4,5,10,30 H-29a/H-29b 1.85 (d, 12.6) 30 2.86 (s) 73.7 1,2,3,8,9,14,29 - 2-OMe 3.48 (s) 53.1 2 - 7-OMe 3.71 (s) 52.7 7 -

Page 39 CHAPTER # 02 RESULTS AND DISCUSSION

2.1.2 Structure Elucidation of Kigelianolide (159)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO 7 H3C 16 6 14 5 9 8 15 10 O O OH OH 29 30 1 4 2 O H3C 3 28

HO 159

The IR spectrum of compound 159 was similar to that of 158. The molecular formula + C27H36O10 was established by HR-EI-MS which showed a molecular ion peak [M] at m/z 518.2315. The 1H NMR spectrum of 159 (Table 2) was also similar to that of 159 except the missing the methoxy signal at δ 3.48, additionally, it displayed two oxygenated methines at δ 4.47 (1H, dd, J = 9.5, 6.5 Hz) and 3.37 (1H, d, J = 6.5 Hz) which were correlated in the COSY spectrum for being vicinal to each other. The 13C NMR spectrum of 159 (Table 2) showed total 27 carbon signals with the two oxymethines at δ 80.1 and 73.9. The above spectroscopic data showed close resemblance to that of reported for khayanolide B (Abdelgaleil et al., 2001). The upfield shift of C-14 (δ 79.3) and the downfield shift of C-8 (δ 88.3) gave a clue about the cleavage of a bond between C-2 and C-14 with the formation of a new four member ring between C-2 to C-8 which was confirmed by HMBC correlations in which the CH3-18 (δ 1.09) showed correlation with C-14 (δ 79.3). Based on these evidences the compound was assigned the structure of 159 and named as kigelianolide. The absolute configuration of (–)-159 is assumed to be that shown in the Scheme, corresponding to its analogs 158 and 161, based on the isolation from the same source and on biogenetic considerations.

Page 40 CHAPTER # 02 RESULTS AND DISCUSSION

1 13 Table 2. H and C NMR data, HMBC and COSY correlations of 159 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) 1 - 85.2 - - 2 4.47 (dd, 9.5, 6.5) 73.9 1,3,4,8,30 H-2/H-3,30 3 3.37 (d, 6.5) 80.1 2,4,5,29,30 H-3/H-2 4 - 43.5 - - 5 3.18 (d, 8.5) 41.9 1,4,6,7,9,10,19,28,29 H-5/H-6 6 4.17 (d, 8.5) 72.3 4,5,7,10 H-6/H-5 7 - 176.9 - - 8 - 88.3 - - 9 2.11 (d, 9.0) 56.9 1,5,8,11,12,14,19,30 H-9/H-11 10 - 60.6 - - 11 2.13 (d, 9.0) 17.4 8,9,10,12,13 H-11/H-12 1.80 (m) 12 1.88 (d, 12.0) 27.6 9,11,13,14,18 H-12/H-11 0.87 (d, 12.0) 13 - 38.9 - - 14 - 79.3 - - 15 3.08 (d, 19.0) 33.2 8,13,14,16 H-15a/H-15b 2.78 (d, 19.0) 16 - 173.5 - - 17 5.76 (s) 82. 6 12,13,14,18,20,21,22 - 18 1.09 (s) 15.3 12,13,14,17 - 19 1.21 (s) 18.3 1,5,9,10 - 20 - 122.2 - - 21 7.50 (s) 142.4 17,20,22,23 - 22 6.45 (s) 111.1 17,20,21,23 - 23 7.46 (s) 144.4 20,21,22 - 28 1.00 (s) 15.2 3,4,5,29 - 29 1.86 (d, 12.0) 46.1 1,3,4,5,10,30 H29a/H-29b 1.34 (d, 12.0) 30 2.60 (d, 9.5) 64.6 1,2,3,8,9,14,29 H-30/H-2 7-OMe 3.73 (s) 52.6 7 -

Page 41 CHAPTER # 02 RESULTS AND DISCUSSION

2.2 STRUCTURE ELUCIDATION OF KNOWN COMPOUNDS

2.2.1 Structure Elucidation of Deacetylkhayanolide E (160)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 5 9 15 10 8 O O OH 29 30 1 4 H C 3 2 O 3 28 H O 160

Deacetylkhayanolide E (160) was purified as white amorphous powder (30 mg). Its IR spectrum showed peaks at 3498, 2951, 1732, 1713, 1464, 1389, 1242, 1148, 1045, 1026, 770 cm-1 indicated the presence of hydroxyl, carbonyl and conjugated double bond where as the UV bands at 213 nm indicated the presence of furan ring. The molecular formula + C27H32O10 was established by HR-EI-MS which showed a molecular ion peak [M] at m/z 516.1995.

The 1H NMR spectrum of 160 was similar to that for 158 except the missing the methoxy signal at δ 3.48. Additionally, it displayed an oxygenated methines at δ 4.33 (1H, d, J = 9.5, Hz) which was correlated in the COSY spectrum appeared at δ 2.80 (1H, d, J = 9.5 Hz).

The 13C NMR spectrum of 160 showed total 27 carbon signals including four methyl (δ 52.6, 18.7, 15.7, 15.3), four methylene (δ 45.3, 34.1, 27.8, 17.4), nine methine (δ 144.1, 142.6, 111.1, 82.6, 71.5, 76.1, 64.6, 56.6, 43.5) and ten quaternary carbons (δ 208.0, 175.6, 173.3, 122.1, 88.50, 85.4, 84.7, 60.9, 50.7, 38.8). The missing of an anomeric quaternary carbon and the appearance of an oxymethine at δ 76.1 indicated the absence of methoxy group at C-2 and rest of the signals were found similar to that for compound

Page 42 CHAPTER # 02 RESULTS AND DISCUSSION

158. The structure was further confirmed by using COSY, HMQC and HMBC correlations. By comparing the above data which showed close resemblance to the data that reported for deacetylkhayanolide E (160) (Zhang et al., 2008). Based on these evidences the compound was assigned the structure of 160 which is known as deacetylkhayanolide E. The absolute configuration of (–)-160 is assumed to be that shown in the scheme, corresponding to its analogs 158 and 159, based on the isolation from the same source and on biogenetic considerations.

Page 43 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.2 Structure Elucidation of 1-O-deacetyl-2α-hyroxykhayanolide E (161)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 5 9 15 10 8 O O OH 29 30 1 4 H C 3 2 O 3 28

O OH 161

1-O-deacetyl-2α-hyroxykhayanolide E (161) was purified as white amorphous powder (25 mg). The IR spectrum showed peaks at 3490, 2952, 1732, 1713, 1464, 1386, 1244, 1139, 1045, 1024, 769 cm-1 indicated the presence of O-H, C=O and C=C functionalities where as the UV bands at 210 nm indicated the presence of furan ring. The molecular formula C27H32O11, was confirmed by HR-EI-MS which showed a molecular ion peak [M]+ at m/z 532.1945. The 1H NMR spectrum of 161 was also similar to that of 158 except the missing the methoxy as well as for oxygenated methines atC-3 and C-2 like compound 159.

The 13C NMR spectrum of 161 showed total 27 carbon signals corroborated the presence of four methyl (δ 52.3,18.6, 15.7, 15.6), four methylene (δ 44.8, 37.7, 28.1, 17.2), eight methane (δ 144.2, 142.5, 111.1, 82.4, 72.7, 71.5, 56.7, 43.4) and eleven quaternary carbon atoms (δ 206.0, 175.6, 174.2, 122.2, 100.3, 88.7, 85.5, 84.7, 61.1, 49.0, 38.7). All data was similar to compound 158 with the difference of the missing of methoxy group. The downfield shift of C-2 (δ 100.3) as compare to 158 confirms the presence of -OH group at this position. The structure was also confirmed by X-ray crystallography (Figure 3). The above spectroscopic data showed close resemblance to that of reported for 1-O- deacetyl-2α-hyroxykhayanolide E (Xue et al., 2009). Based on these evidences the

Page 44 CHAPTER # 02 RESULTS AND DISCUSSION compound was assigned the structure 161 which is known as 1-O-deacetyl-2α- hyroxykhayanolide E.

Figure 3. X-ray drawing of compound 161

Page 45 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.3 Structure Elucidation of Khayanolide B (162)

O 23 21 22 18 20 CH3 OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 5 9 15 10 8 O O OH 29 30 1 4 H C 3 2 O 3 28 HO 162

Khayanolide B (162) was purified as white amorphous powder (25 mg). Its IR and UV were found similar to that for compounds 158-161. The molecular formula C27H34O10, was established by HR-EI-MS which showed a molecular ion peak [M]+ at m/z 518.2152. The 1H NMR spectrum of 162 was also similar to that of 158 with the missing the methoxy signal and addition of two oxygenated methines at δ 4.47 (1H, dd, J = 9.5, 6.5 Hz) and 3.38 (1H, d, J = 6.5 Hz) which were correlated in the COSY spectrum with each other confirming they are vicinal to each other.

The 13C NMR spectrum of 162 showed total 27 carbon signals for four methyl (δ 52.6, 18.3, 15.3, 15.2), four methylene (δ 46.1, 33.2, 27.6, 17.4), nine methine (δ 144.4, 142.4, 111.1, 82.6, 80.1, 73.9, 72.3, 64.6, 56.9, 44.9) and ten quaternary carbons (δ 176.9, 173.5, 122.2, 88.3, 85.2, 84.7, 73.9, 61.1, 43.5, 38.9). The positions of both oxymethines were confirmed through HMBC correlation in which Me-28 (δ 1.00) correlated with carbon resonated at δ 80.1 confirmed its position at C-3 and the COSY correlation between H-3 (δ 3.38) and H-2 (δ 4.47) confirmed the position of second oxymethine (δ 73.9) at C-2. The above spectroscopic data showed close resemblance to the data that reported for khayanolide B (Haque et al., 2008). Based on these evidences the compound was assigned the structure of 162 that was known as khayanolide B.

Page 46 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.4 Structure Elucidation of β-Sitosterol (5)

29 28

21 26

18 20 23 25

12 17 27 19 16 9 14 1 10

3 H 7 H HO 5 5

The compound 5 was purified as colorless amorphous powder (m.p. 143-145 °C). It appeared pink on heating after spraying with ceric sulphate solution. The IR spectrum showed peaks at 3445, 2970, 2868, 1805, 1618, 1452, 1380, 1257, 1021, 985,780 cm-1 characteristic for O-H, C=C and C-H bonds. The EI-MS of compound 5 showed the + molecular ion peak at 414.38 [M] and the molecular formula C29H50O was determined by HR-EI-MS due to the molecular ion peak at m/z 414.3861.

The 1H NMR spectrum of 5 showed a signal at δ 3.17 (1H, m) for oxymethine and δ 5.23 (1H, br s) for a double bond. Six methyl protons showed their presence at δ 1.50, 1.45, 0.95, 0.85, 0.75 and 0.65 among them two were angular which is characteristic in steroids.

The 13C NMR showed twenty nine carbons including six methyl (δ 30.2, 24.4, 18.7, 18.3, 13.3, 11.1), twelve methylene (δ 41.5, 40.6, 37.6, , 33.9, 32.1, 31.9, 27.5, 25.7, 23.2, 22.6, 20.4), ten methines (δ 122.3, 70.1, 56.6, 56.3, 49.9, 36.2, 35.2, 32.5, 28.1) and three quaternary carbons (δ 141.9, 35.6, 42.9). The position of both the double bond and hydroxyl group was confirmed through HMBC correlations in which Me-19 correlated with C-5 (δ 141.5) and COSY correlation of olefinic methine at δ 5.23 with H-7 (δ 2.16) confirm the position of double bond between C-5 and C-6. The oxymethine was placed at position C-3 due to its HMBC correlation with C-1 (δ 36.7) and C-5 (δ 141.5) and its COSY correlation with H-2 (δ 1.73). Based on these results compound 5 have hydroxyl

Page 47 CHAPTER # 02 RESULTS AND DISCUSSION group at C-3 and olefinic bond between C-5 and C-6. The above described data for 5 was completely resembled to the data already reported for β-sitosterol (Kamboj and Saluja, 2011).

Page 48 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.5 Structure Elucidation of β-Sitosterol 3-O--D-glucopyranoside (163)

29 28

21 26

18 20 23 25

12 17 27 19 16 9 14 1 10 H H HO 6' 3 7 O O 5 HO 5' HO 3' 1' OH 163

The compound 163 was purified as colourless amorphous powder which appeared red after spraying with ceric sulphate and after heating. The IR spectrum was similar to that for compound 5. The HR-FAB-MS of compound 163 showed the molecular ion peak of + 577.2645 [M+H] corresponding to the molecular formula C35H61O6.

The 1H NMR spectrum showed a downfield signal at δ 5.23 (1H, d, J = 5.5 Hz) for olefinic methine like that for 5 but with additional signals for five oxyginated methines at δ 4.95, 4.25, 3.65, 3.07, 3.03 and an oxyginated methylene at δ 4.85, 4.65 which are characteristic signals of sugar moiety. Six methyls were appeared at δ 1.50, 1.45, 0.95, 0.85, 0.79 and 0.65 including two angular methyls which are characteristic of steroids.

The 13C NMR showed thirty five carbons including six methyls carbons having chemical shifts at δ 19.7, 19.3, 18.6, 18.4, 12.3 and 11.4. The above data was related to previously discussed -sitosterol (5) with additional signals of glucose moiety at δ 101.4, 76.34, 76.1, 73.7, 70.8, 61.4. The HMBC correlation of anomeric proton at δ 4.95 with C-3 (δ 77.1) confirms the attachment of glucose moiety at C-3. It confirms compound 163 have sugar group at C-3 and all spectral data showed close resemblance to the data reported compound β-sitosterol 3-O--D-glucopyranoside (Haque et al., 2008).

Page 49 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.6 Structure Elucidation of Oleanolic acid (164)

29 30

19 21 12 17 13 28 25 26 H COOH 9 1 15 H 3 5 7 27 HO H 23 24 164

The compound 164 was purified as white needles (m.p. 232-234 °C). It showed light pinkish color on spraying with ceric sulphate solution after heating. The IR spectrum showed peaks at 3445, 3278-2635, 2960, 2868, 1695, 1618, 1452, 1379, 1259, 1041, 975 cm-1 characteristic for carboxylic acid, hydroxyl, carbonyl and double bond. The HR-EI- MS of compound 164 showed the molecular ion peak of 456.3610 [M]+ and molecular formula C30H48O3.

The 1H NMR spectrum showed a signal at δ 5.17 (1H, t, J = 3.8 Hz, H-12) for olefinic methine and δ 3.49 (1H, dd, J = 8.5, 2.0 Hz) for oxyginated methine. Seven methyls appeared at δ 1.29, 1.27, 1.07, 1.06, 1.05, 0.98 and 0.94. These all methyls were singlet and angular methyls which is characteristic of terpenoids. The signal for the carboxylic acid was also appeared at δ 12.8.

The 13C NMR showed thirty carbons indicated seven methyls at δ 34.4, 28.4, 27.6, 23.0, 17.7, 16.2 and 15.8 which are characteristic for terpenoids. The most downfield carbon appeared at δ 179.6 indicating the presence of carboxylic acid. Its position was confirmed by downfield shift of proton H-18 at δ 2.89 (1H, dd, J = 14.2, 4.4 Hz). The HMBC correlation of proton H-18 (δ 2.89) with C-28 (δ 179.6) confirmed the attachment of carboxylic acid at C-17. The HMBC correlation of Me-27 with C-13 (δ 142.1) and 1H-1H

Page 50 CHAPTER # 02 RESULTS AND DISCUSSION

COSY correlation between of H-11 and H-12 confirms the position of double bond between C-12 and C-13. The oxyginated methine was placed at position C-3 due to HMBC correlation with C-1 (δ 38.7), C-5 (δ 54.9), C-23 (δ 28.4), C-24 (δ 16.2) and

COSY correlation with CH2-2 (δ 27.9). Two methyls resonated at δ 1.27 and 1.05 showing HMBC correlation with each other and with C-3 (δ 77.9) and C-5 (δ 54.9) were placed at C-4. In the same way other two methyls at δ 0.98 and 1.07 showing HMBC relation with each other and with C-19 (δ 47.3) and C-21 (δ 33.4) were placed at C-20. Hence compound 164 have an hydroxyl group at C-3, an olefinic bond at C-12,13 and carboxylic acid group at C-17 in terpenoid nucleus. The all spectral data of 164 was closely related to the already reported for oleanolic acid (Seebacher et al., 2003).

Page 51 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.7 Structure Elucidation of Quercitrin (165)

OH 2' 4'

HO 9 O 7 1 6'

3 OH CH3 10 O OH 5 5'' OH OH O 1'' 3'' O 165

The compound 165 was purified as yellow amorphous solid. It appeared yellowish brown on spraying ceric sulphate solution after heating. The IR spectrum showed peaks at 3445, 2960, 2868, 1695, 1618 cm-1 characteristic for hydroxyl, carbonyl and aromatic system and UV band at 325, 288 and 245 nm indicating the presence of aromatic system. The HR-FAB-MS of 165 showed the molecular ion peak of 449.0850 [M+H]+ corresponding to the molecular formula C21H21O11.

The 1H NMR spectrum showed five signals at δ 7.29 (1H, d, J = 2.3 Hz), 7.17 (1H, dd, J = 8.6, 2.3 Hz), 6.89 (1H, d, J = 8.6 Hz), 6.36 (1H, d, J = 2.3 Hz), 5.59 (1H, d, J = 2.3 Hz) in aromatic region indicated the presence of flavanol nucleus. The presence of rhamnose sugar was due to the signals at δ 5.38 for anomeric proton, 3.30-3.90 for four oxygenated methines and δ 0.98 for methyl, respectively.

The 13C NMR showed twenty one carbons including the signals for rhamnose (δ 102.6, 73.9, 73.4, 73.3, 72.3, 16.9). A carbonyl appeared at δ 180.2, four oxygenated quaternary carbons at δ 168.1, 166.9, 150.1 and 147.1 and two aromatic methines at δ 98.3 and 93.5, specific for meta-substituted A ring of flavonoid. The position of all the substituents was fixed by HMBC correlations in which two aromatic methines at position 6 and 8 and the hydroxyl groups were present at C-5 and C-7. The attachment of rhamnose at C-3 was confirmed by HMBC correlation H-1'' (δ 5.38) with C-3 (δ 137.2). The above discussion

Page 52 CHAPTER # 02 RESULTS AND DISCUSSION deduced 165 a flavonoid which is substituted by a rhamnose at C-3 confirming compound 165 as quercitrin (Bose et al., 2013; Hasan et al., 2006).

Page 53 CHAPTER # 02 RESULTS AND DISCUSSION

2.2.8 Structure Elucidation of 1-O-methyl-D-chiro-inositol (166)

OCH3

HO 1 OH

3 5 HO OH

166

Compound 166 was isolated as white crystals. The IR spectrum showed peaks at 3589- 3599, 2960, 2868 cm-1. The HR-EI-MS showed molecular ion peak at 194.1802 [M]+ which confirmed the molecular formula as C7H14O6.

The 1H NMR spectrum showed four signals at δ 3.85 (2H, m), 3.67 (2H, m), 3.52 (1H, m) and 3.18 (1H, dd, J = 8.6, 2.3 Hz). The spectrum also showed for the signal for methoxy at δ 3.23.

The 13C NMR showed signal at δ 80.2, 73.9, 73.8, 73.2, 70.5, 70.4 and 56.9 for altogether seven carbons. The HMBC correlation of methoxy (δ 3.23) with C-1 (δ 85.2) confirm its attachment at C-1. The signals at δ 3.85 showed HMBC correlations with C-4 (δ 73.2) and C-6 (δ 70.5) and signal at δ 3.18 correlated with C-3 (δ 73.8) and C-5 (δ 73.9). The presence of 1H-1H COSY relation between H-5 and H-4,6 and H-2 and H-1,3 indicates that 166 is cyclohexane molecule with five hydroxyls and a methoxy at C-1 which found in the literature by the name 1-O-methyl-D-chiro-inositol (McDonald IV et al., 2012).

