Phytochemical Studies on the Bioactive Constituents of Hypericum Oblongifolium

PHYTOCHEMICAL STUDIES ON THE BIOACTIVE CONSTITUENTS OF HYPERICUM OBLONGIFOLIUM

A thesis submitted to the University of the Punjab For the Award of Degree of Doctor of Philosophy in CHEMISTRY

BY ANAM SAJID

INSTITUTE OF CHEMISTRY UNIVERSITY OF THE PUNJAB LAHORE 2017

DEDICATION

I Dedicate my work To my Parents

My Husband

And My Little Angels Ghanim and Afnan

DECLARATION

I, Anam Sajid d/o Sajid Saddique, solemnly declare that the thesis entitled “Phytochemical

Studies on the Bioactive Constituents of Hypericum oblongifolium” has been submitted by me for the fulfillment of the requirement of the degree of Doctor of Philosophy in Chemistry at Institute of Chemistry, University of the Punjab, Lahore, under the supervision of Dr. Ejaz

Ahmed and Dr. Ahsan Sharif.

I also declare that the work is original unless otherwise referred or acknowledged and has never been submitted elsewhere for any other degree at any other institute.

Anam Sajid Institute of Chemistry, University of the Punjab, Lahore

APPROVAL CERTIFICATE

It is hereby certified that this thesis is based on the results of experiments carried out by Ms.

Anam Sajid and that it has not been previously presented for a higher degree elsewhere. She has done this research work under our supervision. Also we found no typographical and grammatical mistake while reviewing the thesis. She has fulfilled all requirements and is qualified to submit the accompanying thesis for the award of the degree of Doctor of

Philosophy in Chemistry.

Supervisors

Dr. Ejaz Ahmed Institute of Chemistry, University of the Punjab, Lahore, Pakistan.

Dr. Ahsan Sharif Institute of Chemistry, University of the Punjab, Lahore, Pakistan.

Acknowledgement

Saying of Prophet Muhammad (PBUH) ‘a person who is not thankful to his benefactors is not thankful to Allah’ so I would like to thank all the people who contributed in some way to the work described in this thesis. All and every kind of praises is upon ALLAH Almighty the strength of universe, who ever help in darkness and difficulties. I have a great honour to offer my heartiest gratitude to my worthy advisors Dr. Ejaz Ahmed & Dr. Ahsan Sharif for their inspiring guidance without which this work would never been materialized. Sir, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been invaluable. I also want to thank you for letting my defense be an enjoyable moment, and for your brilliant comments and suggestions, thanks to you.

Every result described in this thesis was accomplished with the help and support of HEC and

HEJ so I greatly appreciate their cooperation as they responded promptly and enthusiastically to my requests despite their congested schedules. A special thanks to my husband Ghulam

Mustafa, mother-in-law, father-in-law and mother, words cannot express how grateful I am to my family for all of the sacrifices that they’ve made on my behalf. I greatly benefited from suggestions of my beloved sisters Arfaa and Najam. Their prayer for me was what sustained me thus far. I would also like to thank to my fellows Faiza, Naila, Sumra, Fatima and Zahid.

Thanks to Faiza and Naila for supporting me for everything, and especially I can’t thank you enough for encouraging me throughout this experience. To my beloved kids Ghanim and

Afnan, I would like to express my thanks for being such good guys always cheering me up.

Finally I thank my God, Allah Almighty, for letting me through all the difficulties. I have experienced His guidance day by day. He is the one who let me finish my degree. I will keep on trusting Him for my future.

Anam Sajid

TABLE OF CONTENTS

Chapter 1 Introduction 1-16

1.1 Introduction 1

1.2 Terpenes 4

1.3 Steroids 7

1.4 Phenolics 8

1.5 Alkaloids 10

1.6 Hypericaceae family 13

1.7 Genus Hypericum 13

1.8 Hypericum oblongifolium 15

1.9 Pharmacological importance of H. oblongifolium 15

Chapter 2 Review of Literature 17-68

2.1 Review of Literature of Hypericum oblongifolium 17

2.1.1 Structures of the compounds previously isolated from 25

Hypericum oblongifolium

2.2 Biosynthesis of Terpenes 31

2.2.1 Biosynthetic route for terpenes 31

2.2.2 Cyclization of Squalene 36

2.2.3 Oxidative cyclization of squalene 38

2.2.3.1 Cyclization of squalene epoxide in chair-chair-chair-boat 38

sequence

2.2.3.2 Lupane and Hopane series 44

2.2.4 Non-oxidative cyclization of squalene 46

2.2.5 Cyclization at both ends of squalene molecule 47

2.3 Biosynthesis of steroids 48

2.3.1 Formation of Cholesterol from Lanosterol 49

2.3.2 Stigmasterol and β-Sitosterol 52

2.4 Biosynthesis of Fatty Acids 55

2.5 Biosynthesis of flavonoids 60

2.5.1 Biosynthesis of Flavanone 65

2.5.2 Formation of Isoflavone 66

2.5.3 Formation of flavone 67

2.5.4 Formation of Flavanol 68

Chapter 3 Experimental 69-109

3.1 General Experimental Conditions 69

3.1.1 Physical constants 69

3.1.2 Spectroscopy 69

3.1.3 Chromatography 70

3.1.4 Spray reagent for visualization of spot 71

3.1.4.1 Ceric Sulphate Reagent 71

3.2 Extraction and isolation 71

3.2.1 Plant material 71

3.2.2 Extraction and Isolation 71

3.3 Characterization of new compounds 77

3.3.1 Hyperinoate A (86) 77

3.3.2 Hyperinoate B (87) 79

3.3.3 Hyperinone (88) 81

3.3.4 Hyperinoic acid (89) 83

3.4 Characterization of known compounds 85

3.4.1 4,4-Dimethyl cholesterol (90) 85

3.4.2 β-Sitosterol (91) 87

3.4.3 Lupeol (92) 89

3.4.4 Taraxerol (93) 91

3.4.5 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94) 93

3.4.6 Oleanolic acid (95) 95

3.4.7 Erectasteroid D (96) 97

3.4.8 (S)-4', 5-Dihydroxy-7-methoxyflavanone (97) 99

3.4.9 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98) 101

3.4.10 α-D-Glucopyranosyl-6'-O-hexadecanoate (99) 103

3.4.11 β-sitosterol-3-O-β-D-glucopyranoside (100) 104

3.4.12 Quercetin-3'-O-β-D-glucopyranoside (101) 106

3.5 Biological screening 108

3.5.1 Lipoxygenase Inhibitory Assay 108

3.5.2 Antibacterial assay 108

3.5.3 Antifungal assay 108

Chapter 4 Results and Discussion 110-166

4.1 Hyperinoate A (86) 110

4.2 Hyperinoate B (87) 116

4.3 Hyperinone (88) 121

4.4 Hyperinoic acid (89) 127

4.5 4,4-Dimethyl cholesterol (90) 133

4.6 β-Sitosterol (91) 135

4.7 Lupeol (92) 136

4.8 Taraxerol (93) 138

4.9 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94) 140

4.10 Oleanolic acid (95) 142

4.11 Erectasteroid D (96) 144

4.12 (S)-4', 5-Dihydroxy-7-methoxyflavanone (97) 146

4.13 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98) 148

4.14 α-D-Glucopyranosyl-6'-O-hexadecanoate (99) 150

4.15 β-sitosterol-3-O-β-D-glucopyranoside (100) 152

4.16 Quercetin-3'-O-β-D-glucopyranoside (101) 154

4.17 Biological screening 156

4.17.1 Lipoxygenase inhibitory activity 156

4.17.1.1 Lipoxygenases 156

4.17.1.2 The 5-Lipoxygenase pathway 156

4.17.1.3 Lipoxygenase inhibitory activity of compounds 86-101 159

4.17.2 Antimicrobial activity 161

4.17.2.1 Introduction 161

4.17.2.2 Antibacterial activity 161

4.17.2.3 Antifungal activity 162

4.17.2.4 Antimicrobial activities of compounds 86-101 164

Conclusion 167

References 168

LIST OF TABLES

Table 1 Classification of Terpenes 4

Table 2 Classification of Phenolic compounds 8

Table 3 Classification of Alkaloids 12

Table 4 Compounds previously isolated from Hypericum 21

oblongifolium

1 13 Table 5 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 115

of compound 86, with J values (Hz) in parenthesis.

1 13 Table 6 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 120

of compound 87, with J values (Hz) in parenthesis.

1 13 Table 7 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 126

of compound 88, with J values (Hz) in parenthesis.

1 13 Table 8 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 132

of compound 89, with J values (Hz) in parenthesis.

Table 9 In vitro quantitative inhibition of lipoxygenase by 160

compounds 86-101.

Table 10 Antibacterial and Antifungal activity of compounds 86-90 165

from Hypericum oblongifolium

Table 11 Antibacterial and Antifungal activity of compounds 91-101 166

from Hypericum oblongifolium

LIST OF FIGURES

Figure 1 Structures of some Terpenoids 6

Figure 2 Structures of some common steroids 7

Figure 3 Structures of some common flavonoids 10

Figure 4 Structures of some common alkaloids 11

Figure 5 Different parts of Hypericum oblongifolium 16

Figure 6 Important HMBC correlations in 86 114

Figure 7 Important NOESY correlations in 86 114

Figure 8 Important HMBC correlations in 87 119

Figure 9 Important NOESY correlations in 87 119

Figure 10 Important HMBC correlations in 88 124

Figure 11 Important NOESY correlations in 88 125

Figure 12 Important HMBC correlations in 89 130

Figure 13 Important NOESY correlations in 89 131

Figure 14 The translocation of 5- Lipoxygenase, and cPLA2, to the nucleus, 157

upon cellular, stimulus following by the leukotrienes generation.

Figure 15 Bacterial mode of action 162

LIST OF ABBREVIATIONS

BB Broad Band

CDCl3 Deutrated Chloroform

13C-NMR Carbon-13 nuclear magnetic resonance

CoA Coenzyme A d Doublet dd Doublet of doublet

DEPT Distortionless enhancement by Polarization transformation e.g. For example

EtOAc Ethylacetate

EIMS Electron ionized mass spectrometry

FT-IR Fourier-transform infrared spectroscopy g Gram

1H-NMR Proton Nuclear Magnetic Resonance

2D-NMR Two dimensional nuclear magnetic resonance spectroscopy

HMBC Heteronuclear Multiple Bond Correlation

HMQC Heteronuclear Multiple-Quantum Correlation

H-H-COSY Proton-Proton Correlation Spectroscopy

HREIMS High Resolution Electron Ionization Mass Spectrometry

HR-FAB-MS High Resolution Fast Atom Bombardment Mass Spectrometry

Hz Hertz

IC50 Half maximal inhibitory concentration

i.e. That is to say

IR Infrared

J Coupling constant m Multiplet

[M]+ Molecular ion

MHz Mega Hertz m/z mass to charge ratio max. Maximum

Me Methyl

MeOH Methanol m.p. Melting Point

MS Mass Spectroscopy n Normal

NOESY Nuclear Overhauser Effect Spectroscopy

[α]D Optical rotation rel. int. Relative Intensity

OAc Acetate

OMe Methoxy s Singlet t Triplet

TLC Thin layer chromatography

TMS Tetramethyl silane

UV Ultraviolet

α alpha

β beta

δ Chemical shift

Ʋ Frequency

 Molar absorptivity

 Wavelength

Summary

Hypericum oblongifolium is a flowering plant in Hypericaceae family. It is found at an altitude of 4000-6000 meter especially, in Himalaya, China and northern parts of Pakistan. It has been traditionally used in Chinese herbal medicine for treatment of bacterial and viral infection, burns, hepatitis, swellings, bruises, nasal hemorrhage and inflammations. A series of pharmacological properties, ranging from wound healing and antiseptic to antiviral, anti- inflammatory, anticancer, ethanol intake inhibition and apoptosis-inducing activities have been associated with this plant. This plant has been proved to be act as anti-ulcer, anti-proliferative and anti-inflammatory agent. The plants also contain compounds which are chymotrypsin, urease and lipoxygenase inhibitors. H. oblongifolium have also been reported for its antispasmodic, bronchodilator, hypotensive and anti-myocardial infraction behavior.

