Withania Coagulans Thesis Submitted

Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulans Thesis submitted

for

The Partial Fulfillment of the Degree of DOCTOR OF PHILOSOPHY (Chemistry) by SAIM A M AHE R

H. E. J. Research Institute of Chemistry International Center for Chemical and Biological Sciences University of Karachi, Karachi-75270, Pakistan 2012

Dedicated

To My Mother Whose inspiration and prayer always worked with me

To My Father Whose teaching and blessings have only made this venture possible

To My Husband Whose love, care and invaluable patronage are the most important assets of my life

CERTIFICATE

To Whom It May Concern

It is certified that the thesis entitled, “Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulans,’’ has been submitted by Mrs. Saima Maher to the Board of Advanced Studies and Research, University of Karachi, for the award of the degree of Doctor of Philosophy (Chemistry). She carried out her research under my supervision. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or university for the award of any degree or diploma.

PROF. DR. M. IQBAL CHOUDHARY Hilal-e-imtiaz, Sitara-e-imtiaz, Tamagha-e-imtiaz Director/Research Supervisor

H. E. J. RESEARCH INSTITUTE OF CHEMISTRY International Center for Chemical and Biological Sciences University of Karachi, Karachi-75270, Pakistan

CERTFICATE OF THESIS EVALUATION

This is to certify that the Ph.D. thesis of Ms. Saima Maher d/o Mr Muhammad

Israil,entitled Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulan supervised by Prof. Dr. Prof. Dr. Muhammad Iqbal

Choudhary H.I.,S.I.,T.I. has been checked and evaluated with positive comments by the following faculty members of the H.E.J Reasearch Institute of Chemistry.

1. Prof. Dr. M. Iqbal Choudhary H.I., S.I., T.I. 2. Prof. Dr. Viqar Uddin Ahmed

H.I., S.I.

3. Prof. Dr. Bina Shaheen SiddiquiS.I.,T.I. 4. Prof. Dr. Khalid M. Khan T.I.

International Center for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan

Content List

CONTENTS

(i) ACKNOWLEDGEMENT i (ii) PERSONAL INTRODUCTION iv (iii) SUMMARY v (iv) URDU SUMMARY (Khulasa) x 1.0 GENERAL INTRODUCTION

1.1 INTRODUCTION 1 1.1.1 Medicinal Importance of Plants 1 1.1.2 History of Research on Medicinal Plants 2 1.1.3 Economic Value of Botanicals 3 1.1.4 Drug Discovery from Nature 4 1.1.5 Objectives of the Current Study 6

PART-A 2.0 PHYTOCHEMICAL STUDIES ON BIOACTIVE METABOLITES FROM VISCUM ALBUM L.

2.1 INTRODUCTION 8 2.1.1 The Family Loranthaceace 8 2.1.2 The Genus Viscum L. 7 2.1.3 Previous Phytochemical Investigation on Genus Viscum 9 2.1.4 The Plant Viscum album L. 14 2.1.5 Biological Significance of Viscum album L. 15 2.1.6 Significance of Constituents of Viscum album L. 15 2.1.7 Major Secondary Metabolites of the Plant: Flavonoids 16 2.1.7.1 Biosynthesis of Flavonoids 17 2.1.8 Minor Secondary Metabolites of the Plant: Phenylpropanoids 19 2.1.8.1 Biosynthesis of Phenylpropanoids 19

2.2 RESULTS AND DISCUSSION 22 2.2.1 Isolation of Major, and Minor Constituents from Viscum album 22 2.2.2 New Compounds 1 and 2 23 2.2.2.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone 23 (1) 2.2.2.2 3-(4′-Acetoxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyrnoside (2) 32 2.2.3 Known Compounds 3-10 39

Content List

2.2.3.1 3-(4′-Hydroxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyranoside (3) 39 2.2.3.2 5,7-Dimethoxy-4′-O-β–D-glucopyranosideflavanone (4) 40 2.2.3.3 5,7-Dimethoxy-4′-hydroxyflavanone (5) 41 2.2.3.4 4′,5-Dimethoxy-7-hydroxyflavanone (6) 42 2.2.3.5 7-Hydroxy-8methoxy flavanone (7) 43 2.2.3.6 Betuline (8) 45 2.2.3.7 Gallic Acid (9) 46 2.2.3.8 β-Sitosterol (10) 47

2.3 EXPERIMENTAL 48 2.3.1 General Experimental Conditions 48 2.3.1.1 Physical Constants 48 2.3.1.2 Spectroscopic Techniques 49 2.3.1.3 Purification and Detection of Compounds on Chromatographic Plates 49 2.3.2 Plant Material 49 2.3.3 Extraction and Fractionation of Methanol- Choloroform Soluble Constituents of Viscum 49 album 2.3.3.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone 52 (1) 2.3.3.2 3-(4′-Acetoxy-3′,5′-dimethoxy)-phenyl-2-(E)-propenyl-β-D-gluco-pyranoside 53 (2) 2.3.3.3 3-(4′-Hydroxy-3′,5′-dimethoxy)-phenyl-2E-propenyl-β-D-glucopyranoside 54 (3) 2.3.3.4 5,7-Dimethoxy-4′-O-β–D-glucopyranosideflavanone (4) 55 2.3.3.5 5,7-Dimethoxy-4′-hydroxyflavanone (5) 56 2.3.3.6 4′,5-Dimethoxy-7-hydroxyflavanone (6) 57 2.3.3.7 7,8-Dihydroxyflavanone (7) 58 2.3.3.8 Betuline (8) 59 2.3.3.9 Gallic Acid (9) 60 2.3.3.10 β-Sitosterol (10) 61 SECTION-A 3.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM VISCUM ALBUM L.

3.1 ANTIOXIDANT ACTIVITY 62 3.1.1 Introduction 62 3.1.2 Results and Discussion 63

Content List

3.1.3 Methodology 64

3.2 ANTI-GLYCATION ACTIVITY 65 3.2.1 Introduction 66 3.2.2 Results and Discussion 66 3.2.3 Methodology 68 PART-B 4.0 PHYTOCHEMICAL STUDIES ON SECONDARY METABOLITES FROM WITHANIA COAGULANS DUN. (STOCK.)

4.1 INTRODUCTION 71 4.1.1 The Family Solanaceae 71 4.1.2 The Genus Withania 71 4.1.3 Previous Phytochemical Studies on Genus Withania 72 4.1.4 Major Secondary Metabolites of the Plant: Withanolides 77 4.1.4.1 Biosynthesis of Withanolides 78

4.2 RESULTS AND DISCUSSION 83 4.2.1 Isolation of Major and Minor Constituents from Withania coagulans 83 4.2.2 New Compounds 11-12 84 4.2.2.1 (20R,22R)14,16α,17α,20α-Tetrahydroxy-1-oxo-witha-5,24-dienolide-3-O-β- 84 D-glucopyranoside (11) 4.2.2.2 3β,17β-Dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-β-D- 91 glucopyranoside (12) 4.2.3 Known Compounds 13 -17 98 4.2.3.1 Withanolid J (13) 98 4.2.3.2 Coagulin E (14) 99 4.2.3.3 Withaperuvin C (15) 100 4.2.3.4 27-Hydroxywithanolide I (16) 101 4.2.3.5 Ajugin E (17) 103

4.3 EXPERIMENTAL 104 4.3.1 General Experimental Conditions 104 4.3.1.1 Chromatography 104 4.3.1.2 Purification and Detection of Compounds on Chromatographic Plates 105

4.3.2 Isolation of Major and Minor Constituents from Withania coagulans 105 4.3.3 New Compounds 11 - 12 108

Content List

4.3.3.1 (20R,22R)14,16,17,20-Tetrahydroxy-1-oxo-witha-5,24-dienolide-3-O-β-D- 108 glucopyranoside (11) 4.3.3.2 3β,17β-Dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-β-D- 108 glucopyranoside (12) 4.3.3.3 Withanolid J (13) 109 4.3.3.4 Coagulin E (14) 110 4.3.3.5 Withaperuvin C (15) 111 4.3.3.6 27-Hydroxywithanolide I (16) 112 4.3.3.7 Ajugin E (17) 113 SECTION-B 5.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM WITHANIA COAGULANS DUN. (STOCK.)

5.1 CYTOXICITY ACTIVITY 117 5.1.1 Introduction 117 5.1.2 Results and Discussion 118 5.1.3 Methodology 118 6.0 REFERENCES 121 7.0 GLOSSARY 127 8.0 LIST OF PUBLICATIONS 1 30

Acknowledgement

ACKNOWLEDGEMENT

First of all, I am extremely grateful to the Almighty Allah (SWT), the most merciful and beneficent, for His unlimited guidance and blessings upon me to accomplish my goal.

I pay my gratitude to Prof. Dr. Atta-ur-Rahman F.R.S., N.I.,H.I.,S.I.,T.I. for his dedicated efforts to equip, and modernize the H. E. J. Research Institute of Chemistry (International

Center for Chemical and Biological Sciences).

I wish to express my heartiest, and profound gratitudes to my dearest, lovely, highly respectable, kind hearted, ideal teacher, and supervisor Prof. Dr. Muhammad Iqbal

Choudhary H.I.,S.I.,T.I. Director, ICCBS. His valuable gaudiness has brought success to my efforts. His impulsive, cheerful, endless, and harmonious encouragements lift my spirit, and raised my personality to meet the challenges. His intriguing, unique, and outstanding personality is a great motivator for me. I thank to Allah (SWT) and feel myself extremely fortunate that I am his student. He guided me and helped me a lot, and Inshaallah he will support me in future also. I got the lesson of self-respect from his personality. Whatever I was able to achieve, as presented here, is only due to his keen interest and support.

Special expression to my Dearest Famiy members:

I wish to express my respect to my family for their trust, prayers, and everlasting support.

Really I have wonderful parents. Mrs. Mehrun Nisa, and Mr. Muhammad Israil. I can not do anything without their help, and support. I am thankful for their continuous encouragement and prayers for my success. I have no words to describe my true feelings for them. I am indebted to my dearest sisters, Mrs. Almas, Mrs. Shazia, and Ms.

Acknowledgement

Rumsha, and specially thankful to my loving brothers Mr. Khalid, Mr. Naveed, and

Mr. Junaid who supported me all the way through.

I express my special and deepest gratitude to my loving husband Mr. Shahzad Imran, for his trust, love, and continuous encouragement. His loving care enabled me to continue my studies with full concentration. I am specially thankful to my lovely daughters Yashal

Shahzad, and Fatima Shahzad for their patience.

I am thankful to all the faculty members of the institute, Prof. Dr. Viqar-ud-Din Ahmed

H.I. S.I., Prof. Dr. Bina S. Siddiqui T.I, S.I., Prof. Dr. Khalid M. Khan T.I., S.I., and specially

Dr. Sammer Yousuf, Dr. Syed Ghulam Musharraf, and Dr.Atia-tul-Wahab for their help time to time.

I wish to express my specially thank to Prof. Dr. Fatima Basha T.I for her support, guides, kindness, and loving behavior and care.

I wish to express my sincere thanks to Dr. Achyut Adhikari for his help, support and proficient suggestion and cooperation.

I want to express my sincere gratitudes to my very kind colleague, and seniors

Dr. Ahmad Abbas Khan, and dear friends Dr. Nadra Naheed, Dr. Afshan Begum, Dr.

Sumaira Hareem who has always helped me during my research studies.

I wish to express my deepest gratitude to my lab. Fellows, Dr. Saleem Jan, Dr. Ismail, ii

Dr. Sofia, Dr. Javeria, Dr. Muhammad Abdusalam Dr. Ali Azarpira, and particular I wish to extant my specially thanks to my dearests sweets juniors Ms. Nasreen, Ms.

Naureen, Mrs Shagufta, for their love great support, invaluable care and assistance.

Acknowledgement

I also thank Mr. Abdul Hafeez Sahib and Mr. Faisal Azim, for their kind co-operation and help.

Last but not least, I wish to acknowledge the help and cooperation of colleagues for bioactivity evaluation, specially Ms. Ambreen Khan, Dr. Sajjad Ali and Dr. Samina

Abdul Sattar. I am thankful to all the office and technical staff of the H.E.J. Research

Institute of chemistry for their help, which contributed in the successful completion of this study.

SAIMA MEHER 2012, Karachi

iii

PERSONAL INTRODUCTION

My native town is Nazimabad, in Karachi city (Sindh), Pakistan. I belong to an Urdu speaking Behari family. Teaching is my family occupation. I got my primary and secondary education from the Brilliant Carrier School in Karachi. I fulfilled my higher secondary education requirment from the Abdullah Government Girls College, Karachi.

My graduation i.e. bachelor degree is from the Jinnah University for Women, Karachi. I also completed my Masters program with Chemistry from the same university. As I was doing my post graduation, I read a Jang Newspaper in which a detailed interview of Prof.

Dr. Iqbal Choudhary was published. At that moment, I decided to join him and do Ph. D.

At this moment he became a role model for me. After that I was able to get admission at the premier research center, the H. E. J. Research Institute of Chemistry, for Ph. D. in natural product, chemistry. For the Ph. D. studies, I opted isolation, and structure elucidation of natural products as field of interest, and selected Prof. Dr. M. Iqbal

Choudhary S.I., T.I., H.I., Director, ICCBS as my supervisor. Under his leadership and guidance, I became a member of one of the best research groups in the world and acquired valuable analytical and chromatographic skill in natural product chemistry. I was able to build strong understanding for structure elucidation by interpretation of various spectroscopic results.

I look forward to explore fascinating world of natural products and contribute in exploring bioactive natural molecules found in the rich flora of Pakistan. Beside this, I will also try to bring my understanding, knowledge, and skills to an advanced level to meet the challenges of future, while remain a caring mother, and a responsible wife.

SUMMARY

This thesis is divided into two parts. Part A describes the phytochemical studies on the secondary metabolites of Vicum album L., a Pakistani medicinal plant which is commonly found as a semi parasite on trees of Juglans regia (Walnut). Part B explains the phytochemical studies on Withania coagulans Dun. (Stock.).

PART-A

The Part-A deals with the phytochemical investigations on Vicum album L., which resulted in the isolation and structure elucidation of two new 1-2 and nine known compounds 3-10.

Flavanoid glycosides were identified as main components, of the ethyl acetate and butanolic fractions of the plant.

During the current study, the minor constituents were characterized as members of phenylpropanoides class of secondary metabolites. New metabolites were identified as, 4′-

O-[-D-apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone (1), and 3-(4′- acetoxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyrnoside (2). Known compounds include, 3-(4′-hydroxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyranoside (3), 5,7- dimethoxy-4′-O-β–D-glucopyranosideflavanone (4), 5,7-dimethoxy-4′-hydroxyflavanone

(5), 4′,5-dimethoxy-7-hydroxyflavanone (6), 7,8-dihydroxyflavanone (7), betuline (8), gallic acid (9), and β-sitosterol (10). Compounds 1-6, obtained in good yields, were also evaluated for their potential bioactivites and all of them exhibited a significant antiglycation activity, while compounds 2 and 5 showed a potent antioxidant activity.

Structures of these secondary metabolites were elucidated by using various modern spectroscopic techniques, such as 1D- and 2D-NMR, COSY, NOESY, HMQC, HMBC,

etc. Similarly the high- and low-resolution mass spectrometry (EI-MS, FAB-MS), UV, and

IR spectrophotometery techniques were also employed to elucidate the structures of new compounds.

OH H 6''' H OCH3 4''' 5''' O 3' HO 1''' 3'' 3''' O OH HO 2''' 2' H O 4' 2'' 4'' H O H 8'' 5' O O 1'' 5'' 4'''' HO 1'''' 1' 1 8 OCH 9 6'' 3 5'''' 6' 2 7 7'' 3'''' 3 6 2'''' 10 O HO 4 5 OH

O OH

H OH

4''' 6''' H 5''' O 1 3 2' OCH3 HO 1' 3' HO O O 2''' 2 3''' H OH 1''' 4' H H 6' 5' O

OCH 2 3

H OH

4''' 6''' H 5''' O 1 3 2' OCH3 HO 1' 3' HO O 2''' 2 3''' H OH 1''' 4' H H 6' 5' OH

OCH 3 3

OH H 6'' H 4'' 5'' O 3' HO 1'' O 3'' HO 2'' 2' H OH 4' H 5' O OCH3 1' 1 8 9 6' 2 7 6 3 10 4 5

4 O OCH3

3' 3' OH OCH3

2' 4' 2' 4'

5' H3CO O HO O 5' 8 1 1' 8 1 1' 9 2 9 7 6' 7 2 6' 6 6 10 3 10 3 5 4 5 4

OCH3 O OCH3 O 29 5 6

30 20

3' 19 21 H 22 OCH3 2' 4' 18 12 11 17 5' 25 26 13 HO O CH OH 1' H 2 8 1 28 9 9 14 16 2 6' 7 1 15 6 2 8 10 3 10 5 H 4 27 3 5 7 4 6 HO O H 23 24

7 8 21 29 28 22 18 O OH 20 24 H 23 7 12 27 17 19 11 13 H 16 1 9 14 2 15 26 6 1 2 8 5 10 3 H H 4 HO OH 3 5 7 4 6 HO OH

9 10

vii

PART-B Part-B of the dissertation describes the phytochemical studies on Withania coagulans

Dun. (Stock.) of Pakistani origin. This study has resulted in the isolation of two new compounds 11–12 and five known compounds 13-17.

New compounds include, (20R,22R)14α,16α,17α,20α-tetrahydroxy-1-oxo-witha-5,24- dienolide-3-O-β-D-glucopyranoside (11), 3β,17β-dihydroxy-14,20-epoxy-1-oxowitha-

5,24-dienolide-27-O-β-D-glucopyranoside (12), whereas known constituents were identified as withanolid J (13), coagulin E (14), withaperuvin C (15), 27- hydroxywithanolide I (16), and ajugin E (17).

24 27

23 25 OH 22 26 21 Me O O 20 18 Me H OH 12 17 O 19 11 13 Me 16 OH 9 H 14 OH H 1 10 2 H 8 6' H OH 5' O 3 HO 4' 6 4 O 1' HO 2' 3' H OH H H 11 H H 2 8 Me OH HO 3' 24 27 2' 4' OH

O O 5' 6' 23 25 1' H 21 22 OH Me H O 26 O H 20 18 HO H Me 12 O 17 11 13 O 19 H Me 16 9 14 15 1 2 10 8 H 3 5 7 4 6 HO 12 viii

28 28

24 27 24 27

OH 23 21 23 21 25 25 22 26 20 22 26 18 18 H 20 O O O O H H OH 12 12 O 17 17 O 11 O 19 13 19 11 13 H 16 H 16 9 14 9 14 15 15 1 1 2 10 8 2 10 8 H OH H 3 5 7 3 5 7 4 6 4 6

13 14

28 28

24 27 24 27

23 OH 25 23 OH 21 HO 25 22 26 21 22 26 18 20 O O 18 H 20 O O H 12 OH O 17 12 H 19 11 13 O 17 H 16 19 11 13 16 9 14 H 15 9 14 1 15 1 2 10 8 H OH 2 10 8 3 5 7 H OH 3 4 6 5 7 4 6

15 16

24 27 OH HO 23 25

21 22 26 18 20 O O H 12 OH O 17 19 11 13 H 16 9 14 15 1 2 10 8 H OH 3 5 7 4 6

Urdu Khuasa

1.0 GENERAL INTRODUCTION 1.1 INTRODUCTION x 1.1.1 Medicinal Importantce of Plants

Traditional medicines are used for the treatment of a variety of ailments

throughout the world. People rely on medicinal plants for the relieve from physical

sufferings. The majority of the world’s civilizations had developed their own systems of

the therapeutic uses of medicinal plants, based on centuries of experience. The use of

plant medicines have often guided by the earlier observations, and believe and were

carefully documented in treatise.

Natural products have played an important role in the human well being, survival and

prosperity. They have been extensively employed in the treatment of many diseases.

Natural products, obtained from medicinal plants, and their derivatives are still a major

source of pharmaceutical leads and valuable therapeutic agents, as well as templates for

targeted synthetic modifications.

According to an estimate, about 70% people in the developing world largely depend on

natural products for primary healthcare. More than 75% of traditional medicines contain

the constituents of plant origins. According to another study, the natural products and

associated drugs can be used for the treatment of 87% of all human ailments, including as

antibacterial, antitumer, anticoagulant, anti-parasitic, and immunosuppressant agents.

[Thomas, 2002].

Over 200,000 biochemicals have been isolated from plants. According to an annual report

of the United States Food and Drug Administration (USFDA), about 60% and 70% drugs

in the field of cancers, and infectious diseases, respectively, are of natural origin. Same study showed that natural product-based drugs comprised of 52% of all new chemical entities launched in the markets. Out of which, 24% of new chemical entities are of synthetic origin, while other 28% are natural products. The combined percentage (52%) of total number of drugs discovered from natural sources or derivatives of natural products, suggests that nature remain a major source of new drugs and lead compounds

[Mahidol et al., 1998; Newman et al., 2003].

Large number of chemical constituents, diverse carbon skeletons, and functional groups, and useful physiochemical properties makes plants a very useful source of newest pharmaceuticals, health-promoting substances, flavors, fragrances, fine chemicals, toxins, etc. Plant components therefore have a profound impact on health and wellbeing of human society. It is estimated that about 90% of those drugs which were approved worldwide during 1982 to 2002 were of natural origin or based on their templates. Many natural products have showed excellent potency as anticancer agents, and played a significant role in the development of anticancer drugs [Hussain, 1993].

1.1.2 History of Research on Medicinal Plants

Medicinal plants remained of especial interests from early human history. As early as 5,000 B.C., a lot of plant-based drugs were in use in China. The Babylonians, ancient Hebrews and Assyrians were well-known users of the medicinal plants. In ancient

Egypt, several medicinal plants were in use by physicians. This includes myrrh, opium, cannabis, aloes, cassia and hemlock. The Greeks were familiar of the use of many natural drugs which are used even today. The interest in medicinal plants was particularly

prominent among the early botanists (physicians). The WHO recorded ethno-botanic practices in many region of the world.

Currently the use of plant products or botanicals is increasing with 10-20% annually in

North America, Western Europe, and Japan. About 35,000 to 75,000 plants species are used for medicinal purposes throughout the world.

Over the last decades with the technical advancement, there have been several new discoveries in the field of natural products. Latest tools and methodologies have been developed for phytochemical research and phytometabolomics studies. These development are now transforming a time-consuming natural product research into a fast, high-throughput and target-based drug invention regime. During the last few years, tremendous efforts have been made in the field of natural product-based drug discovery, along with combinatorial synthesis and high-throughput screening [Anonymous, 1996].

Beside all these efforts, millions of compounds are still waiting to be discovered which can serve as potential lead compounds in drug discovery.

1.1.3 Economic Value of Botanicals

The importance of plants in our daily life cannot be underestimated. It fulfills most of the basic requirements of the human beings. Plants play a very important role in food security, health, livelihood, and ecological services. Many plant species have been commonly used for foodstuff, yarn, industrial, cultural and remedial uses. Today, only

300 plant species are used to meet the 85% of the world's foodstuff and energy needs

(Anon., 1996). About 70% of the world’s food dry weight is resulting from four cereals: rice and wheat, maize and barley [Wilson, 1992]. Approximately 150 medicines,

obtained from 100 species of herbal plants, are in the market place for clinical use

[Padulosi et al., 2003].

The flora of Pakistan is rich and diverse due to climatic and soil conditions. The country has over 6,500 species of higher plants, and about 400 to 600 of them are medicinally important. Many plants are also imported as raw materials. According to a study of the

Pakistani Forest Institute (PFI), Peshawar, 75 crude traditional medicines are widely explodes abroad and other 200 are locally traded in Pakistan. In another study, conducted for SDC (Swiss Development Corporation), over 300 medicinal plants were identified as medicinal plants from Malakand division of Khyber Pakhtoon Khwa [Sher et al., 2010].

Native citizens have no guidance in sustainable croping, crop care after harvesting, and storing of herbal plants. From the wild plants they collect 85 % of crude herbs. Such activity has created a frequent decrease of the resources of medicinal plants [Hamayun et al., 2007].

1.1.4 Drug Discovery from Nature

Natural products have been the largest source of molecular diversity for the drug discovery. Many natural products and their derivative have been developed over the years for clinical application. Ethnobotany and traditional and indigenous medicines are the important components of medicinal plant research. The human experiences, well known and described, form the basis of many useful systems of medicines thoughtout the world.

The use of naturally occurring compounds in medicine is not new. The rich structural and stereochemical characteristics of natural products make them make them precious templates for discovering novel molecular therapies against diseases related biological

target [Newman et al., 2003]. Natural products based drugs, such as taxol, digitaline, scopolamine, vinblastine, vincristine, aspirin, amoxycillin, cefaclor, ceftriaxone and lovastatin, have had a great impact on the pharmaceutical production. Many of these compounds are in use for decades are still the essential components of modest therapy. In

1950s, two plant-derived antileukemic agents, vinblastine and vincristine, were extracted from Catharanthus roseus. Vinblastine has been proved to be a successful treatment for

Hodgkin’s disease, and also used for the breast cancer treatment.