Page 54 CHAPTER # 02 RESULTS AND DISCUSSION

2.3 AChE, BChE and LOX inhibitory Activities of Compounds 158-162

All the isolated limonoids (158-162) were evaluated for their enzyme inhibitory potential against enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX) using eserine and baicalein (Aldrich Chem. Co.; Seelze, Germany) as positive controls. The results (Table 3) showed that the tested compounds were found inhibitors against the used enzymes.

Table 3. AChE, BChE and LOX inhibitory activities of compounds 158-162

Compound AChE (%) AChE (IC50) M BChE (%) BChE (IC50) M LOX (%) LOX (IC50) M

158 61.8 ±0.63 225.2±0.22 65.3±0.62 198.7±0.15 52.2±0.63 <400

159 46.6±0.31 <400 60.2±0.91 228.5±0.27 63.0±0.16 281.2±0.11

160 78.6±0.74 137.5±0.05 55.8±0.55 <400 45.2±0.82 <400

161 46.9±0.55 <400 59.9±0.77 241.5±0.11 62.5±0.44 289.6±0.14

162 55.2±0.33 <400 66.7±0.18 185.4±0.38 54.3±0.46 <400

Eserine 91.3±1.17 0.04±0.0001 82.8±1.09 0.85±0.001 - -

Baicalein - - - - 93.8±1.2 22.4±1.3

All compounds were prepared in methanol with a concentration of 0.5mM All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003. Results are presented as mean ± sem.

Page 55 CHAPTER # 03 EXPERIMENTAL

EXPERIMENRAL

Page 56 CHAPTER # 03 EXPERIMENTAL

3.1 General Experimental Procedures

Physical Constants Melting points were determined in glass capillaries using Buchi 535 melting point apparatus. Optical rotation was measured on a JASCO DIP-360 polarimeter (Tokyo,

Japan). IC50 values (concentration at which there is 50% enzyme inhibition) of the isolated compounds were calculated using EZ-Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA). Spectroscopy Ultra Violet (UV) spectra were obtained in methanol on U-3200 Schimadzu UV-240 spectrophotometer (Duisburg, Germany). Infrared (IR) spectra were recorded on Shimadzu 460 spectrometer (Duisburg, Germany). 1H NMR (400, 500, 600 MHz), 13C NMR (100, 125 MHz) and 2D NMR (HSQC, HMBC and COSY; 400, 500, 600 MHz) spectra were recorded on Bruker spectrometer (Zurich, Switzerland). The chemical shift values (δ) are reported in ppm and the coupling constant (J) are in Hz. EI-MS, HR-EI- MS, FAB-MS and HR-FAB-MS were recorded on Finnigan (Varian MAT, Waldbronn, Germany) JMS H × 110 with a data system and JMSA 500 mass spectrometers, respectively. Chromatography Chromatographic separations were carried out using aluminum sheets pre-coated with silica gel 60 F254 (20 × 20 cm, 0.2 mm thick; E. Merck; Darmstadt, Germany) for thin layer chromatography (TLC) and silica gel (230-400 mesh, Darmstadt, Germany) for column chromatography. TLC (Thin Layer Chromatography) plates were visualized under UV at 254 and 366 nm and by spraying with ceric sulphate solution (by heating). Computational Section

ECD spectra were recorded with a Jasco J-715 spectropolarimeter (Duisburg, Germany) with the following conditions: scanning speed, 100 nm/min; time constant, 1 s; band width, 2 nm; 8 accumulations. Solid-state ECD spectra were obtained using the KCl pellet technique (Pescitelli et al., 2009) using ca. 100 µg of compound and ca. 200 mg of

Page 57 CHAPTER # 03 EXPERIMENTAL oven-dried KCl. Rotation-dependent artifacts were checked by recording the spectrum upon four rotations of 90˚ of the disc around the light direction and vertical flip, which resulted in nearly identical ECD curves.

Enzyme Inhibitory Assays

Acetylcholinesterase Inhibition Assay

The Acetylcholinesterase (AChE) inhibition activity was performed according to the method used by Ellman (Ellman et al., 1961) with slight modifications. The percent inhibition was calculated by the help of following equation.

Inhibition (%) = Control – Test × 100 Control Butyrylcholinesterase Inhibition Assay

The Butyrylcholinesterase (BChE) inhibition activity was performed according to the method used by Ellman with slight modifications. The percent inhibition was calculated with the help of following equation.

Inhibition (%) = Control – Test × 100 Control

IC50 values (concentration at which there is 50% enzyme inhibition) of compounds were calculated using EZ–Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA).

Lipoxygenase Inhibition Assay

Lipoxygenase (LOX) activity was assayed according to the reported method (Tappel, 1953) but with slight modifications. All reactions were performed in triplicates. Baicalein (0.5 mM well-1) was used as a positive control. The percentage inhibition was calculated by formula given below.

Inhibition (%) = Control – Test × 100 Control

Page 58 CHAPTER # 03 EXPERIMENTAL

3.2 Plant Material

The aerial part of Kigelia africana Benth was collected from Lal Sohanra (District Bahawalpur, Pakistan) in September 2010 and was identified by Dr. Muhammad Arshad (late), Plant Taxonomist, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan, where a voucher specimen is deposited (KA/CIDS- 404/10).

3.3 Extraction and Isolation

EtOAc/MeOH and MeOH as eluent resulted into six fractions E1 up to 20% EtOAc in n- hexane, E2 up to 50% EtOAc in n-hexane, E3 up to 75% EtOAc in n-hexane, E4 up to

Page 59 CHAPTER # 03 EXPERIMENTAL

Kigelia africana 7 kg Ground, extracted with MeOH, concentrated under reduced pressure Methanolic extract 970 gm Suspended in water and extracted with organic solvents

n-Hexane soluble EtOAc soluble Water soluble 393 gm 195 gm 382 gm

Purification by CC

10% EtOAc 45% EtOAc 55% EtOAc 2% MeOH 10% MeOH in n-hexane in n-hexane in n-hexane in EtOAc in EtOAc 5 158 159,162 163 166 40% EtOAc 50% EtOAc 80% EtOAc 5% MeOH in n-hexane in n-hexane in n-hexane in EtOAc 160 161 164 165

Scheme 1. Isolation scheme of compounds 5, 158-166 from Kigelia africana

Page 60 CHAPTER # 03 EXPERIMENTAL

3.4.1 Characterization of 1-O-deacetyl-2-α-methoxykhayanolide E (158)

Colorless crystalline solid (48 mg). O 23 ° 21 M.p: 106-108 C. 22 18 20 25 CH [α]D + 9.7 (c = 0.0015, MeOH). 3 OH 12 17 19 H 11 13 O H3CO OH UV (CH OH) λ nm: 210 (3.09). 7 H3C 14 16 3 max 6 5 9 15 10 8 O -1 O OH IR (KBr) max cm : 3435, 2954, 1736, 29 30 1 4 2 O H3C 3 1718, 1615, 1459, 1388, 1249, 1026, 983. 28

O OCH3 158

1 H NMR (CD3OD, 500 MHz): δ 2.89 (1H, d, J = 8.4 Hz, H-5), 4.22 (1H, d, J = 8.4 Hz, H-6), 2.28 (1H, d, J = 9.0 Hz, H-9), 1.97 (1H, d, J = 13.8 Hz, H-11a), 1.81 (1H, dd, J = 14.1, 5.4 Hz, H-11b), 1.73 (1H, dt, J = 13.8, 3.0 Hz, H-12a), 0.94 (1H, d, J = 12.0 Hz, H- 12b), 3.19 (2H, s, H-15), 5.51 (1H, s, H-17), 1.07 (3H, s, H-18), 1.32 (3H, s, H-19), 7.54 (1H, s, H-21), 6.46 (1H, s, H-22), 7.49 (1H, s, H-23), 1.01 (3H, s, H-28), 2.10 (1H, d, J = 12.6 Hz, H-29a), 1.85 (1H, d, J = 12.6 Hz, H-29b), 2.86 (1H, s, H-30), 3.48 (3H, s, 2- OMe), 3.71 (3H, s, 7-OMe).

13 C NMR (CD3OD, 100 MHz): δ C-1), 101.9 (C-2), 204.7 (C-3), 50.2 (C-4), 44.1 (C-5), 71.5 (C-6), 175.5 (C-7), 88.3 (C-8), 56.8 (C-9), 61.0 (C-10), 17.2 (C-11), 28.8 (C- 12), 38.7 (C-13), 86.4 (C-14), 37.5 (C-15), 173.9 (C-16), 82.5 (C-17), 15.7 (C-18), 18.7 (C-19), 122.1 (C-20), 142.7 (C-21), 111.1 (C-22), 144.2 (C-23), 15.6 (C-28), 43.8 (C-29),

73.7 (C-30), 53.1 (2-OCH3), 52.7 (7-OCH3).

+ HR-EI-MS m/z: 546.2110 [M] (calcd. for C28H34O11, 546.2105).

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3.4.2 Characterization of Kigelianolide (159)

Colorless amorphous powder (40 mg). O ° 23 M.p: 300 C 21 22 18 20 [α]D – 22.6 (c = 0.0013, MeOH). CH3 OH 12 17 19 H 11 13 O UV (CH OH) λ nm: 211 (3.6). H3CO 3 max 7 H3C 16 6 14 8 15 5 9 O -1 10 IR (KBr) max cm : 3434, 2955, 1735, O OH OH 29 30 1 4 O H C 3 2 1717, 1616, 1459, 1386, 1250, 1025, 3 28 985. HO 159

1 H NMR (CD3OD, 500 MHz): δ 4.47 (1H, dd, J = 9.5, 6.5 Hz, H-2), 3.37 (1H, d, J = 6.5 Hz, H-3), 3.18 (1H, d, J = 8.5 Hz, H-5), 4.17 (1H, d, J = 8.5 Hz, H-6), 2.11 (1H, d, J = 9.0 Hz, H-9), 2.13 (1H, d, J = 9.0 Hz, H-11a), 1.80 (1H, m, H-11b), 1.88 (1H, d, J = 12.0 Hz, H-12a), 0.87 (1H, d, J = 12.0 Hz, H-12b), 3.08 (1H, d, J = 19.0 Hz, H-15a), 2.78 (1H, d, J = 19.0 Hz, H-15b), 5.76 (1H, s, H-17), 1.09 (3H, s, H-18), 1.21 (3H, s, H-19), 7.50 (1H, s, H-21), 6.45 (1H, s, H-22), 7.46 (1H, s, H-23), 1.00 (3H, s, H-28), 1.86 (1H, d, J = 12.0 Hz, H-29a), 1.34 (1H, d, J = 12.0 Hz, H-29b), 2.60 (1H, d, J = 9.5 Hz, H-30), 3.73 (3H, s, 7-OMe).

13 C NMR (CD3OD, 100 MHz): δ 85.2 (C-1), 73.9 (C-2), 80.1 (C-3), 43.5 (C-4), 44.9 (C- 5), 72.3 (C-6), 176.9 (C-7), 88.3 (C-8), 56.9 (C-9), 60.6 (C-10), 17.4 (C-11), 27.6 (C-12), 38.9 (C-13), 79.3 (C-14), 33.2 (C-15), 173.5 (C-16), 82.6 (C-17), 15.3 (C-18), 18.3 (C- 19), 122.2 (C-20), 142.4 (C-21), 111.1 (C-22), 144.4 (C-23), 15.2 (C-28), 46.1 (C-29),

73.7 (C-30), 64.6 (C-30), 52.6 (7-OCH3).

+ HR-EI-MS m/z: 518.2315 [M] (calcd. for C27H36O10, 518.2308).

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3.4.3 Characterization of Deacetylkhayanolide E (160)

White amorphous powder (30mg). O 23 21 [α]D – 26.6 (c = 0.0011, MeOH). 22 18 20 CH UV (CH OH) λ nm: 213 (3.6). 3 3 max OH 12 17 19 H 11 13 O H3CO OH -1 7 H3C 14 16 6 IR (KBr) max cm : 3498, 2951, 1732, 5 9 15 10 8 O O OH 1713, 1464, 1389, 1242, 1148, 1045, 1026, 29 30 1 4 H C 3 2 O 770. 3 28 H O 160

1 H NMR (CD3OD, 500 MHz): δ 4.33 (1H, d, J = 9.5 Hz, H-2), 3.30 (1H, d, J = 8.4 Hz, H-5), 4.21 (1H, d, J = 8.4 Hz, H-6), 2.27 (1H, d, J = 9.0 Hz, H-9), 2.05 (1H, d, J = 13.8 Hz, H-11a), 1.82 (1H, dd, J = 14.1, 5.4 Hz, H-11b), 1.80 (1H, dt, J = 13.8, 3.0 Hz, H- 12a), 0.91 (1H, d, J = 12.0 Hz, H-12b), 3.16 (1H, d, J = 19.2 Hz, H-15a), 2.73 (1H, d, J = 18.6 Hz, H-15b), 5.51 (1H, s, H-17), 1.10 (3H, s, H-18), 1.32 (3H, s, H-19), 7.51 (1H, s, H-21), 6.44 (1H, s, H-22), 7.48 (1H, s, H-23), 0.99 (3H, s, H-28), 2.11 (1H, d, J = 12.6, H-29a), 1.83 (1H, d, J = 12.6 Hz, H-29b), 2.80 (1H, d, J = 9.5 Hz, H-30), 3.70 (3H, s, 7- OMe).

13 C NMR (CD3OD, 100 MHz): δ 85.4 (C-1), 76.1 (C-2), 208.0 (C-3), 50.7 (C-4), 43.5 (C-5), 71.5 (C-6), 175.6 (C-7), 88.50 (C-8), 56.6 (C-9), 60.9 (C-10), 17.4 (C-11), 27.8 (C- 12), 38.8 (C-13), 84.7 (C-14), 34.1 (C-15), 173.3 (C-16), 82.6 (C-17), 15.3 (C-18), 18.7 (C-19), 122.1 (C-20), 142.6 (C-21), 111.1 (C-22), 144.1 (C-23), 15.7 (C-28), 45.3 (C-29),

64.6 (C-30), 52.6 (7-OCH3).

EI-MS m/z (rel. int.): 516 [M]+ (82), 420 (13), 402 (41), 378 (100), 361 (9), 343 (10), 198 (35), 165 (57), 125 (34).

+ HR-EI-MS m/z 516.1995 [M] (calcd. for C27H32O10, 516.1989).

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3.4.4 Characterization of 1-O-deacetyl-2α-hyroxykhayanolide E (161)

White amorphous powder (25mg). O 23 21 [α]D - 28.6 (c = 0.0014, MeOH). 22 18 20 CH3 UV (CH3OH) λmax nm: 210 (3.06). OH 12 17 19 H 11 13 O H3CO OH -1 7 H3C 14 16 6 IR (KBr) max cm : 3490, 2952, 1732, 5 9 15 10 8 O O OH 1713, 1464, 1386, 1244, 1139, 1045, 1024, 29 30 1 4 H C 3 2 O 769. 3 28 OH O 161

1 H NMR (CD3OD, 500 MHz): δ 3.11 (1H, d, J = 9.0 Hz, H-5), 4.22 (1H, d, J = 8.4 Hz, H-6), 2.27 (1H, d, J = 9.0 Hz, H-9), 2.00 (1H, d, J = 13.8 Hz, H-11a), 1.78 (1H, dd, J = 14.1, 5.4 Hz, H-11b), 1.75 (1H, dt, J = 13.8, 3.0 Hz, H-12a), 0.91 (1H, d, J = 12.6 Hz, H- 12b), 3.54 (1H, d, J = 19.2 Hz, H-15a), 3.17 (1H, d, J = 18.6 Hz, H-15b), 5.49 (1H, s, H- 17), 1.07 (3H, s, H-18), 1.33 (3H, s, H-19), 7.50 (1H, s, H-21), 6.43 (1H, s, H-22), 7.47 (1H, s, H-23), 1.02 (3H, s, H-28), 2.12 (1H, d, J = 12.6 Hz, H-29a), 1.81 (1H, d, J = 12.6, H-29b), 2.82 (1H, s, H-30), 3.70 (3H, s, 7-OMe).

13 C NMR (CD3OD, 100 MHz): δ 85.5 (C-1), 100.3 (C-2), 206.0 (C-3), 49.0 (C-4), 43.45 (C-5), 71.5 (C-6), 175.6 (C-7), 88.7 (C-8), 56.75 (C-9), 61.0 (C-10), 17.1 (C-11), 28.1 (C- 12), 38.7 (C-13), 85.2 (C-14), 37.6 (C-15), 174.2 (C-16), 82.3 (C-17), 15.6 (C-18), 18.6 (C-19), 122.2 (C-20), 142.5 (C-21), 111.1 (C-22), 144.2 (C-23), 15.7 (C-28), 44.7 (C-29),

72.7 (C-30), 52.6 (7-OCH3).

EI-MS m/z (rel. int.): 532 (7), 504 (5), 468 (5), 411 (35), 390 (25), 351 (40), 315 (100), 255 (48), 237 (23), 95 (42), 69 (21).

+ HR-EI-MS m/z: 532.1945 [M] (calcd. for C27H32O11, 532.1939).

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3.4.5 Characterization of Khayanolide B (162)

Colorless amorphous powder (35 mg). O 23 ° 21 M.p: 310-311 C 22 18 20 CH [α] - 28.6 (c = 0.0012, MeOH). 3 D OH 12 17 19 H 11 13 O H3CO OH 7 H3C 14 16 6 UV (CH3OH) λmax nm: 213 (3.6). 5 9 15 10 8 O O OH -1 29 30 IR (KBr) max cm : 3430, 2950, 1745, 1 4 2 O H3C 3 1719, 1618, 1452, 1383, 1257, 1021, 985. 28 HO 162

1 H NMR (CD3OD, 500 MHz): δ 4.47 (1H, dd, J = 9.5, 6.5 Hz, H-2), 3.38 (1H, d, J = 6.5 Hz, H-3), 3.17 (1H, d, J = 8.5 Hz, H-5), 4.16 (1H, d, J = 8.5 Hz, H-6), 2.11 (1H, d, J = 9.0 Hz, H-9), 1.27 (1H, d, J = 9.0 Hz, H-11a), 1.31 (1H, m, H-11b), 1.88 (1H, d, J = 12.0 Hz, H-12a), 0.87 (1H, d, J = 12.0 Hz, H-12b), 3.10 (1H, d, J = 19.0 Hz, H-15a), 2.79 (1H, d, J = 19.0 Hz, H-15b), 5.76 (1H, s, H-17), 1.09 (3H, s, H-18), 1.21 (3H, s, H-19), 7.50 (1H, s, H-21), 6.45 (1H, s, H-22), 7.46 (1H, s, H-23), 1.00 (3H, s, H-28), 1.86 (1H, d, J = 12.0 Hz, H-29a), 1.34 (1H, d, J = 12.0 Hz, H-29b), 2.60 (1H, d, J = 9.5 Hz, H-30), 3.73 (3H, s, 7-OMe).

13 C NMR (CD3OD, 100 MHz): δ 85.2 (C-1), 73.9 (C-2), 80.1 (C-3), 43.5 (C-4), 44.9 (C- 5), 72.3 (C-6), 176.9 (C-7), 82.5 (C-8), 56.9 (C-9), 60.6 (C-10), 17.4 (C-11), 27.6 (C-12), 38.9 (C-13), 88.3 (C-14), 33.2 (C-15), 173.5 (C-16), 82.6 (C-17), 15.3 (C-18), 18.3 (C- 19), 122.2 (C-20), 142.4 (C-21), 111.1 (C-22), 144.4 (C-23), 15.2 (C-28), 46.1 (C-29),

73.7 (C-30), 52.6 (7-OCH3).

EI-MS m/z (rel. int.): 518 (5), 476 (5), 386 (15), 380 (100), 255 (48), 223 (23), 177 (29), 95 (42), 69 (21).

+ HR-EI-MS m/z: 518.2152 [M] (calcd. for C27H34O10, 518.2149).

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3.4.6 Characterization of β-Sitosterol (5)

Colorless amorphous powder (35 mg). 29 M.p: 143-145 °C. 28 21 26

18 20 23 25 [α]D – 28.6 (c = 0.0015, MeOH). 17 12 27 19 16 UV (CH3OH) λmax nm: 203 (4.5). 9 14 1 10 -1 H IR (KBr) max cm : 3445, 2970, 2868, 3 7 H 5 5 1805, 1618, 1452, 1380, 1257, 1021, HO 985,780.