In the present study whole plant of H. oblongifolium was selected for the identification and isolation of medicinally important natural products present in it. From the chloroform and ethyl acetate soluble fractions of H. oblongifolium, sixteen compounds have been isolated. Four compounds were considered as new natural products, while twelve compounds were identified as known compounds. Their structures were elucidated by the use of sophisticated modern spectroscopic techniques (1H-NMR, 13C-NMR, HMBC, HMQC, 1H-1H- COSY, NOESY,

HREIMS and HR-FAB-MS) and several chemical tests. Following are the structures of new compounds.

New Compounds isolated from Hypericum oblongifolium

Chemistry of Natural compounds (Submitted)

Journal of Chemical Society of Pakistan, Vol. 40, No. 1, 249-254, 2018.

Chemical and pharmaceutical Bulletin (Submitted)

Known Compounds from Hypericum oblongifolium • 4,4-Dimethyl cholesterol (90) • ß- Sitosterol (91) • Lupeol (92) • Taraxerol (93) • 4,4-Dimethylergosta-8,12,24(28)-triene-3β,12β,17α-triol (94) • Oleanolic acid (95) • Erectasteroid D (96) • (S)-4',5-Dihydroxy-7-mrthoxy isoflavone (97) • 7,4'-Dihydroxy-5,3-dimrthoxy flavanone (98) • α-D-Glucopyranosyl-6'-O-hexadecanoate (99) • ß-sitosterol-3-O-ß-D-glucopyranoside (100) • Quercetin-3'-O-ß-D-glucopyranoside (101)

Journal of Chemical Society of Pakistan, Vol. 40, No. 1, 249-254, 2018.

Chapter 1 Introduction

1.1: Introduction

Human beings have a great connection with nature from their origin. Since that time they are trying to discover nature in different ways for their betterment and humans have always been used natural resources to improve their lifestyle. They trusted on nature for basic need like food production, means of transportation, shelter, clothing, flavors, fragrances and medicines also [1].

Health has always been the most important issue of human beings and they always try to found natural sources to overcome health issues. Nature has been serving as a good source of therapeutic agents for thousands of years and a close relationship of nature and medicines has developed through traditional medicinal system [2]. The history of Chinese, Indian and South

African civilizations provide written evidence for use of natural sources as remedy for different diseases [3]. The first written document is a 4000 years old Sumerian clay tablet used for treatment of various diseases [4].

In a broad sense natural product is a substance that produced by living organism. If we specify the natural product to the field of organic chemistry it may be defined as purified organic compounds obtained from natural sources and are produced by the pathways of primary and secondary metabolites [5]. In medicinal chemistry definition of natural product is limited to secondary metabolites only. So, natural product is a term used for those chemical substances that found in nature and have distinctive pharmacological effects. Natural products may be derived from microorganism, marine and terrestrial organisms (plants and animals). Most of the modern drugs are based on natural sources and many of these based on their use in traditional medicines [6]. As the terrestrial environment has extraordinary life diversity so most 2

of the natural products that act as a good drug or discussed so far are from terrestrial habitats and mostly from plants [7].

Plants are playing a crucial role in the lives of human beings throughout the world. People satisfy their basic needs like food, shelter, clothing and health care from plants. According to an approximation 80 % population of the world is relying on the plants for their health and healing [8]. A plant which contains therapeutic substances in one or more of its organs which can be used as precursor for useful drugs is called medicinal plant. A variety of medicinal plants and their constituents have shown useful therapeutic properties [9]. For example, turmeric was used for blood clotting, endive plant roots were used as remedy for gallbladder disorders, garlic was prescribed for circulatory diseases and mandrake was helpful in reliving from pain. These are still being used in different countries as alternative medication. Medicinal plants have played an important function in the maintenance of human health as well as to improve the quality of human life for thousands of years. Even in the era of combinatorial chemistry and biotechnology, there was a great demand of herbal formulations and medicines.

Despite of overwhelming effect of modern medicine and remarkable advancement in synthetic drugs, still a huge population of the world prefers to use plant originated remedies for various illnesses [10].

The use of medicinal plants to eradicate different diseases is very frequent in the medical tradition of various cultures [11]. The earliest herbal medicines used 6400 BC was an analgesic drug (ethanol) produced by fermentation of sugar in a solution by yeast (Ortega). Quinine an antimalarial drug isolated from cinchona bar in 19th century [12] and in coming year’s use of taxol for the treatment of cancer [13] shows the consistency for drug development from plants.

World Bank reported in 1997 that plant derived medicines have been gaining importance 3

worldwide. About fifty percent of the medicines available in the market are made by basic natural materials. It is interesting to know that medicinal herbs never lost their market value as many of the plant based active ingredients of medicines cannot yet be synthesized [14].

The medicinal properties of the plants depend upon some chemical substances found in plants and these are secondary metabolites of plant. The plants are solar powered biosynthetic and biochemical laboratory which uses water, minerals, sunlight and air to produce both primary and secondary metabolites. The primary metabolites (carbohydrates, fatty acids, amino acid etc.) are extensively distributed in nature as these are required for general and physiological development of plants. While the secondary metabolites (steroids, terpenoids, alkaloids glycosides etc.) are produced from primary ones during biosynthesis pathways. Secondary metabolites are rare in animals but common in bacteria, fungi and plants. These are not required for plant growth but act as defensive or offensive chemicals against predators like insects, herbivores and microorganisms. These are produced to adapt environmental changes and sometimes are considered as waste products of plant metabolism but have pharmaceutical importance. Each family of plants produce specific mixture of secondary metabolites.

Sometimes these metabolites can be used as taxonomic characters for classifying plants [15].

Depending upon the biogenetic route, the plant secondary metabolites can be distributed in three major chemically different classes.

1. Terpenes

2. Phenolics

3. Nitrogen containing and sulfur containing compounds

1.2: Terpenes The terpenes are among the most diverse group of metabolites. There are more than 23000 terpenes reported from plant origin. This is the largest family of natural products having wide range of structure (from linear to polycyclic molecules) and size (from the five carbon hemi- terpenes to natural rubber consisting of thousands of isoprene units). Terpenes are constructed by isoprene units which are joined in head to tail manner to form different compounds.

Terpenes are synthesized from acetate via mevalonic acid pathway [16]. Terpenoids have the general formula (C5H8)n. Their classification depend upon the carbon number in the molecule or on the basis of n (Table 1).

Table 1: Classification of Terpenes

Each of these classes can be subdivided into further subclasses on the basis of ring present in the molecule.

 Acyclic Terpenoids (open structure) 5

 Monocyclic Terpenoids (one ring in the structure)

 Bicyclic Terpenoids (two rings in the structure)

 Tricyclic Terpenoids (Three rings in the structure)

 Tetracyclic Terpenoids (four rings in the structure)

 Pentacyclic Terpenoids (five rings in the structure)

Mono and sesquiterpenes are volatile compounds from the plant. These have lipophilic nature with high vapor pressure. The plant synthesized volatile substances has been exceeded from

1000 in number and this number is growing as the more plants are being examined.

The basic skeleton of diterpenes has 20 carbons and are derived from geranyl diphosphate.

Cyclization reaction of geranyl diphosphate produce different compounds of this class having large polarity range from apolar hydrocarbon (cembrene) to a 14 membered ring structure

(virescenoside).

Sesterpenes is the least common type of terpenes. It originates from geranylfarnesyl diphosphate which cyclizes to produce various skeletal types.

Tertraterpenoids are carotenoids which is considered the biggest group of natural dyes. The precursor of carotenoids is also geranyl diphosphate which originates the basic C40 skeleton which further modifies to various compounds [17]. Some examples of terpenoids are given in

Figure 1. 6

Figure 1. Structures of some Terpenoids

Triterpenoids belongs to a large group of natural products including steroids and sterols.

Squalene (C30) is the biological precursors of all the triterpenoids. Most of the triterpenes are tetracyclic or pentacyclic. Tetracyclic compounds have three six membered and one five membered ring while pentacyclic have four six membered and one five membered ring. 7

Tetracyclic triterpenes are steroid which are found in minor quantity in animals while these are abundant in plants.

1.3: Steroids

Steroids has 1,2-cyclopentanophenanthrene ring as basic skeleton with methyl substituents at

C-10 and C-13. Beyond this a side chain at C-17 is also the part of skeleton of steroids. The length of the side chain and stereochemistry of its stereo centers leads to the diversity in steroid skeleton. Besides this, a Δ5,6 olefinic bond and a hydroxyl moiety biogenetically at carbon 3 are also part of this skeleton. Natural sterols have the cholestane, ergostane or stigmastane

skeleton. “Their classificationis based upontheir” origin as zoosterols, “phytosterols,mycosterols

and marinesterols”. “Some of the plantsterols are naturally present in alittle extent insome fruits,

seeds, vegetables, cereals, nuts, legumesand some other plantsources” [18]. Some examples of steroids are shown in Figure 2.

Figure 2. Structures of some common steroids 8

1.4: Phenolics Phenolic compounds are one of the most widely distributed group of secondary metabolites.

These are derived through three different biogenetic routes in plants, i.e. pentose phosphate, phenylpropanoid and shikimate pathway. Phenolic compounds have great morphological and physiological importance for plants as well as for humans. These play vital role in reproduction and growth of plants and also protect plants from predators and pathogens. For human beings these compounds act as anti-allergic, antioxidant, anti-inflammatory, anti-thrombotic, antimicrobial, anti-artherogenic as well as they have cardio-protective and vasodilatory effects.

Structure of the phenolic compounds based on an aromatic ring having one or more hydroxyl substituents. Phenolics range in structure from simple phenolic compounds to highly polymerized molecules. This structural diversity produces wide range of naturally occurring phenolic compounds which can be classified into various classes as depicted in Table 2.

Table 2. Classification of phenolic compounds

Among all these classes of phenolic compounds flavonoids, phenolic acid and tannins are considered the main dietary components [19].

Flavonoids are most abundant naturally occurring polyphenols that are widely distributed in plant kingdom. These are the derivatives of diphenylpropane and include flavones, flavanones, flavanols and antocyanidines. Flavonoids are an important and frequent part of human diet and have number of pharmacological activities like antioxidant, anti-inflammatory, anti- carcinogenic, anti-proliferative. These are also have antiulcer, antiviral, anti-hepatotoxic, antimicrobial and immune-modulatory activities. The chemical structure of flavonoids based on two benzene rings surrounded with a heterocyclic six membered ring with oxygen in the ring [20]. Some examples of flavonoids are given in Figure 3.

Figure 3. Structures of some common flavonoids

1.5: Alkaloids

The term alkaloid derived from the word alkaline and used for nitrogen containing basis. These are defined as the compounds which have nitrogen in heterocyclic ring, belongs to plants kingdom and are physiological active. A large number of organisms produce alkaloids, for example, fungi, bacteria, animals and plants. Alkaloids form a major group of natural products.

Caffeine, cocaine, morphine, nicotine, and quinine are some common examples of alkaloids. 11

Figure 4. Structures of some common alkaloids

There is wide variety of structures in alkaloid molecules due to which their classification is very complex. Mostly these are classified on the basis of nitrogen containing ring, or on the basis of their biogenetic origin.

Table 3. Classification of alkaloids

Alkaloids have many physiological effects on human body, so play an important role in maintaining human health. Many alkaloids are being used as drugs like, quinine obtained from

Cinchona officinalis has been used as antibiotic especially as anti-malarial drug. Morphine has analgesic properties. Taxol and vinblastine are anticancer drugs. Tubocurarine is a muscle reactant [7].

Medicinal plants have a promising future because there are about half million plants around the world, and medical activities of most of the plants have not investigate yet, and their medical activities could be significant in the treatment of present or future studies [21]. 13

1.6: Hypericaceae family Hypericaceae is a family represented worldwide by 9 genera and 540 species. It includes shrubs to trees but some annual herbs also fall under this family. Taxa that grow in drier regions mostly develop lignotuber for support after fire or drought while those inhabiting swamps have swollen rote with air spaces. Leaves are mostly opposite while rarely are more or less irregularly spiral or whorled [22]. Nine genera under Hypericaceae are

1. Cratoxylum

2. Eliea

3. Harungana

4. Hypericum

5. Lianthus

6. Santomasia

7. Thornea

8. Triadenum

9. Vismia

1.7: Genus Hypericum

Hypericum is a large genus which mostly comprises of herbs and shrubs. Its plant widely found in temperate regions of the world [23]. Members of this genus are habitant of areas ranging from rocky sea-facing cliffs to damp biotopes around mountainous streams [24]. In Pakistan this genus is represented by nine species [25] which are Hypericum choisianum, Hypericum dyeri, Hypericum elodeoides, Hypericum monogynum, Hypericum napaulense, Hypericum oblongifolium, Hypericum perforatum, Hypericum scabrum and Hypericum uralum. These species are found to be the rich source of phenolic compounds which are accumulated in plants 14

with the phloroglucinol substitution pattern and exhibited antiviral [26], antifungal [27], anticancer [28] and antibiotic [29] activities. From the last two centuries Hypericum species are well known as healing herbs because of their different medicinal properties [30].