Semisynthetic anticancer agents, etoposide and teniposide, are derivatives of podophyllotoxin, a natural lignan isolated from Podophyllum peltatum. The anti cancer drug taxol, obtained from bark of Taxus brevifolia, is comparatively a new arrival to the mainstream medicine [Wani et al., 1971]. Taxol was approved for the treatment of refractory ovarian (used in combination in the front-line treatment of patients with ovarian cancer and in the retreatment of platinum-sensitive patients). Only altretamine

(hexamethylmelamine), liposomal doxorubicin, paclitaxel, and topotecan are approved by the (USFDA).

Most of diseases, such as heart diseases, arthritis, diabetes, cancers, viral diseases and antibiotic-resistant infections, still lack effective treatments. As a result, in the last few decades, scientists have focused their attention on traditional medicines and have screened a large number of unexamined plants for new bioactive compounds. In many cases, results have been very promising and interesting molecular leads have been discovered.

1.1.5 Main Objectives of Current Study

Pakistan possesses approximately 1,500 species of medicinal plants. Out of these, about 300 are used by traditional practitioners (hakims) and local healers. On the other hand, a large amount of foreign exchange is spent annually on the import of pharmaceutical raw material for synthetic drugs. In view of the extensive need of medicines against prevailing diseases, establishment of basic drug manufacturing in

Pakistan, both herbal and modern is necessary. Along with the systematic study of the cultivation and propagation of medicinal plants, the development of processes to isolate their active constituents as well as to conduct clinical and toxicological studies is of equally importance.

The medicinal importance of plants, extensively reported in the Flora of Pakistan,

(Ajaibi et al., 2010), motivated us to carry out a detailed phytochemical study on some medicinal plants of Pakistani origin. We focused on the isolation of bioactive chemical constituents from two medicinally important plants of Pakistan, which include Viscum album L. (Part A) and Withania coagulans Dun. Stock. (Part B). This bioassay-guided isolation study has resulted in the isolation of flavanoids from Viscum album and cytotoxic withanolides from Withania coagulans.

PART-A

2.0 PHYTOCHEMICAL STUDIES ON BIOACTIVE METABOLITES FROM VISCUM ALBUM L.

7 Introduction

2.1 INTRODUCTION

2.1.1 The Family Loranthaceace

Loranthaceae is one of the biggest families http://en.wikipedia.org/wiki/Family_(biology) of flowering plants, comprises 75 genera and over 1,000 species. Most of them are hemi- parasites, having mistletoe habit (EMEA/MRL, 680-99 (1999). All mistletoe species are included in Loranthaceae family, although European and North American (the genera

Viscum and Phoradendron), typical Christmas mistletoes, belong to family Santalaceae.

2.1.2 The Genus Viscum L.

Viscum L. (Loranthaceae) is greenish parasitic plant. It broadly distributed all over the tropical regions of Europe and northern part of Asia. The Viscum plants are hemi- parasitic shrubs with branches 15–80 centimeters long. They grow on the host trees. The flowers are greenish-yellow. The fruits are soft white berry, containing many seeds and very sticky. Birds are dispersed out the seeds on tree branches where they can germinate.

Many species of Viscum grow in different host species; mostly occupy several host species [wikipedia.org/wiki/Loranthaceae].

Folk Uses

These plants are also important economically. They have been recognized as a source of traditional treatment of diabetes mistletoe is also valuable for hypotensive, sedative, and anticancer agents (Arndt, 2000). Two main types of Viscum, European (Viscum album) and American (Phoradendron leucarpum), express the same proteins, but are reported to have diverse uses. European Viscum is used for the treatment of cancers, reducing side

8 Introduction effects of cancer therapy and high blood pressure. It also acts as antispasmodic and calmative agents [Lin et al., 1973].

2.1.3 Previous Phytochemical Investigation on Genus Viscum

A number of phytochemical investigations on various species of Viscum have been conducted. Viscum album is known to contain alkaloids, amines, flavonoids, acids and flavonone glycosides as the main components. The minor constituents of Viscum album is phenylpropanoids. The flavonone glycosides contain 37 or less carbon either in free state or substituted with sugars. Phenylpropanoids are 19 carbon skeletons. The isolation of these compounds involves the primary extraction of plant material with aqueous methanol or ethanol, and then column chromatography of crude extracts by using different eluents, such as ethyl acetate, chloroform-methanol-water or chloroform- methanol or BuOH: water to obtain different fractions. Further column chromatography

(silica gel) on these fraction afforded flavonone glycosides and phenylpropanoids.

A large number of flavanone glycosides have been reported from Viscum plants, some of them are listed in Table-1(Page-10).

9 Introduction Table- 1: The summary of results of previous phytochemical studies on Viscum species.

S.No. Name Molecular Molecular Source Refrence Weight Formula 1 Propionylcholine 160.236 C H NO 8 18 2 Viscum album Baker, J. T., et al., Tet. Lett., 1976, 1233-1234.

2 2,6-Diamino-5-hydroxyhexanoic acid 162.188 C H N O Wilding, M. D., et al., Phytochemistry, 1962, 1, 6 14 2 Viscum album 263- 3 Cysteic acid 169.158 C H NO S 3 7 5 Viscum album Vester, F., et al Physiol. Chem., 1960, 322, 273.

4 Viscumitol 208.211 C8H16O6 Viscum album Richter, A., et al., Phytochemistry, 1992, 31, 3925.

5 Kynurenine 208.216 C10H12N2O3 Viscum album Yambe, H. et al., Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 15370-15374.

6 2,3-Butanediol-O--D- 252.264 C10H20O7 Viscum Shindo, M., et al., J.O.C., 1998, 63, 9351- Glucopyranoside coloratum 9357. 7 Viscutin 3 274.273 C H O Viscum 15 14 5 Ghosal, S., et al., J. Chem. Res., 1983, 330. tuberculatum 8 5-[3-(3,4-Dihydroxyphenyl)-propyl]- Viscum Lin, J.-H. et al., J. Nat. Prod., 2002, 65, 638- 292.288 C H O 1,2,3,4-benzenetetrol 15 16 6 angulatum 640. 4',7-Dihydroxy-3',5-dimethoxy- Viscum Leu, Y.-L., et al., Chem. Pharm. Bull., 2006, 9 316.31 C H O flavanone 17 16 6 coloratum 54, 1063-1066. Viscum 10 Mistletonone 328.364 C H O Yao, H., et al., Molecules, 2007, 12, 312-317. 19 20 5 coloratum Fraser, A. W., et al., Phytochemistry, 1974, 13, 11 2',3-Dihydroxy-4,4',6'- 330.337 C18H18O6 Viscum album 1561. trimethoxychalcone

Introduction

Viscum Leu, Y. L. et al., Chem. Pharm. Bull., 2006, 54, 12 Viscolin 348.395 C H O 19 24 6 coloratum 1063-1066. 1,7-Bis(4-hydroxy-3- 13 352.386 C H O Viscum Martin, Cordero, C., et. al., Photochemistry, methoxyphenyl)-1,4,6-heptatrien-3- 21 20 5 cruciatum 2001, 58, 567-569. 10 one

14 2',4'-Di-methyl ether,4-O--D- 462.452 C23H26O10 Viscum album De, Vlaming, P., et al., Phytochemistry, 1976, glucopyranoside chalcone 15, 348.

15 5,7-Di-methylether,4'-O--D- 462.452 C23H26O10 Viscum album Fukunaga, T., et al., Chem. Pharm. Bull., 1987, glucopyranoside 35, 3292-3297. 16 2',4-Dihydroxy-4',6-dimethoxy- 462.452 C H O Viscum album 23 26 10 Bilia, A. R., et al., Tetrahedron, 1994, 50, 5181. chalcone 4-glucoside 2,6-Dimethyl-2,7-octadiene-1,6-diol- 17 Sigstad, E. E. et al., Phytochemistry, 1996, 42, 6-O-[-D-Apiofuranosyl-(16)--D- 464.509 C H O Viscum album 21 36 11 1443-1445. glucopyranoside] Viscum Shimizu, K., et al., Planta Med., 1998, 64, 408- 18 Viscoside A 464.425 C H O 22 24 11 coloratum 412.

19 Viscumside A 464.425 C22H24O11 Viscum Fukunaga, T., et al., Chem. Pharm. Bull., 1988, multinerve 36.

20 3',5,7-Trimethyl ether,4'-O--D- 492.479 C24H28O11 Viscum album Ghosal, S., et al., Phytochemistry, 1978, 17, glucopyranoside 2119.

22 Rhamnazin3-glucoside. 492.435 C23H24O12 Viscum album Lopez-Lzaro, M., et al., Planta Med., 1999, 65, Flavoyadorinin A 777.

Viscum Matsuda, H., et al., Chem. Pharm. Bull., 2002, 23 Viscumneoside VI 506.462 C H O 24 26 12 coloratum 50, 972-975.

11 Introduction

24 Viscutin 1 526.496 C H O Viscum 27 26 11 Kubo, I. et al., Tet. Lett., 1987, 28, 921. tuberculatum

25 Viscumneoside IV 534.473 C25H26O13 Viscum Jer-Huei, L., et al., J. Nat. Prod., 2002, 65 (5), coloratum pp 638–640.

26 7-O-[-D-Apiofuranosyl-(12)--D- Viscum Ma, X.-M. et al., Chem. Biodiv., 2007, 4, 2172- 550.515 C H O glucopyranoside] 26 30 13 articulatum 2181.

27 Viscutin 2 568.533 C29H28O12 Viscum Garo, E. k, et al., Phytochemistry, 1996, 43, tuberculatum 1265-1269.

Viscum 28 Viscumneoside I 596.541 C H O Garo, E., et al., Phytochemistry, 1996, 43, 1265. 27 32 15 coloratum Viscum 29 Viscumneoside III 596.541 C H O Garo, E., et al., Phytochemistry, 1996, 43, 1265. 27 32 15 coloratum 30 Homoflavoyadorinin B 608.552 C H O Ohta, N. et al., Agric. Biol. Chem., 1970, 34, 28 32 15 Viscum album 900

31 3',7-Di-Meether,4'-O-[-D--apio- 610.568 C28H34O15 Viscum Chou, C. J., et al., J. Nat. Prod., 1999, 62, furanosyl-(12)--glucopyranoside alniformosanae 1421-1422. 32 624.551 C H O Viscum Lopez-Lzaro, M., et al., Z. Naturforsch., C, Rhamnazin4'-(2-apiosylglucoside) 28 32 16 alniformosanae 2000, 55, 40. Viscum De Carvalho, M. G., et al., J. Braz. Chem. Soc., 33 Homoeriodictyol 4',7-diglucoside 626.567 C H O 28 34 16 coloratum 1999, 10, 163-166.

34 3',7-Dimethoxy,3,4'-Di-O--D- C H O Viscum album Wollenweber, E., et al., Z. Naturforsch., C, 654.577 29 34 17 glucopyranoside 1984, 39, 303.

Introduction

7-O-[E-Cinnamoyl-(5)--D- 35 Viscum Ma, X. M., et al., Chem. Biodiversity, 2007, 4, 680.661 C H O apiofuranosyl-(12)--D-gluco- 35 36 14 articulatum 2172-2181. pyranoside]

7-O-[-D-Apiofuranosyl-(15)--D- 36 Viscum Leu, Y. L. et al., Chem. Pharm. Bull., 2004, 52, 682.631 C H O apiofuranosyl-(12)--D-gluco- 31 38 17 angulatum 858-860. pyranoside] 2',4'-Di-methylether,4-O-[E- 37 724.714 C H O Viscum album Hunt, G. M., et al., Phytochemistry, 1980, 19, cinnamoyl-(5)--D-apiofuranosyl- 37 40 15 1415. (12)-D-glucopyranoside 5,7-Di-methylether,4'-O-[E- 38 724.714 C H O Viscum album Orhan, D. D., et al., Pharm. Biol., 2002, 40, cinnamoyl-(5)--D-apiofuranosyl- 37 40 15 380-383. (12)- -D-glucopyranoside] Viscum Kong, D., et al., Phytochemistry, 1988, 23, 593- 39 Viscumneoside V 728.657 C H O 32 40 19 coloratum 600

40 Acanthoside D 742.727 C34H46O18 Viscum album Seikel, M.F. et al., Phytochemistry, 1971, 10, 2249-2251.

41 Isorhamnetin-3,7-diglycoside 756.2113 C33H40O20 Viscum album Fukunaga, T., et al., Chem. Pharm. Bull., 1988, 36, 1185-1189.

3-O-[-D-Apiofuranosyl-(15)--D- 42 apiofuranosyl-(12)-[-L- 872.783 C38H48O23 Viscum Harvala, E., et al., J. Nat. Prod., 1984, 47, rhamnopyranosyl-(16)]--D- angulatum 1054. glucopyranoside]

Introduction

2.1.4 The Plant Viscum album L. Subkingdom Tracheobionta (Vascular plants) Super division Spermatophyta (Seed plants) Class Dicotylendons Division: Magnoliphyta (Flowering plants) Family: Loranthaceace Genus: Viscum L. (Mistletoe) Species: Viscum album L. (European mistletoe) Local Name: Guch (Mansehra, Hazara, and Neelam valley, Azad Kahsmir) Occurrence: Pakistan, Kashmir, Europe Occurrence in Pakistan: Mansehra, Hazara, Azad Kashmir Known Natural Compounds:Flavanoids, Phenylpropanoids Collection: Neelam valley (Azad Kahsmir), Pakistan

2.1.5 Biological Significance of Viscum album

Viscum album L. (Mistletoe) is an important medicinal plant. It is widely used as a medication. The chemical components of an extract may diverge according to season, host tree, parts of the plant used and extraction techniques. Mistletoe extracts, exhibit antioxidant antiglycation and increase cytotoxicity effects on different cell lines in vitro.

A number of species of the genus Viscum are utilize in folk medicine for the treatment of diabetes, jaundice, indigestion, common fever and asthma, (Hajto, 2005). Many of these also support macrophage cytotoxicity, stimulate phagocytosis and enhance cytokine secretion.

Introduction

2.1.6 Biological Significance of Constituents of Viscum album

The plants also contain acids, alkaloids, amines, flavonoids, phenylpropanoids, terpenoids and viscotoxins (Renata, 2002). A large number of components of Viscum have showed diverse biological properties, such as anticancer, antitumor, and immunostimulatory activites (Stein, 2002) [Hsu, 1984]. Several of these components have shown a significant antioxidant property [Kishida, 1989].

During the present study, the crude extract of V. album showed a significant antiglycation activity. The methanolic extract of V. album has showed anti-glycation potential (Gray et al., 1999).

Phytochemical Studies on Viscum album L.

Based on the extensive medicinal uses of Viscum plants, we decided to carry out a detailed phytochemical study on Viscum album L. of Pakistani origin, collected from

Neelam valley (Azad Kahsmir, Pakistan).

The present phytochemical study was focused on the isolation, characterization and bioactivity evaluation of purified secondary metabolites of V. album. As a result of this comprehensive study, two main classes of compounds were obtained. Among them, flavonoids were identified as the major components, whereas the minor constituents are phenylpropanoids in nature. The isolation involved the initial extraction of plant material with methanol. Crude methanolic extract was suspended in dist. H2O and extracted with

EtOAc (3 L×3) and n-BuOH (3 L×3) successively, yielding EtOAc and n-BuOH extracts. n-BuOH extracts were loaded to silica gel C.C. and eluted with chloroform and methanol

15 Introduction in a gradient manner to obtained eight fractions (E1-E8). Repeated column chromatography of fraction E-2 (100 mg) over silica gel by using the solvents system

(CHCl3 and MeOH)(10%, 600 mL) afforded flavonoids and phenylpropanoids.

Recycling HPLC was also utilized to isolate these compounds. The structures of compounds were determined by using various techniques (1D and 2D-NMR, COSY,

ROESY, HMQC, HMBC, high- and low-resolution mass spectrometry (EI-MS, FAB-

MS) and IR spectrophotometry.

These compounds showed a strong antiglycation activity. Potent antioxidant activity was also exhibited by a few of compounds, when they were evaluated for their biological activity.

2.1.7 Major Secondary Metabolites of the Plant: Flavonoids

The flavonoids have an important position in the phenolic secondary metabolites. The flavonoids are among the major matabolites of Viscum album plants. Name “flavonoid” has been derived from the Greek word “flavus” (yellow). They are the coloring pigments of the plants (Monitto, 1981). They are also known as anthroxanthins of plants phenolics.

About 2% of all the primary metabolites photosynthesized in plant, are converted into flavonoids (Harborne, 1988).

Structurally flavonoids contain fifteen carbon atoms in their parent nucleus. The common structural feature is two phenyl rings linked through a three carbon chain (diphenyl propane derivatives). This three carbon chain forms a third ring (five-or-six membered) through cyclization with the hydroxyl of one of the phenyl rings.

16 Introduction

The tricyclic compounds which possess a six-membered heterocyclic ring are known as flavonoids. Those flavonoids derived from 1, 2-diphenylpropane or 1, 1-diphenylpropane systems are named as isoflavanoids, isoflavonoids and neoflavonoids (Fig. 1).

2.1.7.1 Biosynthesis of Flavonoids:

Biosynthesis of flavonoids takes place from 4-hydroxy-cinnamoyl-Co-A (18).

Condensation of 4-hydroxy-cinnamoyl-Co-A (18) with three molecules of malonoyl-

CoA, takes place to result an intermediate 19. Enzymatic reduction of this intermediate by NADPH yields intermediate 20. Reaction is catalyzed by reductase enzyme. This intermediate then go through a Claisen condensation and following cyclization through

17 Introduction intermediate chalcones (21), finally lead to flavanone (22). The interconversion between the chalcones (21) and flavanones (22) is catalyzed in vivo by an enzyme, chalcone isomerase (Scheme-1).

OH OH O SCoA CoSA O 3 x Malonoyl-CoA O

O O 4-Hydroxycinnamoyl-CoA (18) 19

NADPH2 (Reductase)

BH: OH OH H O SCoA HO O O Cyclization

OH O O O H A

Chalcone (21) 20

-BH3 Claisen condensation -A

OH OH

HO O HO O

H OH OH O OH H O A: Flavone (22)

Scheme-1: Biosynthesis of flavanonoids.

18 Introduction

2.1.8 Minor Secondary Metabolites of the Plant: Phenylpropanoids

Secondary metabolites, phenylpropanoids, are phenolic compounds contain a three carbon side chain attached to a phenol. Hydroxyl-coumarins, phenylpropenes, and lignans are the common examples of phenylpropanoids. Other examples include derivatives of hydroxycinnamic acids, such as caffeic, ferulic, and coumaric acids. These are generally present in green and roasted coffee beans. The functions of phenylpropanoids are diverse based their structural diversity. They are known as precursors of lignins and are used as pigments, phytoanticipins, UV protectants, and phytoalexins. They also serve as signaling molecules between plants, and microbes.

Phanylpropanoid metabolism is unique in plant, but it is quite complex.

2.1.8.1 Biosynthesis of Phenylpropanoids

The biosynthesis of phenylpropanoids starts from two amino acids, L-phenylalanine, and

L-tyrosine. L-Phenylalanine (23) undergoes an enzyme catalyzed non-oxidative deaminaion reaction in the presence of phenylalanine ammonialyase enzyme to yield trans-cinnamic acid (24). The hydroxylation occurs at C-4 position of t-cinnamic acid, in the presence of cytochrome P450 monooxygenase cinnamate 4-hydroxylase to yield 4- coumaric acid. 4-Coumaric acid is transformed to 4-coumaroyl-CoA by the enzyme hydroxycinnamic acid-CoA ligase (Scheme-2).

In another step, the transamination of L-tyrosine (27), catalyzed by the action of enzyme tyrosine aminotransferase, takes place to obtain 4-hydroxyphenylpyruvic acid (28). 4-

Hydroxyphenylpyruvic acid (28) is reduced to the corresponding

4-hydroxyphenyllactate (29) by an enzyme hydroxyphenylpyruvate reductase (Kim et al.,

2004).

Introduction

COOH COOH

NH 2 Phenylalanine ammonialyase Trans-Cinnamic acid (24) L-Phenylalanine (23)

Cinnamate 4-hydroxylase O

COOH SCoA

HO Hydroxycinnamic HO acid-CoA ligase

4-Coumaroyl-CoA (26) 4-Coumaric acid (25) Scheme-2: 4-Coumaroyl-CoA biosynthesis from L-phenylalanine.

These subsequent ester formation between 29 and coumaroyl-CoA (26) takes place to yield, 4-coumaroyl-4′-hydroxyphenyllactic acid (phenylpropanoids) (Petersen et al.,

1988).

Introduction

2.2 RESULTS AND DISCUSSION

2.2.1 Isolation of Major and Minor Chemical Constituents from Viscum album

The current phytochemical study of whole plants of Viscum album L. has resulted in the isolation of two phenylpropanoids 1-2, along with known flavanone and flavanone glycosides 3-7 and known compounds, betuline (8), gallic acid (9), and β-sitosterol (10).

Silica gel column chromatography was employed for the preliminary fractionation of the ethyl acetate extract of the plants. Column was eluted with CHCl3 and MeOH in a gradient manner to afford eight fractions. Repeated column chromatography on sub- fraction E-2 (100 mg) yielded compounds 6 and 4 as known secondary metabolites, while compounds 11 and 8 were isolated as a white amorphous material and 7 was obtained as a yellow powder. Compound 3 was isolated from the polyamide column chromatography by using 100% chloroform, as eluent, followed by gradual increase of polarity by adding

MeOH. This compound was identified as flavanone glycoside.

The n-butanolic fraction (10 g) of the plant was loaded on a Diaion HP-20 column and two most important fractions were eluted. The sub-fraction (1:1) (H2O-MeOH) was successively processed by different chromatographic procedures, and finally purified from recycling HPLC. This led to the isolation of compounds 1-2 as new constitutents physical and spectral analysis, the flavanone glycosides and phenyl propanoids 3-6 were identified as known compounds. The major and minor components of Viscum album have exhibited a good antiglycation, and antioxidant activities (Tables-5, and 6, Pages-66, 68).

2.2.2 New Compounds

2.2.2.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapylflavanone (1)

Air-dried and whole parts of Viscum album (1.4 Kg) were extracted with 80% methanol- water (15 L) at room temperature. The crude extract was partitioned with n-hexane,

EtOAc, and BuOH. The resulting n-butanolic fraction (2.0 g) was loaded on a silica gel column, and the resulting fractions were rechromatographed over silica gel and polyamide columns, and finally purified by recycling HPLC (see glossary). This afforded compound 1 as a main component (3 mg, 0.5 x 10-3 % yield).

HO 6''' OCH3 4''' 5''' O 2''' 3' HO 1''' O 3'' OH HO 3''' 2' O 4' 2'' 4'' O 8'' 5' O O 1'' 5'' HO 4'''' 1'''' 1' 1 8 OCH 9 6'' 3 5'''' 6' 2 7 7'' 3'''' 3 6 2'''' 10 O HO 4 5 OH

O OH

The UV spectrum (see glossary) showed absorptions at 326 and 283 nm, characteristic of a flavanone skeleton (Mabry et al., 1975). The IR absorptions use at 3412 (OH), 1668

(,-unsaturated carbonyl), and 1606, 1520 cm-1 (C=C), respectively.

The 1H-NMR spectrum of 1 displayed the presence of three mutually coupled proton signals at H 2.64 (dd, J3a, 3b = 17.8 Hz, J3a, 2 = 3.1 Hz), 2.86 (dd, J3b, 3a = 17.8 Hz, J3b, 2 =

12.8 Hz), and 5.23 (dd, J2, 3b = 12.8 Hz, J2, 3a = 3.1 Hz), corresponded to the C-3 methylene and C-2 methine protons of ring C of flavanone, respectively. A 2H doublet at

H 7.02 (J2′, 3′ = 8.6 Hz) were assigned to C-3′ and C-5′ aromatic protons. Similarly, another 2H doublet resonated at H 7.31 (J3′, 2′ = 8.6 Hz), was assigned to C-2′ and C-6′ aromatic protons. Two meta coupled protons of ring A appeared at H 5.97 (d, J 8, 6 = 2.3

Hz) and 6.01 (d, J6, 8 = 2.3 Hz), corresponding to the C-8 and C-6 aromatic protons, respectively.