1 H NMR (CD3OD, 500 MHz): δ 2.35 (2H, m, H-1), 1.73 (2H, m, H-2), 3.17 (1H, m, H- 3), 2.27 (2H, m, H-4), 5.23 (1H, m, H-6), 2.16 (2H, m, H-7), 1.11 (1H, m, H-8), 2.11 (1H, m, H-9), 1.48 (2H, m, H-11), 1.77 (2H, m, H-12), 2.01 (1H, m, H-14), 1.75 (2H, m, H-15), 1.29 (2H, m, H-16), 1.85 (1H, m, H-17), 0.95 (3H, s, H-18), 1.50 (3H, s, H-19), 2.40 (1H, m, H-20), 1.45 (3H, d, J = 6.5 Hz, H-21), 1.79 (2H, m, H-22), 1.72 (2H, m, H- 23), 1.95 (1H, m, H-24), 1.85 (1H, m, H-25), 0.75 (3H, d, J = 6.5 Hz, H-26), 0.65 (3H, d, J = 6.5 Hz, H-27) 1.63 (2H, s, H-28), 0.85 (3H, t, J = 7.0 Hz, H-29).

13 C NMR (CD3OD, 100 MHz): δ 36.7 (C-1), 33.9 (C-2), 70.1 (C-3), 41.5 (C-4), 141.9 (C-5), 122.3 (C-6), 32.1 (C-7), 32.5 (C-8), 49.9 (C-9), 35.6 (C-10), 20.4 (C-11), 40.6 (C- 12), 42.9 (C-13), 56.3 (C-14), 23.2 (C-15), 27.5 (C-16), 56.6 (C-17), 11.1 (C-18), 30.2 (C-19), 36.2 (C-20), 24.4 (C-21), 31.9 (C-22), 22.6 (C-23), 35.2 (C-24), 28.1 (C-25), 18.7 (C-26), 18.3 (C-27), 25.7 (C-28), 13.3 (C-29).

+ EI-MS m/z (rel. int.): 414.4 [M] C29H50O: 414 (4.2), 400 (2.9), 381 (2.8), 275 (2.8), 272 (2.0), 254 (5.9), 230 (4.0), 212 (6.0), 197 (3.7), 173 (5.1), 163 (5.5), 137 (7.9), 120 (9.8), 106 (15.2), 94 (19.9), 83 (16.0), 69 (42.0), 55 (52.0), 43 (100.0).

+ HR-EI-MS m/z: 414.3861 [M] (calcd. for C29H50O), 414.3855.

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3.4.7 Characterization of β-Sitosterol 3-O-β-D-glycopyranoside (163)

Colorless amorphous

29 powder (39 mg). 28 M.p: 279-280 °C. 21 26 18 20 23 25

12 17 27 [α]D - 14.5 (c = 0.0011, 19 16 9 14 MeOH). 1 10 H H HO 6' 3 7 UV (CH3OH) λmax nm: 206 O O 5 HO 5' HO 3' (4.10). 1' OH 163 IR (KBr)  cm-1: 3445, max 2970, 2868, 1805, 1618, 1452, 1380, 1257, 1061, 985,783.

1 H NMR (CD3OD, 500 MHz): δ 1.35 (2H, m , H-1), 1.79 (2H, m, H-2), 3.89 (1H, m, H- 3), 2.47 (2H, m, H-4), 5.23 (1H, d, J = 5.5 Hz , H-6), 1.96 (2H, m, H-7), 1.41 (1H, m, H- 9), 1.98 (2H, m, H-11), 1.17 (2H, d, J = 12.0 Hz, H-12), 1.19 (1H, m, H-14), 1.05 (2H, m, H-15), 1.59 (2H, m, H-16), 1.49 (1H, m, H-17), 0.65 (3H, s, H-18), 1.50 (3H, s, H-19), 1.45 (2H, d, J = 6.0 Hz, H-21), 1.09 (1H, m, H-22), 1.27 (2H, m, H-23), 1.29 (3H, m, H- 24), 1.74 (1H, m, H-25), 0.95 (3H, d, J = 6.1 Hz, H-26), 0.79 (3H, d, J = 6.6 Hz, H-27), 1.15 (2H, m, H-28), 0.85 (3H, d, J = 7.3 Hz, H-29), 4.95 (1H, s, H-1'), 3.65 (1H, m, H- 2'), 3.07 (1H, m, H-3'), 3.03 (1H, m, H-4'), 4.25 (1H, d, J = 5.6 Hz, H-5'), 4.85 (1H, s, H- 6'a), 4.65 (1H, s, H-6'b).

13 C NMR (CD3OD, 100 MHz): δ 35.7 (C-1), 29.9 (C-2), 77.1 (C-3), 40.5 (C-4), 140.9 (C-5), 121.3 (C-6), 34.1 (C-7), 32.5 (C-8), 49.5 (C-9), 36.6 (C-10), 21.4 (C-11), 39.6 (C- 12), 42.1 (C-13), 56.1 (C-14), 24.2 (C-15), 28.5 (C-16), 55.6 (C-17), 12.3 (C-18), 19.3 (C-19), 36.6 (C-20), 18.4 (C-21), 34.9 (C-22), 25.4 (C-23), 44.2 (C-24), 27.8 (C-25), 19.7

Page 67 CHAPTER # 03 EXPERIMENTAL

(C-26), 18.6 (C-27), 23.6 (C-28), 11.4 (C-29), 101.4 (C-1'), 76.1 (C-2'), 73.7 (C-3'), 70.8 (C-4'), 76.34 (C-5'), 61.4 (C-6').

+ HR-FAB-MS m/z: [M+H] 577.2645 (calcd. for C35H61O6) 577.2639.

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3.4.8 Characterization of Oleanolic Acid (164)

White needles (159 mg) 29 30

° M.p: 305-306 C 19 21 12 17 28 [α]D + 78.9 (c = 0.0014, CHCl3) 25 26 13 H COOH 9 1 15 -1 IR (KBr) max cm : 3445, 3278-2635, H 3 5 7 27 2960, 2868, 1695, 1618, 1452, 1379, 1259, HO H 24 164 1041, 975. 23

1 H NMR (CD3OD, 500 MHz): δ 1.04 (1H, dd, J = 12.1, 4.1 Hz, H-1a), 1.54 (1H, d, J = 12.1 Hz, H-1b), 1.79 (2H, m, H-2), 3.49 (1H, dd, J = 8.5, 2.2 Hz, H-3), 1.44 (1H, m, H- 5), 1.56 (2H, m, H-6), 1.54 (2H, m, H-7), 1.88 (d, J = 2.8 Hz, H-9), 1.56 (2H, d, J = 10.2 Hz, H-11), 5.17 (1H, t, J = 3.8 Hz, H-12), 1.41 (2H, m, H-15), 2.12 (1H, m, H-16a), 1.99 (1H, m, H-16b), 2.89 (1H, dd, J = 10.2, 2.5 Hz, H-18), 1.81 (1H, m, H-19a), 1.30 (1H, m, H-19b), 1.47 (1H, d, J = 10.2 Hz, H-21a), 1.27 (1H, d, J = 10.2 Hz, H-21b), 1.68 (1H, m, H-22a), 1.57 (1H, m, H-22b), 1.07 (3H, s, H-23), 0.94 (3H, s, H-24), 0.98 (3H, s, H-25), 1.06 (3H, s, H-26), 1.29 (3H, s, H-27), 1.05 (3H, s, H-29), 0.98 (3H, s, H-30), 12.8 (1H, s, -OH).

13 C NMR (CD3OD, 100 MHz): δ 38.72 (C-1), 27.9 (C-2), 77.9 (C-3), 40.2 (C-4), 54.9 (C-5), 18.3 (C-6), 34.1 (C-7), 39.5 (C-8), 49.1 (C-9), 36.9 (C-10), 23.4 (C-11), 121.6 (C- 12), 142.1 (C-13), 43.1 (C-14), 29.2 (C-15), 23.5 (C-16), 45.6 (C-17), 42.3 (C-18), 47.3 (C-19), 30.6 (C-20), 33.4 (C-21), 32.9 (C-22), 28.4 (C-23), 16.2 (C-24), 15.8 (C-25), 17.7 (C-26), 27.6 (C-27), 179.6 (C-28), 34.4 (C-29), 23.0 (C-30).

+ EI-MS m/z (rel. int.): 456 [M] C30H48O3, 456 (9.2), 248 (2.9), 231 (2.8), 215 (2.8), 203 (100.0), 184 (5.9), 170 (4.0), 118 (6.0), 107 (3.7), 94 (19.9), 82 (16.0), 69 (42.0), 55 (52.0).

+ HR-EI-MS m/z: 456.3610 [M] (calcd. for C30H48O3) 456.3604.

Page 69 CHAPTER # 03 EXPERIMENTAL

3.4.9 Characterization of Quercitrin (165)

Yellow Powder (57 mg). OH M.p: 181-182 °C. OH 2' 4' 25 HO O [α]D -158 (c, 0.0011 in MeOH). 9 7 1 6'

3 OH CH3 UV (CH3OH) λmax nm: 245, 288 and 325. 10 O OH 5 5'' OH -1 OH O 1'' 3'' IR (KBr) max cm : 3445, 2960, 2868, 1695, O 1618. 165

1 H NMR (CD3OD, 500 MHz): δ 5.59 (1H, d, J = 2.3 Hz, H-6), 6.36 (1H, d, J = 2.3 Hz, H-8), 7.29 (1H, d, J = 2.3 Hz, H-2'), 6.89 (1H, d, J = 8.6 Hz, H-5'), 7.17 (1H, dd, J = 8.6, 2.3 Hz, H-6'), 5.38 (1H, br s, H-1''), 3.86 (1H, m, Hz, H-2''), 3.68 (1H, m, H-3''), 3.34 (1H, m, H-4''), 3.18 (1H, m, H-5''), 0.98 (3H, d, J = 6.7 Hz, H-6'').

13 C NMR (CD3OD, 100 MHz): δ 157.9 (C-2), 137.2 (C-3), 180.2 (C-4), 166.9 (C-5), 98.3 (C-6), 168.1 (C-7), 93.5 (C-8), 159.1 (C-9), 106.1 (C-10), 122.4 (C-1'), 115.6 (C-2'), 147.1 (C-3'), 150.1 (C-4'), 114.2 (C-5'), 123.5 (C-6'), 102.6 (C-1''), 72.3 (C-2''), 73.3 (C- 3''), 73.9 (C-4''), 73.4 (C-5''), 16.9 (C-6'').

+ HR-FAB-MS m/z: 449.0850 [M+H] (calcd. for C21H21O11) 449.0845.

Page 70 CHAPTER # 03 EXPERIMENTAL

3.4.10 Characterization of 1-O-methyl-D-chiro-inositol (166)

White crystal (57mg). OCH3 ° HO OH M.p: 207-208 C. 1

3 5 [α]D + 67.7 (c = 0.0012, MeOH). HO OH

-1 OH IR (KBr) max cm : 3589-3599, 2960, 2868, 1695. 166

1 H NMR (CD3OD, 500 MHz): δ 3.18 (1H, dd, J = 8.6, 2.3 Hz, H-1), 3.85 (2H, m, H-2,6),

3.67 (2H, m, H-3,5), 3.52 (1H, m, H-4), 3.23 (3H, s, -OCH3).

13 C NMR (CD3OD, 100 MHz): δ 85.2 (C-1), 70.4 (C-2), 73.8 (C-3), 73.2 (C-4), 73.9 (C-

5), 70.5 (C-6), 56.9 (OCH3).

+ HR-EI-MS m/z: 194.1802 [M] (calcd. for C7H14O6) 194.1797.

Page 71 CHAPTER # 04 INTRODUCTION

Part B: Isolation of Bioactive Constituents from Phlomis stewartii; The Medicinal Plant of Pakistan

Page 72 CHAPTER # 04 INTRODUCTION

INTRODUCTION

Page 73 CHAPTER # 04 INTRODUCTION

4.1 The Family Labiatae

The family Labiatae (Lamiaceae) is a member of order lamiales generally known as mint family which comprises 220 genera and 4000 species. These are mostly herbs, subshrubs, annual, perennial, 4-angeled stems, alternate and opposite leaves, compound inflorescence, solitary flowers. Mostly genera are distributed as cosmopolitan but few like Salvia, Nepeta, Lagochilus, Eremostachys and Phlomis occur in Mediterranean region and Asia (Hedge, 1986; Xing-ke et al., 1994). This family is of great pharmacological importance due to presence of bioactive essential oils and secondary metabolites like diterpenoides. The members belong to this family are used as medicines for culinary diseases and cultivated as herbal medicine, herbal tea and vegetable by the local community (Naghibi et al., 2005). The genus Phlomis is one of its important members of the family Labiatae.

4.2 The Genus Phlomis

The genus Phlomis is comprises more than 100 species which are found commonly in Euro-Asia and North Africa. The plants have opposite leaves, whorled with yellow to pink color flowers, ascending four stamens, forked end anther, nutlets four sided fruits and hairs cover its all parts (Amora et al., 2009).

4.3 Phlomis stewartii

It is a rare species of genus Phlomis and is restricted to Pakistan and Afghanistan which first time discovered in 1885. It is perennial shrub, stem is erect 30-40 cm, leaves are elliptic, petiole, thick in texture and flowers are pink in color (Hooker, 1885).

4.4 Ethnobotanical Importance of the Genus Phlomis

The members of the genus Phlomis are used in folk medicine as herbal tea to cure the gastrointestinal, kidney, heart and bone diseases. Fewer species are used for the treatment of cold, cough, fever, burns, skin infections, allergies, lesions and gastric ulcers (Amora et al., 2009). In Portugal, P. purpurea is used to cure seventeen different diseases as for gastric pain and as an intestinal antispasmodic (Novais et al., 2004). In Spain, some

Page 74 CHAPTER # 04 INTRODUCTION relative species are used to cure prostate and liver diseases (Gonzalez-Tejero et al., 1995). Mostly all parts of plants are used ethnobotanically including seeds, fruits, leaves, roots and flowers as an infusion, decoction and juice or used directly like P. fruticosa leaves are used as sauce paste in Italy (Lentini and Venza, 2007).

4.5 Pharmacological Importance of Genus Phlomis

The genus Phlomis is well known for its pharmacological uses as various species possesses anti-diabetic, anti-hyperglycemic (Sarkhail et al., 2007), anti-nociceptive, analgesic (Sarkhail et al., 2003), anti-ulcerogenic, anti-inflammatory, anti-allergic (Shin and Lee, 2003), anticancer, cytotoxic (Kirmizibekmez et al., 2004), anti-infective, antibacterial (Morteza et al., 2006) and antifungal (Demirci et al., 2008) activities and also used for the protection of vascular system (Amora et al., 2009).

4.6 Literature Survey on Genus Phlomis

The genus Phlomis is rich of secondary metabolites as the following classes of secondary metabolites like essential oils, monoterpenes, sesquiterpenes, triterpenes, nortriterpenes, fatty acids, aliphatic alcohols, flavonoides, iridoids. phenylethylalcohol glycosides etc are reported from this genus. A brief literature review is given in Table 4.

Page 75 CHAPTER # 04 INTRODUCTION

Table 4. Compounds already isolated from the genus Phlomis

Sr. # Name Molecular Formula Source Reference Molecular Mass 1 α-pinene C10H16 P. cretica (Aligiannis et al., 2004) (m/z 136.125) P. fruticosa

2 Limonene C10H16 P. cretica (Aligiannis et al., 2004) (m/z 136.125) P. fruticosa 3 Apigenin C15H10O5 P. lychnitis (Tomas et al., 1986) (m/z 270.057) P. samia 4 Leutuline C15H10O6 P. lychnitis (Kabouche et al., 2005) (m/z 286.048) P. crinita 5 Kaempferol C15H10O6 P. aurea (El-Negoumy et al., 1986) (m/z 286.048) P. floccose 6 Chryseriol C16H12O6 P. lychnitis (Marin et al., 2007) (m/z 300.048) P. samia P. fruticosa 7 Lamiide C18H28O11 P. linearis (Calis et al., 1991) (m/z 420.163) P. aurea 8 Shanzhiside methanoate C17H26O10 P. rotata (Zhang et al., 1991) (m/z 390.153) P. younghusbandii 9 8-O-Acetylshanzhiside C18H26O10 P. rigida (Takeda et al., 2001; methanoate (m/z 402.153) P. spinidens Takeda et al., 2000) 10 Phlomisin C29H46O6 P. umbrosa (Liu et al., 2008) (m/z 490.329) 11 Phlomishexaol A C29H48O6 P. umbrosa (Liu et al., 2008) (m/z 492.345)

12 Phlomishexaol B C29H48O6 P. umbrosa (Liu et al., 2008) (m/z 492.345)

13 Phlomisone C29H46O6 P. umbrosa (Liu et al., 2008) (m/z 490.329)

14 19(18→17)-Abeo-28-nor-12- C29H48O5 P. umbrosa (Liu et al., 2007b) oleanene-2,3,18,23,24-pentol (m/z 476.350)

15 19(18→17)-Abeo-12-methoxy- C30H50O5 P. umbrosa (Liu et al., 2007b) 28-nor-13(18)-oleanene- (m/z 490.366) 2,3,23,24-tetrol 16 19(18→17)-Abeo-12-methoxy- C30H50O6 P. umbrosa (Liu et al., 2007b) 28-nor-13(18)-oleanene- (m/z 506.361) 2,3,23,24,29-pentol 17 Phlomistetraol A C29H48O4 P. umbrosa (Liu et al., 2008) (m/z 460.355)

18 Phlomistetraol C C29H48O4 P. umbrosa (Liu et al., 2008) (m/z 460.355)

19 Phlomisamide C25H47NO6 P. cashmeriana (Hussain et al., 2010) (m/z 457.340)

20 Apigenin 7-glucuronide C21H18O11 P. cashmeriana (Hussain et al., 2010) (m/z 446.085)

21 Benzyl β-sambubioside C18H26O10 P. aurea (Kamel et al., 2000) (m/z 402.153)

22 Brunneogal-eatoside C27H34O13 P. brunneoga-leata (Kirmizibekmez et al., (m/z 566.201) 2004)

23 Chlorotubero-side C17H25ClO11 P. tuberosa (Calis et al., 2005b) (m/z 440.109)

24 Dehydropenste-moside C17H24O11 P. rotata (Zhang et al., 1991) (m/z 404.302)

25 Lamiophlomiol C C11H14O7 P. rotata (Yi et al., 1991) (m/z 258.074)

Page 76 CHAPTER # 04 INTRODUCTION

26 Sesamoside C17H24O12 P. younghusbandi (Kahn and Andrawis, (m/z 420.127) 1987)

27 Kaempferol C23H24O11 P. spectabilis (Kumar et al., 1985) 3-O-β-D-Glucopyranoside (m/z 476.132)

28 Kaemferol-3-O-[4- C32H30O13 P. spectabilis (Kumar et al., 1985) Hydroxycinna-moyl-(→6)-β- (m/z 622.129) D-glucopyranoside 29 Longifloroside A C27H34O11 P. chimerae (Ersoza et al., 2002) (m/z 534.209)

30 Phlomisoside IV C32H48O12 P. younghusbandii (Katagiri et al., 1991) (m/z 624.315)

31 Phlomisoside III C31H46O12 P. younghusbandii, (Katagiri et al., 1991) (m/z 610.299) P. medicinalis

32 Baiyunoside C31H48O11 P. betonicoides (Jun, 1991) (m/z 596.320)

33 Phlomisoside I C32H50O11 P. betonicoides (Jun, 1991) (m/z 610.355)

34 Phlomisoside II C32H50O12 P. betonicoides (Jun, 1991) (m/z 626.330)

35 5,6-Epoxy-3-hydroxy-β-ionol- C19H32O8 P. aurea (Kamel et al., 2000) 9-O-β-D-Glucopyrano-side (m/z 388.321)