After, the discovery of antidepressant activity of Hypericum perforatum, scientists has shown great interest in the study of plants of genus Hypericum [31]. The plants of this genus are also a source of phloroglucinol derivatives of which some are related to the well-known hyperfine, found in H. perforatum while others present a phloroglucinol unit conjugated with a filicinic acid moiety. Antifungal and antibacterial activities were exhibited by phloroglucinol derivatives, found mostly in lipophilic fractions of Hypericum species, against microorganisms for example Staphylococcus aureus, Bacillus cereus, Bacillus subtilis and Nocardia gardenen.

Their presence could justify the popular use of some Hypericum species as wound healing agents and in the treatment of some microbiological diseases. More recently extracts of

Hypericum species have shown a significant antidepressant effect when administered to humans. Numerous clinical trials and meta-analyses assessing the efficacy of Hypericum species for lessening of mild to moderate depression report that extracts are significantly

superior toplacebo, similarly effective asstandard antidepressant drugs ” (such as simiprimine and diazepam), and produce significantly fewer side-effects than the synthetic preparation.

Other substances found in some species of Hypericum are benzopyrans, xanthones, flavonoids and the tannins, have also shown antimicrobial activity against various bacteria and fungi [32,

33].

1.8: Hypericum oblongifolium

Hypericum oblongifolium is a flowering plant in Hypericaceae family. It is an herbaceous plant generally 6-12 meter in height. The leaves of H. oblongifolium are yellowish green in color and 1-2 cm long. It is considered as a native flowering plant of Eurasia. It is found at an altitude of 4000-6000 meter especially, in Himalaya, China and northern parts of Pakistan [34]. In

Pakistan it is found in Kashmir, Hazara and Murree Hills [35].

1.9: Pharmacological importance of H. oblongifolium

A series of pharmacological properties, ranging from wound healing and antiseptic to antiviral, anti-inflammatory, anticancer, ethanol intake inhibition and apoptosis-inducing activities have been described. Several researches have concluded that the total pharmacological activity of

H. oblongifolium preparations may depend not on a single compound, but on the combined activities of several plant constituents. These compounds work synergistically and cannot be separated into active parts.

Hypericum oblongifolium has been traditionally used in Chinese herbal medicine for treatment of bacterial and viral infection, burns, hepatitis, swellings, bruises, nasal hemorrhage and inflammations. It has also been used as remedy for dog bites and bee stings [36-38]. Now days it has been proved to be act as anti-ulcer [36], anti-proliferative [39] and anti-inflammatory

[37] agent. The plants also contain some compounds which act as chymotrypsin inhibitor [34] and some others can act as urease inhibitor [38]. During pharmacological evaluation this plant was reported to have antispasmodic, bronchodilator, hypotensive and cardiovascular inhibitory activity [35].

Figure 5. Different parts of Hypericum oblongifolium

Chapter 2 Review of Literature

2.1: Review of Literature

Raziq et al., (2016) worked on the crude methanolic extract of Hypericum oblongifolium for

investigating its anti-nociceptive, antipyretic and anti-inflammatory activities. “Acetic acid

inducedwrithing test” and hot plate test wereused for anti-nociceptive while antipyretic and anti- inflammatory potential was checked against yeast induced hyperthermia and carrageenan induced paw edema in mice, respectively. The methanolic extract of H.oblongifolium exhibited significant antinociceptive, antipyretic and anti-inflammatoryeffects comparable to standard diclofenac sodium and paracetamol [40].

Raziq et al., (2015) isolated two new compounds from Hypericum oblongifolium one was flavones named as folicitin while second one was a bicyclic conjugates lactone, folenolide.

DPPH radical scavenging assay revealed that folicitin is a good antioxidant while, folenolide was totally inactive against this activity [41].

Ali et al., (2014) found three new xanthones hypericorin C, hypericorin D and 3,4-dihydroxy-

5-methoxyxanthone, in root extract of H. oblongifolium. Except these they also isolated eight

known compounds: “2,3-dimethoxyxanthone”,“3,4-dihydroxy-2-methoxyxanthone”,3,5-

dihydroxy-1-methoxyxanthone”, “3-acetylbetulinic acid”, “10H-[1,3]dioxolo[4,5-b]xanthen-10-

one”, “3-hydroxy-2-methoxyxanthone”, “3, 4, 5-trihydroxy-xanthone” and “betulinic acid”. These compounds were subjected to contemporary assay for urease inhibition activity. Only 3,4- dihydroxy-5-methoxyxanthone and 3, 4, 5-trihydroxy-xanthone showed good activity. The compounds hypericorin D, 2,3-dimethoxyxanthone and 3,5-dihydroxy-1-methoxyxanthone were moderately active against urease. Hypericorin C exhibited very weak activity against urease [36]. 18

Abbas et al., (2013) investigated H. perforatum and H. oblongifolium for their potential antioxidant, antiglycation, anti-lipid peroxidation activity and cytotoxity. n-Butanol fraction of

H. oblongifolium was active against oxidation (IC50 = 215.375±3.562 μg/mL), glycation

(57.250 %) and lipid peroxide (54.219 %). Dichloromethane and methanol fraction only inhibited lipid peroxide 67.206 % and 61.874% respectively. n-Hexane fraction exhibited only anti-glycation activity (50.018 %). All the fractions were largely non-toxic in cytotoxicity assay except dichloromethane fraction which showed mild toxicity [42].

Ali et al., (2013) discovered two new xanthones hypericorin A and hypericorin B in the extract of twigs of H. oblongifolium Wall. They also found five known compounds in the same extract.

These were 4-hydroxy-2,3-dimethoxyxanthone, 1,3-dihydroxy-5-methoxyxanthone, kielcorin,

1,3,7-trihydroxy xanthone and 3,4,5-trihydroxy xanthone. All the seven compounds were screened for in-vitro anti-inflammatory activity using isolated human neutrophils. Compound

1,3-dihydroxy-5-methoxyxanthone was moderately active against inflammation while, compound 4-hydroxy-2,3-dimethoxyxanthone was totally inactive at 1000 μg/mL. Other compounds exhibited significant activities (IC50 = 816.23±73.30, 985.20±55.80,

965.21±65.80, 907.20±50.80 and 975.20±81.10 μM) [37].

Ali et al., (2011) investigated the “antiproliferative activityof hexane, ethyl acetate, butanoland

aqueous extracts”of H. oblongifolium in vitro on the cell lines: “NCI-H460 human non-smallcell

lungcarcinoma”, “HT-29human colonadenocarcinoma”, RXF-393 human renal “cell carcinoma”,

MCF-7 human breast cancer and “OVCAR-3 humanovarian adenocarcinoma” with etoposide as

positive control. Among all extracts thehexane “showed relativelypotent anti-proliferative

activity” (10.55 ± 4.19 µg/mL) on OVCAR-3 human ovarian adenocarcinoma cell growth. They

also isolated seven compounds from this plant. These were identified as “tetracosyl 3-(3,4- 19

dihydroxyphenyl) acrylate”, β-sitosterol, shikimic acid, “1-octatriacontanol”, 18βH-urs-20 (30)- en-3β-ol-28-oic acid, hexacosyl tetracosanoate and β-Sitosterol-3-O-β-D-glucopyranoside

[39].

Arfan et al., (2010) isolated three urease inhibitor 3,4,5-trihydroxyxanthone, tetracosyl 3-(3,4- dihydroxyphenyl) acrylate and 1,3,7-trihydroxy xanthone from H. oblongifolium by bioassay- guided fractionation. Tetracosyl 3-(3,4-dihydroxyphenyl) acrylate depicted a potent enzyme inhibition activity with IC50 = 20.96±0.93. 3,4,5-Trihydroxyxanthone and 1,3,7-trihydroxy xanthone also showed significant enzyme inhibition activity. They also tested sub crude extracts for their urease inhibition activity. Ethyl acetate and water fractions exhibited significant activity (IC50 = 140.37±1.93 and 167.43±3.03 respectively) [38].

Khan et al., (2010) checked H. oblongifolium for its “antispasmodic,bronchodilator and blood

pressurelowering properties” with possible mechanism of action. They found that H. oblongifolium possessed antispasmodic, bronchodilator, cardiac inhibitory activity along wiyh hypotensive and vasodilator effects, mediated possibly through Ca++ antagonism [35].

Ferheen et al., (2006) isolated two new taraxastane type triterpenes hyperinols A and B from chloroform soluble fraction of H. oblongifolium both these compounds depicted chymotrypsin inhibitory activity [34].

Ferheen et al., (2005) discovered eleven compounds in H. oblongifolium as 4-hydroxy-2-

methl-tricos-2-en-22-one, (S)-4',5-dihydroxy-7-methoxyflavanone, “1β,3β,23-trihydroxy-

olean-12-en-28-oic acid”, gernotoxanthone J, 3',5,7-trihydroxy-4'-methoxyisoflavone 3'-O-β-

D-glucopyranoside, 7,4'-dihydroxy-5,3'-dimethoxyisoflavone, “Ophioglonin-7-O-β-D-

glucopyranoside”, Quercitine-3-O-β-D-glucopyranoside, 2-(4-hydroxyphenyl)-ethanol, 2- hydroxybenzyl-β-D-glucopyranoside, 3β-acetoxy-2α-hydroxy-urs-12-en-28-oic acid [43]. 20

The literature survey revealed many “compounds havebeen isolated” from Hypericum

oblongifolium and most of themare Terpenoids, steroids, flavonoidsand xanthones. “An up-to-

datereview” ofcompounds reported from Hypericum oblongifolium is given in Table 4.

Table 4: Compounds previously isolated from Hypericum oblongifolium

Sr. Compound name Mol. Physical state Parts used Ref No. Formula M.P °C Mol. wt

1 Folecitin C21H21O11 Yellow brown Whole plant [41] 449.1083 crystalline solid 187-189

2 Folenolide C7H9O4 White crystalline Whole plant [41] 157.0495 solid 175-176

3 Hypericorin C C26H22O9 White amorphous Roots [36] 479.1359 solid 230-232

4 Hypericorin D C24H20O10 White amorphous Roots [36] 467.1359 solid 250-254

5 3,4-Dihydroxy-5- C14H10O5 Pale yellow Roots [36] methoxyxanthone 258.0528 amorphous solid 230-235

6 2,3- C15H12O4 White crystalline Roots [36] Dimethoxyxanthone 256.2570 solid 145-150

7 3,4-Dihydroxy-2- C14H10O5 Yellowish Roots [36] methoxyxanthone 258.0528 amorphous powder 243-245

8 3,5- C14H10O5 White amorphous Roots [36] Dihydroxy-1- 258.0528 powder methoxyxanthone 354-355 22

9 3-Acetylbetulinic C32H50O4 White needles Roots [36] acid 498.8005 180-182

10 “10H-[1,3]Dioxolo C14H8O4 Crystalline Roots [36] [4,5-b]xanthen-10- 240.0422 217-218

one”

11 3-Hydroxy-2- C14H10O4 Crystalline Roots [36] methoxyxanthone 242.0579 225-230

12 3,4,5- C13H8O5 Yellow Roots, [36- Trihydroxyxanthone 244.0371 amorphous Twigs 38] powder solid 280-283

13 Betulinic acid C30H48O3 Crystalline Roots [36] 456.7003 316-318

14 Hypericorin A C26H22O9 White solid Twigs [37] 478.1270 235-238

15 Hypericorin B C24H20O8 White amorphous Twigs [37] 436.2123 solid 240-243

16 Kielcorin C24H20O8 Crystalline Twigs [37] 436.4110 250-251

17 4-Hydroxy-2,3- C15H12O5 Pale yellow Twigs [37] dimethoxyxanthone 272.2530 rectangular plates 218-219