The presence of a sinapyl moiety in compound 1 was inferred from the doublets of the trans olefinic protons at H 7.59 (J7, ″8″ = 15.7 Hz, H-7″) and 6.31 (J8, ″7″ = 15.8 Hz, H-8″) and a two- proton singlet at H 6.82, was assigned to the C-2″ and C-6″ protons of the phenyl ring. Two methoxy groups were inferred from a 6H singlet at H 3.82. Signals between H 3.40-5.54 were due to the proton of sugar moieties. Two anomeric protons

appeared as doublets at H 5.54 (J1,′′′′2′′′′ = 1.4 Hz), and 4.93 (J1,′′′2′′′ = 7.9 Hz), which were in agreement with those of apiose and glucose sugar moieties, respectively (Hosoya et. al.,

2005), (Leu et. al., 2004).

The 13C-NMR spectrum of 1 showed 37 carbon signals, deduced from DEPT (see glossary), and HMQC techniques (see glossary). These signals were resolved into two methoxy, four methylene, nineteen methine, and twelve quaternary carbons, including two carbonyl carbons at c 162.8 and 197.6, due to ester and ketone groups, respectively.

Among them, 15 signals were due to a flavanone moeity, 11 to sinapyl group, and remaining 11 to the -glucopyranosyl and -D-apiofuranosyl sugars.

1 13 Table-2: H- (300 MHz) and C-NMR (100 MHz) Chemical Shift Data of Compound 1 in CD3OD.

Position H (J= Hz) C Multiplicity 1 - - - 2 5.23 (dd, J = 12.8, 3.1) 79.1 CH

3 2.64 (dd, J = 17.8, 3.1) CH2 2.86 (dd, J = 17.8, 12.8) 44.0 4 - 197.6 C 5 - 168.9 C 6 6.01 (d, J = 2.3 ) 94.8 CH 7 - 164.3 C 8 5.97 (d, J = 2.3) 95.8 CH 9 - 162.6 C 10 - 107.1 C 1′ - 128.7 C 2′ 7.31 (d, J = 8.6) 126.3 CH 3′ 7.02 (d, J = 8.6) 117.6 CH 4′ - 158.9 C 5′ 7.02 (d, J = 8.6) 117.6 C 6′ 7.31 (d, J = 8.6) 126.3 CH 1′′ - 128.6 C 2′′ 6.82 (br. s) 106.2 CH 3′′ - 149.4 C 4′′ - 133.9 C 5′′ - 149.4 C 6′′ 6.82 (br. s) 106.8 CH 7′′ 7.59 (d, J = 15.8) 147.9 CH 8′′ 6.31(d, J = 15.8) 115.5 CH 9″ - 162.8 C 3″/5″- 3.82 (s) 56.5 CH3 OCH3 Glucose C-1′′′ 4.93 (d, J = 7.9) 100.5 CH C-2′′′ 3.64 (dd, J = 9.2, 7.5) 78.1 CH C-3′′′ 3.99 ( t, J = 9.2) 77.7 CH C-4′′′ 3.84 (dd, J = 9.2, 6.8) 71.6 CH C-5′′′ 3.40 (m) 71.4 CH

C-6′′′ 3.67 (dd, J = 14.3, 7.3) 62.3 CH2 Apiose C-1′′′′ 5.54 (d, J = 1.4) 110.3 CH C-2′′′′ 3.91 (d, J = 1.4) 75.8 CH C-3′′′′ - 79.0 C

C-4′′′′ 4.24 (d, J = 11.5) 78.8 CH2 C-5′′′′ 4.39 (d, J = 11.3) 67.5 CH2

All chemical shift assignments are based on 1H-1H COSY, HMBC, HMQC, and DEPT NMR spectroscopic techniques.

Sub-structure Determination: 25

Two-dimensional NMR experiments (COSY-45°, HMQC, and HMBC) techniques were utilized to elucidate the structure of compound 1. A number of structural fragments were initially developed on the basis of COSY-45 and HMQC (see glossary) techniques. The complete three- dimensional structure of 1 was then built-up from structural fragments with the help of HMBC correlations (see glossary).

H O . 1 COSY 2

H 3 HMQC 4 H .

O a

The COSY-45 (see glossary) spectrum of compound 1 was very informative, and showed the

presence of four isolated spins systems in the molecule, in addition to a sugar moiety. Spin

system “a” constitutes on three protons, including an oxo methine (C-2) and methylene protons

(C-3). A double doublet at H 5.23 (J3, 2β = 12.8 Hz, J3, 2 = 3.1 Hz) was assigned to C-2 (CH)

methine proton, which showed cross peaks with C-3 methylene protons (H 2.64, 2.86). The

downfield chemical shift of the C-2 methine proton indicated its proximity to an electron-

withdrawing group, such as an ether carbon. The HMQC spectrum was used to determine the

direct (one- bond) heteronuclear couplings between carbon and protons. The HMBC

experiments were conducted to determine the long-range heteronuclear (1H/13C) correlations.

In the HMBC spectrum, the C-2 methine protons exhibited long-range couplings with carbons,

which resonated at  128.7 (C-1′), 44.0 (C-3) and 197.6 (C-4). On the otherhand, H2-3 showed

HMBC interactions with carbons resonated at  79.1 (C-2), 107.1 (C-10), and 197.6 (C-4).

The downfield chemical shift of aromatic C-5 (168.9) indicated the presence of an OH group.

It was also supported by bathochromic shift of band II (~ 24 nm) in the presence of shift reagent (AlCl3/HCl) in UV spectrum, which indicated C-5 hydroxyl functionality (Mebry et al., 1975). A signal at  94.8 was assigned to C-6 by HMQC (see glossary) analysis, while signals at C 164.3 162.6, and 107.1 were due to C-9, C-7, and C-10, respectively.

In the HMBC spectrum, the C-6 (H 6.01) methine proton exhibited long-range correlations with the carbons resonated at  107.1 (C-10) 164.3 (C-7) and 168.9 (C-5).

3' H 2' 4' 1' HMBC 5'

6' HMQC H

Fragment “b” comprises of parts of six-membered ring C of the flavanone skeleton. The elucidation of this fragment started with the C-2' ortho proton, which resonated at H 7.31

(J2', 6' = 8.6 Hz) coupled with C-3' proton which showed as a doublet at H 7.02 (J3', 5' = 8.7

Hz). A downfield carbon signal at C 126.3 was assigned to C-2′ by HMQC analysis, while signals at C 117.6, and 126.3 were assigned to C-3′ and C-6′, respectively.

The HMBC spectrum (see glossary) showed long-range heteronuclear correlations between the C-6′ proton ( 7.02) with carbons resonating at 128.7 (C-1′), 158.9 (C-4′)

and 117.6 (C-5′). These observations led to the fragment “b” as comprising part of the ring

C of the main flavanone skeleton.

OCH3

5'' H OH 6'' H 4'' COSY 3'' 8'' HMBC 1'' OCH 7'' 2'' 3

O H H

The sinapyl substituent formed the fragment “c”. This was worked out starting from an doublets of the trans olefinic protons at H 7.59 (J7″, 8″ = 15.8 Hz, H-7″) and 6.31 (J8″, 7″ =

15.8 Hz, H-8″), and a two proton singlet at H 6.82, was due to the C-2″ and C-6″ protons of the phenyl moiety. Two methoxy groups were inferred from a singlet at H 3.82 (6H).

The HMQC spectrum showed that C-8′′ methine proton ( 6.31) has a direct 1H/ 13C correlation with a carbon resonated at  115.5, while C-7′′ olefinic proton ( 7.59) was correlated with C-7′′ ( 147.9). This trans coupling was also inferred the COSY-45 spectrum.

The presence of a phenylpropanoid moiety in compound 1 was mainly deduced from the

3 analysis of JCH (HMBC) correlation of the C-2′′ methine proton of aromatic ring resonated at 6.82 which showed long-range heteronuclear correlations with C-1′′ ( 128.6), and C-

3′′ (147.9) and a three-bond coupling with C-2′′ ( 106.2). On the other hand, the C-6′′

3 methine proton of aromatic ring exhibited JCH interactions with C-5′′ ( 149.4), C-1′′ (

128.6) and C-4′′( 133.9). Based on the spectroscopic observation, fragment “c” was

1 deduced. The H-NMR spectrum of 1 also showed signals at H 3.40-5.54 corresponding

to a sugar moiety. Two anomeric protons, appeared as doublets at H 5.54 (J1′′′′, 2′′′′ = 1.4 Hz)

and 4.93 (J1′′′, 2′′′ = 7.9 Hz), were in agreement with those of apiose and glucose moieties, respectively.

The structure of compound 1 was finally deduced by detailed interpretation of 2-D NMR

3 data. In the HMBC spectrum, the signal at H 4.93 (C-1″′) was correlated J with a carbon

3 signal c 158.9 (C-4′), while the apiose anomeric proton (H 5.54) showed a J connectivity with C-2″′ (c 78.1), suggesting the attachment of apiose at C-2″′ of glucose moeity. It was further infered from the downfield shift of C-2 of the glucose moiety (c

79.1).

The nature of glycosides was determined by the hydrolysis of compound 1. Compound 1

(1.3 mg) was dissolved in 5% HCl/H2O (2 mL) and refluxed for 1 hour. The solution was extracted with EtOAc (1 mL × 3) to obtain aglycone, while the water layer was neutralized by NaHCO3 (pH 6), and sugars were identified by paper chromatography, in comparison with the standard sugars in the solvent system EtOAc/AcOH/H2O (5:3:2). The results further supported the presence of apiose and glucose moieties.

Complete Structure Elucidation by Assembling Sub-Structures.

The HMBC technique was used to obtain information about the overall structural assembly. The interactions between the protons and carbons of different fragments were employed to link various fragments together.

The position of sinapyl moiety at C-7 was deduced from the downfield chemical shift of

c 164.3. This seems to be the only position of sinaplyl moiety in ring A (Wei L et al.,

2005). Characteristic signals for α, β-unsaturated carbonyl (c 162.8) and two methine carbons (c 147.9, 115.5) further indicated the presence of a sinapyl moiety.

HO 6''' OCH3 4''' 5''' O HO 3' 2''' 3'' 3''' O OH HO 1''' O 4' 2' 2'' 4'' O 8'' 5' O O 1'' 5'' 4'''' HO 1'''' 1' 1 8 9 OCH3 5'''' 6' 2 7 7'' 6'' 3'''' 3 6 2'''' 10 O HO 4 5 H H OH

O OH

Fig. 2: Key HMBC correlations in 1.

A bathochromic shift of band II (~ 24 nm), in the presence of shift reagent (AlCl3/HCl) in

UV spectrum, indicated C-5 hydroxyl functionality. This substitution was further inferred from the HMBC interaction of C-6 methine proton ( 6.01) with C-5 (δ 94.8). In the

3 HMBC spectrum, the proton signal at H 4.93 (H-1″′) was J correlated with c 158.9 (C-

3 4′), while the apiose anomeric proton (H 5.54) showed a J connectivity with C-2″′ (c

78.1). This suggested the linkage of apiose at C-2″′ of glucose. This was further deduced from the downfield shift of C-2 of the glucose moiety (c 79.1).

Mass Fragment of Compound 1

The HRFAB-MS (+ve) (see glossary) showed the [M+H]+ at m/z 771.2578, resultant to the formula C38H43O17 (calcd. 773.2500). The EI-MS (see glossary) showed the base peak at

+ m/z 286 (C16H14O5 ).

+. HO OCH3 6''' 4''' O 1''' 3' 5'' OH HO O 5''' 2' 6'' 4'' HO 2''' 4' 3''' O 1 8'' O 1' O 9 O 5' 3'' 4'''' 1'''' 8 1'' OCH3 2 2'' 5'''' HO 6' 7 7'' 3 O HO 3'''' 2'''' 6 4 10 OH 5 + O OH m/z 773.107 (M )

OCH3 OH HO 3HC O H HO O OCH3 HO O O O O C H O+ O 8 8 C H O + m/z 120.06 HO 11 12 4 HO + m/z 208 C22H28O12 OH O m/z 484.14 HO

O HO OH OH HO O OH O HO H HO + C11H20O10 + C9H10O3 OH O m/z 312.27 m/z 166.06

Scheme-5: Mass Fragmentation of compound 1.

The presence of a sinapyl moiety was inferred from the fragment ion at m/z 207

+ + (C11H11O4 ), which underwent further fragmentation to yield an ion m/z 167 (C9H11O3) by the cleavage at C-7″/ C-8″ bond. The fragment ion at m/z 120 was due to acetophenone

cation, resulted from the cleavage of flavanone via rearrangement. The appearance of sugar moieties (apiose and glucose) was inferred from the ion at m/z 311 [M-482] +.

Stereochemical assignment of compound 1

The stereochemistry at C-2 position was assigned to be S, by employing circular dichroism

(CD) technique. In CD spectrum compound 1 showed a (+) Cotton value at 331 nm, and (-

Cotton value at 287 nm (Leu et al., 2004)

Biological Activity

Compound 1 showed significant inhibitory activities against the formation of AGEs

(Advanced Glycation End products) (Table-6). It also exhibited the antioxidant activity

(see glossary), against the superoxide anion.

2.2.2.2 3-(4-Acetoxy-3, 5-dimethoxy) phenyl-2E-propenyl-β-D-glucopyrnoside (2)

The methanolic extract of Viscum album was concentrated under vacuum to obtain a crude methanolic extract (45 g), which was suspended in dist. water (1000 L) and fractionated with EtOAc (3 L × 3). The ethyl acetate extract (1.2 g) was further fractionated by loading on a silica gel column to obtain eight main fractions. Fractions 4 and 5, eluted at 30%

MeOH: CHCl3, were mixed together and loaded on a polyamide column and eluted with

100% CHCl3, followed by gradual increase of polarity with MeOH. This led to the separation of five sub-fractions (1-5). Among them, sub-fraction 4 was rechromatographed by silica gel column chromatography by using 30% MeOH in CHCl3 (1000 mL) as eluent to afford compound 2 (5.5 mg).

The UV spectrum of compound 2 exhibited absorption maxima 242 and 203 nm, representing the occurrence of a phenylpropanoid moiety (Mabry et al., 1975). The IR spectrum showed absorptions band at 3385, 1735, 1660 and 1593 cm-1, which indicated the presence of hydroxy, carbonyl ester and an aromatic ring, respectively.

1 The H-NMR spectrum of 2 displayed signals for two trans olefinic protons [H 6.57 (d,

J7, 8 = 15.0 Hz), and 6.36 (dt, J8, 7 = 15.0 Hz, J8, 9 = 5.5 Hz)], allylic methylene protons [H

4.22 (J9a, 9b = 2.0 Hz, J9, 8 = 5.5 Hz)], and a symmetrically tetra-substituted phenyl ring [H

6.75 (s, 2H), 3.85 (s, OCH3 x 6H)]. A downfield methyl singlet at H 1.88 showed the presence of an acetate moiety.

The 13C-NMR spectrum of compound 2 displayed signals for 19 carbons, including three methyl, two methylene, nine methine, and five quaternary carbons. Along with them, six were characteristic of a glucose unit. A downfield signal in 13C-NMR spectrum at c

180.0, along with a methyl signal at c 30.7, supported the presence of an acetate functionality. Three oxygenated quarternary carbons at c 154.4, 154.3 and 135.3 were due to substituted aromatic carbons.

1 13 The H- and C-NMR spectra of compound 2 showed an anomeric signal (H 4.86, d, J1′,

2′ = 7.2 Hz, c 105.3), recognized for -D-glucopyranosyl moiety.

The preliminary spectral examination indicated that compound 2 has a phenylpropanoid moiety, linked to a glucose moiety.

1 13 Table -3: H- (300 MHz) and C-NMR (100 MHz) Chemical Shift Data of Compound 2 in CD3OD.

Position H (J, Hz) C Multiplicity

1 4.22 63.4 CH2 2 6.36 (dt, 15.0, 5.5) 130.0 CH 3 6.57 (d , 15.0) 110.5 CH 1 - 135.8 C 2 6.75 ( s) 105.4 CH 3 - 154.3 C 4 - 135.3 C 5 - 154.4 C 6 6.75 (s) 105.4 CH

COOCH3 - 180 C

COOCH3 1.88 (s) 30.7 CH3 Glucose C-1′′ 4.86 (d, 7.2) 105.3 CH C-2′′ 3.38 (t, 7.2) 75.6 CH C-3′′ 3.38 (t, 7.8) 77.7 CH C-4′′ 3.41 (dd, 7.0, 4.3) 78.4 CH C-5′′ 3.44 (m) 71.4 CH

C-6′′ 3.82 (11.5, 7.5) 62.4 CH2

Elucidation of Partial Structures

Two-dimensional NMR experiments (COSY-45, HMBC, and HMQC) were used to determine the structure of compound 2. A number of structural fragments were initially derived on the basis of COSY-45and HMQC spectra, and the structure of 2 was finally deduced with the help of HMBC technique.

HMBC H3CO O HMQC

O H

a OCH3

contain a symmetrically tetra-substituted phenyl ring system, was started ״a״ Fragment from the C-2, C-6 aromatic protons, which were resonated at δH 6.74. The upfield chemical shift of the C-2/C-6 protons indicated their proximity with OCH3. The HMQC spectrum was used to determine the direct-heteronuclear couplings between carbon and protons. The C-2/C-6 aromatic protons (δ 6.74) showed direct 1H/13C correlations with

C-2/C-6 (δ 105.4). Long-range heteronuclear interactions between the C-2 proton (δ

6.74) and the oxygen-bearing quarternary carbons, resonated at δ 154.3 (C-3) and 135.8

(C-1), were also observed in the HMBC spectrum. This spectral examination led to the

.״a״ identification of fragment

HMBC H 1 3 OGlc 2 1' H 6'

Fragment ˝b˝

Fragment ˝b˝ consisted of a propanoid moiety, attached with a glycoside. This fragment was deduced from C-3 olefinic proton, resonating at  6.57, and showed trans couplings with C-2 vinylic proton at  6.36 (dt, J2, 3 = 15.0 Hz, J2, 1 = 5.5 Hz). The C-1 methylene protons appeared as a doublet of a double doublet at δ 4.22 and showed couplings with C-

2 olefinic proton. The downfield chemical shift of the C-1 methylenic protons supported the attachment of an O-sugar moiety at C-1. Anomeric proton of glucose moiety appeared as a doublet at  4.86, and was characteristic of -D-glucopyranoside. The HMQC (see glossary) spectrum was used to determine the one-bond heteronuclear couplings between carbons and protons. For example, C-1'' anomeric proton exhibited direct 1H/13C correlation with a carbon resonating at  105.3. In the HMBC spectrum, C-1'' proton also exhibited a long-lange correlation with C-1 methylene carbon of the phenylpropanoid moiety which resonated at  63.4. The 1H-NMR spectrum showed signals for protons of glucose moiety [H-2'' ( 3.38), H-3'' ( 3.38), H-4'' ( 3.41), H-5'' ( 3.44), H-6'' ( 3.82)].

Complete Structure Elucidation by Assembling Sub-structural Fragments.

The HMBC experiment was employed to link different fragments together. The C-2' methine proton ( 6.75) which exhibited HMBC correlations with C-1' ( 135.8), and C-3'

( 154.3). This indicated that the methoxy groups were substituted at C-3' and C-5'. The substitution of the acetate moiety at C-4' was inferred from the downfield chemical shift

( 135.3) of C-4. This was also supported from the HMBC interaction between The C-6'

3 methine proton ( 6.75) showed JCH interactions with the C-4' ( 135.3) and C-5' (

154.4) quaternary carbons. The C-2 trans olefinic methylene proton ( 6.36) showed

HMBC interaction with C-3 (c 110.5). The C-3 methylene protons showed 2J correlations with C-1' ( 135.8) of the phenyl ring.

The attachment of sugar moiety with C-1 methylene carbon was deduced from the

HMBC correlation between the anomeric proton of glucose H-1′' ( 4.86) and C-1 methylene carbon ( 63.4) of the phenylpropanoid moiety. The coupling constant of anomeric C-1′' proton (J1′', 2′' = 7.2 Hz) indicated that the glucose was a β-suger. These

HMBC interactions indicated that the fragment "a" is connected to the fragment "b"

through the C-3/C-1' bonds, while attachment of β-D- glucopyranoside was inferred through the C-1′'/C-1 (fragment b).

Mass Fragment of Compound 2

The HREI-MS of 2 showed the molecular ion at m/z 414.1596, corrsponding to the formula C19H26O10 (calcd. 414.1526). A fragment at m/z 180 (C6H12O6), resulted from the cleavage of the C-1/C-9 bond, and indicate the presence of a glucose unit. The ion at m/z

252 (C13H18O4) characterized the remaining half of the molecule. The fragment at m/z

356 may arose by the loss of acetate on moiety from the molecular ion peak (M+).

Biological Activity

Compound 2 was found to exhibit an antioxidant activity in superoxide anion radical scavenging assay. It also showed protein antiglycation property in vitro, reported for the first time here.

2.2.3 Known Compounds 3-11

2.2.3.1 3-(4-Hydroxy-3, 5-dimethoxy)-phenyl-2-E-propenyl-1 -D–glucopyranoside (3)

Compound 3 was isolated by the same procedure as reported for compound 1 (Section

2.3.3.3., Experimental Section, Page-54). The HRFAB-MS (+ve) of 3 displayed the

+ [M+H] ion at m/z 387.1689 (calcd for C18H27O9, 387.1655). The UV spectrum (MeOH) of compound 3 showed absorptions at 326, 220, 266, and 214 nm. The IR (KBr) spectrum supported the presence of OH (3417 cm-1), and aromatic ring (2953 cm-1).

1 The H-NMR (300 MHz, CD3OD) spectrum of 3 displayed a downfield methyl singlet at

δ 3.84 (6H, s, OMe) for the methoxy group, with the corresponding carbon resonated at δ

56.4. The two chemically equivalent protons were appeared at  6.56 (2H, s, H-2 and H-

6). The C-1 proton appeared at  4.31 (2H, dd, J1a, 1b = 5.2 Hz, J2, 1a 1.5 Hz, H2-1). An anomeric proton signal appeared at  4.87 (d, J1'', 2' = 7.8 Hz, H-1′') was indicated a

βglucose moiety.

The DEPT 13C-NMR (see glossary) spectrum showed resonances for 17 carbons, including two methoxy, nine methine, four quaternary carbons, and two methylene carbons. Among them, five carbon signals were due to a glucose moiety. Three oxygenated quaternary carbons at  149.7, 133.3 and 149.7 were due to substituted aromatic carbons. A downfield signal at δ 149.7 was assigned to C-3′ and C-5′. The sugar carbons were appeared at  104.6 (C-1'′), 75.0 (C-2′′), 78.3 (C-3′′), 72.1 (C-4′′), 78.1 (C-

5′′), and 70.2 (C-6′′).

From these spectroscopic evidences, compound 3 was characterized as a known compound, 3-(4′-hydroxy-3′,5′-dimethoxy)-phenyl-2-E-propenyl-1-D-glucopyranoside (3).

(Greca et al., 1988). It showed a significant inhibitory activity against the formation of

AGEs (Advanced Glycation End Products).

2.2.3.2 5,7-Dimethoxy-4′-O-β–D-glucopyranoside flavanone (4)

Compound 4 was isolated from EtOAc extract as colorless needles (Experimental Section

2.3.3.4, Page 55). The FAB-MS (-ve) showed the [M+H]+ at m/z 463.1663, supporting the formula C23H27O10 (Calcd for C23H27O10 = 463.1604). The UV spectrum showed absorption maxima at 313 and 281 nm. The IR spectrum showed the presence of hydroxy

(3370 cm-1) and carbonyl (1645 cm-1) groups.

1 The H-NMR spectrum of 3 (300 MHz, CD3OD) showed signals for two methoxy groups

at δ 3.78, and 3.84. Doublet at δ 6.20 and 6.22 (each 1H, J = 2.3 Hz) were due to meta coupled protons of the ring-A, while two doublets at δ 7.06 and 7.43 (each 2H, J = 8.7

Hz) were due to the ortho coupled protons of ring B. After the hydrolysis of 3, sugar moeity was identified as D glucose.

The 13C-NMR spectral data of 4 supported twenty three carbons, including two methoxy, two methylene, twelve methine and seven quaternary carbons. Five carbon signals were due to the glucose moiety of 5, 7 -dimethoxy-4-O-D-glucopyranoside flavanone. D-

Glucose was identified by the hydrolysis of 4. A downfield signal at δ 187.7 was due to the carbonyl carbon. The comparison of the spectral data with that reported in the literature showed that the compound 4 is 5, 7-dimethoxy-4-O-D-glucopyranoside flavanone, reported previously from Viscum album (Fukunaga, 1987).

2.2.3.3 5,7-Dimethoxy-4′-hydroxy flavanone (5)

Compound 5 is isolated from the ethyl acetate fraction as a yellow gummy material

(Experimental, Section-2.2.3.5, Page-56). The HRFAB-MS (+ve) of 5 displayed the

+ [M+H] ion at m/z 301.1123, in agreement with C17H17O5 (calcd 301.1076). The UV spectrum (MeOH) of compound 5 showed absorptions at 258 and 313 nm. The IR (KBr) spectrum displayed bands at 3290 (hydroxyl group), 1658 (carbonyl), and 1612, 1585

(C=C double bond) cm-1.