36 Penstemoside C17H26O11 P. younghusbandii (Kasai et al., 1994) (m/z 406.148)

37 4-O-p-Coumaroyl-betonicine C16H19NO5 P. brunneogaleata (Kirmizibekmez et al., (m/z 305.125) 2004)

38 3′′′-O-Acetylleucosceptoside B C38H50O20 P. umbrosa (Liu et al., 2009) (m/z 826.801)

39 Integrifolioside B C36H48O19 P. integrifolia (Saracoglua et al., 2003) (m/z 784.761)

40 2′′′,3′′′-Di-O- C40H52O21 P. umbrosa (Liu et al., 2007a) acetylleucosceptoside B (m/z 868.835)

41 3′′′,4′′′-Di-O- C40H52O21 P. umbrosa (Liu et al., 2007a) acetylleucosceptoside B (m/z 868.835)

42 Physocalycoside C43H60O24 P. physocalyx (Ersz et al., 2003) (m/z 960.932)

43 Phlinoside F C36H48O19 P. angustissima (Yalcin et al., 2005) (m/z 784.759)

44 6-O-β-D-Apiofuranosyl- C35H46O19 P. rotata (Yi et al., 1999) cistanoside C (m/z 770.728)

45 Hattushoside C28H36O15 P. pungens (Saracoglu et al., 1998) (m/z 612.583)

46 Phlomisethanoside C27H34O14 P. grandiflora (Takeda et al., 1999) (m/z 582.552)

47 Peteroside C25H30O10 P. lanceolata (Makhmudov et al., 2011) (m/z 490.502)

48 Lunariifolioside C39H52O23 P. lunariifolia (Calis and (m/z 888.821) Kirmizibekmez, 2004)

49 Samioside C34H44O19 P. samia (Kyriakopoulou et al., (m/z 756.714) 2001)

50 Oppositifloro-side C35H46O19 P. oppositiflora (Calis et al., 2005a) (m/z 770.734)

51 Integrifolioside A C35H46O19 P. integrifolia (Saracoglua et al., 2003) (m/z 770.734)

Page 77 CHAPTER # 04 INTRODUCTION

52 Phlinoside A C35H46O20 P. linearis (Calis et al., 1991) (m/z 786.714)

53 Teucrioside C34H44O19 P. armeniaca (Saracoglu et al., 1995) (m/z 756.714)

54 Forsythoside B C34H44O19 P. armeniaca (Saracoglu et al., 1995) (m/z 756.714)

55 Phlinoside C C35H46O19 P. linearis (Calis et al., 1991) (m/z 770.730)

56 Phlinoside E C36H48O19 P. linearis (Calis et al., 1991) (m/z 784.764)

57 Phlinoside B C34H44O19 P. linearis (Calis et al., 1991) (m/z 756.714) P. armeniaca

58 Phlinoside D C35H46O19 P. linearis (Calis et al., 1991) (m/z 770.730)

59 Phlorigidoside B C19H28O13 P. rigida (Takeda et al., 2000) (m/z 464.201)

60 Amanicadol C20H32O P. amanica (Yalcin et al., 2006) (m/z 288.470)

61 Phlorigidoside A C19H28O13 P. rigida (Takeda et al., 2000) (m/z 464.419)

62 7-Epilamalbide C17H26O12 P. medicinalis (Xue et al., 2009) (m/z 422.381)

63 Phlomoside D C24H30O13 P. regelii (Maksudov et al., 1996c) (m/z 526.491)

64 Phlomoside C C26H32O14 P. regelii (Maksudov et al., 1996b) (m/z 568.532)

65 Phlomoside B C21H30O14 P. regelii (Maksudov et al., 1996a) (m/z 506.461) 66 Durantoside IV C26H32O14 P. fruticosa (Marin et al., 2007) (m/z 568.53)

67 Lamiidic acid C16H24O12 P. nissolii (Kırmızıbekmez et al., (m/z 408.357) 2004)

68 Lamiophlomiol A C11H14O6 P. rotata (Yi et al., 1991) (m/z 242.221)

69 Phlorigidoside C C17H24O11 P. tuberosa (Takeda et al., 2000) (m/z 404.371) P. rigida

70 Phlomisionoside C19H32O8 P. spinidens (Takeda et al., 2002) (m/z 388.453)

71 3,19,29-Trihydroxy-28-nor- C29H44O4 P.spectabilis (Kumar et al., 1992) 16,21-oleanadien-23-al (m/z 456.660)

72 Norviscoside C35H52O12 P. viscosa (Calis et al., 2004) (m/z 664.780)

73 Lunaroside C25H44O15 P. lunariifolia (Calis and (m/z 584.613) Kirmizibekmez, 2004)

74 Phlomiol C17H26O10 P. fruticosa (Zhang et al., 1991) (m/z 438.384) P. rotata P. younghus-bandii 75 Phloyoside III C17H25O12Cl P. mongolica (Li and Zhang, 2000) (m/z 456.714)

76 Phlomoside A C17H26O11 P. thapsoides (Maksudov et al., 1995) (m/z 406.384) P. linearis

77 Phlomurin C18H28O11 P. aurea (Kamel et al., 2000) (m/z 420.414)

78 8,10-Dehydropulche-lloside C17H24O12 P. medicinalis (Xue et al., 2009) (m/z 420.361)

Page 78 CHAPTER # 04 INTRODUCTION

79 Phlomisflavo-side A C26H28O16 P. spinidens (Takeda et al., 2001) (m/z 596.439)

80 8-O-Acetylshanzhi-side C18H26O12 P. tuberosa (Takeda et al., 2000) (m/z 434.390)

81 8-Acetylshanzhi-genin C13H18O7 P. umbrosa (Guo et al., 2001) methanoate (m/z 286.282)

82 Shanzhigenin methanoate C11H16O6 P. umbrosa (Guo et al., 2001) (m/z 244.241)

83 Peterin C23H24O11 P. lanceolata (Calis et al., 2004) (m/z 476.438)

84 Phlomisflavo-side B C26H28O15 P. spinidens (Takeda et al., 2001) (m/z 580.489)

85 Viscoside A C36H58O11 P. viscosa (Calis et al., 2004) 29-O-β-D-glucopyranoside (m/z 666.846)

86 Viscoside B C36H58O11 P. viscosa (Calis et al., 2004) 30-O-β-D-glucopyranoside (m/z 666.845)

Page 79 CHAPTER # 04 BIOSYNTHESIS OF 28-NORTERPENOIDS

4.6 Biosynthesis of 28-Nortriterpenoids

The biochemical reactions occurring in natural product are enzyme controlled known as biosynthesis. The triterpenoids are biosynthetically formed by isoprene units (2- methylbuta-1,3-diene) via isoprene rule (Eschenmoser et al., 1955; Ruzicha and Eschenmoser, 1953).

The biosynthesis of terpenoids is divided into three steps

1. Isoprene unit formation

2. Acyclic terpenoid formation from isoprene units

3. Cyclic terpenoid formation from acyclic terpenoids and substitution of different groups

Acetyl CoA is basic precursor for isopentane unit formed by fat or carbohydrate metabolism. Its three molecules undergo Aldol condensation to form α-hydroxy-β- methylglutaryl Co-enzyme A (167) which undergo reduction in the presence of nicotinamide-adenine dinucleodephosphate (NADPH) to form mevalonic acid (MVA) (169). The decarboxylation of mevalonic acid (169) results into isopentane unit (Clayton, 1965; Ferguson et al., 1959; Knauss et al., 1959; Rudney et al., 1966).

Page 80 CHAPTER # 04 BIOSYNTHESIS OF 28-NORTERPENOIDS

2 H O 2 CH3COOH 2 CoA-SH 2 2 CH3CO-SCoA

O O O

H3C C SCoA H3C SCoA CoA-SH

O CH3CO-SCoA O H2C C SCoA H2C C SCoA

OH NADPH NADP

CoA-SH CO2H O SCoA 167

NADPH NADP

CO H CH OH CO2H CHO 2 2 168 169

Mevalonic acid (169) on two continuous phosphorylations followed by loss of water and carbon dioxide results into 3-methylbut-3-enyl pyrophosphate (170) which is known as isoprene unit. 3-methylbut-3-enyl pyrophosphate (170, IPP) undergo isomerization to form 3-methylbut-2-enyl pyrophosphate (171, DMAPP) (Waller, 1969).

OH OH ATP ADP ATP ADP

CO2H CH2OH CO2H CH2OP 169

CH3 CH3 H2O

CH2OPP CO O O 2 CH2OPP CH2OPP H 170 171

These two isomers (170, 171) on condensation to form geranyl pyrophosphate (172, GPP) which on further reaction with another 3-methylbut-3-enyl pyrophosphate (170)

Page 81 CHAPTER # 04 BIOSYNTHESIS OF 28-NORTERPENOIDS forms the cis and trans isomer of fernesyl pyrophosphate (173) (Jacob et al., 1983; Koyama et al., 1980; Poulter et al., 1979; Qureshi and Porter, 1981).

CH3

CH2OPP OPP

H HOPP PPO OPP

H3C CH3 trans 172 cis 172

OPP OPP CH3

CH2OPP

H PPOH2C trans 172 trans 173 cis 173

The squalene (175), rearranged product of presqualene (174), a basic precursor of triterpenes forms by head to head union of two fernesyl pyrophosphate (173, FPP) (Mannito, 1981).

OPP OPP H H PPO

X-Enz trans 173 trans 173

PPO

174 175

Terminal tertiary carbon of squalene on oxidation forms 2,3-epoxide (176) which increase the rate of cyclization to form various kinds of triterpeniods. Epoxy squalene

Page 82 CHAPTER # 04 BIOSYNTHESIS OF 28-NORTERPENOIDS

(176) undergo cyclisation to form dammarene diol (177) (Biellmann, 1966; Tanaka et al., 1968) which on further oxidative cyclisation results in β-amyrin (183) (Atallah et al., 1975; Corey and Cantrall, 1959).

[O] [O]

HO O 177 175 176

OH2

Rearangement - H O H 2 H H

HO HO HO H H 180 H 179 178

H H

H H H

H H H HO HO H HO H H 181 182 183

The β-amyrin (183) on carboxylation forms oleanolic acid (164) which is the precursor for nortriterpenoids. Oleanolic acid (164) on decarboxylation leaves a cation ion which on rearrangement followed by hydroxylation results in stewertiisin A-C (187, 188, 190), phlomispentanol (189) and notohamosin A (191) (Liu et al., 2008).

Page 83 CHAPTER # 04 BIOSYNTHESIS OF 28-NORTERPENOIDS

H H COOH H

Carboxylation - CO2 H H H HO HO HO H H H 183 164 184

OH OH

HO Hydroxylation Hydroxylation H H H

HO HO HO H H H 191 187 185 OH OH OH OH

Hydroxylation

OH OH

HO HO HO OH Hydroxylation H H H HO HO O H H H 189 190 188 OH OH OH OH OH OH

Page 84 CHAPTER # 05 RESULTS AND DISCUSSION

RESULTS & DISCUSSION

Page 85 CHAPTER # 05 RESULTS AND DISCUSSION

5 Results and Discussion

The shade-dried whole plant material of Phlomis stewartii (10 kg) was extracted thrice with methanol (3 × 30 L) at room temperature. The crude methanolic extract (670 g) was suspended in water and extracted with n-hexane and ethyl acetate. The ethyl acetate soluble fraction (70 g) was subjected to column chromatography over silica gel using n- hexane/EtOAc, EtOAc, EtOAc/MeOH and MeOH as eluent resulted into six fractions E1-

E6. The fractions E1 (1.5 g) on gradient elusion using 40% EtOAc in n-hexane to get oleanolic acid (164), 2-hydroxybenzoic acid (192) and 4-hydroxybenzoic acid (193) from the head, middle and tail fractions, respectively. The fraction E2 (1.7 g) on gradient elusion using 60% EtOAc in n-hexane to get caffeic acid (194). The fraction E3 (3.5 g) on gradient elusion using 5% MeOH in EtOA to get tiliroside (195) and isorhamnetin-3-(6- p-coumaroyl)-β-D-glucopyranoside (196), respectively. The fraction E4 (2.9 g) on gradient elusion using 10% MeOH in EtOA to get notohamosin A (190) and stewertiisin

B (188), respectively. The fraction E5 (1.7 g) on gradient elusion using 15% MeOH in EtOA to get stewertiisin A (187), stewertiisin C (189) and phlomispentanol (191) from the head, middle and tail fractions, respectively. The fraction E6 (3.9 g) on gradient elusion using 25% MeOH in EtOA to get stewartiiside (186) and lunariifolioside (197), respectively.

Page 86 CHAPTER # 05 RESULTS AND DISCUSSION

5.1 NEW COMPOUNDS ISOLATED FROM PHLOMIS STEWARTII

5.1.1 Structure Elucidation of Stewartiiside (186)

6 CH3 OH 4 O 5 OH 2 OH 1 3

2 3 OH OH

Compound 186 was obtained as a colorless amorphous powder. Its IR spectrum showed the presence of O-H (3438 cm-1), conjugated C=O (1701 cm-1) and aromatic system -1 (1605, 1520, 1450 cm ). The molecular formula C40H55O23 was determined by HR-FAB- + MS which showed the molecular ion peak [M+H] at m/z 903.3140 (calcd. for C40H55O23, 903.3134).

The 1H NMR spectrum of 186 displayed well-separated eight signals in the aromatic region. Due to COSY spectrum and calculation of coupling constants, the three signals [δ 7.06 (1H, d, J = 2.0 Hz), 6.96 (1H, dd, J = 9.2, 2.0 Hz), 6.80 (1H, d, J = 9.2 Hz)] splitted at ABX pattern were attributed to a tri-substituted benzene ring, whereas, another set of three signals [δ 6.68 (1H, d, J = 8.0 Hz), 6.66 (1H, d, J = 2.0 Hz), 6.55 (1H, dd, J = 8.0, 2.0 Hz)] were identified for another benzene ring. The remaining two signals resonating at δ 7.60 (1H, d, J = 16.0 Hz), 6.28 (1H, d, J = 16.0 Hz) were attested for a conjugated trans-olefinic system due to their coupling constants and chemicals shifts. This data revealed that at least one caffeoyl moiety is present in 186, which was further supported

Page 87 CHAPTER # 05 RESULTS AND DISCUSSION due to the carbon signals at δ 168.1, 149.8, 148.0, 146.0, 127.5, 123.4, 117.1, 116.3 and 116.2 in 13C NMR spectrum. Another set of COSY relatives resonated in 1H NMR spectrum at δ 4.01 (2H, t, J = 7.0 Hz) and 2.80 (2H, t, J = 7.0 Hz) showed its attachment with the aromatic system in HMBC spectrum of 186. This information helped to identify a tyrosol moiety in 186. Besides the above data, the 1H NMR spectrum showed the presence of four sugar moieties as it afforded signals for four anomeric protons at δ 5.26 (1H, br s), 5.20 (1H, d, J = 6.8 Hz), 5.17 (1H, br s) and 4.36 (1H, d, J = 8.2 Hz) together with overlapped signals for oxymethylene and oxymethine at δ 3.30-4.10. The 13C NMR spectra (BB and DEPT) of 186 was fully supportive of the mass and 1H NMR information as it showed 40 carbon signals for two methyl, five methylene, twenty five methine and eight quaternary carbon atoms. The four anomeric carbon signals were observed at δ 111.4, 104.1, 103.0 and 102.1. Careful analysis of both 1H and 13C NMR data for sugar moieties indicated the presence of a glucose unit, two rhamnose moieties and an apiose sugar in 186.

The above spectral data showed close resemblance to the reported data for lunariifolioside (Calis and Kirmizibekmez, 2004) except the presence of a rhamnose instead of an apiose. All structural assignments were accomplished through interpretation of 2D NMR (COSY, HSQC, HMBC) spectroscopic data. The acid hydrolysis of 186 provided a binary mixture of aglycones which could be separated and identified as caffeic acid and 3,4-dihydroxyphenylethanol, respectively from organic layer and glycones could be separated through preparative thin layer chromatography (PTLC) using EtOAc-

MeOH-H2O-HOAc; 4:2:2:2 as developing solvent and subsequently identified as D- glucose, L-rhamnose and D-apiose from aqueous layer through sign of their optical rotations and comparison of retention time of their trimethylsilyl (TMS) ethers with those of standards in gas chromatography (GC). The substitutions and the linkages at various positions in 186 were finally confirmed by HMBC correlations in which the oxymethylene protons at δ 4.01 correlated with the carbon at δ 104.1 (C-1) of the glucose moiety. The H-4 (δ 4.90) of glucose moiety showed correlation with ester

Page 88 CHAPTER # 05 RESULTS AND DISCUSSION carbonyl (δ 168.1) confirmed the attachment of caffeolyl group at C-4. The downfield shift of C-3 (δ 81.6) of glucose and its correlation with anomeric proton of H-1 (δ 5.17) of a rhamnose unit established their ether linkage. Further the downfield shift of C-6 (δ 65.6) of glucose and the HMBC correlation its protons (δ 3.62, 3.28) with C-1 (δ 102.1) of other rhamnose moiety confirmed another ether linkage between glucose and the second rhamnose unit. The apiose sugar was fixed at C-4 (δ 80.1) of the first rhamnose due to the HMBC correlation of H-4 (δ 3.55) with C-1 (δ 111.4) of apiose. Based on these evidences, the structure of 186 was established as 2-(3,4-dihydroxyphenyl)ethyl O- α-rhamnopyranosyl-(1→6)-O-[O-β-apiofuranosyl-(1→4)-α-rhamnopyranosyl-(1→3)]-4- O-(E)-caffeoyl-β-glucopyranoside and named as stewartiiside.