18 1,3-Dihydroxy- C14H10O5 Yellow needles Twigs [37] 5-methoxyxanthone 258.0528 228-229

19 1,3,7-Trihydroxy C13H8O5 Yellow solid Twigs [37, xanthone 244.0371 318-320 38]

20 “18βH-urs-20 (30)-en- C30H48O3 Colorless solid Twigs [39]

3β-ol-28-oic acid” 456.3603 262-265 23

21 Tetracosyl 3-(3,4- C33H56O4 White solid Twigs [38, dihydroxyphenyl) 516.4178 202-205 39] Acrylate

22 Shikimic Acid C7H10O5 White solid Twigs [39] 174.0528 190-193

23 1-Octatriacontanol C38H78O White amorphous Twigs [39] 551.6052 powder solid 63-66

24 Hexacosyl C50H100O2 White amorphous Twigs [39] tetracosanoate 733.3480 powder solid 79-81

25 β-Sitosterol C29H50O Colorless crystals Twigs [39] 414.3851 136-138

26 β-Sitosterol-3-O-β-D- C35H60O6 Colorless crystals Twigs [39] glucopyranoside 576.4389 236-238

27 Hyperinol A C30H46O3 Colorless crystal Whole plant [34] 454.3336 260-262

28 Hyperinol B C30H46O4 Colorless crystal Whole plant [34] 470.3396 246-248

29 4-Hydroxy-2-methyl- C24H42O2 Colorless crystal Whole plant [43] tricos-2-en-22-one 366.3466 145-147

30 (S)-4',5-Dihydroxy-7- C16H14O5 Colorless needles Whole plant [43] methoxyflavanone 286.089 152-154

31 7,4'-Dihydroxy-5,3'- C17H14O6 Crystalline Whole plant [43] dimethoxyisoflavone 314.0780 283-285

32 2-(4-Hydroxyphenyl)- C8H10O2 Colorless needles Whole plant [43] ethanol 138.0659 85-86

33 Gernotoxanthone J C19H18O6 Brownish yellow Whole plant [43] 342.1140 needles 279-280 24

34 “3β-Acetoxy-2α- C30H50O5 Colorless oil Whole plant [43] hydroxy-urs-12-en- 490.3688

28-oic acid”

35 “1β,3β,23-Trihydroxy- C30H48O5 Gummy solid Whole plant [43] olean-12-en-28-oic 488.3501

acid”

36 Quercitine 3-O-β-D- C21H21O12 Yellow Whole plant [43] glucopyranoside 465.1025 amorphous powder 244-246

37 2-hydroxybenzyl β-D- C13H19O7 Amorphous Whole plant [43] glucopyranoside 287.1130 powder 64-65

38 Ophioglonin 7-O-β- C22H20O12 Yellow Whole plant [43] D-glucopyranoside 477.1011 amorphous powder 275-277

39 3',5,7-trihydroxy-4'- C22H23O11 White needles Whole plant [43] methoxyisoflavone 3'- 463.1131 236-237 O-β-D- glucopyranoside

2.1.1: Structures of the compounds previously isolated from Hypericum oblongifolium are given below:

The structures of all the compounds which have been previously isolated from H. oblongifolium are given below;

2.2: Biosynthesis of Terpenes

The biogenetic route for terpenes synthesis started from acetyl-CoA through mevalonic acid pathway, ended with the production of all types of terpenoids and steroids (Scheme 1) [7].

Ruzicka proposed the isoprene rule for terpenoids biosynthesis according to which 2-methyl-

1, 3-butadiene (a five carbon isoprene unit) combine together in head to tail manner to produce various types of terpenes [44].

2.2.1: Biosynthetic route for terpenes

Following three steps are involved in terpenes biosynthesis. 32

1. Formation of isoprene unit fromacetate.

2. Condensation of isoprene unit to produce acyclicterpenoids

3. “Conversionof acyclic terpenes” to cyclicone “and introduction offunctional group”.

The biosynthesis of terpenes started by the production of an isopentane unit, for this production first step is the conversion of acetyl CoA (first biogenetic precursor) to mevalonic acid. Three molecules of acetyl CoA combined to each other to form mevalonic acid. The initial step is the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA (2) through Clasien condensation. In the next step aldol condensation occurs between acetoacetyl-CoA and third molecule of acetyl-CoA affording β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) (3). After the removal of thioester group from 3 by enzymatic reduction, mevalonic acid (4) is produced, which is the main building block of nearly all the isoprenoids (Scheme 2) [45].

Mevalonic acid (4) [6 carbon molecule] is converted to five carbon compound by the loss of one carbon atom. Phosphorylation of 4 produces mevalonic acid-5-phosphate (5) by using one molecule of ATP. Further phosphorylation of 5 produces mevalonic acid-5-pyrophosphate (6), which is converted to 7 [isopentyl pyrophosphate (IPP)] by the removal of CO2 and H2O. Next

step is the isomerization of IPP into DMAPP (“3-methyl-buta-2-enyl(β,β-

dimethylallyl)pyrophosphate”) Scheme 3.

The next step of terpenoids biosynthesis “is the formationof geranylpyrophosphate” (GPP) (10 carbon molecule), by head to tail reaction of nucleophilic IPP and electrophilic DMAPP. “The

GPP is the precursor for all the monoterpenes” (Scheme 4 & 5). 34

GPP (10) further reacts with IPP to form 15 carbon compound [farnesyl pyrophosphate (FPP)]

both in cis and trans form (Scheme 6). “FPP is the main for sesquiterpenes” [46]. 35

FPP (17) reacts with IPP to give GGPP [geranyl-geranyl pyrophosphate (19)]. “ It is the

precursor for diterpenoids” (Scheme 7) [47].

Two molecules of FPP combine in head to head manner to produce presqualene (20), which changes to squalene (21) after rearrangement (Scheme 8).. It also acting as synthetic tool for all terpenoids. Structure of terpenoids formed is dependent to cyclization route of squalene. 36

2.2.2: Cyclization of Squalene

Terminal double bond of squalene undergoes formation of tertiary carbocation which results in cyclization of squalene. Both oxidative and non-oxidative agents can promote this process

(Scheme 9). 37

Primary triterpenes are such compounds which obtained directly either from squalene or its

2,3-epoxide. These are further categorized on the basis of their origin described as following:

2.2.3: Oxidative cyclization of squalene

This process is utilized to produce tetra and pentacyclic triterpenes which depends on squalene epoxide conformation. Enzyme surface is employed to carry this process prior to cyclization.

2.2.3.1: Cyclization of squalene epoxide in chair-chair-chair-boat sequence

Chair-chair-chair-boat sequence of squalene epoxide is adopted on enzyme surface to convert it into dammarenediol (25, 26) after cyclization. Isomerization of non-classical cation 23 into

24 is responsible for production of dammarene 25 or 26 [48]. 39

Dammarenyl cation (23) undergoes enzymatic stabilization and intramolecular rearrangement assisted by enzymes converted 23 to cycloartenol and lanosterol (Scheme 11& 12). Both of these are of great importance regarding to biosynthesis of tetracyclic triterpenes (steroids).

Formation of 23 from oxidative cyclization of 21 further undergoes ring expansion with production of baccharenyl cation (30). This cation undergo many rearrangements and finally 41

produce α-amyrin or β-amyrin (Scheme 13) [49]. α-amyrin relates to ursane series while β- amyrin belongs to oleanane skeleton. 42

Various stereospecific 1, 2-migrations occur in β-Amyrin cation and rearranges into many skeleton like taraxerol (34), glutinol (35) and friedaline (36) (Scheme 14) [50].

2.2.3.2: Lupane and Hopane series

These two are small and biogenetically important groups, studied collectively owing to have five membered ring E. During conversion of dammarenyl cation to β-amyrin, an intermediate lupenyl cation is produced which led to formation of lupine skeleton (37) (Scheme 15).

The squalene molecule undergoes direct cyclization which give hopane skeleton (39) and it is obtained by attacking of protonated oxene (HO+) and H2O on all the chair conformation of 38

(Scheme 16) [51]. 45

Chair-chair-chair-chair-boat (40) conformation produces moretenol (41), whereas chair-chair- chair-chair-boat (42) ends with the production of arborinol (43) (scheme 17). 46

2.2.4: Non-oxidative cyclization of squalene

Non-oxidative cyclization is less common as compared to oxidative one. Trans squalene is involved in this kind of cyclization rather than squalene epoxide. In this type of cyclization triterpenes are produced by proton-induced cyclization of squalene. However, examples are less common such as diploptene and tetrahymanol (Scheme 18) [52]. 47

2.2.5: Cyclization at both ends of squalene molecule

The biosynthesis which involves the squalene attack by two independent electrophiles at its both ends, results in formation of onocerin like triterpenoids (Scheme 19) [53].

2.3: Biosynthesis of steroids

The biosynthesis of steroids also started from acetic acid involving mevalonic acid pathway.

During the year 1942 Baloch gave the evidence that during biogenesis acetic acid converted

into sterols. “This statement was further confirmed by Cornforth in 1953”. During preceding years Tavorina also confirmed the formation of cholesterol from mevalonic acid. Biogenetic route for the conversion of acetic acid to squalene has been already discussed in Scheme 1-8 in terpenoid biosynthesis.

Biosynthesis of sterols occur in plant membrane, involving a series of more than thirty enzymatic reactions. These enzymatic steps can occur either in the presence or absence of photosynthetic apparatus. Cycloartenol is formed from squalene oxide in reaction occur in photosynthetic apparatus, while non photosynthetic fungi produce lanosterolfollowing the formation of ergosterol. Finally, Δ5- 24- alkyl sterols are produced by transformation of lanosterol and cycloartenol [54].

Further studies on steroid biosynthesis were continued and in 1966 and 1967 two groups (Van

Talmelen and Corey) working independently also investigated that 2,3-epoxy-squalene (22) act as intermediate during the formation of 27 and 29. Squalene produces an ionic intermediate

(22a) via oxidative cyclization, this intermediate undergoes 1,2-migration following the loss of proton to afford 27 and 29 (Scheme 20) [55].

2.3.1: Formation of Cholesterol from Lanosterol

Cholesterol is formed from lanosterol after losing methyl groups in a specific manner. First methyl lost is 14α-methyl (47→48), then 4β-methyl is lost (49→51) and finally 51 also loses

4α-methyl to produce 53. Formic acid pathway is involved in the loss of 4α-methyl, other methyls lost as CO2. When 53 exchanges protons with the medium it undergoes phototropic reaction to produce 54. The compound 54 loses two more hydrogens by using molecular oxygen to give 55 (∆5,7diene). During last step of cholesterol biosynthesis 55 take one hydrogen from NADPH reducing it to NADP+ resulting the formation of cholesterol (56) (Scheme 21)

[56]. 50

The route for ergosterol biosynthesis from acetate was similar to cholesterol biosynthetic route as discovered by Baloch in 1951. During 1953 Hanahan and his coworkers discovered that carbon skeleton of ergosterol is also formed by squalene cyclization excep 28-methyl which generated from an independent route [57].

2.3.2: Stigmasterol and β-Sitosterol

β-Sitosterol and stigmasterol are common plant sterols and are biosynthesized from cycloartenol. During this biosynthesis 4α methyl is lost first following the cyclopropane ring opening and loss of 14α methyl.

Phytosterol skeleton has a characteristic feature to convert one or two more carbons as compared to cholestane series. These additional carbon atoms attached at carbon 24 either as methylene or methyl, or as ethyledene or ethyl group resulting in C28 and C29 structure ergostane and stigmastane respectively (Scheme 22 and 23). 53

Some examples of phytosterols are given below.

2.4: Biosynthesis of Fatty Acids

An important source of energy for most of the living organisms is fatty acids, biosynthesized in cytosol. Acetyl CoA is the building block in fatty acid biosynthesis, activated to malonyl

CoA. The end product of the basic fatty acid biosynthesis is a sixteen carbon saturated fatty acid (palmitic acid). Palmitoyl CoA is the basic precursor for all fatty acids either shorter or longer than palmitic acid and reacted under specific enzymatic conditions. Fatty acids CoA unsaturases are important group of enzymes in the biosynthesis of unsaturated fatty acids and has specificity for double bond position [58-59].