1 The H-NMR (300 MHz, CD3OD) spectrum of 5 was closely resembled with a known compound 5,7-dimethoxy-4′-O-β–D-glucopyranosideflavanone (4). The only difference being the appearance of a hydroxy (OH) moiety in 5. The overall spectroscopic data of the compound 5 indicated it to be a known compound, 5,7-dimethoxy-4′-hydroxy flavanone (5), previously obtained from Viscum album (Tanaka, 1989).

2.2.3.4 4′,5-Dimethoxy-7-hydroxyflavanone (6)

Compound 6 was isolated by the same procedure as described for 5,7-dimethoxy-4′- hydroxyflavanone (5) (Experimental, Section-2.2.3.6, Page-57). The HRFAB-MS (+ve)

+ of 6 displayed the [M+H] ion at m/z 301.1123, in agreement with C17H17O5 (calcd

301.1076). The UV spectrum (MeOH) of compound 6 showed absorptions at 280, 258 and 313 nm. The IR (KBr) spectrum showed absorptions at 3620-3100 (OH), and 1680 cm-1 (C=O).

3' OCH3

2' 4'

HO O 5' 8 1 1' 9 7 2 6' 6 10 3 5 4

OCH3 O 6

The spectroscopic data showed almost simillar values as for compound 5. The substitutions pattern was inferred from HMBC interactions. Finally spectroscopic data led to the identification of 4′, 5-dimethoxy-7-hydroxyflavanone (6), previously isolated from Viscum album (Lam, 1975).

2.2.3.5 7-Hydroxy-8-methoxyflavanone (7)

7-Hydroxy-8-methoxyflavanone (7) was isolated, along with compound 3 from the ethyl acetate extract of the plant (Experimental, Section-2.2.3.7, Page-58). The EI -MS of 7

+ showed the M at m/z 270, corresponding to the formula C15H12O4.

The UV spectrum (MeOH) of compound 7 showed absorptions at 289, and 324 nm. The

IR spectrum showed absorptions at 3447, 3355 (OH), and 1698 cm-1 (C=O).

The 1H-NMR spectrum of 7 showed aromatic protons resonated as a multiplets at  8.11

(5H, m, ring B). The downfield signals at δ 6.58 (1H, d, J6, 5 = 8.6 Hz), and 7.55 (1H, d,

J5, 6 = 8.6 Hz), were assigned to C-6 and C-5 methine protons, respectively. A characteristic signal of flavanone skeleton were appeared at δ 5.62 (J2, 3a = 12.3 Hz, J 2, 3b

= 3.1 Hz) as a doublet of a double doublet, and was assigned to C-2 methine proton. The

C-3 methylene proton appeared at δ 3.10 (1H, dd, J3a, 2 = 4.5 Hz, J3a, 3b = 16.6 Hz) and

2.75 (1H, dd, J3b, 2 = 12.3 Hz, J3b, 3a = 16.6 Hz).

The 13C-NMR spectrum of 7 showed sixteen carbon signals, including one methoxy, eight methine, six quaternary and one methylene carbon. Oxygen was found to be attached at C-1 position, A characteristic signal of flavanone skeleton appeared at δ 44.6 which was assigned to C-2. The methoxy carbon appeared at δ 60.2 (C-8).

The spectral data of 7 led to its identification as 7, 8-dihydroxyflavanone (7), previously obtained from Viscum coloratum (Gaydou et al., 1978).

2.2.3.6 Betulin (8)

Betulin (8) was obtained as colorless crystals (Experimental, Section-2.3.3.8, Page-59).

+ The HREI-MS showed the M at m/z 442.3814, in agreement with formula C30H50O2

(calcd for 442.3810). The IR spectrum showed absorption bands at 1635, 880 (terminal methylene group), 3070, and 3435 cm-1 (OH).

30 20

19 21 H 22 18 12 11 13 17 25 26 H CH2OH 9 14 16 28 1 15 2 10 8 H 27 3 5 7 4 6 HO H 23 24 8

The 1H-NMR spectrum of 8 displayed signals for an isopropylene functionality at δ 4.68 as a multiplet, and a singlet at δ 1.68. Five tertiary methyl groups was appeared at δ 0.87,

0.89, 0.92 and 1.02 (3H each) as singlets. The spectrum showed a downfield doublet of a doublet at δ 3.75 (J3,2a = 10.7 Hz, J3,2b = 4.2 Hz) due to C-3 proton, which indicated a 

(equatorial) configuration of geminal hydroxyl group. Doublets at δ 3.81 and 3.42 (1H each, J 28a, 28b = 11.0 Hz), which were ascribed to the hydroxymethylene group (H-28).

The 13C-NMR spectra revealed the presence of six methyl, twelve methylene, six methine, and six quaternary carbons. The spectral data of 8 was in full agreement with the data reported for betulin (Siddiqui et al., 1988).

2.2.3.7 Gallic Acid (9)

Gallic acid (9) was obtained as a white crystalline solid from the ethyl acetate fraction

(Experimental, Section-2.3.3.9, Page-60). The molecular formula of 9 was deduced from the HREI-MS at m/z 170.0219 (C7H6O5), (calcd 170.0215). The IR spectrum of 9 showed absorptions at 3510 (OH), 1705 (C=O), and 1626 cm-1 (aromatic C=C).

O OH

1 2 6 5 3 4 HO OH

OH 9

The 1H-NMR spectrum of 9 displayed only a singlet in aromatic region at δ 7.03 (2H, H-2,

6) and a carboxylic proton as a broad singlet at δ 11.92 (1H).

The DEPT broad-band decoupled 13C-NMR spectrum showed a total of seven carbon signals, six were assigned to the aromatic ring. The downfield signals at δ 178.5, 146.5, and 139.7 were assigned to acid carbonyl and aromatic oxygenated quaternary carbons, respectively. Whereas other signals in the aromatic region (δ 110.0 and 121.4) were assigned to aromatic methine, and quaternary carbon atoms.

On the basis of above evidences, compound 9 was identified as a well known secondary metabolite, gallic acid (Mehta et al., 1998).

2.2.3.8 β-Sitosterol (10)

The compound 10 was isolated from the ethyl acetate extract of the plant (Experimental,

Secation-2.3.3.10, Page-61). The HREI-MS of 10 showed the M+ at m/z 414.3861 in agreement with the formula C29H50O (calcd 414.3262).

21 29 28 22 18 20 24 H 23 12 27 17 19 11 13 H 16 9 14 15 26 1 2 10 8 H H 3 5 7 4 6 HO 10

1 The H-NMR spectrum of 10 (CDCl3, 400 MHz) was characteristic of a steroidal skeleton.

Two 3H singlets at δ 0.68 and 0.98 were due to the quaternary CH3-18 and CH3-19, respectively. A 3H triplet at δ 0.78 was due to the CH3-29.

The olefinic signal, resonating at δ 5.23 (m), was assigned to H-6. The chemical shift and splitting pattern of the signal integrating for 1H at δ 3.46 was consistent with the H-3of ahydroxy function. Spectraldata of 10 revealed its identification as a well known secondary metabolite, β-sitosterol (Sadikum et al., 1996).

.3 EXPERIMENTAL

2.3.1 General Experimental Conditions

2.3.1.1 Physical Constant

Melting points were determined on a Yanaco MP-S3 apparatus. Optical rotations were

measured on a digital JASCO DIP-360 polarimeter in MeOH by using 5 cm cell.

2.3.1.2 General Methods

UV Spectra were recorded on a Shimadzu UV240 machine in MeOH at max nm (log

). The IR spectra were taken as KBr discs on a JASCO A-302 spectrometer and

presented in cm-1. 1H- (300 MHz) and 13C-NMR (100 MHz) spectra were recorded in

CD3OD solutions on a Bruker AV-500 machine with reference to the residual solvent

proton signal (CD3OD/ CDCl3), and the data is given in  (ppm). 2D NMR spectra were

recorded on a Bruker AMX 500 NMR spectrometer. EI-MS were recorded at 70 eV on a

Finnigan MAT-112 or MAT-312 instruments and main ions are presented as m/z (%).

FAB-MS were taken in glycerol matrix on a JEOL HX-110 mass spectrometer. TLC

Purification was carried out on pre-coated silica gel cards (E. Merck) and the spots were

visualized first under UV light (254 nm), and then sprayed with cerium (IV) sulfate

reagent to develop the colors for different classes of natural products. The recycling high

performance liquid chromatography (HPLC) was used for final purification (JAI, LC-

908W, Japan Analytical Industry Co. Ltd) with a column YMC ODS H-80 or L-80

(YMC, Japan).

2.3.1.3 Purification and Detection of Compounds on Chromatographic Plates

Column chromatography (CC) was performed by using silica gel (E. Merck, type 70-230 and 230-400 M mesh), polyamide, Diaion HP-20 resin and Sephadex LH-20 as absorbent. The compounds were purified by preparative recycling HPLC by using L-80 or H-80 columns (YMC, Co. Ltd). Thin layer chromatography (TLC) was carried out on precoated silica gel TLC (GF 254 of E. Merck). The samples were checked on the TLC card for purity. Separations were examined under the ultraviolet light at 254 nm for fluorescence spots and 366 nm for fluorescence spots. Ceric sulphate was used as the spraying reagent to identify the spots.

2.3.2 Collection of Plant Material

The plant of Viscum album L. was collected from walnut (Juglans regia) trees from

Kundershah, Neelum valley, Azad Kashmir, on 15th of August 2002, and recognized by

Prof. Shafiq-ur-Rehman. A voucher specimen (No: Azbuherb 231) was deposited in the

Herbarium of University of Azad Jammu and Kashmir, Muzaffarabad.

2.3.3 Extraction and Fractionation of Methanol-Chloroform Soluble

Constituents of Viscum album.

Air-dried whole plants of Viscum album (1.5 Kg) were extracted three times with methanol. The resultant methanolic extract was evaporated under vacuum to obtain a crude MeOH fraction (45 g). The MeOH extract was partitioned with EtOAc (3 L × 3) and n-BuOH (3 L × 3), successively to obtain EtOAc (1.2 g), and n-BuOH extracts (19.4 g). The ethyl acetate extract was loaded onto a silica gel column and eluted with chloroform and methanol in a gradient manner to obtain seven fractions (E1a-E7g). further column chromatography of fraction E-2b (100 mg) over silica gel by using the

solvents system CHCl3 and MeOH (10%, 600 mL) yielded compounds 6 (5 mg), and 4 (6 mg). The third fraction E-3c (60 mg) was rechromatographed on a silica gel column and eluted with 20% MeOH-CHCl3, (600 mL) to obtain compound 5 (3 mg).

Fractions E-4d (23 mg) and E-5e (45 mg) were mixed up and loaded to a polyamide column and eluted with 100% CHCl3, followed by gradual raise of polarity with MeOH.

This led to five sub-fractions (1-5). Among these, sub-fraction 4d was again loaded on a silica gel column with 30% MeOH in CHCl3 (1000 mL) to obtain compounds 2 (5.5 mg),

11 (3.5 mg), 7 (6.5 mg), and 8 (5.6 mg), and 2 (5.5 mg), respectively.

A part of butanolic fraction (10 g) was loaded on a Diaion HP-20 column and eluted to obtain three main fractions with 100% H2O (2.7 g), H2O-MeOH (1:1, 3.5 g), and 100%

MeOH (2.0 g). The fraction obtained after elution with H2O-MeOH (1:1) was loaded to polyamide column and eluted with 15% MeOH-CHCl3 which yielded two most important fractions (B1, 1g and B2, 575 mg).

The sub-fraction B1 was again loaded on Sephadex LH-20 column chromatography and eluted with H2O-MeOH (1:1), to obtain 3 main fractions (7-10). Fraction 7 (15 mg) was subjected to RPHPLC (L-80, H2O: MeOH, 1:1, 4 mL/min) to yield compound 1 (3 mg), and 3 (3 mg). Sub fraction B2 (577 mg) was loaded on a polyamide column, and eluted with methanol-chloroform mixture (20-30%).

It was again subjected to column chromatography over HP-20 column with gradients of

MeOH-water. Three main fractions (BVA-1, BVA-2 and BVA-3) were obtained.

The main fraction BVA-2 (3.5 g) was loaded on a polyamide column by elution with 5% and 10% methanol: chloroform to obtain six main sub fraction BVA-4 (45 mg) which was subjected to RPHPLC to obtain compounds 9 and 10.

2.3.3.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone (1)

The methanolic extract of V. album (45 g) was suspended in dist. H2O and extracted with ethyl acetate and n-butanol to afford ethyl acetate and n-butanol fractions. A part of n-BuOH fraction

(10 g) was loaded on Diaion HP-20 column, and eluted with 100% water (2.7 g), water-methanol

(1:1, 3.5 g), and 100% methanol (2.0 g). Fraction obtained after elution with water-methanol (1:1) was loaded to polyamide, and Sephadex LH-20 column subsequently, followed by reverse phase

HPLC (L-80, H2O: MeOH, 1:1, 4 mL/min) to obtain compound 1 (3 mg).

Physical Constant and Spectral Data of 1

State: Yellowish powder

Percentage yield: 3 mg (1.0 x 10-4%)

25 [] D : +8.4 (c= 3.5, MeOH).

UV (MeOH) max (log : 324 (2.75), 284 (3.04), 215 (3.33), 207 (3.35)

−1 IR (KBr) νmax cm : 3412 (OH), 1667, 1605 and 1521 (Aromatic ring)

+ + EI-MS m/z 207 (C11H11O4 ): 311 [M-462] sugar moieties (apiose and glucose)

+ HRFAB-MS (+ve) m/z: [M+H] 771.2578, inagrement with to the formula C38H43O17

(calcd. 773.2500).

1 H-NMR (300 MHz, CD3OD): See Table-3, Page-25.

13 C-NMR (75 MHz, C5D5N): See Table-3, Page-25.

2.3.3.2 3-(4-Acetoxy-3, 5-dimethoxy)-phenyl-2E-propenyl-β-D-glucopyranoside

(2)

The EtOAc fractions was loaded to silica gel column (c.c.) and eluted with chloroform and methanol in a gradient manner to achieve seven sub fractions E1a-E-7h. The fractions E-4d (23 mg) and E-5e (45 mg) were mixed together, and loaded on a polyamide column and eluted with 100% CHCl3, followed by gradual increase of polarity with MeOH. This led to four main sub-fractions (1-4). Sub-fraction 4 was loaded to a silica gel column and eluted with 30% MeOH in CHCl3 (1000 mL) as an eluent to obtain compound 2 (5.5 mg).

Physical Constant and Spectral Data 2

State: White amorphous powder

Percentage yield: (5.5 mg, 2.5 x 10-4%).

UV λmax (MeOH) nm (log ): 262 (3.45), 243 (3.77), 204 (4.05), 187 (4.07)

-1 IR νmax (KBr) cm : 3386 (hydroxy)

HREI-MS m/z: 414.1596 (calcd. for C19H26O10 , 414.1526).

1 H-NMR (300 MHz, CD3OD): See Table-2, Page-34.

13 C-NMR (75 MHz, C5D5N): See Table-2, Page-34.

2.3.3.3 3-(4-Hydroxy-3,5-dimethoxy)-phenyl-2-E-propenyl-1-D– glucopyranoside (3)

Ethyl acetate soluble portion was loaded to a silica gel column to obtain seven fractions

E1a- E-7g. The fraction E-4d (23 mg) and E-5e (45 mg) were combined, loaded on a polyamide column chromatography and eluted with 100% CHCl3, followed by gradual increase of polarity with MeOH to obtain four main sub-fractions (1-4). Among these, sub-fraction 4 was loaded on a silica gel column chromatography by using 30% MeOH in

CHCl3 (1000 mL) as an eluent to afford compound 3 (10 mg).

Physical Constant and Spectral Data

State: White powder

Percentage yield: (10 mg, 3.33 x 10-4%)

-1 IR νmax (KBr) cm : 3417 (OH), 2953 (CH2)

+ HRFAB-MS (+ve) m/z: [M+H] 387.1689 (calcd for C18H27O9, 387.1655).

1 H-NMR (300 MHz, CD3OD): H 6.57 (2H, s, H-2 and H-6), 6.37 (1H, d, J3, 2 = 16.0

Hz, H-3), 5.80 (1H, dt, J3, 2 = 16.0 Hz, J2, 1 = 5.2 Hz, H-2), 4.23 (2H, dd, J1, 2 = 5.2 Hz , J

1, 2 = 1.5 Hz, H-1), 3.85 (6H, s, OMe), 4.86 (1H, d, J1’’, 2’’ = 7.8 Hz, H-1′′), 3.65 (1H, m,

H-2′′), 3.46 (1H, m, H-3′′), 3.40 (1H, m, H-4′′), 3.25 (1H, m, H-5′′), 3.68 (1H, dd, J6′a, 5’ =

10.5 Hz , 6.7 Hz, H-6′′), 3.70 (1H, dd, J6’’a, 6’’b = 10.5 Hz , J = 2.1 Hz, H-6′′).

13 C-NMR (75 MHz, C5D5N): δC 76.8(C-2), 45.6 (C-3), 188.7 (C-4), 164.0 (C-5), 92.7 (C-

117.1 ,(׳C-2) 128.8 ,(׳C-7), 94.6 (C-8), 162.7 (C-9), 106.3 (C-10), 133.0 (C-1) 166.2 ,(6

.(׳C-6) 126.8 ,(׳C-5) 117.1 ,(׳C-4) 156.3 ,(׳C-3)

.(O--D-glucopyranoside (4-׳5,7-Dimethoxyflavanone -4 2.3.3.4

The ethyl acetate fraction was loaded on silica gel column, and eluted with chloroform and methanol (10% methanol-chloroform, 20% methanol-chloroform, 30% methanol- chloroform, 50% methanol-chloroform, and 100% methanol) to obtain six fractions, E1a-

E-6f. Repeated column chromatography of fraction E-2b (100 mg) over silica gel with the solvent system chloroform-methanol (10%, 600 mL) resulted in the isolation of compounds 4 (6 mg).

Physical Constant and Spectral Data

State: White crystal m.p.: 184-187 ◦C

Percentage yield: 6 mg (2.0 x 10-4%)

-1 IR νmax (KBr) cm : 3370 (OH), 1645 (C=O)

HRFAB-MS (+ve) m/z: 463.1663, C23H27O10 (Calcd for C23H27O10 = 463.1604).

1 H-NMR (300 MHz, CD3OD): H 5.38 (1H, dd, J2, 3a = 12.5 Hz, J2, 3b = 2.8 Hz, H-2),

2.52(1H, dd, J3a, 2 = 16.3 Hz, J2a, 2b = 2.8 Hz, H-3), 3.16 (1H, dd, J3a, 2 = 16.3 Hz, J3b, 2 =

׳ ׳ 12.5 Hz, H-3), 6.10 (2H, d, J6, 8 = 2.3 Hz, H-6), 7.05 (1H, d, J2, 6 = 8.7 Hz, H-2, -3 ), 7.42

׳ ׳ (1H, d, J3, 5 = 8.7 Hz, J = 8.7 Hz, H-5 , H-6 ).

13 C-NMR (75 MHz, C5D5N): δC 128.0 (C, C-1), 104.4 (C-2 and C-6), 150.5 (C-3 and C-

5), 134.2 (C-4), 133.41 (C-7), 127.0 (C-8), 71.0 (C-9), 55.0 (OMe), 107.7 (C-1), 76.0 (C-

2′), 79.3 (C-3′), 73.1 (C-4′), 77.1 (C-5′), 71.2 (C-6′).

2.3.3.5 5,7-Dimethoxy-4′-hydroxyflavanone (5)

Compound 5 was isolated from E-2b (100 mg) over silica gel by using the solvent system

CHCl3 and MeOH (10%, 600 mL) which yielded seven fractions (E1a-E-7g). The third fraction E-3c (60 mg) was rechromatographed over a silica gel column by using different solvents, and finally compound 5 (3 mg) was purified with 20% MeOH-CHCl3 (600 mL).

Physical Constant and Spectral Data

State: White powder

Percentage yield: (3 mg, 1.0 x 10-4%)

25 [] D : -65.8 (c= 0.09 MeOH)

-1 IR νmax (KBr) cm : 3290 (OH), 1658 (C=O), 1612, 1585 (C=C)

EI-MS(ve) m/z : 300 (M+), 268 (100), 167 (50)

+ HRFAB-MS (+ve): [M+H] 301.1123, in agreement with C17H17O5 (calcd 301.1076)

1 H-NMR (C5D5N): H 2.56 (1H, dd, J3a, 2 = 3.0 Hz, J3a, 3b = 16.0 Hz, H-3a), 2.80 (1H, dd,

J3b, 2 = 12.0 Hz, J3b, 3a = 16.0 Hz, H-3b), 5.63 (1H, dd, J2, 3a = 3.0 Hz, J2, 3b= 12.5 Hz, H-

2), 3.80 (3H, s, 5-OMe), 3.66 (3H, s, 4′-OMe),

13 C-NMR (C5D5N): δC 187.6 (C-4), 165.6 (C-7), 166.9 (C-9), 164.9 (C-5), 160.5 (C-4′),

133.3 (C-1′), 127.7 (C-2′/6′), 115.9 (C-3′/5′), 107.2 (C-10), 98.3 (C-8), 97.0 (C-6), 55.3

(5-OMe), 56.7 (4′-OMe), 78.7 (C-2), 44.6 (C-3).

2.3.3.6 4′,5-Dimethoxy-7-hydroxyflavanone (6)

Compound 6 (5 mg) was purified from fraction E-2b (100 mg) by repeated CC of sub fraction E-2 (silica gel) with the solvent system chloroform-methanol (10%, 600 mL) compounds 6.

Physical Constant and Spectral Data

State: Yellow powder

Percentage Yield: (5 mg, 1.6 x 10-4%)

25 [] D : +7.0 (c= 0.46, MeOH):

-1 IR νmax (KBr) cm : 3630-3100 (hydoxyl group), 1655 (carbonyl moiety)

EI-MS m/z : 300 (M+), 287 (17), 256 (19).

+ HRFAB-MS (+ve) m/z: [M+H] 301.1123, in agreement with C17H17O5 (calcd 301.1076).

1 H-NMR (C5D5N): H 7.52 (2H, d, J2′, 3′ = 8.6 Hz, H-2′/6′), 7.01 (2H, d, J5′, 6′ = 8.6 Hz, H-

3′/5′), 6.52 (1H, d, J8, 6 = 2.0 Hz, H-8), 6.47 (1H, d, J6, 8 = 2.0 Hz, H-6), 5.52 (1H, dd, J2, 3a

= 12.8 Hz, 2.8 Hz, H-2), 3.81 (3H, s, 5-OMe), 3.67 (3H, s, 4′-OMe), 3.22 (1H, dd, J3, 2 =

16.2 Hz, J3a, 3b = 12.8 Hz, H-3ax), 2.92 (1H, dd, J3b, 2 = 16.2 Hz, J3b, 3a = 2.8 Hz, H-3eq).

13 C-NMR (C5D5N): δC 193.3 (C-4), 159.1 (C-7), 158.9 (C-9), 142.1 (C-5), 130.4 (C-4′),

135.0 (C-1′), 130.0 (C-2′/6′), 127.6 (C-3′/5′), 137,0 (C-10), 100.6 (C-8), 111.9 (C-6), 56.3

(5-OMe), 55.7 (4′-OMe), 79.7 (C-2), 44.9 (C-3).

2.3.3.7 7-Hydroxy, 8-methoxy flavanone (7)

7-Hydroxy,8-methoxyflavanone (7) was isolated along with compound 3 from the ethyl acetate extract of the plant

Physical Constant and Spectral Data

State: Pale yellow powder;

Percentage yield: (6.5 mg, 2.11 x 10-4%)

25 [] D : +7.0 (c= 0.46, MeOH):

-1 IR νmax (KBr) cm : 3620-3100 (OH), 1658 (C=O)

UV λmax (MeOH) nm (log ): 289 (4.24) 324 (3.73)

FAB-MS (-ve) m/z : 269

HRFAB-MS (+ve) m/z: 271.0920 (Calcd for C15H12O4, 270.0921)

1 H-NMR (400 MHz, DMSO): H 7.37 (5H, m, proton of ring B), 6.58 (1H, d, J6, 5 = 8.5

Hz, H-6), 7.55 (1H, d, J5, 6 = 8.5 Hz, H-5), 5.62 (1H, dd, J2, 3a = 12.3 Hz, J2, 3b = 4.5 Hz,

H-2), 3.71 (3H, s, OMe), 2.75 (1H, dd, J3a, 2 = 12.3 Hz, J3a, 3b = 16.6 Hz, H-3a), 3.10 (1H, dd, J3b, 2 = 4.5 Hz, J3b, 3a = 16.6 Hz, H-3b).