Page 89 CHAPTER # 05 RESULTS AND DISCUSSION

1 13 Table 5. H and C NMR data, HMBC and COSY correlations of 186 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) Aglycone 1 - 131.5 - - 2 6.66, d (2.0) 116.5 1,4,6,β H-2/H-6 3 - 146.8 - - 4 - 144.6 - - 5 6.68, d (8.0) 114.6 1,3 H-5/H-6 6 6.55, dd (8.0, 2.0) 121.3 4,5,β H-6/H-5,2 Α 4.01, t (7.0) 72.5 1,1ꞌ H-α/H-β Β 2.80, t (7.0) 36.5 α,2,6 H-β/H-α Glucose 1ꞌ 4.36, d (8.2) 104.1 α,2ꞌ,3ꞌ,5ꞌ H-1ꞌ/H-2ꞌ 2ꞌ 3.37, t (8.2) 76.1 1ꞌ,3ꞌ,4ꞌ H-2ꞌ/H-1ꞌ,3ꞌ 3ꞌ 3.79, t (8.2) 81.6 1ꞌ,2ꞌ,4ꞌ,5ꞌ,1ꞌꞌ H-3ꞌ/H-2ꞌ,4ꞌ 4ꞌ 4.90, t (8.2) 70.1 2ꞌ,3ꞌ,4ꞌ,6ꞌ,C=O H-4ꞌ/H-3ꞌ,5ꞌ 5ꞌ 3.71, m 75.0 1ꞌ,3ꞌ,4ꞌ,6ꞌ H-5ꞌ/H-4ꞌ,6ꞌ 6ꞌ 3.62, 3.28, m 65.6 4ꞌ,5ꞌ,1ꞌꞌꞌ H-6ꞌ/H-5ꞌ Rhamnose 1ʹʹ 5.17, br s 103.0 3ꞌ,2ꞌꞌ,3ꞌꞌ,5ꞌꞌ H-1ꞌꞌ/H-2ꞌꞌ 2ꞌꞌ 3.90, m 72.3 1ꞌꞌ,3ꞌꞌ,4ꞌꞌ H-2ꞌꞌ/H-1ꞌꞌ,3ꞌꞌ 3ꞌꞌ 3.71, m 72.0 1ꞌꞌ,2ꞌꞌ,4ꞌꞌ,5ꞌꞌ H-3ꞌꞌ/H-2ꞌꞌ,4ꞌꞌ 4ꞌꞌ 3.55, m 80.1 2ꞌꞌ,3ꞌꞌ,5ꞌꞌ,6ꞌꞌ,1ꞌꞌꞌꞌ H-4ꞌꞌ/H-3ꞌꞌ,5ꞌꞌ 5ꞌꞌ 3.48, m 68.7 1ꞌꞌ,3ꞌꞌ,4ꞌꞌ,6ꞌꞌ, H-5ꞌꞌ/H-4ꞌꞌ,6ꞌꞌ 6ꞌꞌ 1.11, d (6.4) 18.4 4ꞌꞌ,5ꞌꞌ H-6ꞌꞌ/H-5ꞌꞌ Rhamnose 1ꞌꞌꞌ 5.26, br s 102.1 6,2ꞌꞌꞌ,3ꞌꞌꞌ H-1ꞌꞌꞌ/H-2ꞌꞌꞌ 2ꞌꞌꞌ 3.82, m 72.3 1ꞌꞌꞌ,4ꞌꞌꞌ H-2ꞌꞌꞌ/H-1ꞌꞌꞌ,3ꞌꞌꞌ 3ꞌꞌꞌ 3.89, m 72.1 1ꞌꞌꞌ,2ꞌꞌꞌ,4ꞌꞌꞌ,5ꞌꞌꞌ H-3ꞌꞌꞌ/H-2ꞌꞌꞌ,4ꞌꞌꞌ 4ꞌꞌꞌ 3.61, m 74.6 2ꞌꞌꞌ,3ꞌꞌ,5ꞌꞌꞌ,6ꞌꞌꞌ H-4ꞌꞌꞌ/H-5ꞌꞌꞌ,3ꞌꞌꞌ 5ꞌꞌꞌ 3.54, m 70.4 1ꞌꞌꞌ,3ꞌꞌꞌ,4ꞌꞌꞌ H-5ꞌꞌꞌ/H-6ꞌꞌꞌ,4ꞌꞌꞌ 6ꞌꞌꞌ 1.07, d (6.2) 18.7 4ꞌꞌꞌ,5ꞌꞌꞌ H-6ꞌꞌꞌ/H-5ꞌꞌꞌ Apiose 1ꞌꞌꞌꞌ 5.20, d (6.8) 111.4 4ꞌꞌ,2ꞌꞌꞌꞌ,3ꞌꞌꞌꞌ,4ꞌꞌꞌꞌ H-1ꞌꞌꞌꞌ/H-2ꞌꞌꞌꞌ 2ꞌꞌꞌꞌ 3.62, d (6.8) 78.6 1ꞌꞌꞌꞌ,3ꞌꞌꞌꞌ,4ꞌꞌꞌꞌ,5ꞌꞌꞌꞌ H-2ꞌꞌꞌꞌ/H-1ꞌꞌꞌꞌ 3ꞌꞌꞌꞌ - 80.6 - - 4ꞌꞌꞌꞌ 3.71, s 75.1 1ꞌꞌꞌꞌ,2ꞌꞌꞌꞌ,3ꞌꞌꞌꞌ,5ꞌꞌꞌꞌ - 5ꞌꞌꞌꞌ 3.28, s 65.6 2ꞌꞌꞌꞌ,3ꞌꞌꞌꞌ,4ꞌꞌꞌꞌ - Caffeoyl 1ꞌꞌꞌꞌꞌ - 127.5 - - 2ꞌꞌꞌꞌꞌ 7.06, d (2.0) 116.2 6ꞌꞌꞌꞌꞌ,4ꞌꞌꞌꞌꞌ H-2ꞌꞌꞌꞌꞌ/H-6ꞌꞌꞌꞌꞌ 3ꞌꞌꞌꞌꞌ - 146.0 - - 4ꞌꞌꞌꞌꞌ - 149.8 - - 5ꞌꞌꞌꞌꞌ 6.80, d (9.2) 117.1 1ꞌꞌꞌꞌꞌ,3ꞌꞌꞌꞌꞌ H-5ꞌꞌꞌꞌꞌ/H-6ꞌꞌꞌꞌꞌ 6ꞌꞌꞌꞌꞌ 6.96, dd (9.2, 2.0) 123.4 4ꞌꞌꞌꞌꞌ,2ꞌꞌꞌꞌꞌ H-6ꞌꞌꞌꞌꞌ/H-5ꞌꞌꞌꞌꞌ,2ꞌꞌꞌꞌꞌ αꞌ 6.28, d (16.0) 116.3 1ꞌꞌꞌꞌꞌ H-αꞌ/H-βꞌ βꞌ 7.60, d (16.0) 148.0 2ꞌꞌꞌꞌꞌ,6ꞌꞌꞌꞌꞌ,C=O H-βꞌ/H-αꞌ C=O - 168.1 - -

Page 90 CHAPTER # 05 RESULTS AND DISCUSSION

5.1.2 Structure Elucidation of Stewertiisin A [(17R)-19(18→17)-abeo-3α,18β,23,24- tetrahydroxy-28-norolean-12-ene] (187)

OH 22 21 12 30 18 20 17 25 11 26 19 29 1 9 14 16 10 H 27 3 5 7 HO H 24 23 OH OH 187

Compound 187 was isolated as white amorphous powder, whose IR spectrum exhibited absorption bands for hydroxyl (3410 cm-1) and olefinic (1635 cm-1) functions. The EI-MS showed molecular ion at m/z 460.35, whereas, high resolution (HR-EI-MS) analysis of the same ion (m/z 460.3550) depicted the molecular formula as C29H48O4 with six double bond equivalent (DBE).

The 1H NMR spectrum of 187 displayed signals for five tertiary methyl (δ 1.13, 1.06, 1.00, 0.99 and 0.94), two oxygenated methylene [δ 3.83 (2H, br s), 3.85 and 3.75 (1H each, d, J = 10.0 Hz)], two oxygenated methine [δ 4.05 (1H, d, J = 2.0 Hz), 3.86 (1H, s)], a broad singlet olefinic methine (δ 5.72) along with several aliphatic methylenes and methines between δ 0.75-2.19. The resonance of two oxymethylenes clearly indicated that two tertiary methyl must have been oxidized to alcohols.

The 13C NMR spectra (BB and DEPT) of 187 supported the above data as it displayed 29 carbon resonances for five methyl (δ 30.4, 30.3, 23.4, 18.03, 18.02), twelve methylene (δ 67.3, 63.5, 52.8, 42.9, 42.2, 36.7, 35.1, 30.8, 29.5, 27.8, 24.5, 21.5), five methine (δ 119.8, 76.4, 72.5, 48.8, 45.6) and seven quaternary carbons (δ 142.5, 48.5, 45.3, 43.6, 41.3, 39.8, 38.5). The above spectral data closely related with the data of 28- norterpenoids reported from Phlomis umbrosa (Liu et al., 2007b; Liu et al., 2008), especially the spiro quaternary carbon displayed its position at δ 43.6 and a methylene of

Page 91 CHAPTER # 05 RESULTS AND DISCUSSION cyclopentano-system appeared at δ 52.8 as a characteristic feature of such a system (Liu et al., 2007b). The position of double bond at C-12 was confirmed due to RDA fragments at m/z 240.1730 (C14H24O3) and 220.1830 (C15H24O) in HR-EI-MS spectrum of 187. The HMBC correlation of two oxymethylens (δ 3.75, 3.85 H-24 and 3.83 H-23) with each other and with the carbons at δ 72.5 (C-3), 48.5 (C-4) and 45.6 (C-5) confirmed that geminal methyl at C-4 are oxidized to alcoholic groups. The HMBC correlation of H-12 with oxymethine resonated at δ 76.4 confirmed the presence of a hydroxyl function at C- 18. The smaller coupling constant of H-3 (J = 2.0 Hz) and its NOESY correlation with

OCH2-24 (δ 3.75, 3.85) confirmed hydroxyl group at C-3 as axial and α in orientation. The geometry of OH at C-18 was confirmed as β and equatorial due to NOESY correlation of H-18 with Me-27 (δ 1.13) (Luo et al., 2003) and through molecular model. The above discussion led to the structure of 187 as (17R)-19(18→17)-abeo-3α,18β,23,24- tetrahydroxy-28-norolean-12-ene and named as stewertiisin A.

Page 92 CHAPTER # 05 RESULTS AND DISCUSSION

1 13 Table 6. H and C NMR data, HMBC and COSY correlations of 187 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) 1 1.65, m 42.2 2,3,5,25 H-1/H-2 2 1.57, m 29.5 1,3,4,10 H-2/H-1,3 1.32, m 3 4.05, d (2.0) 72.5 1,5,23,24 H-3/H-2 4 - 48.5 - - 5 1.91, m 45.6 1,3,4,6,7,10,23,24 H-5/H-6 6 1.51, m 21.5 4,5,8,10 H-6/H-5,7 7 1.38, m 35.1 5,6,9,26 H-7/H-6 8 - 41.3 - - 9 1.70, m 48.8 1,5,8,11,12,14 H-9/H-11 10 - 38.5 - - 11 1.99, m 24.5 8,9,10,12,13 H-11/H-9,12 12 5.72, br s 119.8 9,11,13,14,18 H-12/H-11 13 - 142.5 - - 14 - 45.3 - - 15 1.04, m 27.8 8,13,14,16 H-15/H-16 16 1.61, m 36.7 14,15,18,19,22 H-16/H-15 17 - 43.6 - - 18 3.86, s 76.4 12,13,14,17 - 19 1.96, d (12.2) 52.8 16,18,21,22,29,30 - 1.13, d (12.2) 20 - 39.8 - - 21 1.48, m 42.9 17,19,29,30 H-21/H-22 1.38, m 22 1.28, m 30.8 16,18,20 H-22/H-21 23 3.83, br s 67.3 3,4,5,24 - 24 3.85, d (10.0) 63.5 3,4,5,23 - 3.75, d (10.0) 25 1.06, s 18.02 1,5,9,10 - 26 0.94, s 18.03 7,8,9,14 - 27 1.13, s 23.4 8,13,14,15 - 29 1.00, s 30.4 19,20,21,30 - 30 0.99, s 30.3 19,20,21,29 -

Page 93 CHAPTER # 05 RESULTS AND DISCUSSION

5.1.3 Structure Elucidation of Stewertiisin B [(17R)-19(18→17)-abeo- 2α,16β,18β,23,24-pentahydroxy-28-norolean-12-ene-3-one] (188)

OH 22 21 12 30 11 18 20 25 26 17 19 29 9 14 16 HO 1 10 OH H 27 3 5 7 O H 24 23 OH OH 188

Stewartiisin B (188) was also isolated as white amorphous powder. In addition to alcoholic and olefinic absorption bands, the IR spectrum afforded a strong band for -1 carbonyl function at 1719 cm . The molecular formula C29H46O6 of 188 was deduced by HR-EI-MS due to molecular ion peak at m/z 490.3290 with eight DBE.

Most of the signals appeared in 1H NMR spectrum of 188 were similar to the signals observed for 187 with few differences. The 1H NMR spectrum of 188 showed three oxymethine protons at δ 3.80 (1H, dd, J = 9.2, 3.8 Hz), 3.55 (1H, s) and 3.35 (1H, br s) instead of two. The cleavage of ring C through RDA fragmentation resulted into fragments at m/z 254.1520 (C14H22O4) and 236.1780 (C15H24O2) indicated the presence of four oxygen atoms in ring A or B and two in ring D or E. The 13C NMR spectra of 188 was also supportive of these differences as it displayed three oxymethines at δ 78.1, 76.2 and 67.0, whereas, the signal for a ketonic function appeared at δ 208.4. However, the spectrum afforded two methylene less when compared to that of the data of compound 187.

The HMBC correlation of two oxymethylene (δ 3.82, H-23 and 3.86, H-24) with the carbonyl carbon (δ 208.4) revealed that 3-OH has been oxidized to ketonic function. The HMBC correlations of H-5 (δ 1.58) with carbonyl carbon confirmed the above observation. Further the long range interaction of an oxymethine resonating at δ - with carbonyl carbon and two quaternary carbons at δ C-4) and 38.8 (C-10) fixed

Page 94 CHAPTER # 05 RESULTS AND DISCUSSION one hydroxyl group at C-2, whereas, other two alcoholic functions were fixed at C-16 and C-18 due to the HMBC correlations of H-16 (δ  and H- δ with the spirocarbon (C-17). Further, H-16 exhibited HMBC interaction with C-18 (δ 76.2) and H- 18 interacted with C-16 (δ 78.1) and C-12 (δ 119.0).

The relative stereochemistry at various chiral centers was established through NOESY spectrum, in which H-2 (δ 3.80) correlated with Me-25 (δ 1.10), H-16 (δ 3.35) with H-18 (δ 3.55) and Me-27 (δ 1.17) indicated the orientation of OH-2 as α and equatorial, whereas, OH-16 and OH-18 were placed as β and equatorial. These deductions were further substantiated with the help of molecular model. The above discussion and comparative study finally led to the structure of compound 188 as (17R)-19(18→17)- abeo-2α,16β,18β,23,24-pentahydroxy-28-norolean-12-ene-3-one, which is named as stewertiisin B.

Page 95 CHAPTER # 05 RESULTS AND DISCUSSION

1 13 Table 7. H and C NMR data, HMBC and COSY correlations of 188 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) 1 1.85, m 43.0 2,3,5,10 H-1/H-2 1.37, m 2 3.80, dd (9.2, 3.8) 67.0 4,10 H-2/H-1,3 3 - 208.4 - - 4 - 59.8 - - 5 1.58, m 48.2 1,3,7,23,24 H-5/H-6 6 1.15, m 19.1 4,8,10 H-6/H-5,7 1.12, m 7 1.28, m 35.1 5,9,26 H-7/H-6 1.01, m 8 - 40.2 - - 9 1.35, m 43.6 1,5,7,12,25 H-9/H-11 10 - 38.8 - - 11 2.0, d (10.0) 24.9 8,9,10,12,13 H-11/H-9,12 12 5.78, s 119.0 9,11,13,14,18 H-12/H-11 13 - 143.6 - - 14 - 45.2 - - 15 1.60, m 28.1 8,13,14,16 H-15/H-16 16 3.35, br s 78.1 14,18,19,22 H-16/H-15 17 - 44.1 - - 18 3.55, s 76.2 12,14,16,19,22 - 19 1.12, m 53.0 16,18,21,22,29,30 - 20 - 30.5 - - 21 1.35, m 42.2 17,19,29,30 H-21/H-22 22 1.28, m 30.8 16,17,18,19,20 H-22/H-21 23 3.82, br s 66.2 3,4,5,24 - 24 3.86, s 62.2 3,4,5,23 - 25 1.10, s 18.1 1,5,9,10 - 26 0.98, s 17.5 7,8,9,14 - 27 1.17, s 26.5 8,13,14,15 - 29 1.07, s 17.6 19,20,21 - 30 1.02, s 30.3 19,20,21 -

Page 96 CHAPTER # 05 RESULTS AND DISCUSSION

5.1.4 Structure Elucidation of Stewertiisin C [(17R)-19(18→17)-abeo-3α,18β,23,24- tetrahydroxy-28-norolean-11,13-diene] (189)

22 21 12 18 30 11 20 25 26 17 19 29 9 14 16 HO 1 10 H 27 3 5 7 HO H 24 23 OH OH 189

Compound 189 was also found to be a spiro-nor-triterpenoid as most of its spectral data were comparable with that of compounds 187 and 188. The notable difference was observed through UV spectrum that exhibited an absorption peak at 236 nm for a heterodienic system. The 1H NMR spectrum also attested this information as it displayed three olefinic methines at δ 5.90 (1H, d, J = 8.0 Hz), 5.55 (1H, d, J = 8.0 Hz) and 5.36 (1H, s). Although, the 1H NMR spectrum of 189 afforded signals for two oxymethines as were observed in 188, but they were found vicinal to each other at C-2 and C-3 due to their COSY correlations with each other and HMBC interaction with C-4 (δ 48.5). Further, H-3 (δ 3.83) was correlated in HMBC spectrum with C-4 and two oxymethylenes at δ 72.3 (C-23) and 62.3 (C-24). The NMR data of 189 looked similar to the data reported for notohamosin A (190) (Luo et al., 2003). The conjugated double bonds were fixed at C-11 and C-13 due to various HMBC interactions. The stereochemistry of 2-OH and 3-OH was established due to their coupling constants, molecular model and NOESY spectrum in which H-2 (δ 3.89) and H-3 (δ 3.83) were correlated with each other as well as with Me-24 (δ 3.78) and Me-25 (δ 1.02) confirming OH-2 as α and equatorial and OH-3 as α and axial in orientation. Finally, compound 189 was identified as (17R)-19(18→17)-abeo-2α,3α,23,24-tetrahydroxy-28-norolean-11,13- diene and named as stewertiisin C.

Page 97 CHAPTER # 05 RESULTS AND DISCUSSION

1 13 Table 8. H and C NMR data, HMBC and COSY correlations of 189 (CD3OD)

Position δH (J in Hz) δC HMBC (H→C) COSY (H→H) 1 1.85, m 42.4 2,3,5,10 H-1/H-2 1.35, m 2 3.89, m 66.9 4,10 H-2/H-1,3 3 3.83, br s 72.0 1,5,23,24 H-3/H-2 4 - 48.5 - - 5 1.58, m 47.0 1,3,4,6,7,10,23,24 H-5/H-6 6 0.95, m 20.3 4,8,10 H-6/H-5 0.92, m 7 1.35, m 33.5 5,6,8,9,26 H-6/H-7 8 - 41.8 - - 9 2.14, d (9.0) 55.7 1,5,8,11,12,14 H-9/H-11 10 - 39.0 - - 11 5.55, d (8.0) 126.3 8,9,10,12,13 H-11/H-9,12 12 5.90, d (8.0) 131.7 9,11,13,14,18 H-12/H-11 13 - 139.7 - - 14 - 41.1 - - 15 1.69, m 27.2 8,13,14,16 H-15/H-16 16 1.63, m 34.3 14,18.19,22 H-16/H-15 17 - 45.5 - - 18 5.36, s 137.5 12,13,14,17 - 19 1.51, m 57.0 16,18,21,22,29,30 - 1.34, m 20 - 40.2 - - 21 1.44, m 41.4 17,19,29,30 H-21/H-22 22 1.57, m 40.5 16,18,19,20 H-22/H-21 23 4.30, d (10.0) 72.3 3,4,5,24 - 4.00, d (10.0) 24 3.78, br s 62.3 3,4,5,23 - 25 1.02, s 19.4 1,5,9,10 - 26 0.75, s 17.0 7,8,9,14 - 27 0.94, s 20.4 8,13,14,15 - 29 1.35, s 31.4 19,20,21 - 30 1.36, s 30.7 19,20,21 -

Page 98 CHAPTER # 05 RESULTS AND DISCUSSION

5.2 STRUCTURE ELUCIDATION OF KNOWN COMPOUNDS

5.2.1 Structure Elucidation of Notohamosin A (190)

21 30 18 11 13 OH 25 26 17 9 19 29 HO 1 10 15 H 3 5 27 7 HO H 24 23 OH OH 190

Compound 190 was isolated as white powder. Its IR spectrum showed the peaks at 3415, 1635, 1378, 1249 and 1035 cm-1. The HR-EI-MS confirmed its molecular formula as + C29H46O5 due to the molecular ion peak at m/z 474.3350 [M] (calcd. for C29H46O5, 474.3345).

The 1H NMR spectrum of 190 displayed the signals for four tertiary methyls at δ 1.05, 0.97, 0.95 and 0.73 (3H, s each), three oxygenated methylenes at δ 3.90 (1H, d, J = 11.1 Hz), 3.70 (1H, d, J = 11.1 Hz), 3.65 (1H, d, J = 11.5 Hz), 3.58 (1H, d, J = 11.5 Hz), 3.29 (2H, s, H-30) and three olefinic methines at δ 5.90 (1H, dd, J = 10.2, 2.8 Hz), 5.56 (1H, d, J = 10.2 Hz) and 5.33 (1H, s, H-18).