FAS (Fatty Acid Synthase) is a complex of all the required enzyme used in fatty acid biosynthesis. FAS is a polypeptide chain with multiple domains and each domain has characteristic enzyme activities that are used for the synthesis of fatty acid. ACP (Acyl carrier protein) is a vital part of fatty acid synthase (FAS) act as activator for biosynthesis of fatty acid. The CoA group of Acyl carrier protein (ACP) is attached to acyl group by a thioester linkage. β-ketoacyl synthase (K-SH) which is condensing enzyme is another important part of

FAS (fatty acid synthase) having cysteine sulfhydryl (-SH) group that combine a thioester linkage with carboxylate group of fatty acid molecule [60].

The biosynthesis of fatty synthesis initiates from the methyl group of fatty acid and leads to carboxylic acid group of the molecule. By this way, carbon-15 and carbon-16 are added up first while carbon-1 and carbon-2 are last in the formation of fatty acid molecule. By carboxylation acetyl CoA is converted to malonyl CoA (65) (Scheme 24) but during the process of condensation for extension, added CO2 is lost. At each step, two carbon atoms are added to the chain. Biotin is the required cofactor for carboxylation [59]. 56

In start of fatty acid biosynthesis, acyl group of acetyl CoA converted to pantothenate of acyl

carrier protein (ACP) and further stepis the transfer of acetylgroup from “pantothenate of acyl

carrierprotein” (ACP) to cysteine sulfhydryl group of K-SH (β-ketoacyl synthase). Now the malonyl group of malonyl CoA is free to combine with pantothenate-SH (Scheme 25).

In the presence of enzyme β-ketoacyl synthase, the process of condensation between the malonyl group attached with pantothenate-SH and acetyl group attached with cysteine-SH ends of fatty acid synthase (FAS). Acetoacetyl ACP (68) is the final prodsuct (Scheme 26 & 27)

[59]. 57

In the further step β-ketoacid is reduced to butyric group. The whole process is accompanied in three steps. In first step acetoacetyl-ACP (68) reduces to β-3-hydroxybutyryl-ACP (69) in the presence of β-ketoacid derivative and NADPH. In the second step Crotonyl-ACP (70) is formed by the loss of water molecule and in the final step the unsaturated structure of Crotonyl-

ACP (70) is further reduced to Butyryl-ACP in the presence of NADPH and 2,3-trans-enoyl-

ACP reductase (Scheme 28).

This is a whole cycle for the biogenesis of fatty acids. As a result of this first cycle, a four carbon chain linked with acyl carrier protein (ACP) end of fatty acid synthase (FAS) is formed.

The chain transferred to the K arm and new malonyl CoA is introduced to the ACP arm of fatty acid synthase (FAS). The complete cycle comprising of four reactions again continued as before. Two new carbon atoms are added to the chain during each cycle and subsequent structure is two carbon elongated than previous one. After the completion of seven cycles, a sixteen carbon chain attached to acyl carrier protein (ACP) site called palmitoyl ACP (acyl carrier protein) (72) hydrolyzed from acyl carrier protein (ACP) producing a free palmitate

(73) molecule (Scheme 29) [58].

Biosynthesis of flavonoids:

Flavonoids are one of the important classes of natural products. These are the coloring co- pigments of the plant which give different colors to the flowers like yellow, red, purple etc.

These are also called anthoxanthins. These are responsible for protection of leaf cells from photo-oxidative damage. These secondary metabolites are further divided into six subclasses.

These subclasses are flavones, flavondiols, chalcones, flavanols, proanthocyanidins

(condensed tannins) and anthocyanins. Some species also contain a seventh group named aurones. Some legume plants as well as non-legume plants produce some special flavonoids called isoflavonoids (Scheme 30) [61]. 61

Flavonoids have fifteen carbon atoms in their molecule and contains “twophenyl ringslinked by

three carbonchain”. This chain can be converted into a third ring (5 or 6 membered) through oxygen on one of the phenyl rings.

The biosynthesis of flavonoids depends upon the construction of intermediate fifteen carbon chalcone molecule. Number of scientists approved the central role of chalcone during biosynthesis of flavonoids [62]. The precursor for biosynthesis of chalcone (Scheme 33) are

4-coumaroyl-CoA (hydroxycinnamic acid CoA ester) and malonyl-CoA. These precursors are produced from carbohydrates. The production of 4-coumaroyl-CoA involves shikimate 62

pathway, the mainroute to tyrosine, aromaticamino acid and “phenylalaninein higherplants”

(Scheme 31 & 32) [63]. While malonyl CoA is a product of acetyl CoA and CO2 catalyzed by acetyl CoA carboxylase. Two molecules of malonyl CoA combines to produce (77). Which reacts with 4-hydroxy coumaric acid ester to form (78) with the removal of carbon dioxide.

This product undergoes some important changes in the presence of chalcone synthase and finally chalcone (79) is produced (Scheme 33). Chalcone is the precursor for biogenesis of all the classes of flavonoids. 63

2.5.1: Biosynthesis of Flavanone

Chalcone (79) undergoes enzymatic isomerization to produce flavanones. This conversion requires only simple isomerization because the enzyme chalcone isomerase help flavanones and their corresponding chalcones to exit in equilibrium state in vitro and in vivo [47]. The 66

enzyme is stereospecific and produces (S) configuration at C-2 of flavanone derivatives.

Chalcones which have “at least twofree hydroxyl groups at Carbon-2 and Carbon-6” readily and speedily converted to flavanone as in aqueous medium the equilibrium shifted towards flavanones (Scheme 34). If only one free hydroxyl group is present in chalcone then the system will not eclipsed and remained open so no flavone formation will occur.

2.5.2: Formation of Isoflavone:

The equilibrium between chalcone and flavanone mostly favors towards flavanone formation.

So the flavanone further reacts in various conditions to produce compounds related to other classes of flavonoids. Isoflavones are formed from flavanones and the most important stepin

this“formation is the 2,3” shift of aryl moiety in the molecule. This transformation accomplished in two enzymatic processes. In the first step oxidation followed by rearrangement of flavanone 67

take place in the presence of NADPH and molecular oxygen. The resulting molecule is 2- hydroxyisoflavone (82) which in the second steps losses one water molecule in the presence of enzyme and converted to isoflavone (83) (Scheme 35) [64].

2.5.3: Formation of flavone

The conversion of flavanone into flavone involves reduction process which occurs in the presence of a microsomal enzyme needing NADPH as a co-factor. The steps involved in the formation of double bond are not cleared yet. However it is suggested that in the first step formation of 2-hydroxyflavanone occurs and then water molecule eliminated in the second step via a dehydratase (Scheme 36). Though, no such intermediate is still found [65]. 68

2.5.4: Formation of Flavanol

Biosynthesis of flavanol from flavanone is “catalyzedby a soluble 2-oxoglutaratedependent

oxygenase”.Most likely this process proceeds through a “2-hydroxyintermediate” followed by dehydration resulting in the respective flavanols (85) (Scheme 37).

Chapter 3 Experimental

3.1. General Experimental Conditions

3.1.1 Physical constants

Melting points were examined, in glass capillaries using Buchi 535 melting point apparatus.

Optical rotations were determined using, JASCO DIP-360, digital polarimeter. For determination of antimicrobial and enzyme inhibition activities (lipoxygenase), all chemicals and enzymes were purchased from Sigma (St. Louis, MO, USA). .

3.1.2 Spectroscopy

Ultraviolet (UV) spectra were obtained in methanol on Hitachi U-3200 spectrophotometer.

Infrared (IR) spectra were recorded on JASCO A-302 Infrared spectrophotometer. Proton

1 nuclear magnetic resonance ( H-NMR) spectra were recorded in CD3OD using TMS as internal standard at 400 MHz and 500 MHz on Bruker AM-400 and AM-500 nuclear magnetic resonance spectrometers with Aspect 3000 data systems at a digital resolution of 32K. The

13 C-NMR spectra were recorded in CDCL3, CD3OD at 100 and 125 MHz on the same instruments. The 2D NMR (HMQC, HMBC, 1H-1H COSY and NOESY) spectra were recorded in CDCL3, CD3OD at 400 MHz and 500 MHz on the same instruments.

The pulse conditions were followings for the 1H-NMR spectra, spectrometer frequency (SF)

400.03 MHz, acquisition time (AQ) 2.916 s, number of transients (NS) 128, receiver gain (RG)

80, temperature (TE) 297 K, dwell time (DW) 69.6 μs, per scan delay (DE) 10 μs, dummy scans (DS) O; for the 13C-NMR spectrum, SF 100.61 MHz, AQ 0.819s, NS 34389, RG 1600,

TE 297K, DW 19.1μs, DE 38μs, DS 2; and for the HMQC spectrum, SF 400.03 MHz, AQ

0.14177 μs, NS 64, DS 16, DE 10.0μs, DW 138.4 μs, RG 16384, TE 300k.

For NOE measurements, the sample was frozen under liquid nitrogen and degassed. A lower decoupler power of maximum 0.2 watts with 35 attenuations in dbs was used. The pre- 70

irradiation time was 11 sec which is the sum of three delays as used in the NOE difference programme of Bruker. The impulse length of 10 microseconds was maintained to avoid saturation. The two-dimensional 1H-1H COSY-45° experiments were acquired at 400 MHz with a sweep width of 4000 Hz (2K data points) in ω2 and 2000 Hz (256 t1 values zero-filled

1 13 to 1K) in) ω1. The hetreronuclear H- C chemical shift correlation experiments were carried out at 400 MHz with a sweep width of 12820 Hz (2K data points) in ω2 and 1024 Hz (256 t1 values zero-filled to 2K) in ω1. In both the 2D experiments a 2 sec relaxation delay was used, and 16 transients were performed for each t1 value.

Low-resolution electron impact mass spectra were recorded on a Finnigan MAT 311 with

MASSPEC data system. High resolution mass measurements and fast atomic bombardment

(FAB) mass measurements were carried out on Jeol JMS HX 110 mass spectrometer using glycerol and thioglycerol as the matrix and cesium iodide (CsI) as internal standard for accurate mass measurements.

3.1.3: Chromatography

Column chromatography was performed over silica gel (E-Merck, 230-400 mesh). Pre-coated silica gel GF-254 preparative plates (20×20 cm, 0.5 mm thick, E-Merck) were used for preparative thick layer chromatography.

3.1.4 Spray reagent for visualization of spot

3.1.4.1 Ceric Sulphate Reagent

Ceric sulphate (0.1g) and trichloroacetic acid (1g) were dissolved in 4ml distilled water. The solution was boiled and conc. H2SO4 was added drop wise until the disappearance of turbidity.

The TLC plates were visualized by spraying this reagent.

3.2: Extraction and isolation

3.2.1: Plant material

The “whole plantof Hypericum oblongifolium” (10 kg) was collected from Swat, Malakand, in

April 2013 by Dr. Mumtaz Ali from “Department ofChemistry, Universityof Malakand, KPK”,

Pakistan. The plant was identified from Botany Department, University of Malakand, KPK,

Pakistan.

3.2.2: Extraction and Isolation

Air dried plant material (10 Kg) was crushed, ground, and “extracted with methanol”. Methanolic

“extract wasevaporated” under reduced pressure and the gummy material (0.7 Kg), was partitioned between water, n-butanol, ethyl acetate chloroform, and n-hexane soluble fractions.

The chloroform fraction was“chromatographed oversilica gel” in a columneluting with n-

hexane-chloroform and chloroform-methanol mixtures “inincreasing order ofpolarity to obtain”

12 major fractions labelled as A-L. The fractionB eluting with n-hexane-chloroform (8:2) was subjected to column chromatography again and obtained several semi pure fractions. The fraction at 7.8:2.2 (n-hexane-chloroform) was subjected to Preparative TLC using solvent system n-hexane-acetone (4:1) to afford 90 (33 mg), 91 (22 mg) 92 (19 mg) and 93 (14 mg).

Similarly, the fraction C (n-hexane-chloroform; 7:3) “wasagain chromatographed oversilica gel” 72

column chromatography eluting withn-hexane-acetone “in increasing orderof polarity” to give 4 sub-fractions C1-C4. The fraction C3 showed several prominent spots on TLC and was again chromatographed over silica gel to obtain semi-pure fractions. The fined polarity at 7.2:2.8 gave all these three compounds, compound 86 (13 mg) and 87 (9 mg) from the top fraction and compound 89 (15 mg) from the tail fractions.