13 C-NMR (100 MHz, DMSO): δC 189.89 (C=O, C-4), 145.7 (C-9), 139.0 (C-1′),137.3

(C-7), 135.20 (C-8), 128.5 (C-2′/C-6′), 126.3 (C-3′/5′), 128.3 (C-4′), 126.8 (C-5), 112.6

(C-10), 114.8 (C-6), 79.12 (C-2), 60.2 (OCH3), 44.4 (C-3).

2.3.3.8 Betuline (8)

Compound 8 was obtained along with compound 3 from a part of n-BuOH fraction extract of the plant

Physical Constant and Spectral Data

State: White colorless powder

Percentage yield: (6.5 mg, 2.11 x 10-4%)

25 [] D : 20.4 (c = 0.42, MeOH)

-1 IR νmax (KBr) cm : 3435, 3070, 1635, 880

EI-MS m/z: [M]+ 442 (14), 424 (100), 406 (14), 218 (22), 206 (28), 205 (11).

HREI-MS m/z: 442.3814 (calcd. for C30H50O2 , 442.3810).

1 H-NMR (400 MHz, DMSO-d6): H 4.68 (2H, m, Hz, H-29), 3.75 (1H, dd, Jax, ax = 10.7

Jax, eq = 4.2 Hz, H-3), 3.81, 3.42 (1H, each, d, Jgem = 11.0 Hz, H-28), 1.68 (3H, br s, CH3-

30), 0.98 (3H, s, CH3-27), 0.92 (3H, s, CH3-24), 0.89 (3H, s, CH3-23), 0.89 (3H, s, CH3-

25).

13 C-NMR (100 MHz, DMSO-d6): δC 150.6 (C-20), 109.6 (C-29), 78.9 (C-3), 60.4 (C-28),

55.2 (C-5), 50.4 (C-1), 48.7 (C-18), 47.9 (C-19), 47.9 (C-17), 42.8 (C-14), 40.8 (C-8),

38.8 (C-4), 38.6 (C-1), 37.4 (C-13), 37.2 (C-10), 34.2 (C-7), 33.9 (C-22), 29.8 (C-21),

29.1 (C-16), 28.2 (C-23), 27.4 (C-2), 27.1 (C-15), 25.2 (C-12), 20.8 (C-11), 19.1 (C-30),

18.3 (C-6), 16.1 (C-25), 16.0 (C-26), 15.3 (C-24), 14.7 (C-27).

2.3.3.9 Gallic Acid (9)

Compound 9 was isolated from the BuOH sub-fractions PVAB-2 (3.5 g) which was

loaded on polyamide column by using 100% chloroform (1 g, fr. 1-6), and methanol-

chloroform (5%-30%) mixtures as eluent. The sub-fraction PVAB-4 PVAB-4 (45 mg)

was purified by using recycling preparative HPLC to obtain 9 (10 mg).

Physical Constant and Spectral Data

State: Crystalline solid

Percentage yield: (8.5 mg, 4.11 x 10-4%)

-1 IR νmax (KBr) cm : 3510, 3320 (OH), 1705 (C=O), 1626 (Ar. C=C)

UV λmax (MeOH) nm (log ): 275 (3.77)

HREI-MS m/z: 170.0219 (C7H6O5), (calcd 170.0215).

1 H-NMR (400 MHz, DMSO-d6): H 11.92 (1H, s, carboxylic O-H), 7.03 (2H, s, H-2,6),

13 C-NMR (100 MHz, DMSO-d6): δC 178.5 (C-7), 146.5 (C-3, 5), 139.7 (C-4), 121.4 (C-

1), 110.0 (C-2, C-6)

2.2.3.10 -Sitosterol (10)

The ethanolic extract of V. album was loaded on silica gel column and several oily

fractions (E-4, E-5) were obtained on elution with chloroform and hexane (20%). These

sub-fractions were combined and further loaded on a polyamide column, and eluted with

100% CHCl3 to obtain five sub fractions. Sub fraction 4 was further purified by using

silica gel column and 100% CHCl3 eluent to obtain a known compound, β-sitosterol (10).

Physical Constant and Spectral Data

State: White amorphous powder

Percentage yield: (7.5 mg, 2.11 x 10-4%)

-1 IR νmax (CHCl3) cm : 3402 (OH), 2901 (C-H),1641 (C=C)

UV λmax (MeOH) nm (log ): 208 (2.416)

EI-MS m/z [M]+ : 414 (100), 398 (40)

HREI-MS m/z: 414.3861 (C29H50O, calcd 414.3262).

1 H-NMR (400 MHz, DMSO-d6): H 0.68 (3H s, H-18), 0.98 (3H, s, H-19), 1.00 (d, J21a, 22

= 6.5 Hz, H-21), 5.23 (br. s, H-6), 3.46 (m, H-3).

SECTION-A

3.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM

VISCUM ALBUM L.

3.1 ANTIOXIDANT ACTIVITY

The antioxidant is a substance that prevents the utilization of molecular oxygen. An antioxidant inhibits or decrease the rate of oxidation. All living organisms maintain a reducing environment inside their cells, and all cells are equipped with complex systems of antioxidants to protect the chemical damage to the cells, components by oxidation.

These antioxidants contain glutathione and ascorbic acid, and are substrates for enzymes such as peroxidases, and dismutases.

Antioxidants are generally used as ingredients in dietry supplements to protect from cancer, and heart diseases [Matill et al., 1947]. Antioxidants are molecules which can safely interact with the free radicals and terminate the chain reaction before vital biomolecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient (vitamin) antioxidants are vitamin

E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal, is also required for proper functioning of the body's antioxidant enzyme systems. The body cannot manufacture these micronutrients and thus must be supplied in the diet.

Free radicals are unstable compounds in the body that will "do anything" to become stable. In the process, they make the other biomolecules unstable, and thus cause tissue damage, which must be repaired to maintain the normal health and functioning of the body. Free radical reactions are involved in the pathogenesis of many human diseases,

including cancers, cardiovascular diseases, immune system decline, aging process, inflammation, diabetes, immunodepression, neurodegenerative conditions, cancer, diabetes, arthritis and other diseases. Reactive Oxygen Species (ROS) such as superoxide

• 1 (O 2), hydrogen peroxide (H2O2), hydroxyl radicals (OH) or singlet oxygen ( O2), are products of normal metabolism and are capable of attacking biological molecules leading to cell or tissue injury.

ROS Play an important role in on set and pregression of a number of diseases, such as cancer, diabetes, artherosclerosis and chronic inflammation. In diabetic conditions, different sources of ROS are invoved, like xanthine oxidases, NADPH oxidases, uncoupling of nitric oxide synthase (eNOS) and mitochondrial fuel oxidative processes.

In modern drug discovery, program discovery of antioxidant, which can act against superoxides, has a special significance. The potency of a sample to scavenge NO• radicals, in a chemical based in-vitro assay, is measured to study the potential of subject compound for scavenging property.

3.1.2 Results and Discussion

The antioxidant activity were performed by Mr. Sajjad Ali in the Antioxidant Research

Lab. of the H. E. J. Research Institute of Chemistry (International Center for Chemical and Biological Sciences), University of Karachi. The assays were employed to determine the antioxidant activity of compounds 2 and 5. Compounds 2 and 5, isolated from n-

BuOH extract, were screened for superoxide anion scavenging activity. The antioxidant activity of compounds 1-6 was evaluated by using a superoxide anion-scavenging assay.

The antioxidant potential of 5, 7-dimethoxy-4′-hydroxyflavanone (5) was determined to

be more significant than the standard n-propylgallate (Table-4), but only 3-(4-acetoxy-3,

5-dimethoxy) phenyl-2E-propenyl-β-D-glucopyrnoside (2) exhibited the strongest activities.

3.1.3 Methodology

In Vitro Superoxide Anion Scavenging Assay:

The superoxide anion radicals are produced by using phenazine methosulfate (PMS),

NADH system, in the presence of molecular oxygen (air). The production of superoxide was determined by adding the nitroblue tetrazolium salt to the reaction mixture. The protocol used here was based on the method reported by Ferda, 2003. In the presence of

- air, PMS is reduced by NADH, which in turn reduces dioxygen to O2 .

Each reaction well contain 40 L of100 nicotinamide adenine dinucleotide reduced form NADH, 40 L of 80 nitro blue tetrazolium (NBT), 20L

8phenazine methosulphate (PMS)L of 1 mM sample and 90 L of 0.1 M phosphate buffer (pH 7.4). The reagents were dissolved in buffer and test sample in

DMSO. The experiment was carried out in 96-well microtitre plate at room temperature.

The absorbance was measured at 560 nm. The production of superoxide was estimated by measuring the formation of blue Formazan dye. Absorbance is inversely related with the scavenging activity of sample. Lower the absorbance, higher the antioxidant activity of sample. The Radical Scavenging Activity (% RSA) by samples was calculated by the comparison of a control:

% RSA = 100 – (OD Test compound / OD control) x 100

Table-4: Superoxide anion scavenging activity of compounds of V. album.

Compounds (%) Inhibition IC50 ( µM )± sem

2 7 4.75% 211.7 ± 7.0

5 95.40% 58.5 ± 3.0

n Propylgallateb 85.9% 67.5 ± 1.0

b Standard ± SEM  = Results are reported in ± standard error of mean of three experiments

3.2 ANTI-GLYCATION ASSAY

3.2.1 Introduction

Glycation of protein is a spontaneous non-enzymatic reaction of amino groups of protein with carbonyl group of glucose and different reducing sugars [Rahbar et al., 2003]. A

French biochemist, Louis Comalle Millard, in 1912 described the process of protein glycation which is known as Millard’s reaction. This reaction is also known as browning reaction due to the generation of brown color products called Advanced Glycation End products (AGEs), which are fluorescent materials. Presences of reactive oxygen species

(ROS) are responsible of further complications due to the formation of degradetive and rearranged products.

The protein glycation play a vital role in the progress of various diseases related in the aging, and pathogenic age realated disorders. Diabetic complications such as retinopathy, neuropathy and cataract formation, atherosclerosis, last stage renal diseases, rheumatoid arthritis and neurodegenerative diseases are caused as a result of protein glycation.

Previously various attempts have been made to pharmacologically influence the process of protein glycation by preventing or slowing down the formation of AGEs (Ahmed et al., 2005). Hyperglycemia, a clinical hallmark of poorly controlled diabetes, is actually responsible for the formation of AGEs. Therefore protein glycation is also considered to be an important therapeutic target for the treatment of diabetic complications.

Non-enzymatic glycation of proteins is a process that impairs the functioning of various biomolecules, leads to a variety of endocrine disorders, and generates advanced glycation endproducts (AGEs) in the body. AGEs predispose a person to chronic difficulty such as type-II diabetes, early aging and Alzheimer’s disease. Free radicals are also very important in the formation of AGEs. To control the progression of protein glycation is therefore a significant therapeutic approach towards the treatment of protection against diabetic complications.

Antiglycation and antioxidant properties of natural and synthetic products have attracted considerable scientific interest. The reduced toxicity and enhanced antiglycation activities are highly desirable. The screening of compounds of both natural and synthetic origins against a variety of biological target has emerged as a practical approach for new drug developments. The antiglycation assays play an important role in the discovery of new candidates for the treatment of delayed diabetic difficulties.

3.2.2 Results and Discussion

The antiglycation activity of different secondary metabolites were evalvated by a colleague Miss. Ambreen Khan at the Antiglycation Research Lab. of the H. E. J.

Research Institute of Chemistry (International Center for Chemical and Biological

Sciences), University of Karachi. The methanolic extract of V. album showed a significant anti-glycation potential with 72.5% inhibition (IC50 = 199.85 ± 0.067 µM).

Compounds 1-6 showed significant inhibitory activities as compared to the standard inhibitor, rutin, against the formation of AGEs (Table-6). In conclusion, anti-glycation properties of the plant phenolics from V. album is reported here for the first time.

Table-5: Anti-glycation activity of various extracts of V. album.

Samples ( % ) Inhibition IC50 (µM ) ± SEM

1 Methanolic extract 72.5 % 199.8 ± 0.1 2 Ethyl acetate extract 66.5 % 270.7 ± 0.4

3 Butanolic extract 61.45 % 689.4 ± 0.5 4 Rutin a 85.9% 67.5 ± 0.9

a Standard ± SEM  = Results are reported in ± standard error of mean of three experiments

Table-6: Anti-glycation activity of compounds of V. album.

Compounds (% ) Inhibition IC50 (µM ) ± SEM 1 74.50% 264.5 ± 0.9 2 71.36% 255.4 ± 0.5 3 72.92% 413.9 ± 0.5 4 71.4% 345.6 ± 0.8 5 74.62% 264.5 ± 0.7 6 73.80% 405.8 ± 0.8 Rutin a 85.90% 67.5 ± 0.9

a Standard ± SEM  = Results are reported in ± standard error of mean of three experiment

3.2.3 Methodology

Material and Methods

The anti-glycation assays on different secondary metabolites of the plant V. album were carried out by the most recent method of McPherson.

Chemicals: Bovine Serum Albumin (BSA) was purchased from Research Organics

(Cleveland, USA), while other reagents, standard glucose, trichloroacetic acid (TCA)

sodium azide (NaN3), dimethyl sulfoxide (DMSO), sodium dihydrogen phosphate

(NaH2PO4), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), and sodium hydroxide (NaOH) were purchased from Sigma Aldrich. Sodium phosphate buffer (pH

7.4) was prepared by mixing Na2HPO4 and NaH2PO4 (67 mM) containing sodium azide

(3 mM). Phosphate buffer saline (PBS) was prepared by mixing NaCl (137 mM),

Na2HPO4 (8.1 mM), KCl (2.68 mM) and KH2PO4 (1.47 mM), and pH 10 was adjusted with NaOH (0.25 mM). BSA (10 mg/mL) and anhydrous glucoses (50 mg/mL) solutions were prepared in sodium phosphate buffer. Test samples were dissolved in DMSO (1 mM).

Preparation of Sample: Samples were dissolved in DMSO, for crude extract (2 mg/mL) and pure component (1000 μM).

In Vitro Glycation Assay: The sample was prepared in a flat bottom 96-well plate, each well contained 60 µL reaction mixtures (20 µl BSA (10 mg/mL), 20 µL of anhydrous glucose (50 mg/mL and 20 µL examination sample). Glycated control sample contained

20 L BSA, 20 L glucose and 20 L sodium phosphate buffer, whereas control

contained 20 L BSA and 40 L sodium phosphate buffer and then incubated at 37  C for 7 days. After the competion of the incubation process, the sample dish was placed out at normal temperature and 6 µL TCA 100% were mixed very well and then centrifuged

(15,000 rpm) for four minutes at 4 °C. After centrifugation, the plate with 60 µL 5%

TCA. The supernatants including glucose, test compounds and inquisitive material were discarded and pellet, containing AGE-BSA, were dissolved in 60 L PBS. Fluorescence spectrum (ex. 370 nm) and variation in fluorescence intensity (exitation 370 nm to emission 440 nm), was measured to monitor the AGEs formation, were monitored by using spectrofluorimeter RF-1500 (Shimadzu, Japan).

Percent inhibition was determined by following formula:

Percent inhibition calculation = 100 - [(Fluorescence of sample) X 100] Fluorescence of glycated protein

PART-B

4.0 PHYTOCHEMICAL STUDIES ON SECONDARY METABOLITES FROM WITHANIA COAGULANS DUN. (STOCK.)

4.1 INTRODUCTION

4.1.1 The Family Solanaceae

Solanaceae is a large family of herbs or shrubs or trees, often strongly scented and sometimes narcotic or poisonous. Spread all over the drier areas of India and Pakistan.

This is a big family (Chadha, 1976) with approximately 2,000-3,000 species in 90 different genera, originated from most temperate, and tropical areas with a large number found in Australia, and Central and South America. The calyx is synsepalous, ranging from tubular to deeply cleft. The corolla is sympetalous and ranges from a short tube to quite long, reflexed lobes to forms with a long tube and short lobes. The flowers are bisexual and actinomorphic or only a little zygomorphic. Many well-known food plants, including potatoes, tomatoes, aubergines, and peppers, belong to this family. There are quite a few poisonous species, as well as Deadly Nightshade (Atropa belladonna),

Henbane (Hyoscyamus niger), and Thorn Apple (Datura stramonium). This family also include many other important economic plants, such as tobacco (Nicotiana tabacum), which contains the highly toxic alkaloid, nicotine (Ebadi, 2002).

4.1.2 The Genus Withania

Withania coagulans belongs to family Solanaceae. It is a small genus of without arms shrubs, which grow between the Mediterranean regions to South Asia. Two species are found in Pakistan, Withania coagulans, and W. somnifera. It is also found in Afghanistan and East India. Withania has the property of coagulating milk (Watt et al., 1972), (Atal et al., 1961). The major ingredients of Withania berries are free amino acids, fatty oils, and

withanolides (Atal et al., 1963). The fruits are sweet and are reported to be sedative,

emetic, alterative, and diuretic (Garg et al., 1967). W. coagulans is a 60-120 cm high

plant which occurs in the drier parts of the Punjab province (Pakistan). Leaves of the

plants are generally lanecolate-oblong, the berriers are globose red or brownish in color.

The seeds of plant are dark brown ear-shaped. The pulp is brown in color and fruity in

odor (Sathi, 1970). Flowering of the plants is in November to April, while the berries

mature during January-May.

4.1.3 Previous Phytochemical Studies on Genus Withania

Previous phytochemical work on Withania coagulans has resulted in the isolation of

thirty nine withanolides and derivatives (Table-7, Page-73).

Table- 7: The summary of results of previous phytochemical studies on Withania coagulans.

S. No. Name Molecular Molecular Source Refrence Weight Formula 1 Ergosta-5,25-diene-3,24-diol 414.67 C28H46O5 W. coagulans Velde, V.V. et al., Phytochemistry, 1983, 22, 2253-2257. 2 20-Hydroxy-1-oxowitha- 438.606 C28H38O7 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 2003, 63, 387-390. 2,5,24-trienolide

3 14,20-Epoxy-17-hydroxy-1- 452.589 C28H36O5 W. coagulans Atta-ur-Rahman et al., Phytochemistry, oxowitha-3,5,24-trienolide 2003, 63, 387-390.

4 Withacoagulin 452.589 C28H36O5 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 2003, 63, 387-390.

5 Withacoagin 454.605 C28H38O5 W. coagulans Neogi, P. et al., Bull. Chem. Soc. Jpn., 1988, 61, 4479.

6 Coagulin F 452.589 C28H36O5 W. coagulans Atta-ur-Rahman et al., J. Nat. Prod., 1998, 61, 812-814.

7 Coagulin G 468.589 C28H36O6 W. coagulans Atta-ur-Rahman et al., J. Nat. Prod., 1998, 61, 812-814.

8 Withanolide A 470.605 C28H38O6 W. coagulans Haensel, R. et al., Arch. Pharm. 1975, 308, 653-654.

9 Coagulin R 470.605 C28H38O6 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 1999, 52, 1361-1364.

10 Coagulin J 470.605 C28H38O6 W. coagulans Atta-ur-Rahman et al., Heterocycles, 1998,

48, 1801-1811.

11 6,7-Epoxy-5,27-dihydroxy-1- 470.605 C28H38O6 W. coagulans Haensel, R. et al., Arch. Pharm. 1975, 308, 653-654. oxowitha-2,24-dienolide.27-

Hydroxywithanolide B

12 17-Hydroxywithanolide K 470.605 C28H38O6 W. coagulans Choudhary, M. I. et al., Phytochemistry, 1995, 40, 1243-1246.

13 3,20R,22R,27-Trihydroxy-1- 470.605 C28H38O6 W. coagulans Atta-ur-Rahman, et al., phytochemistry, oxowitha-5,14,24-trienolide 1999, 52, 1361-1364.

14 Coagulanolide 486.604 C28H38O7 W. coagulans Maurya, R. et al., Bioorg. Med. Chem. Lett., 2008, 18, 6534-6537

15 Coagulin I 486.604 C28H38O7 W. coagulans Atta-ur-Rahman et al., Heterocycles, 1998, 48, 1801-1811.

16 Coagulin M 488.62 C28H40O7 W. coagulans Atta-ur-Rahman et al., Chem. Pharm. Bull., 1998, 46, 1853-1856.

17 3,14,20,27-Tetrahydroxy-1- 488.62 C28H40O7 W. coagulans Ramaiah, P.A. et al., Phytochemistry, 1984, 23, 143-149. oxo-5,24-withadienolide

18 Coagulin M 488.62 C28H40O7 W. coagulans Atta-ur-Rahman et al., Chem. Pharm. Bull., 1998, 46, 1853-1856

19 3,14,20R,22R-Tetrahydroxy-1- 488.62 C28H40O7 W. coagulans Ramaiah, P. A. et al., Phytochemistry, 1984, 23, 143-149. oxo-5,24-withadienolide

20 3,14,17,20R,22R - 488.62 C28H40O7 W. coagulans Velde, V. V. et al., Phytochemistry, 1983, 22, 2253. Tetrahydroxy-1-oxo-5,24-

withadienolide

21 Coagulanolide 486.604 C28H38O7 W. coagulans Maurya, R. et al., Bioorg. Med. Chem. Lett., 2008, 18, 6534-6537.

22 Coagulin S 538.634 C28H42O10 W. coagulans Nur-e-Alam, M. et al., Helv. Chim. Acta, 2003, 86, 607-614.

23 Coagulin H 520.619 C28H40O9 W.coagulans Atta-ur-Rahman et al., Heterocycles, 1998, 48, 1801-1811

24 Coagulin K 616.747 C34H48O10 W. coagulans Atta-ur-Rahman et al., Heterocycles, 1998, 48, 1801-1811.

25 Coagulin Q 620.779 C34H52O10 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 1999, 52, 1361-1364

26 Coagulin Q 620.779 C34H52O10 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 1999, 52, 1361-1364.

27 Coagulin P 632.747 C34H48O11 W. coagulans Atta-ur-Rahman et al., Phytochemistry, 1999, 52, 1361-1364

28 Coagulin P 632.747 C34H48O11 W. coagulans Atta-ur-Rahman, et al.,Phytochemistry, 1999, 52, 1361-1364.

29 Coagulin O 634.762 C34H50O11 W. coagulans Atta-ur-Rahman et al., Chem. Pharm. Bull., 1998, 46, 1853-1856

30 Coagulin N 648.746 C34H48O12 W. coagulans Atta-ur-Rahman et al., Chem. Pharm. Bull., 1998, 46, 1853-1856.

31 Coagulin L 650.762 C34H50O12 W. coagulans Atta-ur-Rahman et al., Heterocycles, 1998, 48, 1801-1811.

4.1.4 Major Secondary Metabolites of the Plant: Withanolides

Withanolides are steroidal lactones, derived from an ergostane-type skeleton in which C-

22 and C-26 are properly oxidized in order to from a six-membered lactone ring. The basic structure 30 comprise on withanolide skeleton (Lavia et al., 1968). The structure 30 has been generally named as withanolide, although in the chemical abstract (CA), withanolide are named as ergostan-22-hydroxyeorgastan-26-oic acid -26, 22-lactone.

The name ergostan-26, 22-olide is based on IUPAC rules, majority of the withanolides isolated so far are 2-ene-1-ones, and have unsaturated lactone side–chain. A number of compounds, such as 31 have saturated ring A with C-1-OH instead of the resultant ketone.

Certain compounds contain saturated (Kirson et al., 1970 and Glotter et al., 1973) or lower oxidation level 22-26-lactol (Barata et al., 1970), instead of lactones side chain.

Analysis has also led to the discovery of various biogenetically related compounds in which either the carbocyclic skeleton, or the side chain, or both are modified. Among them are 13, 14-seco derivatives and ring D aromatic compounds. Bicyclic structures and epoxylactols in the side chain are important. Majority of these unmodified and modified ergostans derivatives are produced by Solanaceae plant, belonging to the genera

Withania, Acnistus (Dunalia), Physalis, Jaborosa, Datura, and Lycium, although withanolides have also been isolated from some marine sources.

4.1.4.1.1 Biosynthesis of Withanolides

The chemical constituents of Withania coagulans include steroidal lactones called withanolides. The biosynthetic precursor of all the steroids and terpenoids, including cholesterol, is the C6-compounds called 3R-(+)-mavalonic (32) acid. The details study about the biosynthetic pathway of withanolides showed that the key precursor of withanolides is 24-methylenecholestrol (Lockley et al., 1974). A number of publications

reported this precursor. Various steps are involved in the biosynthetic conversion of mavalonic acid to 24-methylenecholestrol via squalene (33), protosterol (34), and lanosterol (35), (Stone et al., 1969; Cornforth et al., 1965; Cornforth et al., 1966; Poulter et al., 1979; Jacob et al., 1983; Koyama et al., 1980).