The 13C NMR spectra of compound 190 showed twenty nine carbon signals at δ 140.1, 136.8, 131.7, 126.3, 73.8, 71.7, 68.7, 66.9, 63.9, 55.6, 52.7, 47.8, 45.5, 45.4, 44.7, 41.9, 41.2, 40.5, 38.7, 36.2, 33.7, 33.3, 27.1, 26.7, 20.4, 19.5, 19.4 and 17.1. The absence of one carbon from whole triterpenoid skeleton indicates the presence of nor- triterpenoid nature. The two double bond was placed at C-11/12 and C-13/18 in conjugation due to the HMBC correlations H-19 (δ 1.61) and methyl-27 with C-18 (δ 136.8) and C-13 (δ 140.1) and COSY correlations of these olefinic methines with each other. Two oxyginated methylenes were placed at C-23 and C-24 due to their HMBC correlations with each other and with C-3 (δ 73.8), C-4 (δ 47.8) and C-5 (δ 44.7). The remaining oxyginated methylene δ 3.29 was at C-30 due to its HMBC relation with C-19

Page 99 CHAPTER # 05 RESULTS AND DISCUSSION

(δ 52.7) and C-21 (δ 36.2). Both the oxymethines were placed at adjacent position at C-2 and C-3 because both were found correlated both in COSY and HMBC spectra. The above data was found similar to the data already reported for notohamosin A (Luo et al., 2003).

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5.2.2 Structure Elucidation of Phlomispentanol (191)

OH 22 21 12 30 18 25 26 13 17 9 19 29 HO 1 10 15 H 3 5 7 27 HO H 24 23 OH OH 191

Compound 191 was isolated as colorless amorphous powder. The IR spectrum showed the signal at 3405, 2930, 1455, 1378, 1279 and 1038 cm-1. The molecular formula + C29H48O5 was deduced by HR-EI-MS due to the molecular ion peak [M] at m/z 476.3076

(calcd. for C29H48O5, 476.3081).

The 1H NMR spectrum of 191 was closely related with that of notohamosin A (190) except the absence of two olefinic methines and an oxymethylene and appearance of an additional oxyginated methine due to the signal at δ 3.62 (1H, s).

The 13C NMR spectra of compound 191 almost similar signals except the missing of two olefinic methines and an oxygenated methylene and appearance a methylene (δ 24.2) oxygenated methine (δ 76.9) and a methyl (δ 31.5). The only double bond was placed at C-12/13 due to COSY and HMBC spectrum. The coming hydroxyl group was placed at C-18 due to HMBC correlations of H-18 (δ 3.62) was neighboring carbons. Rest of the data found similar as discussed for compound 190.

Searching the above data with the literature showed that this data closely related to the spectral data reported for phlomispentanol already reported from the genus Phlomis (Liu et al., 2008).

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5.2.3 Structure Elucidation of Oleanolic acid (164)

29 30

19 21 12 17 13 28 25 26 H COOH 9 1 15 H 3 5 7 27 HO H

23 24 164

Compound 164 was purified as white needles. It gave pink coloration using ceric sulphate as locating reagent and after heating. The IR spectrum of 164 showed peaks at 3445, 3278-2635, 2960, 2868, 1695, 1618, 1452, 1379, 1259, 1041, 975 cm-1. The HR-EI-MS of compound 164 showed the molecular ion peak [M]+ at m/z 456.3673 deduced the molecular formula as C30H48O3.

The 1H NMR spectrum displayed a signal at δ 3.49 (1H, dd, J = 10.5, 3.8 Hz) for an oxymethine and δ 5.17 (1H, dd, J = 10.2, 2.8 Hz) for an olefinic double bond. It also showed the signals for seven tertiary methyls at δ 1.29, 1.27, 1.07, 1.06, 1.05, 0.98 and 0.94 (3H each, s) and the signal for carboxylic acid was appeared at δ 12.8 (1H, s).

The 13C NMR spectra (BB and DEPT) of 164 disclosed highly resolved 30 carbon signals including seven methyl (δ 34.4, 28.4, 27.6, 23.0, 17.7, 16.2, 15.8), ten methylene (δ 47.3, 38.76, 34.1, 33.4, 32.9, 29.2, 27.9, 23.5, 23.4, 18.3), five methine (δ 122.0, 77.6, 54.9, 49.1, 42.3) and seven quaternary carbon atoms (δ 144.3, 45.6, 43.1, 40.2, 39.5, 36.9, 30.6). The downfield carbon resonated at δ 183.6 indicating the presence of carboxylic acid and its position at C-17 was confirmed by downfield shift of H-18 at δ 2.88 (1H, dd, J = 14.2, 2.5 Hz) and which was further confirmed through the HMBC correlation of H- 18 (δ 2.88) with C-28 (δ 179.6). The HMBC of Me-27 (δ 1.29) with C-13 (δ 144.3) and the COSY correlation of H-11 (δ 1.56) with H-12 (δ 5.17) confirmed the presence of double bond between C-12 and C-13. The hydroxyl group was placed at C-3 position due

Page 102 CHAPTER # 05 RESULTS AND DISCUSSION to the HMBC correlation of H-3 (δ 3.28) with Me-23 (δ 28.4) and Me-24 (δ 16.2) and its

COSY correlation with CH2-2 (δ 1.79). The relative stereochemistry at C-3 was deduced through 1H NMR spectrum in which the larger coupling constant of H-3 (δ 3.49, dd, J = 10.5, 3.8 Hz) confirmed H-3 as axial and α and OH-3 as equatorial and β in orientation.

Based on these evidence the structure of compound 164 accomplished as it have hydroxyl group at C-3, double bond between C-12/13 and a carboxylic acid at C-17 in a triterpene nucleus. By comparing the above discussed data with the literature it was found that the above data closely related to the data reported for oleanolic acid (Seebacher et al., 2003).

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5.2.4 Structure Elucidation of 2-Hydroxybenzoic acid (192)

7 COOH

1 OH

5 3

192

Compound 192 was purified as colorless needles. The IR spectrum showed the absorption peaks at 3408, 3246-2657, 1704, 1626 and 812 cm-1. The HR-EI-MS showed molecular ion peak at m/z 138.0316 having the molecular formula C7H6O3 (calcd. for

C7H6O3, 138.0311).

The 1H NMR of 192 showed four aromatic signals at δ 7.85 (1H, d, J = 8.5 Hz), 7.44 (1H, t, J = 8.4 Hz), 6.90 (1H, t, J = 9.0 Hz) and 6.83 (1H, d, J = 8.4 Hz) and all these protons showed the connectivity in their 1H-1H COSY spectrum confirming their neighborhood.

The 13C NMR spectrum of 192 showed seven carbon signals corroborated the presence of four methine (δ 135.9, 131.4, 120.0, 117.9) and three quaternary carbons (δ 175.5, 161.5, 112.6). The downfield signals at δ 175.5, 161.5, 112.6 were due to the presence of a carboxylic acid, oxygenated aromatic and aromatic quaternary carbons, respectively. The position of both substituents were confirmed by 1H NMR (through splitting pattern and coupling constant) and HMBC correlation in which H-6 (δ 7.85) correlated with C-1 (δ 112.6), C-2 (δ 161.5) and C-7 (δ 175.5). The above discussed was compared with the literature and found similar to that reported for 2-hydroxybenzoic acid (Aldrich Library, 1992a).

 

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5.2.5 Structure Elucidation of 4-Hydroxybenzoic acid (193)

7 COOH

5 3

OH 193

Compound 193 was purified as colorless needles. Its IR spectrum showed the peaks at 3509, 3331-2707, 1704, 1626 and 812 cm-1. The HR-EI-MS showed molecular ion peak at m/z 138.0309 which depicted the molecular formula as C7H6O3 (calcd. for C7H6O3, 138.0316) having five double bond equivalence.

The 1H NMR spectrum of 193 displayed two doublets at δ 7.92 (2H, d, J = 8.4 Hz), 6.78 (2H, d, J = 8.4 Hz) and a broad singlet at δ 11.9 (1H, s). The former two doublets were found double in intensity when compared with the broad singlet.

The 13C NMR spectrum of 193 showed altogether five carbon signals for seven carbons including three quaternary (δ 179.9, 159.9, 122.6) and two aromatic methines (δ 131.5, 117.0) carbons. Both the NMR spectra indicated the presence of two magnetically equivalent centers which was further confirmed through 1H-H COSY correlation of two doublets and HMBC correlation of two doublets at δ 7.92 and δ 6.78 (each H, d, J = 8.4 Hz), in addition to other carbons, found correlated with their respective carbons. On searching the above data discussed for 193 in literature and found completely overlapped to that reported for 4-hydroxybenzoic acid (Aldrich Library, 1992b).

 

Page 105 CHAPTER # 05 RESULTS AND DISCUSSION

5.2.6 Structure Elucidation of Caffeic acid (194)

7 9 5 1 OH

HO 3 OH 194

Compound 194 was isolated as crystalline needles. Its IR spectrum showed the peaks at 3499, 3331-2707, 1706 and 1626 cm-1 characteristic for hydroxyl group, carboxylic acid and aromatic system. The HR-EI-MS showed molecular ion peak at m/z 180.0314 used to deduce the molecular formula C9H8O4 (calcd. for C9H8O4, 180.0309).

The 1H NMR spectrum of 194 showed two doublets at δ 7.51 (1H, d, J = 15.5 Hz) and 6.21 (1H, d, J = 15.5 Hz) indicating trans geometry of the double bond. It also showed an ABX-splitting pattern at δ 7.02 (1H, d, J = 2.0 Hz), 6.90 (1H, dd, J = 8.0, 2.0 Hz), 6.76 (1H, d, J = 8.0 Hz) indicated the presence of 1,3,4-trisubstituted benzene ring which further confirmed through 1H-H-COSY correlations in which the coupling of two trans- doublets [δ 7.02 (d, J = 2.0 Hz, H-2), 6.90 (dd, J = 8.0, 2.0 Hz, H-6)] and [6.90 (dd, J = 8.0, 2.0 Hz, H-6), 6.76 (d, J = 8.0 Hz, H-5), 7.51 (d, J = 16.0 Hz, H-7), 6.21 (d, J = 15.5 Hz, H-8)] were found.

The 13C NMR spectra (BB & DEPT) of 194 showed altogether nine resonances for five methines (δ 146.7, 122.7, 115.9, 115.1, 116.0) and four quaternary carbons (δ 171.0, 149.0, 147.0, 125.6). The signal appeared at δ 171.0 were assigned to carboxylic acid, where as the signals at δ 147.0 and 149.0 were due to the presence of aromatic oxygenated quaternary carbons. The resonances at δ 146.7 and 115.9 were due to trans double bond. The substitutions at various positions were finally confirmed by HMBC correlations. By comparing the discussed data with literature found close resemblance with the data reported for caffeic acid (Olennikov et al., 2012).

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5.2.7 Structure Elucidation of Tiliroside (195)

3' OH

1' 5' HO 7 8a O 1

3 4a 4 5 O 3''' HO OH O

5''' 1''' 9''' O 195 7''' O 6'' O HO 5'' HO 1'' 3'' OH

The compound 195 was isolated as yellow powder. The IR spectrum showed the characteristic signals at 3325 (O-H), 1675 (C=O) and 1535 (aromatic C=C) where as the UV band appeared at 318, 268, 259 nm indicated the presence of flavones skeleton. The molecular formula C30H27O13 was deduced by HR-FAB-MS through molecular ion peak [M+H]+ at m/z 595.1370.

The 1H NMR spectrum of compound 195 showed two doublet at δ 6.32 (1H, d, J = 1.6 Hz) and 6.13 (1H, d, J = 1.6 Hz). Their COSY correlations and coupling constants (1.6 Hz) confirmed their meta-orientation in benzene ring. The HSQC spectrum showed their correlations with the carbons at δ 94.8 and 100.2, respectively characterizing them for C- 6 and C-8 in flavonoid skeleton, having two hydroxyl groups at C-5 and C-7 in ring A. The same spectrum showed two AAꞌBBꞌ patterns at δ 7.99 (2H, d, J = 8.2 Hz), 6.84 (2H, d, J = 8.2 Hz) and at δ 7.32 (1H, d, J = 9.2 Hz), 6.80 (1H, d, J = 9.2 Hz) together with a trans-double bond at δ 7.38 (1H, d, J = 16.0 Hz) and 6.10 (1H, d, J = 16.0 Hz) indicated the presence of a p-hydroxy phenyl and a caffeoyl moiety in 196. An anomeric proton was resonated at δ 5.30 (1H, d, J = 7.2 Hz) with oxymethylene and oxymethines at δ 4.30-3.25 characteristic for glucose moiety.

Page 107 CHAPTER # 05 RESULTS AND DISCUSSION

The 13C NMR spectra of compound 195 disclosed 26 carbon signals at δ 64.8 for methylene, δ 146.5, 132.2, 131.2, 116.8, 116.1, 114.2, 103.8, 100.4, 95.0, 73.8, 78.1, 71.8, 75.8 for methine and δ 181.3, 169.1, 166.9, 162.8, 161.0, 161.2, 159.8, 159.1, 131.2, 129.1, 127.0, 104.0 for quaternary carbons. The signals at δ 181.3, 166.9, 162.8, 161.2, 159.8, 159.1, 132.2, 131.2, 127.0, 116.1, 104.0, 100.4, 95.0 were due to kaempferol nucleus where as the signals at δ 169.1, 161.0, 146.5, 131.2, 129.1, 116.8, 114.2, 103.8, 78.1, 75.8, 73.8, 71.8 and 64.8 was due the presence of p- hydroxycinnamoyl and glucose, respectively, in 195.

All substitutions were confirmed with HMBC correlations in which proton at δ 7.99 showed HMBC correlation with C-2 and C-6' indicating the presence of p-hydroxyphenyl moiety. The anomeric proton (δ 5.30) showed HMBC correlation with C-3 (δ 131.2) and oxymethlene (δ 4.26) of glucose correlated with carbonyl of caffeoyl (δ 169.1) confirming the attachment of sugar at C-3 and caffeoyl at C-6 of the sugar moiety. By comparing the above discussed data with the literature it was found that the spectral data discussed for 195 was closely related with the data already reported for tiliroside (Kaouadji, 1990).

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5.2.8 Structure Elucidation of Isorhamnetin 3-(6-p-coumaroyl)-β-D-glucopyranoside (196)

OCH3 3' OH

1' 5' HO 7 8a O 1

3 4a 4 5 O 3''' HO OH O

5''' 1''' 9''' O 196 7''' O 6'' O HO 5'' HO 1'' 3'' OH

The compound 196 was also isolated as yellow powder (77 mg). The IR spectrum showed the peaks at 3305, 2920, 1668, 1650 and 1608 cm-1 where as the UV band appeared at 315, 265, 254 nm indicated the presence of substituted flavones skeleton. The molecular formula C31H29O14 was confirmed by HR-FAB-MS due to its molecular ion peak [M+H]+ at m/z 625.1480.

The 1H NMR spectrum of 196 was very similar to that for 195 except the missing of one AAꞌBBꞌ pattern and the appearance of ABX pattern δ 7.85 (1H, d, J = 1.6 Hz), 7.54 (1H, dd, J = 8.4, 2.0 Hz), 7.30 (1H, d, J = 6.0 Hz) indicated the presence of 1,3,4-trisubstituted instead of 1,4-disubstitued benzene ring. It also showed the signal for methoxy group at δ 3.90.

The 13C NMR spectra showed total 31 which were divided into flavonoid nucleus (δ 181.3, 166.9, 162.8, 159.7, 159.2, 131.2, 104.0, 100.3, 94.9; 151.5, 149.0, 127.1 123.9, 116.0, 114.6), p-hydroxycoumaroyl (δ 169.2, 161.2, 146.6, 131.2, 127.0, 116.8, 114.2) and sugar moiety (δ 103.9, 78.0, 75.8, 73.8, 71.8, 64.4). The position of all the substituents were confirmed through HMBC correlations especially the methoxy group in which methoxy group (δ 56.8) correlated with C-3 (δ 149.0).

Page 109 CHAPTER # 05 RESULTS AND DISCUSSION

On comparison of above discussed data with literature showed its resemblance with the data reported for isorhamnetin 3-(6-p-coumaroyl)-β-D-glucopyranoside (Joua et al., 2004).

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5.2.9 Structure Elucidation of Lunariifolioside (197)

O O 4''' 1''' 6' HO 5'''

3''' OH OH O HO ' 5' O O OH 3''''' 1''''' '  1 O O  3 3' 1' HO OH 5''''' 5 OH 1'' 5'' O CH3 3'' O

OH OH

4'''' 1'''' HO 5''''

3'''' OH OH

197

Compound 197 was isolated as pale yellow amorphous powder. Its IR spectrum showed the peaks at 3432, 1701, 1605, 1520 and 1450 cm-1 where as The UV spectrum showed the bands at 219, 328 indicated the presence of substituted aromatic system. The + molecular formula C39H52O23 was deduced by HR-FAB-MS at m/z 889.2910 [M+H]

(calcd. for C39H52O23, 889.2905).

The 1H NMR spectrum of compound 197 showed two ABX systems in aromatic region, out of which one is for trisubstituted trans-cinnamoyl moiety at δ 7.06 (1H, d, J = 2.0 Hz), 6.96 (1H, dd, J = 9.2, 2.0 Hz), 6.80 (1H, d, J = 9.2 Hz), 7.60 (1H, d, J = 16.0 Hz), 6.28 (1H, d, J = 16.0 Hz) with their respective carbons at δ 168.1, 149.8, 148.0, 146.0, 127.5, 123.4, 117.1, 116.3 and 116.2 and the second one for p-hydroxyphenylethanol at δ 6.68 (1H, d, J = 8.0 Hz), 6.66 (1H, dd, J = 2.0 Hz), 6.55 (1H, dd, J = 8.0, 2.0 Hz), δ 4.01 (2H, t, J = 7.0 Hz)and 2.8 (2H, t, J = 7.0 Hz) having together with their carbons at δ

Page 111 CHAPTER # 05 RESULTS AND DISCUSSION

146.8, 144.6, 131.5, 121.3, 116.5, 114.6, 72.5 and 36.5. In addition, the 1H NMR spectrum displayed signals for four sugar moieties at δ 4.36 (1H, d, J = 8.2 Hz), 3.37 (1H, t, J = 8.2 Hz), 3.79 (1H, t, J = 8.2 Hz), 4.90 (1H, t, J = 8.2 Hz), 3.71 (1H, m), (3.62 1H, m), 3.28 (1H, m) for glucose; 5.17 (br s), 3.90 (m), 3.71 (m), 3.55 (m), 3.48 (m), 1.11 (3H, d, J = 6.4 Hz) for rhamnose; 4.89 (1H. s), 3.61 (1H, d, J = 6.8 Hz), 3.71 (2H, s), 3.34 (2H, s) and 5.20 (1H, d, J = 6.8 Hz), 3.62 (1H, d, J = 6.8 Hz), 3.71 (2H, s), 3.28 (2H, s) for two apiose unit, respectively. The acid hydrolysis of 197 confirms these sugars as rhamnose, glucose and two apiose.

The positions of all the substituents in 197 were confirmed by HMBC correlations as cinnamoyl at C-1ꞌ, rhamnose at C-3ꞌ, phenylethanol at C-4ꞌ and one apiose at C-6ꞌ of glucose and other apiose at C-4ꞌꞌ of rhamnose at C-3ꞌ. This data was closely related to already reported for stewartiiside (Calis and Kirmizibekmez, 2004).

 

Page 112 CHAPTER # 05 RESULTS AND DISCUSSION

5.3 α-Glucosidase Inhibition of compounds 164, 186-197

All the isolates 164, 186-197 were evaluated for their enzyme inhibition activity against enzyme α-glucosidase. Nearly all the tested compounds exhibited good to moderate activity (Table 9) with IC50 values ranging between 14.5-355.4 µM. Compounds 186,

187, 194, 195 and 197 showed better potential (IC50 = 26.1, 37.9, 26.5, 14.5, 27.4 µM, respectively) than the standard drug whereas compounds 189 and 190 were least active.

Tiliroside (195) was the most active (IC50 = 14.5 µM) where as its methoxy derivative 3- (6-p-coumaroyl)-β-D-glucopyranoside (196) showed least activity which clearly indicated that the presence of hydroxyl group in ring B of 195 has important role in enzyme inhibition. The activity of stewartiiside (186, IC50 = 21.1 µM) is comparable with that of lunariifolioside (197, IC50 = 26.5 µM), which revealed that the glycone part is not playing important role in enzyme inhibition. Stewertiisin A (187, IC50 = 37.9 µM) was found to be a good inhibitor among other analogues (188, 189, 190, 191). This indicated that 3-OH and 18-OH play important role in enzyme inhibition.