The fraction C-4 was subjected to Preparative silica gel TLC using solvent system n-hexane- acetone (8:2) to afford compound 88, (13 mg), 94 (15 mg), 95 (40 mg) and 96 (11 mg). The

fraction H (n-hexane-chloroform, 1:4) was again columnchromatographed over silicagel” using solvent system n-hexane-ethyl acetate in increasing order of polarity. Repeated column chromatography by same solvent system (n-hexane-ethyl acetate, 1:4) to give compound 97

(10 mg). The fraction K (chloroform-methanol, 9:1) was further purified “over silicagel column

chromatography elutingwith chloroform-methanol (9.7:0.3) to” afford compound 100 (26 mg).

The ethylacetate fraction of H. oblongifolium was also chromatographed oversilica gel column with eluents n-hexane-chloroform, chloroformand chloroform-methanol inorder of increasing polarity toobtain eight fractions[(Fraction 1 = 6:4, n-hexane -CHCl3), (Fraction 2 = 8:2, n- hexane-CHCl3), (Fraction 3 = 100 % CHCl3), (Fraction 4 = 9:1, CHCl3-MeOH), (Fraction 5 =

8:2, CHCl3-MeOH), (Fraction 6 = 7:3, CHCl3-MeOH), (Fraction 7 = 6:4, CHCl3-MeOH) and

(Fraction 8 = 5:5, CHCl3-MeOH). The fraction 4 obtained from CHCl3-MeOH 9:1 “was

subjected tocolumn chromatography” using“silica gel and eluted” with increasing polarity of chloroform-methanol mixtures and the sub-fractions showed prominent spots on TLC. The eluent fractions collected from CHCl3-MeOH (9:1) were combined again and subjected to preparative TLC (CHCl3-MeOH 8:2) which resulted 98 (21 mg). 73

Fraction 5 was chromatographed over silica gel using solvent CHCl3-MeOH in increasing order of polarity. A fined polarity (8.5:1.5) was maintained to obtain semi-pure fractions. These semi-pure fractions were combined and rechromatographed by preparative HP-TLC (RP-18,

MeOH-H2O 8:2) to afford 99 (32 mg) and 101 (14 mg).

A schematic view of extraction, fractionation and isolation has shown in Scheme 38-40. 74

Scheme 39 76

Scheme 40

3.3: Characterization of new compounds

3.3.1: Hyperinoate A (86)

Physical state: Colorless crystalline solid

Melting Point: 171-172 °C

23 [α]D : + 56.5 (c 1.05, CHCl3)

-1 FT-IR: ʋmax cm 3452 (OH), 2935 (C-H str), 1730 (acetate), 1170 (C-O), 1370 (gem dimethyl)

1 “ H-NMR (CDCl3, 500 MHz)”

δ: 0.81 (“3H, d, J = 6.5 Hz, Me-21”), 0.84 (3H, d, J = 6.3 Hz, Me-26), 0.85 (“ 3H, d, J = 6.3 Hz,

Me-27”), 0.87 (3H, s, Me-18), 0.88 (3H, s, Me-19), 0.92 (3H, s, Me-28), 2.01 (3H, s, Me-30),

3.65 (1H, dt, J = 7.0, 4.5 Hz, H-6), 4.95 (“1H, m, H-3”).

13 “ C-NMR (CDCl3, 125 MHz)”

δ: 35.4 (C-1), 28.8 (C-2), 72.3 (C-3), 30.5 (C-4), 44.0 (C-5), 71.1 (C-6), 33.2 (C-7), 42.7 (C-

8), 48.4 (C-9), 34.3 (C-10), 19.7 (C-11), 21.2 (C-12), 44.8 (C-13), 45.9 (C-14), 33.4 (C-15),

31.6 (C-16), 50.9 (C-17), 15.0 (C-18), 15.9 (C-19), 35.8 (C-20), 18.5 (C-21), 36.3 (C-22), 23.6

(C-23), 39.1 (C-24), 27.5 (C-25), 22.7 (C-26), 20.3 (C-27), 13.9 (C-28), 170.8 (C-29), 20.8 (C-

30).

+ HREIMS: [M] at m/z 460.3911 (Calcd. for C30H52O3 460.3916)

EIMS [M]+ m/z (rel int %): 460 (11), 442 (26), 427 (28), 417 (40), 412 (80), 401 (25), 382

(55), 347 (44), 331 (30), 317 (30), 306 (60), 154 (78), 129 (81), 113 (51), 59 (95), 43 (100).

3.2.2: Hyperinoate B (87)

Physical state: Colorless solid

Melting Point: 205-206 °C

23 [α]D : + 61.0 (c 1.10, CHCl3)

-1 FT-IR: ʋmax cm 3440-3390 (OH), 2935 (C-H str), 1729 (C=O), 1230 (C-O), 1090 (C-C), 870

(C-H bending).

1 “ H-NMR (CDCl3, 500 MHz)”

δ: 0.81 (“3H, d, J = 6.5 Hz, Me-21”), 0.84 (“3H, d, J = 6.3 Hz, Me-26”), 0.85 (“ 3H, d, J = 6.3 Hz,

Me-27”), 0.87 (“3H, s, Me-18”), 0.88 (3H, s, Me-19), 0.92 (3H, s, Me-28), 2.01 (3H, s, Me-30),

3.65 (1H, dt, J = 7.0, 4.5 Hz, H-6), 4.95 (“1H, m, H-3”).

13 “ C-NMR (CDCl3, 125 MHz)”

δ: 35.4 (C-1), 28.8 (C-2), 72.3 (C-3), 30.5 (C-4), 44.0 (C-5), 71.1 (C-6), 33.2 (C-7), 42.7 (C-

8), 48.4 (C-9), 34.3 (C-10), 19.7 (C-11), 21.2 (C-12), 44.8 (C-13), 45.9 (C-14), 33.4 (C-15),

31.6 (C-16), 50.9 (C-17), 15.0 (C-18), 15.9 (C-19), 35.8 (C-20), 18.5 (C-21), 36.3 (C-22), 20.5 80

(C-23), 43.8 (C-24), 70.2 (C-25), 29.7 (C-26), 27.3 (C-27), 13.9 (C-28), 170.8 (C-29), 20.8 (C-

30).

+ HREIMS: [M] at m/z 476.3862 (Calcd. for C30H52O4 476.3865)

EIMS [M]+ m/z (rel int %): 476 (10), 458 (60), 440 (55), 417 (35), 399 (40), 390 (35), 381

(30), 347 (52), 170 (84), 129 (90), 86 (45), 59 (100).

3.2.3: Hyperinone (88)

Physical state: Colorless needles

Melting Point: 246-248 °C

20 [α]D : + 55.5 ° (c 1.08, CHCl3)

-1 FT-IR: ʋmax cm 2950 (CH- stretching), 1755 (γ-lactone), 1695 (C=O), 1650, 895 (terminal double bond).

1 H-NMR (CDCl3, 500 MHz)

δ: 0.76 (“3H, s, Me-24”), 0.94 (“3H, s, Me-25”), 0.95 (“3H, s, Me-26”), 0.97 (“3H, s, Me-27”), 1.04

(3H, s, Me-23), 1.10 (“3H, d, J = 6.9 Hz, Me-29”), 2.02 (“1H, dq, J = 6.9, 4.4, H-19”), 2.22 (“1H,

d, J = 4.6 Hz, H-18”), 5.10, 4.66 (1H each, s, H-30).

13 “ C-NMR (CDCl3, 125 MHz)”

δ: 36.1 (C-1), 29.4 (C-2), 216.5 (C-3), 40.6 (C-4), 53.0 (C-5), 16.5 (C-6), 57.8 (C-7), 42.7 (C-

8), 51.3 (C-9), 33.9 (C-10), 15.7 (C-11), 29.6 (C-12), 89.9 (C-13), 40.6 (C-14), 23.4 (C-15),

19.4 (C-16), 42.1 (C-17), 53.2 (C-18), 32.8 (C-19), 151.9 (C-20), 30.6 (C-21), 28.1 (C-22),

22.3 (C-23), 20.7 (C-24), 14.2 (C-25), 16.2 (C-26), 17.2 (C-27), 178.4 (C-28), 14.4 (C-29),

106.8 (C-30).

+ HREIMS: [M] at m/z 452.3265 (Calcd. for C30H44O3 452.3290)

EIMS: [M]+ m/z (rel int %): 452 (8), 438 (15), 424 (30), 409 (100), 394 (40), 379 (35), 369

(65), 355 (20), 348 (10), 97 (45), 83 (15), 67 (80), 53 (69).

3.2.4: Hyperinoic acid (89)

Physical state: Colorless solid

Melting Point: 210-211 °C

23 [α]D : + 19.5 (c 1.05, CHCl3)

-1 FT-IR: ʋmax cm 3450 (OH), 3051 (C-H str), 1697 (C=O), 1634 (C=C), 1160 (C-O), 810 (C-

H bending)

1 H-NMR (CDCl3, 500 MHz)

δ: 0.89 (“3H, d, J = 6.5 Hz, Me-30”), 0.90 (“3H, s, Me-26”), 0.93 (“3H, s, Me-25”), 0.96 (“3H, d, J

= 7.0 Hz, Me-29”), 1.03 (“3H, s, Me-27”), 1.15 (“3H, s, Me-24”), 1.20 (“3H, s, Me-23”), 3.27 (“1H,

dd, J = 9.1, 6.3 Hz, H-3”), 3.81 (“1H, dt, J = 9.8, 8.7, 7.4 Hz, H-21”), 5.37 (“1H, t, J = 3.5, H-12”).

13 “ C-NMR (CDCl3, 125 MHz)”

δ: 38.0 (C-1), 26.5 (C-2), 77.3 (C-3), 39.2 (C-4), 54.3 (C-5), 17.8 (C-6), 32.3 (C-7), 38.9 (C-

8), 46.8 (C-9), 36.2 (C-10), 22.7 (C-11), 124.6 (C-12), 137.8 (C-13), 41.9 (C-14), 27.4 (C-15),

25.5 (C-16), 45.6 (C-17), 52.5 (C-18), 36.5 (C-19), 48.1 (C-20), 70.2 (C-21), 40.4 (C-22), 22.3

(C-23), 20.2 (C-24), 15.1 (C-25), 16.2 (C-26), 23.4 (C-27), 180.1 (C-28), 17.3 (C-29), 14.0 (C-

30).

+ HREIMS: [M] at m/z 472.3549 (Calcd. for C30H48O4, 472.3552)

EIMS [M]+ m/z (rel int %): 472 (9), 264 (35), 251 (32), 221 (60), 219 (45), 208 (100), 133

(60).

3.3: Characterization of known compounds

3.3.1: 4,4-Dimethyl cholesterol (90)

Physical state: Colorless crystalline solid

Melting Point: 109-110 °C

20 [α]D : + 20.5 (c 0.50, CHCl3)

-1 FT-IR: ʋmax cm 3437 (OH), 2950 (C-H stretching), 1610 (C=C), 1485 (gem dimethyls),

1038 (C-C), 812 (C-H bending),

1 H-NMR (CDCl3, 500 MHz)

δ: 0.74 (“3H, s, Me-18”), 1.01 (“3H, d, J = 6.5 Hz, Me-21”), 1.12 (“3H, m, Me-19”), 1.34 (“3H, s,

Me-28”), 1.37 (“1H, m, H-20”), 1.55 (“3H, s, Me-29”), 1.67 (“6H, d, J = 6.9 Hz, Me-26, 27”), 3.69

(“1H, dd, J = 7.1, 2.9 Hz, H-3”), 5.38 (“1H, dd, J = 5.1 Hz, H-6”).

13 C-NMR (CDCl3, 100 MHz)

δ: 32.5 (C-1), 24.8 (C-2), 72.1 (C-3), 33.7 (C-4), 140.7 (C-5), 121.6 (C-6), 42.9 (C-7), 40.8 (C-

8), 43.4 (C-9), 37.5 (C-10), 29.7 (C-11), 31.6 (C-12), 45.8 (C-13), 55.6 (C-14), 40.4 (C-15),

22.3 (C-16), 51.8 (C-17), 15.9 (C-18), 17.7 (C-19), 36.7 (C-20), 16.2 (C-21), 21.3 (C-22), 25.6

(C-23), 34.4 (C-24), 36.5 (C-25), 13.8 (C-26), 15.3 (C-27), 20.2 (C-28), 24.9 (C-29).