HR H3C OH SH HOOC OH HS SH RH HR 3R-Mavalonic acid (32)

H H

Squalene (33)

Me H Me R H

HO Me Me

H R H Me Me

Protosterol (34)

Me H Me Me Me Me Me

HO H

Me Me H Lanosterol (35)

Two biogenetic processes have been identified to explain the biosynthesis of withanolides, one relates to the development of lactone containing side chain, and the second to the substitution pattern of rings A, and B. These sequences were discovered

through several cross breedings of different chemotypes of W. somnifera, a plant well- known to contain withanolides.

24-Methylenecholesterol (Scheme-9) has been shown to be a precursor of withanolides

(Lockley et al., 1974). It has been proposed that compound b (Scheme-10) is one of the intermediates leading to the lactone moiety. In this case, compound b should undergo 22- hydroxylation to c, a well known reaction in the steroidal biosynthesis (Glotter et al.,

1978).

From this point, two alternative hypotheses were presented, as shown in Scheme-10. One involves cyclization of d, and oxidation to e, while the second is through the oxidation of g to allylic isomer f, followed by cyclization to e. The latter compound, a lactone, has been found to be present in a number of withanolides (Begley et al., 1972). Final oxidation can lead to the -lactone h (36).

In the substitution pattern of rings A and B of the withanolides (Scheme-11), the first step is presumably the hydroxylation at C-1 from the rear and less hindered side of the molecule.

The intermediate 5-ene-1, 3diol is then selectively oxidized to the corresponding ketone (38), which undergoes elimination to yield the 2, 5-diene-1-one structure 39, found in most of the withanolides.

4.2 RESULTS AND DISCUSSION

4.2.1 Isolation of Major and Minor Constituents from Withania coagulans

During the current phytochemical study on the methanolic extracts of the whole plants of

Withania coagulans, seven withanolides 11-17 were obtained and structurally characterized. Two withanolide glycosides 11-12 were identified as new compounds, whereas five compounds 13-17 have been identified as known components.

Preliminary solvent–solvent extraction of the methanolic extract of Withania coagulans

(ca. 120 g) has resulted in the isolation of four major fractions (hexane, ethyl acetate, n- butanol, and water) (Scheme-12, Page-108). The chloroform and butanol extracts was subjected to column chromatography on silica gel for the primary fractionation. Five withanolides 13-17 were obtained from the chloroform fraction (pH-3). While the other two withanolide glycosides 11-12 were obtained from n-butanolic fraction of the plants by using silica gel column chromatography, followed by reverse phase column chromatography. Finally recycling HPLC was employed for the purification of withanolide glycosides from Withania coagulans (Experimental, Scheme-13, Page-109).

All structures were elucidated on the basis of spectroscopic data. This indicated that compounds 11-12 were new withanolides, whereas the spectral and physical data of known compounds were in agreement with that reported in the literature. Cytotoxity assays were performed on major withanolides, obtained during this study.

4.2.2 New Compounds

4.2.2.1 (20R, 22R) 14, 16, 17, 20-Tetrahydroxy-1-oxo-witha-5, 24-dienolide- 3-O-β-D-glucopyranoside (11)

24 27

23 25

OH 22 26 21 O O 20 18 H 17 OH 12 O 11 13 19 16 OH 9 H 14 OH 1 10 2 H 8 OH O 3 4 6 HO O HO OH 11

Compound 11 was obtained from BuOH extract of W. coagulans, which was loaded on a

HP-20 column, and eluted with H2O-MeOH to obtain three fractions. The water/MeOH fraction (1:1) was subjected to polyamide column chromatography, which resulted into five main fractions. The sub Fr C of the butanolic extract on repetitive column chromatography on silica gel (Experimental, Section-4.3.3.1, Page-108) yielded compound 11 as a white powder. The molecular formula of compound 11 was deduced as

C34H50O13 on the basis of HRFAB-MS (+ve) which showed pseudomolecular ion peak

+ [M+H] at m/z 667.3338 (calcd for C34H51O13 667.3330). The EI-MS showed characteristic fragmentation of withanolides (Scheme-1.2, Page-85). The UV spectrum of

11 showed an absorption band at 225 nm for -unsaturated lactone. The IR spectrum

(KBr) showed stretching bands at 1704 (carbonyl), 1680 (-unsaturated lactone), and

3412 (hydroxyl) cm-1.

The 1H-NMR spectrum of compound 11 (Table-8, Page-89) showed five singlets at δ

1.91, 1.26, 1.80, 1.92, and 1.71, each intergrating for three protons, assigned to Me- 18,

Me-19, Me- 21, Me- 27, and Me-28, respectively. The 1H-NMR spectrum also showed three oxygenated methine signals at δ 4.01 (m, H-3), 3.55 (dd, J16, 15 = 6.5 Hz, J16, 15

= 15.4 Hz, H-16) and 5.33 (dd, J22, 23a = 10.9 Hz, J22, 23b = 3.2 Hz, H-22). Signals for C-6 olefinic proton appeared as a doublet at δ 5.61 (d, J6, 7a = 5.2 Hz). A multiplet at δ 1.52 was assigned to the C-8 methine proton. The C-9 methine proton appeared at δ 1.60 (m).

The signals at δ 5.01 (d, J1' , 2' = 7.9 Hz, H-1′), 4.38 (dd, J2', 1' = 9.2 Hz, J 2', 3' = 7.5

Hz, H-2′), 4.01 (t, J = 9.2 Hz, H-3′), 3.90 (m, H-4′), 4.25 (m, H-5′), and 4.51 (dd, J6′,, 5′ 

= 14.3, J6′, 5′ = 7.3 Hz, H-6), were assigned to -protons of the D-glycopyranosyl moeity, subtituted at C-3 of compound 11.

The 13C-NMR spectrum (Table-7, Page-92) of compound 11 indicated the presence of 39 carbons, including five methyls, seven methylene, six methine, and ten quaternary carbons. Along with this, ten quaternary carbons were resonated at δ 211.2 (C-1), 135.3

(C-5), 53.4 (C-13), 82.8 (C-14), 89.1 (C-17), 79.4 (C-20), 150.0 (C-24), 121.4 (C-25), and 166.8 (C-26). Six methine carbons resonated at δ 75.3 (C-3), 126.3 (C-6), 32.8 (C-8),

36.2 (C-9), 76.3 (C-16), and 81.7 (C-22). Seven methylene carbons were appeared at δ

43.4 (C-2), 38.5 (C-4), 26.1 (C-7), 22.7 (C-11), 32.6 (C-12), 43.4 (C-15), and 35.6 (C-

23). Five methyl carbons were resonated at δ 20.1 (C-18), 18.5 (C-19), 20.8 (C-21), 19.5

(C-27), and 12.5 (C-28), respectively. The glucose anomeric carbon appeared at δ 102.8

(C-1′). Other carbons of the glucose moiety were resonated at δ 78.7 (C-2′), 76.3 (C-3′),

75.1 (C-4′), 71.2 (C-5′), and 62.1 (C-6′).

The 1H-1H COSY spectrum of the compound 11 exhibited that H-3 (δ 4.01) was coupled with C-2 protons at δ 3.0. It was also coupled with protons resonating at δ 2.67 and 2.59

(H-4). The vinylic H-6 showed connectivity with the C-7 proton (δ 2.40). The C-22 methine proton, resonated at δ 5.33, showed coupling with the C-23 methylene protons resonating at δ 2.72 (H-23).

The one-bond 1H/13C chemical shift correlations of 11 were determined by HMQC spectrum. The carbon at δ 46.5 (C-2) was coupled with geminal pair of protons at δ 3.01

(H-2), while sugar–bearing C-3 was resonated at δ 75.3 and correlated with H-3 at δ 4.0.

The C-4 methylene protons (δ 2.67) showed HMQC with a carbon at δ 38.5 (C-4). This connectivity was further indicated the substitution pattern of ring A. The anomeric carbon showed one-bond 1H/13C connectivity with proton at δ 5.01 (H-1′). Similarly, the C-7

resonated at δ 26.1 showed one-bond heteronuclear interaction with a proton at δ 2.42 (H-

7), while carbon at δ 76.3 (C-16) showed interaction with a proton signal at δ 3.55 (H-

16). Another interaction was observed between the carbon resonating at δ 81.7 (C-22), and the proton at δ 5.33 (H-22). The downfield methine proton at δ 5.61 (H-6) showed correlations with the C-6, resonated at δ 126.3.

The HMBC data were used to connect different structural fragments together. Long-range

13C/1H correlations showed that C-19 methyl protons (δ 1.26) have correlations with C-1

(δ 211.2), C-5 (δ 135.3), and C-10 (δ 54.6). C-18 Methyl protons (δ 1.91) showed correlations with C-13 (δ 53.4), C-14 (δ 87.3), and C-17 (δ 78.1). The C-21 methyl protons (δ 20.8) exhibited long-range couplings with C-17 (δ 89.1). The C-21 protons exhibited HMBC couplings with C-17 (δ 89.1), and C-20 (δ 79.4), and C-22 (δ 81.7).

Long-range interactions were also observed between Me-27 protons (δ 1.92), and C-23 (δ

35.6), C-25 (δ 121.4), C-24 (δ 150.0), while C-28 methyl protons displayed long-range

interactions with C-23 (δ 35.6), C-25 (δ 121.4), and C-24 (δ 150.0). The downfield methine protons, resonated at δ 5.61 (H-6), displayed connectivites with C-10 (δ 54.6), C-

4 (δ 38.5), and C-7 (δ 26.1). The long-range interactions were observed between proton signal at δ 5.33 (H-22) and C-20 (δ 79.4), C-17 (δ 89.1), and C-23 (δ 35.6).

The substitution of sugar at C-3 was inferred from the 3J coupling of H-3 (δ 4.01) with anomeric carbon (δ 102.8), whereas anomeric H-1′ (δ 5.01) also showed 3J correlation with C-3 (δ 75.3) of steroidal skalton. The stereochemistry at various asymmetric centers of compound 11 was deduced on the basis of the chemical shift comparison with the reported withanolides, as well as on biogenetic basis. The relative configuration of glucose was determined by the analysis of vicinal coupling constants for the sugar proton signals, based on well-known dependence of J (coupling constant) in pyranoses. Thus the large coupling constant (J1′, 2′ = 7.9 Hz) showed a diaxal relationship between the H-1′

Table-8: 1H- (400 MHz) and 13C- NMR (100 MHz) Chemical Shift Data of Compound 11 in Pyridine-d5.

Position H (J =Hz) C Multiplicity 1 - 211.2 C 2 3.01 (d, J = 20.0, J= 4.0) 46.5 CH2 2.72 (dd, J = 20.0, J= 7.0) 3 4.01 (m) 75.3 CH 4 2.67 (dd, J = 21.4, J= 4.8 ) 38.5 CH2 3.28 (br, d, J = 21.4, 5 - 135.3 C 6 5.61 (d, J = 5.2) 126.3 CH 7 2.42 d (J = 16.6) 26.1 CH2 8 1.52-1.99 (m) 32.8 CH 9 1.60-2.17* (m) 36.2 CH 10 - 54.6 C 11 1.75 (m) 22.7 CH2 12 2.76 (m) 32.6 CH2 13 - 53.4 C 14 - 82.8 C 15 2.91(m) 43.4 CH2 3.65 (d, J = 5.8) 16 3.55 (dd, J = 6.6, J = 15.4) 76.3 CH 17 - 89.1 C 18 1.91 (s) 20.1 CH3 19 1.26 (s) 18.5 CH3 20 79. 4 C 21 1.80 (s) 20.8 CH3 22 5.33 (dd, J = 10.9, J = 3.2) 81.7 CH 23 2.72* (m) 35.6 CH2 24 - 150.0 C 25 - 121.4 C 26 - 166.8 C 27 1.92 19.5 CH3 28 1.71 12.5 CH3 Glucose C-1′ 5.01 (d, J = 7.9) 102.8 CH C-2′ 4.38 (dd, J = 9.2, 7.5) 78.7 CH C-3′ 4.02 (t, J = 9.2) 76.3 CH C-4′ 3.9* (m) 75.1 CH C-5′ 4.25 (m) 71.2 CH C-6′ 4.51 (dd, J = 14.3, 7.3) 62.1 CH2

a Signal pattern unclear due to overlapping,*Overlapped signals

and H-2′, indicating and a pyranose form of the glucose. The configuration of C-17, and C-20 hydroxyl groups was assigned as  on the basis of the chemical shifts of the

Me-18, Me-21, and H-22. The chemical shift of Me-18 (δc 20.1), Me-21 (δc 79.4) and C-

22 (δc 81.7) were found to be similar to those reported for coagulin S, a known withanolide (Atta-ur-Rahman et al, 2003).

NOE Spectrum and Perspective View of Compound 11

The C-16 epimer of compound 11 has an oriented C-16 OH which showed an NOE interaction between C-16 methine proton (δ 2.95) and C-18 methyl protons (δ 1.36). This interaction suggested that the C-16 OH is oriented. This was inagreement with the known withanolides, exodeconolides B, and coagulin S. The configuration of C-22 was therefore assigned to be R.

18 CH3 21 CH 19 HO 3 CH OH

3 11 12 O H 13 28 20 CH 14 H 17 3 2 8 15 24 1 10 23 9 3 4 5 7 H 16 22 HO 25 27 Oglc OH O 6 CH3 H 26

O Fig. 4: Key NOE interactions in compound 11.

4.2.2.2 3β, 17β-Dihydroxy-14R, 20R-epoxy-1-oxowitha-5, 24-dienolide-27-O-β-D- glucopyranoside (12)

The whole dried plants of Withania coagulans (120 g) were extracted with methanol and then partitioned by solvent-solvent extraction into four main extracts (Experimental,

Section-4.3.3.2, Page-109). Compound 12 was isolated as a colorless solid from BuOH fraction by using column chromatography (silica gel).

28 H HO H H 27 3' 24 1' 2' OH O 5' 23 25 21 O 4' OH H 20 22 26 6' H 18 HO O O O OH 12 O 17 19 11 13 H 16 9 14 15 1 2 10 8 H 3 5 7 4 6 HO 12

The molecular formula of compound 12 was deduced as C34H48O12 on the basis of HREI-

MS at m/z 648.3140 (calcd for C34H48O12 = 648.3146). The EI-MS of compound 12

+ + + showed peaks at m/z 184.07 (C9H12O4 ), 141 (C7H9O3 ), 486 (C28H38O7 ), and 484

+ (C28H36O7 ).

The UV spectrum of 12 showed absorption at 226 nm (-unsaturated lactone). The IR spectrum (KBr) showed absorptions peaks at 1718 (carbonyl), 1684 (-unsaturated lactone), and 3490 cm-1 (hydroxyl).

HO OH O OH O HO OH O O OH O OH O O O H H O O O + H C6H7O3 C8H10O4 m/z 127.04 m/z 170.06 HO C34H48O12 m/z 648.31

OH OH HO O O HO O O O O O H O -H20 H H H HO C H O 28 38 7 C28H36O6 m/z 486.26 m/z 468.25

Scheme-13 : Mass fragmentation pattern in compound 12.

The 1H-NMR spectrum of 12 displayed four quaternary methyl signals, and five methine signals including two oxygenated methine, one olefinic, and nine methylene carbons. The

1H-NMR spectrum showed methyl signals at δ 1.42, 1.27, 1.54, and 1.94, which were assigned to the C-18, C-19, C-21, and C-28, respectively. Two oxygenated methine protond at δ 4.01 (m), and 5.11 (dd, J22, 23ax = 12.1 Hz, J22, 23eq = 4.12 Hz) were assigned to H-3 and H-22, respectively. The downfield olefinic proton appeared as a doublet at δ

5.57 (d, J6, 7a = 5.2 Hz, H-6). The other downfield methine protons appeared as a multiplets at δ 1.71 (H-8) and 1.87 (H-9). The C-2 methylene protons signals were resonated at δ 2.86 (dd, J2a, 3 = 8.1 Hz, J2a, 2b = 15.5 Hz), and 2.64 (dd, J2b, 3 = 3.4 Hz, J2a,

2b = 15.5 Hz), whereas a signal at δ 2.09 (m) was assigned to C-4 methylene protons. The other methylene proton signals at δ 2.09 (overlap) were assigned to the C-7 protons. The remaining methylene protons signals at δ 1.75 m (H-11), 2.58 (dd, J12a, 12b = 12.0, J12a, 11a

= 4.5 Hz, H-12), 2.32 (m, H-15), 2.41 (m, H-16), and 2.76 (m, H-23). Two AB doublets

at δ 4.78 and 4.67 (d, J= 11.4 Hz), (d, J= 8.6 Hz) were due to the geminally coupled C-27 hydroxymethylene protons. The signals at δ 5.01 (d, J'' = 7.9 Hz, H-1′),

4.38 (dd J'' = 11.7 Hz, J'3′ = 5.5 Hz, H-2′), and 4.01 (m, H-4′), and methylene sugar protons at δ 4.51 (dd, J6′6′ = 11.5 Hz, J6′5′ = 7.3 Hz, H-6′) were assigned to protons of -D-glucopyranoside moiety, substituted at C-3. The 13C-NMR spectrum of compound 12 (Table-9, Page-97) showed the presence of 34 carbons, including four methyls, five methine, nine methylene and ten quaternary carbons, remaining carbons signals which were characteristic for glucose moiety. Signals for quaternary carbons at δ

210.8 (C-1), 136.0 (C-5), 53.3 (C-10), 52.2 (C-13), 86.0 (C-14), 77.8 (C-17), 88.5 (C-20),

154.9 (C-24), 126 (C-25), and 166.2 (C-26). The methylene carbons were resonated at δ

38.5 (C-2), 38.5 (C-4), 27.1 (C-7), 21.6 (C-11), 27.3 (C-12), 33.2 (C-15), 34.4 (C-16),

33.2 (C-23), and 56.1 (C-27). Five methine carbons appeared at δ 75.3 (C-3), 126.7 (C-

6), 36.4 (C-8), 35.5 (C-9), and 81.7 (C-22). Four methyl signals were at δ 1.42 (CH3-8),

1.27 (CH3-9), 1.54 (CH3-21), and 1.94 (CH3-27).

The 13C-NMR spectrum of 12 showed two oxygen-bearing carbons in ring D resonated at

C-14, and C-17. This indicated the presence of an ether linkage either between C-14/C-

17, or between C-14/C-20. The model of 12 indicated that an ether linkage is only possible between C-14 and C-20, because the joining of C-14 with C-17 would afford four-member cyclic ether within a five-membered ring which would be too strained to exist. The HMBC interactions of CH3-18 with C-14, C-17 and C-20 indicated the presence of a cyclic ether linkage between C-14 and C-20. This was also confirmed from the mass and 13C-NMR spectroscopy.

The COSY spectrum of 12 exhibited that H-3 (δ 4.0) is coupled with a signal resonated at

δ 2.86 (H-2). The vinylic H-6 showed cross peaks with the H-7 proton (δ 2.29). The C-22 methine proton (δ 5.11) showed coupling with the C-23 methylene protons (δ 2.76). The geminal coupling were observed between the C-28 methylene protons, resonated at δ

4.78 (H-28), and 4.67 (H-28).

The one-bond 1H/13C chemical shift correlations in 12 were determined by HMQC spectrum. The carbon resonated at δ 38.5 (C-2) was coupled with the proton at δ 2.86 (H-

2), while the sugar-bearing carbon at δ 75.3 (C-3) was correlated with H-3 (δ 4.0). A carbon signal at δ 38.5 (C-4) showed correlations with protons at δ 2.09 (H-4). Carbon resonated at δ 27.1 (C-7) showed one-bond heteronuclear interaction with the H-7 at δ

2.29, while carbon resonated at δ 34.4 (C-23) was correlated with the proton signal at δ

2.76. The downfield methine protons at δ 5.57 (H-6), and 5.11 (H-22) showed one-bond

heteronuclear connectivities with the carbons at δ 126.7 (C-6), and 80.8 (C-22), respectively.

The HMBC corelations were used to combine different fragments together and to confirm the chemical shift assigments. The HMBC spectrum of 12 showed the C-18 protons at δ

1.27 have 3J interactions with C-1 (δ 210.8), C-5 (δ 136.0), and C-10 (δ 53.3). The

HMBC interactions were observed between C-18 methyl protons (δ 1.42) and C-13 (δ

52.2), C-14 (δ 86.0), and C-17 (δ 77.8). The C-21 methyl protons exhibited long-range couplings with C-17 (δ 77.8), C-20 (δ 88.5), and C-22 (δ 81.7). Long-range couplings were also observed between C-27 methylene protons (δ 4.78) and C-25 (δ 126.7), C-24 (δ

154.9) and C-26 (δ 166.2), while C-28 methyl protons showed interactions with C-23 (δ

33.2), C-25 (δ 126.7), and C-24 (δ 154.9). A downfield methine proton at δ 5.57 (H-6) showed HMBC connectivities with C-10 (δ 53.3), C-4 (δ 38.5), and C-7 (δ 27.1). The important long-range interactions were observed between 22-H (δ 5.11) and C-20 (δ

88.5), C-17 (δ 77.8), and C-23 (δ 33.2). The position of sugar moiety was inferred, when

C-27 proton at δ 4.78 was found to exhibit 3J couplings with anomeric C-1 (δ 102.8), C-

25 (δ 126.7), and C-26 (δ 166.2), while anomeric H-1′ (δ 5.0) displayed 3J correlations with C-27 (δ 56.1).

The stereochemistry at various asymmetric centers was assigned on the basis of biogenetic consideration, and chemical shift comparison with the known withanolides.

The stereochemistry of H-3 in ring A was based on comparison of the NMR data with previously reported withanolide, ajugin.

The 14R, 20R--epoxide between C-14 and C-20 was also present in compound 12, and as previously reported by Kirson (1980). According to Kirson, the oxides bridge is formed by the attack of the C-14OH on C-20 carbonium ion. Rotation about the C-

17/C-20 bond then facilitates the back side attack at C-20 to yield the 14R, 20R configuration.

The -orientation of the C-17 OH group was inferred from the study of the 1H-NMR spectrum of 12 in pyridine-d5. The prydine is known to influence the chemical shifts of neighbouring protons of the OH substituent (Demarco, Farkas, Doddrell, Mglari 1986).

Further elaborated the configuration of the C-17- OH group was deduced from the chemical shifts of the Me-18, Me-21, and H-22 by the pyridine-induced chemical shift values. It was observed that the 17-OH strongly deshields the signals of the Me-21 and

Me-18 protons (Gottlieb, Kirson, 1981), and thus an -oriented ether bridge was predicted between C-14 and C-20.

Table-9: 1H- (300 MHz) and 13C- NMR (100 MHz) Chemical Shift Data of Compound 12 in Pyridine-d5.

Position H (J = Hz) C Multiplicity 1 - 210.8 C 2 2.86 (dd, J = 15.5, J = 3.4) 38.5 CH2 2.64 (dd, J = 15.5, J = 8.1) 3 4.0 (m) 75.3 CH 4 2.09-3.31 (m) 38.5 CH2 5 - 136.0 C 6 5.57 (d, J = 5.2) 126.7 CH 7 1.99-2.29 (overlap) 27.1 CH2 8 1.71 (m) 36.4 CH 9 1.87 (m) 35.5 CH 10 - 53.3 C 11 1.75 m 21.6 CH2 12 2.58 (dd, J = 10.0, J = 4.5) 27.3 CH2 1.97 (dd, J = 10.0, J = 7.5)

13 - 52.2 C 14 - 86.0 C 15 1.51-2.32 (m) 33.2 CH2 16 2.41 (dd, J = 14.2, J = 1.9) 34.4 CH2 17 - 77.8 C 18 1.42 (s) 19.9 CH3 19 1.27 (s) 18.8 CH3 20 - 88.5 C 21 1.54 (s) 21.6 CH3 22 5.11 (dd, J = 12.1, 4.1) 81.7 CH 23 2.76* (m) 33.2 CH2 24 - 154.9 C 25 - 126.7 C 26 - 166.2 C 27 4.78 (d, J = 11.4) 56.1 CH2 4.67 (d, J = 8.6) 28 1.94 20.0 CH3 Glucose C-1′ 5.0 (d, J = 7.9) 102.8 CH C-2′ 4.38 (dd, J = 11.7, 5.5) 78.7 CH C-3′ 3.32 (t, J = 9.2) 75.3 CH C-4′ 4.01* (m) 75.1 CH C-5′ 4.25 (m) 71.2 CH C-6′ 4.51 (dd, J = 11.5, 7.3) 62.1 CH2 a Signals unclear due to *overlapping.

4.2.3 Known Compounds 13-17

4.2.3.1 Withanolid J (13)

Compound 13 was isolated from the chloroform extract (pH-3) of Withania coagulans

(Experimental, Section-4.3.3.3, Page-109), as a white solid. The [M+H]+ was observed at m/z 471.2789 in the HRFAB-MS (+ve), which was in agreement with the formula

C28H39O6 (Calcd. 471.2747). Its IR spectrum showed absorptions at 1706 (carbonyl),

1692 (-unsaturated lactone), and 3384 cm-1 (hydroxyl). Absorption band at 224 in the

UV spectrum indicated the presence of an unsaturated lactone in the molecule.