Page 113 CHAPTER # 05 RESULTS AND DISCUSSION

Table 9. -Glucosidase Inhibition of Compounds 164, 186-197

Compound Percentage Inhibition (%) Conc (mM) IC50 (µM) 186 95.8 ± 0.3 0.5 26.1 ± 0.3 187 97.0 ± 0.8 0.25 38.0 ± 0.2 188 79.6 ± 0.4 0.5 315.8 ± 0.2 189 95.2 ± 1.6 0.5 355.4 ± 0.9 190 42.1 ± 1.0 0.5 318.5± 0.3 191 20.2 ± 0.75 0.5 >500 192 41.5 ± 0.1 0.5 290.7 ± 0.0 193 41.1 ± 0.1 0.5 297.7 ± 0.0 194 98.1 ± 0.2 0.5 27.4 ± 0.2 195 99.0 ± 0.7 0.5 14.5 ± 0.1 196 90.8 ± 1.3 0.5 305.2 ± 0.1 197 95.4 ± 0.3 0.5 26.6 ± 0.2 164 88.4 ± 0.1 0.5 231.3 ± 0.1 Methanolic 80.2 ± 1.2 - - extract* Acarbose 92.23±0.14 0.5 38.25±0.12 All compounds were prepared in methanol with a concentration of 0.5mM All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003. Results are presented as mean ± sem. 1.0 mg/ml methanolic extract/assay volume

Page 114 CHAPTER # 06 EXPERIMENTAL

EXPERIMENTAL

Page 115 CHAPTER # 06 EXPERIMENTAL

6.1 Plant Material

The whole plant material of Phlomis stewartii Hk. was collected from District Ziarat (Baluchistan, Pakistan) in September 2011 and was identified by Prof. Dr. Rasool Bakhsh Tareen, Plant Taxonomist, Department of Botany, Baluchistan University Quetta, Quetta, Pakistan where a voucher specimen is deposited (PS-91/11).

6.2 Extraction and Isolation

Page 116 CHAPTER # 06 EXPERIMENTAL

Phlomis stewartii 10 kg

Ground, extracted with MeOH, concentrated under reduced pressure

Methanolic extract 670 gm

Suspended in water and extracted with organic solvents

n-Hexane soluble EtOAc soluble Water soluble 280 gm 170 gm 220 gm

Purification by CC

40% EtOAc 60% EtOAc 5% MeOH 10% MeOH 15% MeOH 25% MeOH in n-hexane in n-hexane in EtOAc in EtOAc in EtOAc in EtOAc 164,192,193 194 195,196 188,190 187,189,191 186,197

Scheme 2. Isolation scheme of compounds 164, 186-197 from Phlomis stewartii

Page 117 CHAPTER # 06 EXPERIMENTAL

6.3 Acid hydrolysis of Compounds 186 and 197

Separately, compound 186 and 197 (8 mg each) were dissolved in 2 ml 2N HCl and heated at 100 ˚C for 3h, cooled and extracted with EtOAc two times (each 8 ml). The aqueous phase was neutralized passing through Dowex (Cl− form) and evaporated to dryness which confirmed as glucose, rhamnose and apiose; (1:1:1) on TLC using EtOAc-

MeOH-AcOH-H2O (4:2:2:2) as mobile by using standards of these sugars, respectively. 20 These sugars were further confirmed by comparing signs of their optical rotation ([α]D 20 20 + 51.0˚, [α]D + 8.0˚, [α]D + 4.0˚,) and retention times (D-glucose 7.8 min, L-rhamnose 8.6 min, D-apiose 5.7 min) in GC with the standards.

6.4 α-Glucosidase Inhibition Assay

The α-glucosidase inhibition assay was performed with slight modifications as done by Pierre et al. Total volume of 100 µL reaction mixture contained 70 µL 50 mM phosphate buffer, pH 6.8, 10 µL (0.5 mM) test compound, followed by the addition of 10 µL (0.0234 units, Sigma Inc.) enzyme. The contents were mixed, preincubated for 10 min at 37ºC and pre-read at 400 nm. The reaction was initiated by the addition of 10 µL of 0.5 mM substrate (p-nitrophenyl glucopyranoside, Sigma Inc.). After 30 min of incubation at 37 ˚C, absorbance of the yellow color produced due to the formation of p-nitrophenol was measured at 400 nm using Synergy HT (BioTek, USA) using 96-well microplate reader. Acarbose was used as positive control. The percent inhibition was calculated by the following equation

Inhibition (%) = (abs of control – abs of test / abs of control) × 100

IC50 values were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).

Page 118 CHAPTER # 06 EXPERIMENTAL

6.5.1 Characterization of Stewartiiside (186)

Pale yellow amorphous powder 6 CH3 OH 4 O (48 mg). 5 OH 2 OH 1 3 20 []D − 80 (c = 0.0012, MeOH). O O 2  OH 4 6 2 5 OH O O  1 3 1  UV (CH3OH) max nm: 219 (3.7), O O  3 2 1 6 3 4 OH 6 4 OH 5 328 (3.09). 1 5 6 5 OH O CH3 3 O -1 2 IR (KBr)  cm : 3438, 1701, 4 max OH 186 OH O 4 1605, 1520, 1450. 1 OH 5

2 3 OH OH

1 H NMR (CD3OD, 500 MHz): δ 6.66 (1H, d, J = 2.0 Hz, H-2), 6.68 (1H, d, J = 8.0, Hz, H-5), 6.55 (1H, dd, J = 8.0, 2.0 Hz, H-6), 4.01 (2H, t, J = 7.0 Hz, H-α), 2.8 (2H, t, J = 7.0 Hz, H-β), 4.36 (1H, d, J = 8.2 Hz, H-1), 3.37 (1H, t, J = 8.2 Hz, H-2), 3.79 (1H, t, J = 8.2 Hz, H-3), 4.90 (1H, t, J = 8.2 Hz, H-4), 3.71 (1H, m, H-5), 3.62 (1H, m, H-6a), 3.28 (1H, m, H-6b), 5.17 (1H, br s, H-1), 3.90 (1H, m, H-2), 3.71 (1H, m, H-3), 3.55 (1H, m, H-4), 3.48 (1H, m, H-5), 1.11 (3H, d, J = 6.4 Hz, H-6), 5.26 (1H, br s, H-1), 3.82 (1H, m, H-2), 3.89 (1H, m, H-3), 3.61 (1H, m, H-4), 3.54 (1H, m, H-5), 1.07 (3H, d, J = 6.2 Hz, H-6), 5.20 (1H, d, J = 6.8 Hz, H-1), 3.62 (d, J = 6.8 Hz, H-2), 3.71 (2H, s, H-4), 3.28 (2H, s, H-5), 7.06 (1H, d, J = 2.0 Hz, H-2), 6.80 (1H, d, J = 9.2 Hz, H-5), 6.96 (1H, dd, J = 9.2, 2.0 Hz, H-6), 6.28 (1H, d, J = 16.0 Hz, H-α), 7.60 (1H, d, J = 16.0 Hz, H-β).

13 C NMR (CD3OD, 100 MHz): δ 131.5 C-1), 116.5 (C-2), 146.8 (C-3), 144.6 (C-4), 114.6 (C-5), 121.3 (C-6), 72.5 (C-α), 36.5 (C-β), 104.1 (C-1), 76.1 (C-2), 81.6 (C-3), 70.1 (C-4), 75.0 (C-5), 65.6 (C-6), 103.0 (C-1), 72.3 (C-2), 72.0 (C-3), 80.1 (C-4), 68.7 (C-5), 18.4 (C-6), 102.1 (C-1), 72.3 (C-2), 72.1 (C-3), 74.6 (C-4), 70.4 (C- 5), 18.7 (C-6), 111.4 (C-1), 78.6 (C-2), 80.6 (C-3), 75.1 (C-4), 65.6 (C-

Page 119 CHAPTER # 06 EXPERIMENTAL

5), 127.5 (C-1), 116.2 (C-2), 146.0 (C-3), 149.8 (C-4), 117.1 (C-5), 123.4 (C-6), 116.3 (C-α), 148.0 (C-β), 168.1 (C=O).

+ HR-FAB-MS m/z: 903.3140 [M+H] (calcd. for C40H54O23, 903.3134).

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6.5.2 Characterization of Stewertiisin A [(17R)-19(18→17)-abeo-3α,18β,23,24- tetrahydroxy-28-norolean-12-ene](187)

Amorphous white powder (40 mg).

25 OH 21 []D  42.6 (c = 0.0011, MeOH). 22 12 30 18 20 17 -1 25 11 26 IR (KBr)  cm : 3410, 1635, 1378, 1249, 19 29 max 1 9 14 16

10 H 1034. 27 3 5 7 HO H 24 23 OH OH 187

1 H NMR (CD3OD, 500 MHz): δ 1.65 (2H, m, H-1), 1.57 (1H, m, H-2a), 1.32 (1H, m, H- 2b), 4.05 (1H, d, J = 2.0 Hz, H-3), 1.91 (1H, m, H-5), 1.51 (2H, m, H-6), 1.38 (2H, m, H- 7), 1.70 (1H, m, H-9), 1.99 (2H, m, H-11), 5.72 (1H, br s, H-12), 1.04 (2H, m, H-15), 1.61 (2H, m, H-16), 3.86 (1H, s, H-18), 1.96 (1H, d, J = 12.2 Hz, H-19a), 1.13 (1H, d, J = 12.2 Hz, H-19b), 1.48 (1H, m, H-21a), 1.38 (1H, m, H-21b), 1.28 (2H, m, H-22), 3.83 (2H, br s, H-23), 3.85 (1H, d, J = 10.0 Hz, H-24a), 3.75 (1H, d, J = 10.0 Hz, H-24b), 1.06 (3H, s, H-25), 0.94 (3H, s, H-26), 1.13 (3H, s, H-27), 1.00 (3H, s, H-29), 0.99 (3H, s, H- 30).

13 C NMR (CD3OD, 100Mz): δ 42.2 (C-1), 29.5 (C-2), 72.5 (C-3), 48.5 (C-4), 45.6 (C-5), 21.5 (C-6), 35.1 (C-7), 41.3 (C-8), 48.8 (C-9), 38.5 (C-10), 24.5 (C-11), 119.8 (C-12), 142.5 (C-13), 45.3 (C-14), 27.8(C-15), 36.7 (C-16), 43.6 (C-17), 76.4 (C-18), 52.8 (C- 19), 39.8 (C-20), 42.9 (C-21), 30.8 (C-22), 67.3 (C-23), 63.5 (C-24), 18.02 (C-25), 18.03 (C-26), 23.4 (C-27), 30.4 (C-29), 30.3 (C-30).

+ HR-EI-MS m/z: 460.3550 [M] (calcd. for C29H48O4, 460.3544).

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6.5.3 Characterization of Stewertiisin B [(17R)-19(18→17)-abeo-2α,16β,18β,23,24- pentahydroxy-28-norolean-12-ene-3-one] (188)

Amorphous white powder (40 mg). OH 22 21 12 30 25 18 20 []D − 52.6 (c = 0.0014, MeOH). 11 25 26 17 19 29 HO 9 14 16 -1 1 OH IR (KBr) max cm : 3435, 1719, 1638, 10 H 27 3 5 7 1378, 1249, 1034. O H 24 23 OH OH 188

1 H NMR (CD3OD, 500 MHz): δ 1.85 (1H, m, H-1a), 1.37 (1H, m, H-1b), 3.80 (1H, dd, J = 9.2, 3.8 Hz, H-2), 1.58 (1H, m, H-5), 1.12 (1H, m, H-6a), 1.15 (1H, m, H-6b), 1.01 (1H, m, H-7a), 1.28 (1H, m, H-7b), 1.35 (1H, m, H-9), 2.00 (2H, d, J = 10.0 Hz, H-11), 5.78 (1H, s, H-12), 1.60 (2H, m, H-15), 3.35 (1H, br s, H-16), 3.55 (1H, s, H-18), 1.12 (2H, m, H-19), 1.35 (2H, m, H-21), 1.28 (2H, m, H-22), 3.82 (2H, br s, H-23), 3.86 (2H, s, H-24), 1.10 (3H, s, H-25), 0.98 (3H, s, H-26), 1.17 (3H, s, H-27), 1.07 (3H, s, H-29), 1.02 (3H, s, H-30).

13 C NMR (CD3OD, 100Mz): δ 43.0 (C-1), 67.0 (C-2), 208.4 (C-3), 59.8 (C-4), 48.2 (C- 5), 19.1 (C-6), 35.1 (C-7), 40.2 (C-8), 43.6 (C-9), 38.8 (C-10), 24.9 (C-11), 119.0 (C-12), 143.6 (C-13), 45.2 (C-14), 28.1 (C-15), 78.1 (C-16), 44.1 (C-17), 76.2 (C-18), 53.0 (C- 19), 30.5 (C-20), 42.2 (C-21), 30.8 (C-22), 66.2 (C-23), 62.2 (C-24), 18.1 (C-25), 17.5 (C-26), 26.5 (C-27), 17.6 (C-29), 30.3 (C-30).

+ HR-EI-MS m/z: 490.3290 [M] (calcd. for C29H46O6, 490.3284).

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6.5.4 Characterization of Stewertiisin C [(17R)-19(18→17)-abeo-3α,18β,23,24- tetrahydroxy-28-norolean-11,13-diene] (189)

Amorphous white powder (40 mg). 22 21 12 18 30 25 11 20 [α]D − 50.0 (c = 0.0012, MeOH). 25 26 17 19 29 9 14 16 HO 1 10 UV (CH3OH) λmax nm: 236 (1.6). H 27 3 5 7 HO -1 H IR (KBr)  cm : 3415, 1635, 1378, 23 max 24 1249, 1035. OH OH 189

1 H NMR (CD3OD, 500 MHz): δ 1.85 (1H, m, H-1a), 1.35 (1H, m, H-1b), 3.89 (1H, m, H-2), 3.83 (1H, br s, H-3), 1.58 (1H, m, H-5), 0.92 (1H, m, H-6a), 0.95 (1H, m, H-6b), 1.35 (2H, m, H-7), 2.14 (1H, d, J = 9.0 Hz, H-9), 5.55 (1H, d, J = 8.0 Hz, H-11), 5.90 (1H, d, J = 8.0 Hz, H-12), 1.69 (2H, m, H-15), 1.63 (2H, m, H-16), 5.36 (1H, s, H-18), 1.51 (1H, m, H-19a), 1.34 (1H, m, H-19b), 1.44 (2H, m, H-21), 1.57 (2H, m, H-22), 4.00 (1H, d, J = 10.0 Hz, H-23a), 4.30 (1H, d, J = 10.0 Hz, H-23b), 3.78 (2H, s, H-24), 1.02 (3H, s, H-25), 0.75 (3H, s, H-26), 0.94 (3H, s, H-27), 1.35 (3H, s, H-29), 1.36 (3H, s, H- 30).

13 C NMR (CD3OD, 100 MHz): δ 42.4 (C-1), 66.9 (C-2), 72.0 (C-3), 48.5 (C-4), 47.0 (C- 5), 20.3 (C-6), 33.5 (C-7), 41.8 (C-8), 55.7 (C-9), 39.0 (C-10), 126.3 (C-11), 131.7 (C- 12), 139.7 (C-13), 41.1 (C-14), 27.2 (C-15), 34.3 (C-16), 45.5 (C-17), 137.5 (C-18), 57.0 (C-19), 40.2 (C-20), 41.4 (C-21), 40.5 (C-22), 72.3 (C-23), 62.3 (C-24), 19.4 (C-25), 17.0 (C-26), 20.4 (C-27), 31.4 (C-29), 30.7 (C-30).

+ HR-EI-MS m/z: 458.3432 [M] (calcd. for C29H46O4, 458.3427).

Page 123 CHAPTER # 06 EXPERIMENTAL

6.5.5 Characterization of Notohamosin A (190)

Amorphous white powder (40 mg). 21 30 18 25 13 OH [α]D − 50.0 (c = 0.0013, MeOH). 25 11 26 17 9 19 29 HO 1 UV (CH3OH) λmax nm: 236 (2.6). 10 H 15 3 5 27 7 -1 HO IR (KBr) max cm : 3415, 1635, 1378, 1249, H 24 23 1035. OH OH 190

1 H NMR (CD3OD, 500 MHz): δ 1.84 (1H, dd, J = 12.1, 4.1 Hz, H-1a), 1.34 (1H, d, J = 12.1 Hz, H-1b), 3.89 (1H, m, H-2), 4.02 (1H, d, J = 2.2 Hz, H-3), 1.64 (1H, m, H-5), 1.50 (2H, m, H-6), 1.34 (2H, m, H-7), 2.18 (1H, d, J = 2.8 Hz, H-9), 5.56 (1H, d, J = 10.2 Hz, H-11), 5.90 (1H, dd, J = 10.2, 2.8 Hz, H-12), 1.41 (2H, m, H-15), 1.52 (2H, m, H-16), 5.33 (1H, s, H-18), 1.61 (1H, d, J = 13.2 Hz, H-19a), 1.21 (1H, d, J = 13.2 Hz, H-19b), 1.27 (2H, m, H-21), 1.68 (1H, m, H-22a), 1.57 (1H, m, H-22b), 3.90 (1H, d, J = 11.1 Hz, H-23a), 3.70 (1H, d, J = 11.1 Hz, H-23b), 3.65 (1H, d, J = 11.5 Hz, H-24a), 3.58 (1H, d, J = 11.5 Hz, H-24b), 0.97 (3H, s, H-25), 0.73 (3H, s, H-26), 0.95 (3H, s, H-27), 1.05 (3H, s, H-29), 3.29 (2H, s, H-30).

13 C NMR (CD3OD, 100 MHz): δ 42.2 (C-1), 66.9 (C-2), 73.8 (C-3), 47.8 (C-4), 44.7 (C- 5), 19.5 (C-6), 33.3 (C-7), 41.2 (C-8), 55.6 (C-9), 38.7 (C-10), 126.3 (C-11), 131.7 (C- 12), 140.1 (C-13), 41.9 (C-14), 27.1 (C-15), 33.7 (C-16), 45.4 (C-17), 136.8 (C-18), 52.7 (C-19), 45.5 (C-20), 36.2 (C-21), 40.5 (C-22), 68.7 (C-23), 63.9 (C-24), 19.4 (C-25), 17.1 (C-26), 20.4 (C-27), 26.7 (C-29), 71.7 (C-30).

+ HR-EI-MS m/z: 474.3350 [M] (calcd. for C29H46O5, 474.3345).

Page 124 CHAPTER # 06 EXPERIMENTAL

6.5.6 Characterization of Phlomispentanol (191)

Amorphous powder (54 mg) OH 25 22 21 12 [α]D + 6.0 (c = 0.0015, CH3OH) 30 18 25 26 13 17 -1 9 19 29 IR (KBr) max cm : 3405, 2930, 1455, 1378, HO 1 10 15 H 1279, 1038. 3 5 7 27 HO H 24 23 OH OH 191

1 H NMR (CD3OD, 500 MHz): δ 1.66 (1H, m, H-1a), 1.36 (1H, m, H-1b), 3.88 (1H, m, H-2), 4.02 (1H, d, J = 2.5 Hz, H-3), 1.57 (1H, m, H-5), 1.31 (1H, m, H-6a), 1.35 (1H, m, H-6b), 1.39 (2H, m, H-7), 1.65 (1H, d, J = 9.0 Hz, H-9), 1.97 (2H, m, H-11), 5.77 (1H, dd, J = 8.0 Hz, H-12), 1.69 (2H, m, H-15), 1.60 (2H, m, H-16), 3.62 (1H, s, H-18), 1.13 (2H, m, H-19), 1.35 (1H, m, H-21a), 1.32 (1H, m, H-21b), 1.57 (2H, m, H-22), 3.66 (2H, br s, H-23), 3.75 (2H, s, H-24), 1.02 (3H, s, H-25), 0.75 (3H, s, H-26), 1.12 (3H, s, H-27), 1.00 (3H, s, H-29), 0.99 (3H, s, H-30).