+ HREIMS: [M] at m/z 414.3845 (Calcd. for C29H50O, 414.3862)

EIMS [M]+ m/z (rel int %): 414 (15), 317 (25), 303 (32), 301 (67), 260 (23), 203 (63), 154

(53), 113 (42), 97 (71), 57 (100).

3.3.2: β-Sitosterol (91)

Physical state: White solid

Melting Point: 135°C

25 [α]D : +36.4 (c 1.0, CHCl3)

-1 FT-IR: ʋmax cm 3450 (OH), 2942 (C-H str), 1660 (C=C), 1074 (C-C), 814 (C-H bending)

1 “ H-NMR (CDCl3, 400 MHz)”

δ: 0.68 (“3H, s, Me-18”), 0.81 (“3H, d, J = 6.5 Hz, Me-26”), 0.84 (“3H, d, J = 6.5 Hz, Me-27”),

0.86 (“3H, t, J = 7.0 Hz, Me-29”), 0.92 (“3H, d, J = 6.2 Hz, Me-21”), 1.03 (“3H, s, Me-19”), 3.36,

(“1H, m, H-3”), 5.14 (“1H, m, H-6”).

13 “ C-NMR (CDCl3, 100 MHz)”

δ: 37.2 (C-1), 32.4 (C-2), 72.5 (C-3), 43.0 (C-4), 140.5 (C-5), 121.7 (C-6), 33.1 (C-7), 32.2

(C-8), 51.4 (C-9), 37.2 (C-10), 21.5 (C-11), 41.1 (C-12), 42.3 (C-13), 57.0 (C-14), 25.0 (C-

15), 27.9 (C-16), 55.8 (C-17), 12.2 (C-18), 19.8 (C-19), 37.1 (C-20), 19.0 (C-21), 34.1 (C-22),

30.2 (C-23), 51.0 (C-24), 25.8 (C-25), 19.0 (C-26), 20.4 (C-27), 22.6 (C-28), 12.7 (C-29).

+ HREIMS: [M] at m/z 414.3851 (Calcd. for C29H50O, 414.3861) 88

+ “EIMS m/z (rel int %)”: [M] 414 (16), 396 (17), 399 (15), 381 (76), 329 (31), 275 (12), 273

(21), 255 (29).

3.3.3: Lupeol (92)

Physical state: Colorless crystals

Melting Point: 215-216°C

20 [α]D : + 26.9 (c 1.0, CHCl3)

-1 FT-IR: ʋmax cm 3475 (OH), 3081 (C-H), 1645, (C=C), 1045 (C-C)

1 H-NMR (CDCl3, 400 MHz)

δ: 0.75 (“3H, s, Me-24”), 0.78 (“3H, s, Me-28”), 0.83 (“3H, s, Me-25”), 0.94 (“ 3H, s, Me-27”), 0.96

(“3H, s, Me-23”), 1.03 (“3H, s, Me-26”), 1.36 (“3H, s, Me-30”), 3.22 (“1H, dd, J = 9.9, 4.5, H-3”),

4.57 and 4.69 (“1H, each s, H-29”).

13 C-NMR (CDCl3, 100 MHz)

δ: 38.7 (C-1), 27.6 (C-2), 78.8 (C-3), 38.8 (C-4), 55.3 (C-5), 18.4 (C-6), 34.3 (C-7), 40.9 (C-

8), 50.5 (C-9), 37.2 (C-10), 21.2 (C-11), 25.3 (C-12), 38.5 (C-13), 42.9 (C-14), 27.4 (C-15),

35.6 (C-16), 93.3 (C-17), 48.3 (C-18), 47.8 (C-19), 39.6 (C-20), 29.9 (C-21), 150.7 (C-22), 90

28.4 (C-23), 15.5 (C-24), 16.2 (C-25), 15.8 (C-26), 14.6 (C-27), 18.7 (C-28), 109.5 (C-29),

19.6 (C-30).

+ HREIMS: [M] at m/z 426.3835 (“Calcd. for C30H50O 426.3861”)

EIMS [M]+ m/z (rel int %): 426 (20), 411 (25), 408 (30), 393 (35), 385 (15), 220 (80), 218

(55), 207 (25), 189 (100), 139 (70).

3.3.4: Taraxerol (93)

Physical state: White crystals

Melting Point: 275-277°C

20 [α]D : + 0.72 (c 1.15, CHCl3)

-1 FT-IR: ʋmax cm 3584 (OH), 2850 (C-H str), 1638 (C=C), 1035 (C-C), 818 (C-H bending)

1 H-NMR (CDCl3, 400 MHz)

δ: 0.82 (“3H, s, Me-24”), 0.85 (“3H, s, Me-28”), 0.89 (“3H, s, Me-25”), 0.93 (“3H, s, Me-30”), 0.95

(“3H, s, Me-23”), 0.97 (“3H, s, Me-29”), 1.13 (“3H, s, Me-26”), 1.19 (“3H, s, Me-27”), 3.38 (“1H, t, J

= 8.2 Hz, H-3”), 5.54 (“1H, dd, J = 7.1, 4.2 Hz, H-15”).

13 “ C-NMR (CDCl3, 100 MHz)”

δ: 38.3 (C-1), 27.8 (C-2), 79.4 (C-3), 39.9 (C-4), 55.5 (C-5), 19.3 (C-6), 35.4 (C-7), 39.3 (C-

8), 48.5 (C-9), 38.1 (C-10), 17.8 (C-11), 36.2 (C-12), 38.2 (C-13), 158.3 (C-14), 117.3 (C-15),

36.7 (C-16), 38.1 (C-17), 49.2 (C-18), 41.4 (C-19), 30.4 (C-20), 34.3 (C-21), 33.5 (C-22), 28.4 92

(C-23), 15.6 (C-24), 15.3 (C-25), 30.3 (C-26), 26.5 (C-27), 30.2 (C-28), 33.7 (C-29), 21.7 (C-

30)

+ HREIMS: [M] at m/z 426.3826 (Calcd. for C30H50O, 426.7261)

EIMS [M]+ m/z (rel int %): 426 (5), 408 (10), 393 (15), 363 (20), 348 (17).

3.3.5: 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94)

Physical state: Colorless powder

23 [α]D : -15.2° (c, 2.8, MeOH)

-1 FT-IR: ʋmax cm 3572-3420 (OH), 3039 (C-H), 1631 (C=C), 1480 (gem dimethyl), 1085 (C-

C), 864 (C-H bend).

1 “ H-NMR (CDCl3, 500 MHz)”

δ: 0.82 (3H, s, Me-29), 0.86 (3H, s, Me-18), 1.01 (3H, s, Me-30), 1.03 (3H, d, J = 7.0 Hz, Me-

27), 1.04 (3H, d, J = 7.0 Hz, Me-26), 1.07 (3H, s, Me-19), 1.16 (3H, d, J = 6.5 Hz, Me-21),

1.67 (1H, m, H-20), 1.69, (2H, m, H-22), 2.19 (2H, m, H-23), 2.28 (1H, sept, J = 7.0 Hz, H-

25), 3.03 (2H, dd, J = 19.0, 3.5 Hz, H-16), 3.18, (1H, dd, J = 10.0, 8.0 Hz, H-3), 3.93 (1H, dd,

J = 10.5, 6.0 Hz, H-12), 4.72 (1H, br.s, H-28), 5.44 (1H, t, J = 2.5 Hz, H-15).

13 “ C-NMR (CDCl3, 125 MHz)”

δ: 37.1 (“C-1”), 28.3 (“C-2”), 79.1 (“C-3”), 40.5 (“C-4”), 51.4 (C-5), 19.2 (C-6), 28.6 (C-7), 124.5 (C-

8), 142.6 (C-9), 38.5 (“C-10”), 31.7 (“C-11”), 70.3 (“ C-12”), 58.7 (C-13), 152.4 (C-14), 118.1 (C-

15), 41.3 (C-16), 96.6 (C-17), 14.4 (C-18), 20.8 (C-19), 40.5 (C-20), 15.4 (C-21), 32.3 (C-22), 94

34.4 (C-23), 157.7 (C-24), 34.6 (C-25), 22.2 (C-26), 22.5 (C-27), 106.7 (C-28), 16.1 (C-29),

28.6 (C-30).

+ HREIMS: [M] at m/z 456.3632 (“Calcd. for C30H48O3, 456.3603”)

EIMS [M+] m/z (rel int %): 456 (72), 438 (34) 420 (20), 397 (12), 394 (19), 379 (31), 369

(33), 327 (60), 301 (15), 300 (72), 273 (24), 271 (29).

3.3.6: Oleanolic acid (95)

Physical state: Colorless needles

Melting Point: 305-306°C

20 [α]D : + 78.9° (c 1.07, CHCl3)

-1 FT-IR: ʋmax cm 3420-3285 (OH), 2894 (C-H str), 1707 (C=O), 1637 (C=C), 1465 (gem dimethyl), 1205 (C-O), 1035 (C-C) 815 (C-H bending).

1 “ H-NMR (CDCl3, 400 MHz)”

δ: 0.82 (3H, s, Me-25), 0.92 (3H, s, Me-24), 0.93 (3H, s, Me-29), 0.96 (3H, s, Me-26), 0.99

(3H, s, Me-30), 1.01 (3H, s, Me-23), 1.10 (3H, s, Me-27), 3.62 (1H, dd, J = 9.9, 4.1, Hz, H-

3), 5.23 (1H, t, J = 3.4 Hz, H-12).

13 C-NMR (CDCl3, 100 MHz)

δ: 38.5 (C-1), 27.3 (C-2), 79.2 (C-3), 38.9 (C-4), 55.6 (C-5), 18.8 (C-6), 32.7 (C-7), 39.3 (C-

8), 47.7 (C-9), 37.4 (C-10), 23.6 (C-11), 122.3 (C-12), 146.8 (C-13), 41.5 (C-14), 27.9 (C-15),

23.3 (C-16), 46.4 (C-17), 41.4 (C-18), 46.1 (C-19), 30.5 (C-20), 33.7 (C-21), 32.5 (C-22), 28.2 96

(C-23), 15.6 (C-24), 15.4 (C-25), 17.2 (C-26), 25.7 (C-27), 183.7 (C-28), 33.3 (C-29), 23.6 (C-

30).

+ HREIMS: [M] at m/z 456.3603 (Calcd. for C30H48O3, 456.3610)

EIMS [M]+ m/z (rel int %): 456 (4), 248 (98), 208 (12), 203 (60), 133 (53).

3.3.7: Erectasteroid D (96)

Physical state: Gummy solid

24 [α]D : + 29.0° (c 1.17, CHCl3)

-1 FT-IR: ʋmax cm 3414 (OH), 2938 (C-H str), 1725 (C=O), 1665 (C=C), 1454 (gem dimethyl),

1249 (C-O), 1037 (C-C), 787 (C-H bending).

1 H-NMR (CDCl3, 500 MHz)

δ: 0.75 (3H, s, Me-18), 0.84 (6H, d, J = 6.1 Hz, Me-26, 27), 1.02 (3H, d, J = 6.5 Hz, Me-21),

2.01 (3H, s, Me-29), 3.67 (1H, m, H-3), 3.89 (2H, d, J = 11.5 Hz, H-19), 4.94 (1H, br.d, J =

8.5 Hz, H-7), 5.20 (1H, dd, J = 15.0, 7.8 Hz, H-22), 5.24 (1H, dt, J = 15.0, 7.8 Hz, H-23), 5.55

(1H, br.s, H-6).

13 C-NMR (CDCl3, 125 MHz)

δ: 33.4 (C-1), 31.6 (C-2), 70.9 (C-3), 41.3 (C-4), 140.2 (C-5), 126.5 (C-6), 75.6 (C-7), 37.7

(C-8), 48.4 (C-9), 41.3 (C-10), 21.4 (C-11), 39.9 (C-12), 42.7 (C-13), 56.5 (C-14), 25.2 (C-

15), 28.7 (C-16), 55.5 (C-17), 62.7 (C-18), 12.5 (C-19), 39.7 (C-20), 20.5 (C-21), 137.4 (C- 98

22), 126.5 (C-23), 41.4 (C-24), 28.4 (C-25), 22.3 (C-26), 22.6 (C-27), 171.8 (C-28), 21.5 (C-

29).