The 1H-NMR spectrum of compound 13 displayed five quaternary methyl signals at δ

1.13, 1.42, 1.45, 1.88, and 1.94 assigned to CH3-18, CH3-19, CH3-21, CH3-27, and CH3-

28, respectively. The C-21 methyl protons appeared as a singlet (δ 1.45) indicated an oxygen substituent at the vicinal C-20. The downfield signals of C-27 and C-28 and δ

1.88, and 1.94, indicated that these two methyls are substituted on a double bond. Two mutually coupled proton, appeared as double doublet at δ 5.93 (1H, d, J2, 3 = 10.2 Hz, H-

2), and a multiplet at δ 6.78 were due to the C-2 and C-3 olefinic protons, respectively.

Other methine double doublet at δ 5.92 (d, J6, 5 = 5.9 Hz) was assigned to C-6 olefinic

proton. A downfield methine double doublet at δ 4.36 (J22, 23 = 13.4 Hz, J22, 23 = 3.5

Hz) were due to the oxygen-containing methine proton of the lactone moiety (H-22).

The 13C-NMR broad-band spectrum of compound 13 showed signals for 35 carbons,

including ten quaternary, six methine, seven methylene, and five methyl carbons. The

downfield signals at δ 203.4, 168.2, 150.7, 143.2, 138.2, 131.5, and 121.4 were assigned

to C-1, C-26, C-24, C-3, C-5, C-6, and C-25, respectively. The carbon signals appeared at

δ 80.1, 81.9, and 88.0, were assigned to the carbon substituted with OH C-22, C-14 and

C-17, respectively. Compound 13 thus identified as the known compound, withanolide J,

previously isolated from Withania somnifera (Kamernitskii et al., 1977).

4.2.3.1 Coagulin E (14)

Compound 14 was obtained from fraction Fr-A (3 g) of the chloroform extract of

Withania coagulans by repeated column chromatography on silica gel (Experimental,

Section-4.3.3.4, Page-110). The HRFAB-MS (+ve) of compound 14 showed the [M+H]+

at m/z 437.2683, corresponding to the formula C28H36O4 (Calcd 436.2613). The UV

absorption (MeOH) at 223 nm was characteristic of the unsaturated lactone. The IR

spectrum of compound 14 displayed absorption bands at 1703 (carbonyl), 1698 (-

unsaturated lactone), and 3372 cm-1 (hydroxyl).

The 1H-NMR spectrum of 14 (Experimental, Scetion-4.3.3.4, Page-114) showed a distinctly similar 1H-NMR data to compound 14, expect for the appearance of an additional original of two methylene proton for the C-2 methylene protons at δ 5.93 which showed also allylic coupling with the C-4 vinylic proton (δ 5.81). The C-3 and C-4

vinylic protons also exhibited strong vicinal coupling with each other. The C-6 vinylic proton was resonated at δ 5.67 (J6, 7a = 6.8 Hz, J6, 7b = 4.3 Hz) as a double doublet.

The 13C-NMR broad-band decoupled spectrum of compound 14 showed 28 carbon

signals, including ten quaternary, six methine, seven methylene, and five methyl carbons.

The presence of a cyclic ether linkage between C-14 and C-20 was infered from the

chemical shift of carbons resonated at δ 84.0, and 78.3, and assigned to C-14 and C-20,

respectively. These are the characteristic signals of withanolides. On the basis of above

mentioned spectral data, the compound 14 was identified as known coagulin E,

previously isolated from Withania coagulance (Atta-ur-Rahman et al., 1998).

4.2.3.2 Withaperuvin C (15)

Compound 15 was isolated from the sub fraction Fr-A (3 g) of the chloroform extract (45

g). Chromatography over silica gel column yielded compound 15 (Experimental, Section

-4.3.3.5, Page-111). The [M+H]+ in the HRFAB-MS (+ve) appeared at m/z 471.2764,

+ corresponding to the formula C28H38O6 (calcd for C28H38O6 H = 471.2747).

100

The 1H-NMR and 13C-NMR spectra (Experimental, Page-117) of compound 15 was identical to a known compound withapuruvin C, which was earlier isolated from Physalis peruviana (Sahai et al., 1982). The 13C-NMR spectrum of 15 showed signals for C-2, C-3, and C-4 at δ 6.03, 6.88 and 6.14, assigned to H-2, H-3 and H-4, respectively. In the

HMQC spectrum, the proton signal at H 6.88 was correlated with the carbon resonated at

δC 142.7 (C-2), while in the HMBC spectrum this proton was correlated with the carbon signals at δC 160.2 (C-5), 74.7 (C-6), and 55.5 (C-10). The four hydroxyl-bearing carbons appeared at δ 74.7, 84.5, 88.3, and 79.6, which were identified as the C-6, C-14, C-17, and C-20, respectively. The spectral data of 15 was identical to that of a known compound, withaperuvin C (Anjana et al., 1984).

4.2.3.3 27-Hydroxywithanolide I (16)

Compound 16 was isolated from chloroform extracts (pH= 3) of the plants by MPLC.

The three main fractions (MC= 1, 2 and 3), were obtained on elution with 100%

chloroform, 30% chloroform / hexane and 70% chloroform / hexane. Fraction (MC = 2)

30% chloroform / hexane was further subjected to silica gel column chromatography, and

eluted to obtain different fractions (Fr = A-G). Finally compound 16 was isolated from

101

chloroform (pH= 3) fraction. The HRFAB-MS (+ve) showed the molecular ion peak at m/z 471.2659, which was in agreement with the formula C28H38O6 (Calcd C28H38O6 + H =

471.2692). The IR spectrum showed absorptions at 3400 (hydroxyl), 1660 (conjugated

-1 carbonyl), and 1700 cm (six-membered cyclic ketone). 471.2659 (calcd for C28H38O6

471.2692).

The 1H-NMR spectrum of 16 (Experimental, Section-4.3.3.6, Page-112) showed four methyl singlets at  0.93, 1.15, 1.28, and 2.01. Two mutually coupled olefinic signals resonated at  5.61 (multiplet) and 6.02 (dd, J4, 3 = 10.2 Hz, J4, 2 = 2.2 Hz), were assigned to C-3 and C-4 vinylic protons, respectively. Downfield C-6 olefinic signal at  5.66 (dd,

J6, 7 = 5.2 Hz, J6, 7b = 2.1 Hz) showed vicinal couplings with C-7 methylene protons (

2.20, and 1.86). The H-22, resonated at  4.05 (AB, dd, J22, 23a = 12.5 Hz, J 22, 23b = 4.0

Hz), exhibited cross peak with the H2-23 signals at  2.40 and 2.05. Downfield AB doublets, resonating at  4.19 and 4.12 (J27a, 27b = 11.5 Hz) were due to the C-27 hydroxymathylene protons. The configuration of C-22 was assigned to be R on biogenetic ground.

The 13C-NMR spectra (BB and DEPT) of compound 16 showed the presence of four methyl, eight methylene, seven methine, and nine quaternary carbons. A notable feature

102

was the appearance of downfield signals for the quaternary carbons at  82.4, and 74.7, which were assigned to the - oriented epoxy-bearing C-14 and C-20, respectively. This spectroscopic data led to the identification of compound 16 as 27-Hydroxywithanolide I, previously isolated from Withania somnifera (Velde et al., 1981).

4.2.3.5 Ajugin E (17)

Compound 17 was obtained from chloroform sub fraction Fr-E (9 g), by MPLC and characterized as a known compound ajugin E (Experimental Section 4.3.3.7, Page-113).

The HRFAB-MS showed the [M+H]+ peak at m/z 487.2698, corresponding to the molecular formula C28H39O7 (Calcd 486.2697).

The 1H-NMR spectra of 17 was closely resembled with the 16. The only difference between compounds 16 and 17 was an additional hydroxyl at C-17, while in compound

17 there is an ether linkage between C-14 and C-20. The presence of a hydroxyl group at

C-17 was inferred from the mass spectrometry. The hydroxyl group was placed at C-17 on the basis of an HMBC experiment which showed 3J correlations with  0.98 (Me- 18), and 1.10 (Me-21). The data compared well with the reported data for ajugin E (Pir et al.,

1999).

103

4.3 EXPERIMENTAL

4.3.1 General Experimental Conditions

UV Spectra were measured on a Shimadzu UV 240 machine in MeOH solutions as max nm (log ). IR Spectra were recorded as KBr discs on a JASCO A-302 spectrometer and presented in cm-1. 1H- (300 MHz) and 13C-NMR (100 MHz) spectra were recorded in

CD3OD solutions on a Bruker AV-500 machine, referenced with respect to the residual solvent proton signal (CD3OD/ CDCl3), and the data is given in (ppm). 2D NMR spectra were recorded on a Bruker AMX 500 NMR spectrometer. Electron impact mass spectra

(EI-MS) were measured at 70 eV on a Finnigan MAT-112, or MAT-312 instruments and major ions are presented as m/z (%). Fast atom bombardment mass spectra (FAB-MS) were measured as glycerol matrix on a JEOL HX-110 mass spectrometer. TLC purification was carried out on pre-coated silica gel cards (E. Merck), and the spots were observed, first the under UV light (254 nm), and then sprayed with cerium (IV) sulfate reagent, and heated until coloration developed. Recycling preparative high performance liquid chromatography (RPHPLC) was used for the final purification (JAI, LC-908W,

Japan Analytical Industry Co. Ltd.), with a column YMC ODS H-80 or L-80 (YMC,

Japan).

4.3.1.1 Chromatography

Column chromatography (CC) was carried out on silica gel (E. Merck, type 70-230 and

230-400 M mesh), MPLC column chromatography (Silica Wallogel C-300 HG)

Polyamide, and Diaion HP-20 resins. Final purification of compounds was achieved by recycling preparative HPLC (RPHLC) on a JAI LC-908W (Japan Analytical Industry).

104

Thin layer chromatography (TLC) was performed on precoated silica gel TLC (GF 254,

E. Merck).

4.3.1.2 Purification and Detection of Compounds on Chromatographic Plates

The purity of the samples was checked on the precoated TLC card. Spots were viewed under the ultraviolet light at 254 nm for fluorescence quenching spots, and 366 nm for fluorescent spots. Spraying reagents such as Dragendrorff”s and ceric sulphate were used to detect withanolides.

4.3.2 Isolation of Major and Minor Constituents from Withania coagulans

The phytochemical study on the methanolic extracts of the whole parts of Withania coagulans (Stock.) has resulted in the isolation of nine withanolides. The MeOH extract

(ca. 120 g) of the plant was partitioned with hexane, chloroform, n-butanol and water. pH was maintained before the extraction with chloroform. This procedure was repeated three times. At three different pH, fractions were made (pH = 3, 7 and 10). The chloroform

(pH-3) extracts was subjected to MPLC column chromatography with chloroform

/hexane. The three main fractions (MC= 1, 2, and 3), eluted with at 100% chloroform,

30% chloroform / hexane and 70% chloroform / hexane. Fraction (MC = 2) 30% chloroform / hexane was further subjected to silica gel column chromatography to eluteluate different fractions (Fr = A-G). Fr = A proceeds to column chromatographic procedures and finally to the recycling (MeOH) HPLC. This led to the isolation of withanolid J (14), coagulin E (14), and withaperuvin C (15).

Whereas, 27-hydroxywithanolide I (16), and ajugin E (17) were isolated from the same chloroform (pH = 3) fraction by the same procedure.

105

Withania coagulans Whole Plant (2 kg)

Methanolic Extract (120 g)

-Dissolved in Dist-H2O (3x 3000) -Fractionation with various organic solvent

H2O-Insoluble Ethyl acetate Butanol H2O-Soluble

Partitioned maintained pH= 3 fractionated with Hexane Ext. (2 g) CHCl3 Ext. (100 g) ( 3x5000 mL) (3x 5000 mL)

emulsion formed showed good activity in immunomodulatory assay

CHCl3 H2O layer

pH= 7

CHCl3 H2O layer pH= 10 showed good activity in immunomodulatory assay

CHCl3 H2O layer

Scheme-14: General scheme for the fractionation of Withania coagulans.

Compounds 11-12 were isolated from the BuOH extract (20 g) as a light brown gummy material. Preliminary studies on these compounds suggested the presence of monosaccharide, identified as glucose. The n-BuOH extract was fractionated on HP-20

106

column and water/MeOH (1:1) soluble part was subjected to polyamide column chromatography, and eluted with CHCl3/MeOH. Four sub fractions were obtained by polyamide column chromatography. The eluted fraction WC=2 (20% CHCl3 /MeOH) was loaded to a silica gel column, and five main fraction (Fr = A-E) were obtained. Fraction C was further subjected to column chromatography on silica gel, and resulting fraction were rechromatographed over silica gel, and finally purified by silica gel column

107

chromatography with 8% CHCl3/MeOH. The compounds 11-12 were identified as new, while compounds 13-17 were cheracteristic as known withanolides.

4.3.3.1 (20R,22R)14,16,17β,20β-Tetrahydroxy-1-oxo-witha-5,24-dienolide-3- O- β- D-glucopyranoside (11)

Compound 11 was obtained from BuOH fraction by the procedure described on (Scheme page- 99. It was isolated from the semi pure sub fraction (Fr = WC), along with compounds 12. Compound 11 was found to be a new withanolide, which was identified as (20R, 22R) 14,16,17β,20β-tetrahydroxy-1-oxo-witha-5,24-dienolide-3-O-β-D- glucopyranoside (11).

Physical Data

State: Colourless solid

Percentage yield: 2.1 mg (1.0 x 10-4%)

25 [] D : -61 (c= 0.2, MeOH).

UV (MeOH) max nm (log : 226 ( unsaturated lactone)

−1 IR (KBr) νmax cm : 1718 (carbonyl), 1684 (-unsaturated lactone), 3490 (hydroxyl).

+ HRFAB MS (+ve) m/z: [M+H] 667.3338 (calcd for C34H50O13 +H = 667.3330).

1 H-NMR (300 MHz, CD3OD): δ See Table-8, Page 89.

13 C-NMR (75 MHz, C5D5N): δ See Table-8, Page 89.

108

4.3.3.1 3β,17β-Dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-β-D- glucopyranoside (12)

Compound 12 was isolated from the butanolic extract. The extract was loaded on polyamide column, and eluted with 100% chloroform, 10% methanol-chloroform, 20% methanol-chloroform, and 100% methanol to collect four sub fractions. 20% Methanol- chloroform fraction was further fractionated into five sub fractions (Fr =A to Fr =E).

Fraction Fr = C was further rechromatgraphed over silica gel column to obtain four sub- fractions (Fr = Wb- 1, Fr = Wb-2, Fr = WC, and 100% methanol). The semi pure sub- fraction Fr = WC (1.6 g) when loaded on a silica gel column afforded a new compound

12. It was identified as 3β,17β-dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-

β-D-glucopyranoside.

Physical Data

State: White powder

Percentage yield: 2 mg (1.0 x 10-4%)

25 [] D : -62 (c= 0.2, MeOH).

UV (MeOH) max nm (log : 226 (unsaturated lactone)

−1 IR (KBr) νmax cm : 1718 (carbonyl), 1684 (-unsaturated lactone), 3490 (hydroxyl).

HREI-MS m/z : 648.3140 (calcd for C34H48O12 = 648.3146)

1 H-NMR (300 MHz, CD3OD): δ See Table-9, Page 97.

13 C-NMR (75 MHz, C5D5N): δ See Table-9, Page 97.

109

Known Compounds 13 -17

4.3.3.3 Withanolid J (13)

The CHCl3-soluble extract obtained at pH-3 was loaded onto a big silica gel column.

Elution was made with n-hexane-chloroform which yielded Fr = A (3 g). This fraction was loaded onto a silica gel column which yielded a pure compound which was finally purified by MeOH recycling HPLC.

Physical Data

State: White solid

Percentage yield: 5 mg (1.0 x 10-4%)

25 [] D : -61 (c= 0.0137, MeOH).

UV (MeOH) max nm (log : 224 (unsaturated lactone)

−1 IR (KBr) νmax cm : 1706 (carbonyl), 1692 (-unsaturated lactone), 3384 (hydroxyl).

HRFAB-MS (+ve) m/z : 471.2789 (calcd for C28H39O6 471.2747).

1 H-NMR (400 MHz, CD3Cl3): H 1.23 (3H, s, CH3-18), 1.37 (3H, s, Me-19), 1.23 (3H, s,

CH3-21), 2.03 (3H, s, CH3-27), 2.00 (3H, s, CH3-28), 4.25 (1H, dd, J22, 23a = 12.9Hz, J22.,

23B = 3.5 Hz, H-22), 6.76 (1H, ddd, J3, 2 = 10.1 Hz, , J3, 4b= 5.0 Hz, H-3), 2.33 (1H, m, H-

4) 5.79 (1H, m, H-6), 1.56-2.01 (1H, m, H-7), 2.03 (1H, m, H-8),

13 C-NMR (125 MHz, C5D5N): δC 207.1 (C, C-1), 166.2 (C, C-26), 146.9 (C, C-24),

136.3 (C, C-5), 34.2 (CH, C-4), 124.7 (CH, C-6), 122.0 (C, C-25), 126.0 (CH2, C-2),

147.0 (CH, C-3), 84.0 (C, C-14 ), 81.1 (CH, C-22), 78.3 (C, C-20), 52.1 (C, C-10), 53.1

(C, C-13), 42.6 (CH, C-16), 38.7 (CH, C-9), 37.5 (CH, C-8), 33.8 (CH2, C-7), 26.7 (CH2,

110

C-15), 26.3 (CH2, C-12), 22.3 (CH2, C-11), 18.8 (CH3, C-19), 18.0 (CH3, C-28), 19.0

(CH3, C-21), 18.3 (CH3, C-18).

4.3.3.4 Coagulin E (14)

Silica gel column was used for the purification of sub-fraction chloroform Fr-A (3 g), obtained from fraction 30% MeOH /CHCl3 (Scheme-13, Page-111). The structure was identified as a known compound, coagulin E.

Physical Data

State: Greenish gummy

Percentage yield: 5 mg (1.0 x 10-4%)

25 [] D : +111 (c= 0. 26, CHCl3).

UV (MeOH) max nm (log : 230 (characteristic of unsaturated -lactones)

−1 IR (KBr) νmax cm : 3400 (hydroxyl), 1660 (conjugated carbonyl), 1700 (six-membered cyclic ketone).

HRFAB-MS (+ve) m/z : 451.2588 (calcd for C29H38O4 451.2773).

1 H-NMR (400 MHz, DMSO-d6): H 1.33 (3H, s, CH3-18), 1.30 (3H, s, CH3-19), 1.61

(3H, s, Me-21), 1.98 (3H, s, CH3-27), 1.90 (3H, s, CH3-28), 5.59 (1H, dd, J6, 7a = 6.3 Hz,

J6, 7b = 4.0 Hz , H-6), 1.69 (1H, m, H-8), 4.36 (1H, dd, J22, 23a = 13.3 Hz, J22, 23b = 3.5 Hz,

H-22), 3.32, 3.01 (2H, m H-2), 5.57 (1H, m, H-3), 6.87 (1H, m, H-4).

13 C-NMR (100 MHz, DMSO): δC 210.0 (C, C-1), 1665.1 (C, C-26), 148.8 (C, C-24),

135.7 (C, C-5), 126.4 (CH, C-4), 123.0 (CH, C-3), 121.1 (C, C-25), 125.0 (C, C-6), 85.0

(C, C-14), 80.1 (CH, C-22 ), 88.8 (C, C-20), 51.4 (C,C-17), 54.0 (C, C-10), 49.0 (C, C-

13), 38.0 (CH2, C-2), 30.3 (CH, C-8), 38.4 (CH, C-9), 33.9 (CH2, C-23), 30.7 (CH2, C-7),

111

42.6 (CH2, C-16), 28.6 (CH2, C-15), 23.5 (CH2, C-12), 24.7 (CH2, C-11), 19.4 (CH3, C-

27), 20.4 (CH3, C-28), 20.0 (CH3, C-21), 18.9 (CH3, C-18), 20.8 (CH3, C-19).

4.3.3.5 Withaperuvin C (15)

Compound 15 was obtained from chloroform fraction (pH= 3). Along with compound 14

the same procedure was apply to isolate the known withanolide 15

Physical Data

State: Yellow powder

Percentage yield: 3.5 mg (1.9 x 10-4%)

25 [] D : +111 (c= 0. 25, MeOH).

UV (MeOH) max nm (log : 311, 312 (characteristic of unsaturated -lactones)

−1 IR (KBr) νmax cm : 3400 (hydroxyl), 1660 (conjugated carbonyl), 1690 (-

unsaturated -lactone).

HRFAB-MS (+ve) m/z : 471.2764 (calcd for C28H38O6 +H = 471.2747).

1 H-NMR (400 MHz, DMSO-d6): H 1.23 (3H, s, CH3-18), 1.29 (3H, s, CH3-19), 1.21

(3H, s, Me-21), 1.97 (3H, s, CH3-27), 1.80 (3H, s, CH3-28), 6.69 (1H, m, H-2), 6.60 (1H,

m, H-3), 6.68 (1H, m, H-4), 3.69 (1H, overlap, H-6), 1.69 (1H, m, H-8), 4.22 (1H, dd, J22,

23a = 13.3 Hz, J22, 23b = 3.5 Hz, H-22).

13 C-NMR (100 MHz, DMSO-d6): δC 208.0 (C, C-1), 165.1 (C, C-26), 152.8 (C, C-24),

121.7 (C, C-25), 161.6 (C, C-5), 123.0 (CH, C-2), 148.0 (CH, C-3), 127.9 (CH, C-4),

75.6 (C, C-6), 84.0 (C, C-14), 81.1 (CH, C-22 ), 82.8 (C, C-20), 88.4 (C,C-17), 53.0 (C,

C-10), 51.0 (C, C-13), 21.3 (CH2, C-8), 22.4 (C-9), 35.8 (CH2, C-23), 30.7 (CH2, C-7),

112

40.7 (CH2, C-16), 26.6 ( CH2, C-15), 21.7 (CH2, C-12), 22.9 (CH2, C-11), 19.0 (CH3, C-

28), 19.8 (CH3, C-21), 20.9 (CH3, C-18), 18.8 (CH3, C-19).

4.3.3.6 27-Hydroxywithanolide I (16)

Compound 16 was isolated from the sub fraction (FR-5) of CHCl3 fraction by using 20% acetone in hexane and loaded on silica gel column chromatography.

Physical Data

State: Greenish gummy

Percentage yield: 5 mg (2.0 x 10-4%)

25 [] D : -62 (c= 0.026, CH3Cl3).

UV (MeOH) max nm (log : 230 (unsaturated lactone)

−1 IR (KBr) νmax cm : 3400 (hydroxyl), 1666 (conjugated carbonyl), 1700 (six-member cyclic ketone).

HRFAB-MS (+ve) m/z : 471.2659 (calcd for C28H38O6 471.2692).

1 H-NMR (400 MHz, CD3Cl3): H 1.20 (3H, s, CH3-18), 1.37 (3H, s, Me-19), 1.24 (3H, s, CH3-21), 4.78, 4.67 (2H, AB, doublet, J 27a, 27b = 11.4, and 8.6, H-27), 4.23 (1H, dd, J22,

23a = 13.4 Hz, J22, 23b = 3.6 Hz, H-22), 5.52 (1H, m H-3), 5.67 (1H, dd, J6, 7a = 6.8 Hz, J6, 7b

= 4.3 Hz, H-6), 5.06 (1H, d, H-4).

13 C-NMR (125 MHz, C5D5N): δC 212.1 (C, C-1), 167.8 (C, C-26), 141.9 (C, C-24),

136.7 (C, C-5), 121.0 (CH, C-4), 125.5 (CH, C-6), 124.0 (C, C-25), 123.07 (CH, C-3),

86.9 (C, C-14 ), 80.1 (CH, C-22), 79.6 (C, C-20), 54.1 (C, C-10), 53.1 (C, C-13), 42.6

(CH, C-16), 20.7 (CH, C-9), 22.5 (CH, C-8), 33.1 (CH2, C-7), 39.9 (CH2, C-2), 27.8

113

(CH2, C-15), 22.1 (CH2, C-12), 24.3 (CH2, C-11), 57.6 (CH2, C-27), 18.8 (CH3, C-19),

19.5 (CH3, C-28), 20.8 (CH3, C-21), 18.9 (CH3, C-18).

4.3.3.7 Ajugin E (17)

A fraction from chloroform Fr-E (9 g) was subjected to MPLC column chromatography by using n-hexane–acetone (3%) as the mobile phase. Eluted five main fractions, Fr-8 and Fr-9 were combined and rechromatographed on MPLC CC by using acetone hexane

(40%) to obtain compound 17 (4 mg), which was characterized as a known compound, ajugin E.