13 C NMR (CD3OD, 100 MHz): δ 43.0 (C-1), 68.0 (C-2), 74.0 (C-3), 48.0 (C-4), 45.1 (C- 5), 17.9 (C-6), 35.2 (C-7), 40.7 (C-8), 48.4 (C-9), 39.0 (C-10), 24.2 (C-11), 119.0 (C-12), 143.5 (C-13), 45.2 (C-14), 28.1 (C-15), 36.9 (C-16), 51.0 (C-17), 76.9 (C-18), 53.1 (C- 19), 30.8 (C-20), 42.8 (C-21), 29.6 (C-22), 69.7 (C-23), 64.4 (C-24), 19.4 (C-25), 17.7 (C-26), 19.5 (C-27), 30.3 (C-29), 31.5 (C-30).

+ HR-EI-MS m/z: 476.3506 [M] (calcd. for C29H48O5, 476.3501).

Page 125 CHAPTER # 06 EXPERIMENTAL

6.5.7 Characterization of Oleanolic acid (164)

White Needles (70 mg). 29 30

° M.p: 305-306 C 19 21 12 17 [α]D + 78.9 (c = 0.0012, CHCl3) 13 28 25 26 H COOH 9 -1 1 15 IR (KBr) max cm : 3445, 3278-2635, 2960, H 3 5 7 27 2868, 1695, 1618, 1452, 1379, 1259, 1041, 975. HO H

23 24 164

1 H NMR (CD3OD, 500 MHz): δ 1.04 (1H, dd, J = 12.1, 4.1 Hz, H-1a), 1.54 (1H, d, J = 12.1 Hz, H-1b), 1.79 (2H, m, H-2), 3.28 (1H, d, J = 2.2 Hz, H-3), 1.44 (1H, m, H-5), 1.56 (2H, m, H-6), 1.54 (2H, m, H-7), 1.88 (d, J = 2.8 Hz, H-9), 1.56 (2H, d, J = 10.2 Hz, H- 11), 5.17 (1H, dd, J = 10.2, 2.8 Hz, H-12), 1.41 (2H, m, H-15), 2.12 (1H, m, H-16a), 1.99 (1H, m, H-16b), 2.88 (1H, dd, J = 10.2, 2.5 Hz, H-18), 1.81 (1H, m, H-19a), 1.30 (1H, m, H-19b), 1.47 (1H, d, J = 10.2 Hz, H-21a), 1.27 (1H, d, J = 10.2 Hz, H-21b), 1.68 (1H, m, H-22a), 1.57 (1H, m, H-22b), 1.07 (3H, s, H-23), 0.94 (3H, s, H-24), 0.97 (3H, s, H-25), 1.06 (3H, s, H-26), 1.29 (3H, s, H-27), 1.05 (3H, s, H-29), 0.98 (3H, s, H-30), 12.8 (1H, s, -OH).

13 C NMR (CD3OD, 100Mz): δ 38.7 (C-1), 27.9 (C-2), 77.6 (C-3), 40.2 (C-4), 54.9 (C-5), 18.3 (C-6), 34.1 (C-7), 39.5 (C-8), 49.1 (C-9), 36.9 (C-10), 23.4 (C-11), 122.0 (C-12), 144.3 (C-13), 43.1 (C-14), 29.2 (C-15), 23.5 (C-16), 45.6 (C-17), 42.3 (C-18), 47.3 (C- 19), 30.6 (C-20), 33.4 (C-21), 32.9 (C-22), 28.4 (C-23), 16.2 (C-24), 15.8 (C-25), 17.7 (C-26), 27.6 (C-27), 179.6 (C-28), 34.4 (C-29), 23.0 (C-30).

+ EI-MS m/z (rel. int.): 457 [M] C30H48O3, 456 (9.2), 248 (2.9), 231 (2.8), 215 (2.8), 203 (100.0), 184 (5.9), 170 (4.0), 118 (6.0), 107 (3.7), 94 (19.9), 82 (16.0), 69 (42.0), 55 (52.0).

+ HR-EI-MS m/z: 456.3610 [M] (calcd. for C30H48O3) 456.3604.

Page 126 CHAPTER # 06 EXPERIMENTAL

6.5.8 Characterization 2-Hydroxybenzoic acid (192)

Colorless Needles (48 mg). 7 COOH ° M.p: 159-161 C. 1 OH

5 3 UV (CH3OH) max nm: 234 (3.69), 302 (3.97).

-1 192 IR (KBr) max cm : 3408, 3246-2657, 1704, 1626,812.

1 H NMR (CD3OD, 500 MHz): δ 6.83 (1H, d, J = 8.4 Hz, H-3), 7.44 (1H, t, J = 8.4 Hz, H-4), 6.90 (1H, t, J = 9.0 Hz, H-5), 7.85 (1H, d, J = 8.5 Hz, H-6). 13 C NMR (CD3OD, 100 MHz): δ 112.6 (C-1), 161.5 (C-2), 117.9 (C-3), 135.9 (C-4), 120.0 (C-5), 131.4 (C-6), 175.5 (C-7). + HR-EI-MS m/z: 138.0316 [M] (calcd. for C7H6O3, 138.0311).

Page 127 CHAPTER # 06 EXPERIMENTAL

6.5.9 Characterization 4-Hydroxybenzoic acid (193)

Crystalline solid (68 mg). 7 COOH M.p: 212-214 °C. 1

5 3 UV (CH3OH) max nm: 224 (3.89), 312 (3.95).

OH -1 IR (KBr) max cm : 3509, 3331-2707, 1704, 1626, 812. 193

1 H NMR (CD3OD, 500 MHz): δ 7.92 (2H, d, J = 8.4 Hz, H-2,6), 6.78 (2H, d, J = 8.4 Hz, H-3,5), 11.9 (1H, s, COOH). 13C NMR (MeOD, 100 MHz):C-1), 131.5 (C-2,6), 117.0 (C-3,5), 159.9 (C-4), 179.9 (C-7). + HR-EI-MS m/z: 138.0309 [M] (calcd. for C7H6O3, 138.0303).

Page 128 CHAPTER # 06 EXPERIMENTAL

6.5.10 Characterization Caffeic acid (194)

Crystalline needles (68 mg). O M.p: 222-224 °C. 7 9 5 1 OH

UV (CH3OH) λmax nm: 234 (3.89), 302 (3.95). HO 3 -1 OH IR (KBr) max cm : 3499, 3331-2707, 1706, 1626. 194

1 H NMR (CD3OD, 500 MHz): δ 7.02 (1H, d, J = 2.0 Hz, H-2), 6.76 (1H, d, J = 8.0 Hz, H-5), 6.90 (1H, d, J = 8.0, 2.0 Hz, H-6), 7.51 (1H, d, J = 15.5 Hz, H-7), 6.21 (1H, d, J = 15.5 Hz, H-8). 13 C NMR (CD3OD, 100 MHz): δ 125.6 (C-1), 115.1 (C-2), 147.0 (C-3), 149.0 (C-4), 116.0 (C-5), 122.7 (C-6), 146.7 (C-7), 115.9 (C-8), 171.0 (C-9). + HR-EI-MS m/z: 180.0314 [M] (calcd. for C9H8O4, 180.0309).

Page 129 CHAPTER # 06 EXPERIMENTAL

6.5.11 Characterization of Tiliroside (195)

Yellow Powder (67mg). 3' OH

UV (CH OH) λ nm: 318 (3.20), 268 (3.71), 1' 5' 3 max HO 7 8a O 1 259 (4.20). 3 4a 4 5 O 3''' -1 HO OH O IR (KBr) max cm : 3325, 1675, 1653, 1535, 1''' 5''' 9''' O 195 1500. 7''' O 6'' O HO 5'' HO 1'' 3'' OH

1 H NMR (CD3OD, 500 MHz): δ 6.13 (1H, d, J = 1.6 Hz, H-6), 6.32 (1H, d, J = 1.6 Hz, H-8), 7.99 (2H, d, J = 9.2 Hz, H-2',6'), 6.84 (2H, d, J = 9.2 Hz, H-3',5'), 5.30 (1H, d, J = 7.2 Hz, H-1''), 3.45 (1H, m, H-2''), 3.62 (1H, t, J = 5.6 Hz, H-3''), 3.28 (1H, m, H-4''), 3.42 (1H, m, H-5''), 4.26 (2H, d, J = 11.6 Hz, H-6''), 7.32 (1H, d, J = 9.2 Hz, H-2''',3'''), 6.80 (2H, d, J = 9.2 Hz, H-3''',5'''), 6.10 (1H, d, J = 16.0 Hz, H-α), 7.38 (1H, d, J = 16.0 Hz, H-β). 13 C NMR (CD3OD, 100MHz): δ 159.8 (C-2), 131.2 (C-3), 181.3 (C-4), 104.0 (C-4a), 162.8 (C-5), 100.4 (C-6), 166.9 (C-7), 95.0 (C-8), 159.1 (C-8a), 127.0 (C-1), 132.2 (C- 2',6'), 116.1 (C-3,5), 161.2 (C-4), 103.8 (C-1''), 73.8 (C-2''), 78.1 (C-3''), 71.8 (C-4''), 75.8 (C-5''), 64.8 (C-6''), 129.1 (C-1'''), 131.2 (C-2''',6'''), 114.2 (C-3''',5'''), 161.0 (C-4'''), 116.8 (C-α), 146.5 (C-β), 169.1 (C=O). + HR-FAB-MS m/z: 595.1370 [M+H] (calcd. for C30H27O13) 595.1365.

Page 130 CHAPTER # 06 EXPERIMENTAL

6.5.12 Characterization of Isorhamnetin 3-(6-p-coumaroyl)-β-D-glucopyranoside (196)

Yellow Powder (77mg).

OCH3 ° M.p: 180-181 C. 3' OH

1' 5' HO 7 8a O 1 [α]D – 53.6 (c = 0.0014, MeOH). 3 4a 4 5 O UV (CH3OH) λmax nm: 315 (3.4), 265 (2.9), 3''' HO OH O

254 (4.20). 1''' 5''' 9''' O 196 7''' O 6'' O -1 HO 5'' IR (KBr) max cm : 3305, 2920, 1668, 1650, HO 1'' 3'' OH 1608.

1 H NMR (CD3OD, 500 MHz): δ 6.12 (1H, d, J = 1.6 Hz, H-6), 6.28 (1H, d, J = 1.6 Hz, H-8), 7.85 (1H, d, J = 1.6 Hz, H-2'), 6.83 (1H, d, J = 9.2 Hz, H-5'), 7.54 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 5.30 (1H, d, J = 7.2 Hz, H-1''), 3.43 (1H, m, H-2''), 3.62 (1H, t, J = 5.6 Hz, H-3''), 3.28 (1H, m, H-4''), 3.42 (1H, m, H-5''), 4.28 (2H, d, J = 11.6, H-6''), 7.30 (2H, d, J = 6.0 Hz, H-2''',3'''), 6.82 (2H, d, J = 9.2 Hz, H-3''',5'''), 6.10 (1H, d, J = 16.0 Hz, H-α),

7.40 (1H, d, J = 16.0 Hz, H-β), 3.90 (3H, s, OCH3).

13 C NMR (CD3OD, 100 MHz): δ 159.7 (C-2), 131.2 (C-3), 181.3 (C-4), 104.0 (C-4a), 162.8 (C-5), 100.3 (C-6), 166.9 (C-7), 94.9 (C-8), 159.2 (C-8a), 127.1 (C-1), 116.0 (C- 2), 149.0 (C-3), 151.5 (C-4), 114.6 (C-5), 123.9 (C-6), 103.9 (C-1), 73.8 (C-2), 78.0 (C-3), 71.8 (C-4), 75.8 (C-5), 64.4 (C-6), 127.0 (C-1), 131.2 (C-2,6), 114.2 (C-

3,5), 161.2 (C-4), 116.8 (C-α), 146.6 (C-β), 169.2 (C=O), 56.8 (-OCH3).

+ HR-FAB-MS m/z: 625.1480 [M+H] (calcd. for C31H29O14) 625.1475.

Page 131 CHAPTER # 06 EXPERIMENTAL

6.5.13 Characterization of Lunariifolioside (197)

Pale yellow amorphous powder (38 mg). O O 4''' 1''' 6' HO 5''' 20 3''' [α]D − 88 (c = 0.001, MeOH) OH OH O HO ' 5' O O OH 3''''' 1''''' '  1 O O  3 3' 1' UV (CH3OH) λmax nm: 219 (4.01), 328 HO OH 5''''' 1'' 5'' 5 OH O CH3 (3.09). 3'' O OH OH O

-1 4'''' 1'''' IR (KBr) max cm : 3432, 1701, 1605, HO 5'''' 3'''' OH OH 1520, 1450. 197

1 H NMR (CD3OD, 500 MHz): δ 6.66 (1H, d, J = 2.0 Hz, H-2), 6.68 (1H, d, J = 8.0 Hz, H-5), 6.55 (1H, dd, J = 8.2, 2.0 Hz, H-6), 4.01 (2H, t, J = 7.0 Hz, H-α), 2.8 (2H, t, J = 7.0 Hz, H-β), 4.36 (1H, d, J = 8.2 Hz, H-1ꞌ), 3.37 (1H, t, J = 8.2 Hz, H-2ꞌ), 3.79 (1H, t, J = 8.2 Hz, H-3ꞌ), 4.90 (1H, t, J = 8.2 Hz, H-4ꞌ), 3.71 (1H, m, H-5ꞌ), 3.62 (1H, m, H-6ꞌa) 3.28 (1H, m, H-6ꞌb), 5.17 (1H, br s, H-1ꞌꞌ), 3.90 (1H, m, H-2ꞌꞌ), 3.71 (1H, m, H-3ꞌꞌ), 3.55 (1H, m, H-4ꞌꞌ), 3.48 (1H, m, H-5ꞌꞌ), 1.11 (3H, d, J = 6.4 Hz, H-6ꞌꞌ), 4.89 (1H, s, H-1ꞌꞌꞌ), 3.61 (1H, d, J = 6.8 Hz, H-2ꞌꞌꞌ), 3.71 (2H, s, H-4ꞌꞌꞌ), 3.34 (2H, s, H-5ꞌꞌꞌ), 5.20 (1H, d, J = 6.8 Hz, H-1ꞌꞌꞌꞌ), 3.62 (1H, d, J = 6.8 Hz, H-2ꞌꞌꞌꞌ), 3.71 (2H, s, H-4ꞌꞌꞌꞌ), 3.28 (2H, s, H-5ꞌꞌꞌꞌ), 7.06 (1H, d, J = 2.0 Hz, H-2ꞌꞌꞌꞌꞌ), 6.80 (1H, d, J = 9.2 Hz, H-5ꞌꞌꞌꞌꞌ), 6.96 (1H, d, J = 5.6 Hz, H-6ꞌꞌꞌꞌꞌ), 6.28 (1H, d, J = 16.0 Hz, H-αꞌ), 7.60 (1H, d, J = 16.0 Hz, H-βꞌ).

13 C NMR (CD3OD, 100 MHz): δ 131.5 (C-1), 116.5 (C-2), 146.8 (C-3), 144.6 (C-4), 144.6 (C-5), 121.3 (C-6), 72.5 (C-α), 36.5 (C-β), 104.1 (C-1ꞌ), 76.1 (C-2ꞌ), 81.6 (C-3ꞌ), 70.1 (C-4ꞌ), 75.0 (C-5ꞌ), 65.6 (C-6ꞌ), 103.0 (C-1ꞌꞌ), 72.3 (C-2ꞌꞌ), 72.0 (C-3ꞌꞌ), 80.1 (C-4ꞌꞌ), 68.7 (C-5ꞌꞌ), 18.4 (C-6ꞌꞌ), 111.0 (C-1ꞌꞌꞌ), 75.1 (C-2ꞌꞌꞌ), 80.4 (C-3ꞌꞌꞌ), 75.0 (C-4ꞌꞌꞌ), 65.5 (C- 5ꞌꞌꞌ), 111.4 (C-1ꞌꞌꞌꞌ), 78.6 (C-2ꞌꞌꞌꞌ), 80.6 (C-3ꞌꞌꞌꞌ), 75.1 (C-4ꞌꞌꞌꞌ), 65.6 (C-5ꞌꞌꞌꞌ), 127.5 (C- 1ꞌꞌꞌꞌꞌ), 116.2 (C-2ꞌꞌꞌꞌꞌ), 146.0 (C-3ꞌꞌꞌꞌꞌ), 149.8 (C-4ꞌꞌꞌꞌꞌ), 117.1 (C-5ꞌꞌꞌꞌꞌ), 123.4 (C-6ꞌꞌꞌꞌꞌ), 116.3 (C-αꞌ), 148.0 (C-βꞌ), 168.1 (C=O).

+ HR-FAB-MS m/z: 889.2910 [M+H] (calcd. for C39H52O23, 889.2905).

Page 132 CHAPTER # 07 REFERENCES

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LIST OF PUBLICATIONS

1. M. A. Naveed, N. Riaz, M. Saleem, S. Mussadiq, Bushra Jabeen, M. M. Ahmed and A. Jabbar. Isolation and characterization of secondary metabolites from Tribulus longipetalus. Rec. Nat. Prod., (submitted) 2. M. A. Naveed, N. Riaz, M. Saleem, Bushra Jabeen, M. Ashraf, U. Alam and A. Jabbar. -Glucosidase Inhibitory Constituents from Ficus bengalensis. Pak. J. Pharm. Sci., (submitted). Impact Factor: 3. M. A. Naveed, N. Riaz, M. Saleem, Bushra Jabeen, M. Ashraf, T. Ismail and A. Jabbar. Longipetalosides A-C, new steroidal saponins from Tribulus longipetalus. Steroids (submitted). Impact Factor: 4. N. Riaz, A. Tabussum, M. Saleem, S. Parveen, M. Ashraf, S. A. Ejaz, Bushra Jabeen, I. Ahmad, A. Malik and A. Jabbar. Bioactive Secondary Metabolites from Chrozophora plicata. J. Chem. Soc. Pak., (in press). Impact Factor: 5. N. Riaz, A. Tabussum, M. Saleem, M. Ashraf, R. Nasar, Bushra Jabeen, A. Malik and A. Jabbar. New Lipoxygenase Inhibitory Sphingolipids from Chrozophora plicata. J. Asian Nat. Prod. Res., 15, 1080-1087 (2013). Impact Factor: 0.948 6. Bushra Jabeen, N. Riaz, M. Saleem, M. A. Naveed, M. Ashraf, U. Alam, H. M. Rafiq, R. B. Tareen and A. Jabbar. Isolation of natural compounds from Phlomis stewartii showing α-glucosidase inhibitory activity. Phytochemistry 96, 443-448 (2013). Impact Factor: 3.050 7. A. Tabussum, N. Riaz, M. Saleem, M. Ashraf, M. Ahmad, U. Alam, Bushra Jabeen, A. Malik and A. Jabbar. α-Glucosidase inhibitory constituents from Chrozophora plicata. Phytochemistry Lett., 6, 614-619 (2013). Impact Factor: 1.179 8. Bushra Jabeen, N. Riaz, M. Saleem, M. A. Naveed, M. N. Tahir, G. Pescitelli, M. Ashraf, S. A. Ejaz, I. Ahmed and A. Jabbar. Isolation and characterization of limonoids from Kigelia Africana. Z. Naturforsch. B, 68b, 1041-1048 (2013). Impact Factor: 0.899 9. N. Riaz, K. Feroze, M. Saleem, S. Musaddiq, M. Ashraf, U. Alam, R. Mustafa, Bushra Jabeen, I. Ahmad and A. Jabbar. Oligocephlate, a new α-glucosidase

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inhibitory neohopane triterpene from Vernonia oligocephala. J. Chem. Soc. Pak., 35, 972-975 (2013). Impact Factor: 10. N. Riaz, M. A. Naveed, M. Saleem, Bushra Jabeen, M. Ashraf, S. A. Ejaz, A. Jabbar and I. Ahmad. Cholinesterase Inhibitory constituents from Ficus bengalensis. J. Asian Nat. Prod. Res., 14, 1149-1155 (2012). Impact Factor:0.948

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