Physical Data

State: Amorphous solid

Percentage yield: 3.7 mg (1.0 x 10-4%)

25 [] D : +125 (c= 0. 058, MeOH).

UV max (MeOH) nm (log : 223

−1 IR (KBr) νmax cm : 3455, 1716, and 1704

HRFAB-MS (+ve) m/z : 487.2697 (calcd for C28H38O7, 486.2695).

1 H-NMR (400 MHz, CDCl3 + CD3OD): H 1.27 (3H, s, CH3-18), 1.57 (3H, s, Me-19),

1.41 (3H, s, CH3-21), 2.10 (3H, s, CH3-28), 4.11 (1H, d, J 27a, 27b = 12.0 Hz, H-27), 4.22

(1H, dd, J22. 23a = 12.6 Hz, J22, 23b = 3.5 Hz, H-22), 5.51 (1H, br, d, J6, 7 = 5.1 Hz, H-6),

5.76 (1H, m, H-3), 5.55 (1H, d, J4, 3 = 9.8 Hz, H-4).

13 C-NMR (125 MHz, CDCl3 + CD3OD): δC 210.5 (C, C-1), 165.5 (C, C-26), 155.8 (C,

C-24), 136.2 (C, C-5), 121.2 (C, C-6), 126.4 (C, C-3), 121.3 (CH, C-4), 122.2 (C, C-25),

125.2 (CH, C-6), 84.6 (CH, C-17), 85.5 (C, C-14 ), 81.1 (CH, C-22), 79.3 (C, C-20), 54.7

114

(C, C-10), 54.7 (C, C-13), 34.1 (CH, C-8), 30.6 (CH, C-9), 21.7 (CH2, C-7), 39.5 (CH2,

C-23), 31.9 (CH2, C-16), 24.0 (CH2, C-12), 32.4 (CH2, C-15), 21.0 (CH2, C-11), 20.6

(CH3, C-18), 20.1 (CH3, C-19), 19.4 (CH3, C-21), 20.6 (CH3, C-28).

115

SECTION-B

5.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM WITHANIA COAGULANS DUN. (STOCK.)

116 Cytoxicity Activity

5.1 CYTOTOXICITY EVALUATION:

5.1.1 Introduction

Cyotoxicity is the substance's ability of being poisonous to cells. Cytotoxicity is critical to the body's immune system, but it is also used in pharmaceutical research as a means for killing the unwanted cells in a tissue samples that has been infected with alien genetic material. Cytotoxicity is a subject of major pharmaceutical attention, particularly in the area of cancer drug discovery and development. Low cytotoxicity to healthy cells, and high cytotoxicity to cancerous cells is the ultimate goal of many anticancer chemotherapeutic agents drugs (Dholwani et al., 2008).

Medicinal plants can serve as the source of potential new drugs, and initial research is generally focuses on the isolation of bioactive lead compound(s). Preclinical in vitro screening against human cell lines, followed by in vivo testing, then identifies the most promising drug candidates. There are various classes of recently discovered compounds that possess potent cytotoxicity. These compounds have been obtained through bioassay- guided isolation and characterization strategies.

Diterpenes (Pan et al., 1990), kansuiphorins (Wu et al., 1991), peroxytriterpene (Chen et al., 1990), dilactones (Shi et al., 1992), , triterpenes (Kashiwada et al., 1992), triterpene glucosides (Kuo et al., 1990), quassinoids (Kuo et al., 1989), sesquiterpene alkaloids

(Fujioka et al., 1996), bisdesmosides (Lee et al., 1981), flavonoids, and napthoquinones.

(Hayashi et al., 1987) are cytotoxic compounds obtained from various medicinal plants.from W. coagulans cytotoxic constituents such as withacoagin, dimeric lignan, bispicropodophyllin glucoside, coagulin S (Nur-e-Alam et al., 2003) coagulin-H (Atta-ur

117 Cytoxicity Activity

Rahman et al., 1998), and withanolides E and S (Yu-Hsuan et al., 2009) have been reported.

During the current study, compounds 4, 3, 7, and 8 were isolated in good yields. These compounds were evaluated for their cytotoxicity against 3T3 fibroblast cells and their activity was compound with the standard drug, cycloheximide. Only a moderate cytotoxicity was exhibited by the known compound 8, all other compounds showed a only a weak cytotoxicity (Table-9).

5.1.2 Results and Discussion

Compounds 2-8 were evaluated for their cytotoxicity. The assay was performed by Ms.

Samina Abdul Sattar in Anti-cancer Research Lab. of the Dr. Panjwani Center for

Molecular Medicine and Drug Research (ICCBS), University of Karachi, Karachi.

Table-10: Cytotoxicity of Compounds 4, 3, 7 and 8.

a Compounds IC50 (M) ± (S.D.).

4 52.38 ± 2.20

3 >100

7 >100

8 19.51 ± 5.3

Cycloheximideb 0.216 ± 0.12

b) Standard drug.

5.1.3 Methodology

Chemicals: Mouse fibroblast (3T3) was obtained from the European American Culture

Collection (EACC). DMEM and foetal bovine serum were purchased from Gibco-BRL

(Grand Island, NY, USA). MTT (3-[4, 5-dimehtylthiazole-2-yl]-2, 5-diphenyltetrazolium

118 Cytoxicity Activity bromide) was obtained from Amresco (Ind. PKWY, Solon, Ohio, USA). Pencilline and streptomycin were purchased from Sigma-Aldrich Chemicals (St. Louis, Mo, USA).

Cytotoxicity Asay: Cytoxicity of compounds was evaluated in 96-well flat-bottom micro-plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) colorimetric assay. For this purpose, 3T3 cells (Mouse Fibroblasts) were cultured in Dulbecco’s Modified Eagle’s Medium, supplemented with 5% of foetal bovine serum (FBS), 100 IU/mL of pencillin, and 100 g/mL of streptomycin by using

2 o 25 cm flask, in 5% CO2 incubator at 37 C. Exponentially growing cells were harvested, counted with haemocytometer, and diluted with a particular medium. Cell culture with the concentration of 3 x 104 cells/mL was prepared and was plated (100 L/well) onto

96-well plates. After overnight incubation, medium was removed and 200 L fresh medium was added with different concentrations of compounds (1-100 M). After 72 h,

50 L MTT (2 mg/mL) was added to each well and incubation was continued for further

4 hrs. Subsequently 100 L of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by the measurement of the absorbance at 540 nm by a microplate Spectra Max 340 (Molecular Devices, CA, USA).

The cytotoxicity was recorded as concentration causing a 50% growth inhibition.

119

6.0 REFERENCES

120

6.0 REFERENCES

 Ahmed, N., Res. Clin. Pract., 2005, 67, 3-21.

 Anjana, B., Partha, N., Mahendra, S., Anil B., Ray, Y., Oshima and Hiroshi H.,

Phytochemistry, 1984, 23, 853-855.

 Anonymous, Report on the State of the World's Plant Genetic Resources for Food and

Agriculture, Rome, Italy. 1996, 511.

 Arndt, B., The Genus Viscum Medicinal and Aromatic Plant, Industrial Profiles.

Harwood Academic, 2002, 45.

 Atal, C. K., Sath, P. D., Indian J. Pharm., 1961, 23, 7.

 Atal, C. K., Sathi, P. D., Indian. J. Pharm., 1963, 25, 163.

 Atta-ur rehman, Choudhary, M. I., Qureshi, S., Gul, W., Yousaf, M., J. Nat. Prod,

1998, 61, 812-814.

 Atta-ur-Rehman, Yousaf, M., Gul, W., Qureshi, S., Choudhary, M. I., Voelter, W.,

Jens, F., Naz, A., Heterocycle, 1998, 48, 1801-1811.

 Ayabe, S., Kobayashi, M., Hikichi, M., Matsumoto, K., Furuya, T., Phytochemistry,

1980, 19, 2179.

 Barata, L., Mors, W. B., Kirson, I., Lavie, D., An. Acad Bros Genic. 1970, 42, 401.

 Begley, M. J., Cronbie, L., Ham, P. J., Whiting, D. A., J. Chem. Soc., Perkin Trans I.,

1976, 364, 296.

 Chadha, Y. R., The Wealth of India, New Dehli Publications and Informations

Directorate, CSIR, 1976, 10, 582.

 Chen, G. F., Li, Z. L., Chen, K., Tang, C. M., He, X., Pan, D. J., Hu, C. Q., Mcphail, D.

R., Mcphail, A. T., Lee, K. H. J., Chem. Soc. Chem. Commun., 1990, 1113.

121

 Conforth, J. W., Conforth, R. H., Popjak, G., Yengoyan, L. J., Biol. Chem. Soc., 1966,

101, 6761.

 Ebadi, M., Pharmacodynamic Basis of herbal Medicine, chap 2002, 13.

 EMEA/MRL., (The European Agency for the Evalution of Medicinal Products

Veterinary Medicines Evalution Unit). 1999, 680-99.

 Frison, E., H., Omont, S., Padulosi, GFAR and International Cooperation on

Commodity Chains. Synthesis paper for presentation to the GFAR- Conference,

Dresden, Germany, May 2000, 21-23.

 From Wikipedia, the free encyclopedia wikipedia.org/wiki/Loranthaceae.

 Fujioka, T., Kashiwada, Y., Okabe, H., Mihashi, K., Lee, K. H., Bioorg. Med. Chem.

Let., 1996, 6, 2807.

 Fukunaga, T., Kajikawa, I., Nishiya, K., Watanabe, Y., Takeya, I. H., Chem. Pharm.

Bull., 1987, 35, 3292–3297.

 Garg, K. N., Budhiraja, R. D., Indian. J. Pharm., 1967, 29, 185.

 Gaydou, E. M., Bianchini, J. P., Bull. Soc. Chim., 1978, 11, 43.

 Glotter, E., Nat. Prod. Rep., 1991, 8, 415.

 Gray, A. M., Flatt, P. R., Endocrinology, 1999, 160, 409–414.

 Greca, M. D., Ferrara, M., Fiorentino A., Monaco, L., Previtera L., Phytochemistry,

1988, 49, 1299–1304.

 Hajto, T., Hostanska, K., Berki, T., Palinkas, L., Boldizsar, F., Nemeth, P., eCam.,

2005, 2, 59–67.

 Hayashi, T., Smith, F. T., Lee, K. H., J. Med. Chem., 1987, 30.

 Hussain, S. F., Medicinal an Aromatic Plants in Pakistan. In Chomchalow, N. Henle

H.V. (eds) Medicinal and Aromatic Plants in Asia, RAPA Publication Bangkok,

Thailand, 1993, 145-152. 122

 Hamayun, M., Indian journal of traditional knowledge 2007, 6, 631-641.

 Hyder, N., Bouhlel, I., Skandrani, I., Kadri, M., Steiman, R., Guiraud, P., Mariotte, M.

A., Ghedira, K., Dijoux-Franca, M. G., Chekir-Ghedira, L., Toxicology in Vitro, 2008,

22, 567–581.

 Jacob, J. M., Pfeiffer, B., Rolando, C., Tetrahedron Lett., 1983, 24, 4327.

 Kamernitskii, A. V., Reshetova, I. G., Krivoruchko, V. A., J. Chem. Nat. Comp., 1971,

13.

 Kashiwada, Y., Fujioka, T., Chang, J. J., Chen, I. S., Mihashi, M., Lee, K. H., J. Org.

Chem., 1992, 57, 6946.

 Khan, L., Ahmed, N., Ahmed, K. D., Kifayatullah, Q., M., Arfan, M., International

Journal of Pharmacognosy, 1995, 33, 344-45.

 Kim, K. H., Janiak, V., Peterson, M., Plant. Mol. Biol., 2004, 54, 311-23.

 Kirson, I., Glotter, E., Abraham, A., Lavie, D., Tetrahedron, 1970, 26, 2209.

 Kirson, I., Goitlieb, H. E., J. Chem. Res., 1980, 338, 4275.

 Kirson, I., Gunzberg, G., Gottlieb, H. I., J. Chem. Soc. Perkin., Trans. I., 1980, 30, 531.

 Koyama, A., Saito, K., Ogura, S., Seta, J. Am. Chem. Soc., 1980, 102, 3614.

 Kuo, Y. H., Chen, C. H., Yang-Kuo L. M., King, M. L., Wu, T. S. Lu, S. T., Chen, I.,

Mcphail, D. R., Mcphail, A. T., Lee, K., Heterocycles, 1989, 29, 1465.

 Kuo, Y. H., Chen, C. H., Yang-Kuo, L. M., King, M. L., Wu, T. S., Haruna, M., J. Nat.

Prod., 1990, 53, 422.

 Lam, J., Wrang, P., Phytochemistry, 1975, 14, 1621–1623.

 Lavie, D., Abraham, A., And Lavie, D., Tetrahedron, 1973, 29, 1353.

 Lavie, D., Kirson, I., and Glotter, F., Isr. J. Chem., 1968, 6, 671.

 Lee, K. H., Imakura, Y., Sumida, T., Wu, R. Y., Hall, I. H., Huang, H. C., J. Org.

Chem., 1979, 44, 2180. 123

 Lee, K. H., Tagahara, K., Suzuki, H., Wu, R. Y., Huang, H. C., Ito, K., J. Nat. Prod.,

1981, 44, 530.

 Leu, L. Y., Kuo, M. S., Hwang, T. L., Chiu, S. T., Chem. Pharm. Bull., 2004, 52, 858–

860.

 Lin, J. Y., Nature, 1973, 24, 524.

 Lokley, W. J. S., Roberts, D. P., Rrrs, H. H., Goodwin, T. E., Tetrahedron Lett., 1974,

3773.

 Mahidol, C., Ruchirawat, S., Prawat, H., Pisutjaroenpong, S., Engprasert, S., Chumsri,

P., Tengchaisri, T., Siri-inha, S., Pichas, P., Pure Appl. Chem., 1998, 70, 2065-2072.

 Matill, H. A., Annu. Rev. Bio chem., 1947, 16, 177-192.

 Mebry, T. J., Markham, K. R., Thomas, M. B,. “The Systematic Identification of

Flavonoids”. Springer-Verlag; New York, 1975, 33–35.

 Mehta, B. K., Phytochemistry, 1988, 27, 3004.

 Monitto, P., Biosynthasis of Natural products, Eilis Harwood limited, john willey and

sonds, New york, 1981, 400-401.

 Newman, J. D., Cragg, G. M., Snader, K. M., J. N. Prod., 2003, 66, 1022-1029.

 Padulosi, S., A. Giuliani1, N. J., International Workshop on Underutilized Species,

Leipzig, Germany, 2003, 6-8.

 Pan, D. J., Li, Z. L., Hu, C. Q., Chen, K., Chang, J. J., Lee, K. H., Planta Med., 1990,

56, 383.

 Peterson, M., Alfermann, A. W., Z. Naturforsch., 1998, 43, 501-504.

 Pir, M. K., Saeed, A., Hafiz, R., Malik, A., Phytochemistry, 1999, 51, 669-671.

 Rahber, S., Figarola, J. L., Arcives of Biochemistry and Biophysics, 2003, 419, 63-73.

 Renata, J., Piotrowski, A., Can. J. Plant Pathol., 2002, 24, 21–28.

 Sadikum, A., Aminah, I., Ismail, N., Ibrahim, P., Nat. Prod. Sci., 1996, 2, 19-23.

124

 Sathi, P. D., Indian J. Hosp. Pharm., 1970, 7, 219.

 Shi, Q., Chen, K., J. Nat. Prod., 1992, 55, 1488.

 Siddiqui, S., J. Nat. Prod., 1988, 51, 229-233.

 Stein, G. M., Pfuller, U., Schietzel, M., Bussing, A., Anticancer Res., 2002, 20, 2987–

2994.

 Stone, K. J., Roeske, W. R., Clayton, R. B., Van Tamelen, E. E., Chem. Commun.,

1969, 530.

 Sher, H., Al-Yemeni, M. N., Sher, H., J. Med. Plants Res., 2010, 18, 1853-1864.

 Tanaka, R., Matsunaga, S., Sasaki, T., Planta Medica, 1989, 55, 570-571.

 The Flavonoids advance is research since 1986 edited by J. B. Harborne published in

by Champman and Hall. London. 1993.

 Thomas, S. C. Li., Medicinal plants, CRC Press, USA, 2002, 70, 114.

 Watt, G. A., ‘Dictionary of the Economic Products of India, Cosmo Publications,

Delhi-6, India, 1972, 6, 309.

 Wei, L., Koike, K., Tatsuzaki, M., Koide, A., Nikaido, T., Cucurbitosides F-M., J. Nat.

Prod., 2005, 68, 1754–1757.

 Wilson, E. O., The Diversity of Life. Penguin, London, UK. 1992, 432.

 Wu, T. S., Lin, Y. M., Haruna, M., Pan, D. J., Shingu, T., Chen, Y. P., “Antitumor

Agents, 119. Kansuiphorins A and B, J. Nat. Prod., 1991, 54, 823.

 Yu-Hsuan, L., Fang-Rong, C., Mei-Jung, P., Chin-Chung, Wu., Shu-Jing, Wu., Su-Li,

C., Shyh-Shyan, W., Ming-Jung, Wu., and Yang-Chang, Wu., Food Chemistry, 2009,

116, 2, 462-469.

125

7.0 GLOSSARY

126

7.0 GLOSSARY

1. Anti-bacterial: A chemical or drug with a proven ability to kill bacteria in a selective manner.

2. Anti-cancer: A chemical substance used to inhibit the rapid growth of cancer cells that result in inhibiting the formation of malignant tumors.

3. Anti-oxidant: A substance which prevent or slow down the oxidation process in organisms to avoid or minimize the ageing and oxidative stress.

4. Anti-antiglycation: Non-enzymatic reaction of sugars with proteins. Chemical glycation is also very important in the damage done to diabetics when their sugar levels rise above normal, and in damage done to critical proteins of long-lived nerve cells in aging.

5. Assay: The testing or measuring the activity of a drug or a biochemical in an organic sample.

6. Base Peak/Parent Peak: A peak with 100% intensity in the mass spectrum which arises due to the most stable ion formed by the fragmentation of parent molecule in an ionization chamber. The intensities of other peaks in the mass spectrum are determined, relative to the base peak. 7. Bioassay: Determination of the activity of a sample by noting its effect on a live animal or an isolated organ preparation, or against a purified biochemical, and compared with the effect of a standard preparation. It is also called biological assay.

8. Biosynthesis: The route of the formation of organic molecules from basic building blocks in living matter.

9. Broad-Band (BB) 13C-NMR Spectrum: It is a fully decoupled 13C-NMR spectrum, which provides information about the resonances of all the carbons present in a molecule without any coupling interactions.

10. Chemical Shift: In NMR spectroscopy, chemical shift refers to the difference between the precession frequency of a particular nucleus, and the signal for a reference. It is expressed in ppm (parts per million) and is represented by a symbol .

11. Chromatogram: It is a plot or detector signal output versus time or elution volume during the chromatographic process.

127 126 126

12. Column chromatography: A column is tubular structure containing solid stationary substance which is used for the separation, and purification of chemical entities from complex mixtures.

13. COSY-45o Spectrum: A homonuclear two-dimensional NMR spectrum showing 1H- 1H couplings correlations of geminal and vicinal protons in a molecule.

14. Coupling Constant: Scalar coupling is a through-bond interaction between nuclear spins. One manifestation of the J-coupling is the splitting of resonance lines. The magnitude of the splitting is known as the coupling constant "J" and is expressed in cycles/second or Hz. It is independent of the strength of the applied magnetic field but depends on the molecular stereochemistry and dihedral angle between the coupled protons. The J value may be either positive or negative in sign.

15. 1H-NMR Spectrum: A one-dimensional spectrum proton NMR, which provides information about the electronic environment of the protons present in a molecule. The spectral range for 1H-NMR spectrum is 1-12 ppm.

16. Cytotoxicity: Cytotoxicity is the ability of a substance to be toxic to particular cells.

17. Distortionless Enhancement by Polarization Transfer (DEPT): The multi-pulse 13C-NMR experiment at the angles of = 45o, 90o, and 135o to differentiate between primary, secondary, and tertiary carbons.

18. Electron Impact Mass Spectrum (EI-MS): A plot of m/z versus intensities containing information about the structural features through fragmentation pattern of the compounds by application of a high energy electron beam.

19. Eluate: The combination of mobile phase and solute exiting from a column.

20. Eluent: Mobile phase used to elute the sample from a column in order to achieve separation.

21. Elution: The process of passing a mobile phase through the column to separate the sample components.

22. Fast Atom Bombardment Mass Spectrum (FAB-MS): The soft ionization technique used in mass spectrometry to locate the molecular ion peak for the determination of molecular formula.

23. Flow rate: The volumetric flow of mobile phase per unit time through an LC column.

128

24. Gradient elution: A method of chromatographic separation by which the polarity of the mobile phase increases gradually to decrease the separation time.

25. Heteronuclear Multiple Bond Connectivity (HMBC): It is an inverse heteronuclear two-dimensional NMR technique. In these experiments, the magnetization of 13C nuclei is detected through the 1H nuclei in order to increase sensitivity of signals. This technique is used to investigate the long-range heteronuclear interactions between carbons and protons (up to four-bonds).

26. Hetronuclear Multiple Quantum Coherence (HMQC): It is an inverse hetronuclear two-dimensional NMR technique employed to establish the direct 1H/13C one-bond shift correlations. In this experiment, the 13C nuclei are detected through their effects on 1H nuclei.

27. High-resolution Electron Impact Mass Spectrum (HREI-MS): A method of exact mass measurements for direct determination of elemental composition. A double focusing mass spectrometer is normally required to record the HREI-MS.

28. HPLC: Liquid chromatographic technique used for the separation of sample mixture under the influence of high pressure at constant flow rate.

29. Immunomodulator: A substance that influences the immune system of an organism.

30. Infrared Spectroscopy (IR): The technique used to determine the functionalities present in a molecule. Infrared spectra are produced by the absorption of infra-red radiations (350-4200 cm-1) by organic molecules.

31. In-vitro: The experiments done outside the body of living organisms.

32. Isocratic: A constant composition of mobile phase used in liquid chromatography.

33. Mobile phase: The solvent/gas that moves the solutes through a column.

34. Molecular Ion (M+): The ion formed from the molecule by loss of only one electron. The m/z ratio of the molecular ion gives the molecular weight of the compound.

35. Nuclear Overhauser Enhancement Difference Spectrum (NOE): It is a 1D NMR experiment used to determine the stereochemistry of organic molecules with the help of signal enhancement caused by dipolar coupling of nuclei in proximity.

36. Nuclear Overhauser Enhancement Spectroscopy (NOESY): The 2D version of NOE used to determine the stereochemistry of molecule with the help of interacting nuclei through space.

129

37. Octadecylsilane (ODS): Modified silica gel by attachment of octadecyl carbon-chain to silica. It is used as adsorbent in HPLC.

38. Optical Rotation: It is the physical property of the compound to rotate the plane of polarized light, either clockwise or counter-clockwise.

39. Rearrangement: A class of organic reactions where the carbon frame work undergoes skeletal changes without gain or loss of any atom/group.

40. Retention Time (TR): The time difference between the injection and peak maximum of a certain sample. It represents the time taken by certain molecule to pass through the whole length of the column.

41. Reversed-phase Chromatography: A liquid chromatographic technique that uses a non-polar stationary phase and a polar mobile phase. It is the most common method of separation in HPLC.

42. Stereochemistry: It refers to a three-dimensional arrangement of atoms/groups in a space.

43. Stereogenic Center/Chiral Center: Any point in a molecule bearing groups such that an interchange of any two groups which lead to stereoisomers.

44. Thin-Layer Chromatography (TLC): A qualitative/quantitative method of sample analysis used to separate mixtures. Thin-layer chromatography can be employed on aluminum foil, glass plate or plastic sheet with a coated adsorbent. TLC can be developed in a tank containing a solvent mixture. The components in a sample move

with different flow rate (Rf) by the capillary action against the gravity.

45. Ultraviolet Spectrum (UV): It is a plot of wave length of ultraviolet radiations vs absorbance. The absorption spectrum (UV spectrum) of an organic compound is presented in a graph of  or log  against wavelengths expressed in nanometer (nm).

130

8.0 LIST OF PUBLICATIONS

131

8.0 LIST OF PUBLICATIONS

1. Choudhary, M. I., Maher, S., Abbaskhan, A., Begum, Khan, Ambreen., Ali, Sjjad.,

Atta-ur-Rahman. Characterization and Antiglycation Activity of Phenolic

Constituents from Viscum album (European Mistletoe) Chem. Pharm. Bull., 58 980-

982 (2010).

2. Choudhary, M. I., Maher, S., Abbaskhan, A., Atta-ur-Rahman. New Cytotoxic

Withania glucosides from Withania coagulans (in progress).

132