+ HREIMS: [M] at m/z 458.3396 (Calcd. for C29H46O4, 458.3398)

EIMS [M+] m/z (rel int %): 458 (4), 375 (9), 341 (5), 316 (11), 279 (7), 252 (14), 237 (14),

149 (43), 83 (64), 69 (83), 57 (100), 59 (32), 43(94)

3.3.8: (S)-4', 5-Dihydroxy-7-methoxyflavanone (97)

Physical state: Colorless needles

Melting Point: 152-154°C

20 [α]D : -8.0 (c 1.0, MeOH)

UV-Vis.: λmax nm (MeOH) (log ε) 286 (4.40), 329 (4.12)

-1 FT-IR: ʋmax cm 3434 (OH), 2950 (C-H str), 1725 (C=O), 1625 (C=C), 1265 (C-O), 1025 (C-

C), 810 (C-H bending).

1 H-NMR (CDCl3, 400 MHz)

δ: 2.86 (1H, dd, J = 17.5, 2.8 Hz, H-3eq), 3.21 (1H, dd, J = 17.5, 13.5 Hz, H-3ax), 3.67 (3H, s, OMe), 5.54 (1H, dd, J = 13.5, 2.8 Hz, H-2), 6.41 (1H, d, J = 2.1, H-6), 6.66 (1H, d, J = 2.2,

H-8), 7.27 (2H, dd, J = 8.2, 2.3 Hz H-3', 5'), 7.69 (2H, dd, J = 8.2, 2.3 Hz H-2', 6').

13 C-NMR (CDCl3, 100 MHz)

δ: 78.7 (C-2), 46.5 (C-3), 192.9 (C-4), 161.7 (C-5), 99.7 (C-6), 164.1 (C-7), 96.2 (C-8), 158.5

(C-9), 108.5 (C-10), 128.9 (C-2', 6'), 118.4 (C-3', 5'), 56.3 (CH3O) 100

+ HREIMS: [M] at m/z 286.0980 (Calcd. for C16H14O5 286.0961)

EIMS [M]+ m/z (rel int %): 286 (45), 194, (48), 178 (55) 166 (48), 148 (73), 120 (54), 94 (93).

101

3.3.9: 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98)

Physical state: Colorless crystals

Melting Point: 283-285°C

UV-vis.: λmax nm (MeOH) (log ε) 258 (4.4), 285 (4.1), 372 (3.6)

-1 FTIR: ʋmax cm 3483-3345 (OH), 2947 (C-H str), 1665 (C=O), 1272 (C-O), 1089 (C-C), 812

(C-H bending)

1 “ H-NMR (CDCl3, 400 MHz)”

δ: 3.64 (3H, s, OMe), 3.77 (3H, s, OMe), 6.15 (1H, d, J = 2.2 Hz, H-8), 6.30 (1H, d, J = 2.2

Hz, H-8), 6.79 (1H, d, J = 7.2 Hz, H-5'), 7.55 (1H, dd, J = 7.2, 2.1 Hz, H-6'), 7.69 (1H, d, J =

2.1 Hz, H-2'), 8.08 (1H, s, H-2), 9.1 (1H, br, OH), 10.67 (1H, br, OH).

13 “ C-NMR (CDCl3, 100 MHz)”

δ: 150.6 (C-2), 124.7 (C-3), 173.8 (C-4), 96.6 (C-6), 162.4 (C-7), 94.8 (C-8), 159.1 (C-9),

108.2 (C-10), 123.6 (C-1'), 113.8 (C-2'), 147.2 (C-3'), 146.6 (C-4'), 115.1 (C-5'), 121.8 (C-6').

102

+ HREIMS: [M] at m/z 314.0780 (Calcd. for C17H14O6, 314.0790)

EIMS m/z (rel int %): [M]+ 314 (12), 296 (43), 283 (21), 278 (19), 265 (25), 252 (30), 247

(25), 216 (45), 191 (39), 173 (28), 160 (41), 142 (58), 123 (34), 105 (33), 92 (20), 74 (65).

103

3.3.10: α-D-Glucopyranosyl-6'-O-hexadecanoate (99)

Physical state: White powder

Melting Point: 106-107°C

18.6 [α]D : +67.7 (c 1.3, pyridine)

-1 FT-IR: ʋmax cm 3453 (OH), 1735 (C=O), 1172 (C-O), 824 (C-H bending)

1 H-NMR (Pyridine-d5, 400 MHz)

δ: 0.84 (3H, t, J = 6.8Hz, Me-16), 1.22 (24H, brs, H-4 to H-15), 1.58 (2H, m, H-3), 2.31 (2H, t, J = 7.5 Hz, H-2), 4.12 (1H, dd, J = 8.8, 9.3 Hz, H-3'), 4.23 (1H, dd, J = 3.6, 9.3 Hz, H-2'),

4.77 (1H, t, J = 7.2 Hz H-5'), 4.82-4.89 (2H, m, H-6´), 4.93 (1H, m, H-4'), 5.88 (1H, d, J = 3.6

Hz, H-1').

13 C-NMR (Pyridine-d5, 100 MHz)

δ: 173.5 (C-1), 34.1 (C-2), 24.8 (C-3), 29.1-29.7 (C-4-C-13), 22.6 (C-14), 31.8 (C-15), 13.8

(C-16), 94.0 (C-1'), 74.0 (C-2'), 75.2 (C-3'), 70.6 (C-4'), 71.8 (C-5'), 64.9 (C-6')

- HRFABMS: [M-H] 417.2847 (Calcd. for C22H41O7 417.2851)

FABMS: m/z 417 [M-H]-1 (60), 255 [M-162-H]- (100) 104

3.3.11: β-sitosterol-3-O-β-D-glucopyranoside (100)

Physical state: White solid

Melting Point: 279-280°C

25 [α]D : -14.5 (c 1.9, CHCl3)

-1 FT-IR: ʋmax cm 3478-3395 (OH), 3038 (=C-H), 1649 (C=C) and 1598

1 “ H-NMR (CDCl3, 400 MHz)”

δ: 0.80 (3H, s, Me-18), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.83 (3H, d, J = 6.5 Hz, Me-26), 0.86

(3H, t, J = 7.0 Hz, Me-29), 0.94 (3H, d, J = 6.2 Hz, Me-21), 1.04 (3H, s, Me-19), 3.85, (1H, m, H-3), 3.84  4.46 (m, Glc-H), 5.15 (1H,br.d, J = 5.4 Hz, H-6), 5.37 (1H, d, J = 7.2 Hz, H-

1').

13 C-NMR (CDCl3, 100 MHz)

δ: 37.4 (C-1), 28.6 (C-2), 81.7 (C-3), 41.3 (C-4), 141.7 (C-5), 121.6 (C-6), 33.4 (C-7), 32.6

(C-8), 50.2 (C-9), 36.6 (C-10), 22.4 (C-11), 41.7 (C-12), 42.6 (C-13), 57.4 (C-14), 24.7 (C-

15), 30.6 (C-16), 56.7 (C-17), 13.8 (C-18), 18.4 (C-19), 36.7 (C-20), 19.1 (C-21), 40.3 (C-22), 105

30.7 (C-23), 51.1 (C-24), 2.6 (C-25), 17.8 (C-26), 19.4 (C-27), 22.5 (C-28), 12.5 (C-29), 102.4

(C-1'), 74.2 (C-2'), 78.6 (C-3'), 72.3 (C-4'), 78.7 (C-5'), 61.6 (C-6').

+ HRFABMS: [M] at m/z 576.4386 (Calcd. for C35H60O6 576.4389)

EIMS [M]+ m/z (rel int %): 576 (16), 396 (13), 381 (75), 329 (31), 256 (41), 179 (21).

106

3.3.12: “Quercetin-3'-O-β-D-glucopyranoside” (101)

Physical state: “Amorphous powder”

Melting Point: 244-246°C

25 [α]D : -20.0 (c 1.01, MeOH)

UV-Vis. λmax nm (MeOH) (log ε) 255 (4.5), 279 (3.9), 364 (4.2).

-1 FTIR: ʋmax cm 3478-3250 (OH), 1736 (C=O), 1630 (C=C), 1310 (C-O) 1015 (C-C), 860 (C-

1 “ H-NMR (CDCl3, 400 MHz)”

δ: 3.61-4.40 (6H, m, H-2'', H-3'', H-4'', H-5'', H-6''), 5.02 (1 H, d, J = 7.4 Hz, H-1''), 6.38 (1H, d, J = 2.1 Hz, H-6), 6.22 (1H, d, J = 2.1 Hz, H-8), 6.77 (1 H, d, J = 7.8 Hz, H-5'), 7.74 (1 H, dd, J = 7.8, 2.2 Hz, H-6'), and 7.98 (1 H, d, J = 2.2 Hz, H-2').

13 C-NMR (CDCl3, 100 MHz) δ: 147.5 (C-2), 137.6 (C-3), 177.3 (C-4), 162.7 (C-5), 99.3 (C-6), 165.9 (C-7), 94.8 (C-8),

158.6 (C-9), 104.5 (C-10), 124.3 (C-1'), 118.3 (C-2'), 146.9 (C-3'), 150.7 (C-4'), 117.3 (C-5'),

125.1 (C-6'), 105.3 (C-1''), 72.5 (C-2''), 74.5 (C-3''), 70.1 (C-4''), 77.6 (C-5''), 62.4 (C-6'').

+ HRFABMS: [M+ H ] at m/z 465.1038 (Calcd. for C21H21O12 465.1033) 107

EIMS [M]+ m/z (rel int %): 464 (12), 285 (33), 193 (39) 180 (45), 139 (47), 92 (53).

108

3.4: Biological screening

3.4.1: Lipoxygenase Inhibitory Assay

The lipoxygenase inhibition potential of all the isolated compounds was performed according to Tappel method [66] with minor, amendments. A mixture was prepared by adding 10μL test compound in 150μL sodium phosphatebuffer, (100mM, pH8.0) along with 15μL purified lipoxygenase enzyme. After incubation of mixture at room temperature (10 minutes) absorption was measured at 234 nm. Then the reactionwas initiated by theaddition of 25μL linoleic acid (substrate) solution and change in absorbance at same wavelength was checked after six minutes. The control (baicalein) and test compounds were dissolved in methanol.

Experiments were run in triplicates. IC50 values “of compoundswere calculated, usingEZ-Fit

Enzymekinetics software” (Perella Scientific Inc.Amherst, USA).

3.4.3: Antibacterial assay

All the isolated compounds were tested for their antibacterial potential using disc diffusion assay. A mixture of bacterial suspension (100 µL) and liquid nutrient agar medium (100 mL) was prepared to make agar plates. Then, sterilized filter paper discs (6mm diameter) containing

10 µL of tested compound were placed on these agar plates. All plates were incubated (28 ºC) for twenty four hours. After incubation, the diameter of inhibition zone was measured in millimeters. Imipenem was used as reference standard and experiments were performed thrice

[67].

3.4.2: Antifungal assay

Agar well diffusion method was used to test the antifungal potential of isolated compounds against various fungal strains.30 mL PDA (Potato Dextrose Agar) solution was autoclaved and 109

aseptically transferred to petri plates which were solidified at 25 ºC. A hole of 6 mm diameter was punched using sterile cork borer. Tested compound was placed in this well, while fungal spores were placed in the middle of the plates. The plates were rapped with thin paraffin film and incubated (28 ºC) for three days. After incubation, the diameter of inhibition zone was measured in millimeters. The experiments were run in triplicate. Standard drug used was

Miconazole [68].

Chapter 4 Results & Discussion

110

4.1: Hyperinoate A (86)

The compound 86 was isolated as colorless crystalline solidfrom chloroform soluble fraction of Hypericum oblongolium. It behaved positively against LiebermannBurchard and Salkowski testsfor steroids [69]. The molecular formula C30H52O3 with five degrees of unsaturation was established with the help of HREIMS, produced molecularion peak at m/z 460.3911 (calcd. for

C30H52O3 460.3916). The FT-IRspectrum displayed bands at 3452 (OH), 1730 (C=O), 1170

(C-O) and 1370 (gem dimethyl).

In EIMS the ions at m/z 113 and 154 were diagnostic ofsteroid bearinga side chainat C-17 [70].

A peak at m/z 442 was originated from molecular ion peak (m/z 460) and was due tothe loss of H2O molecule from the compound. Similarly a peakat m/z 59 was originated due tothe loss of acetyl group. The loss of side chain and the ring D fission supported the presence of oxygen functionalities in the steroidal skeleton (Scheme 41).

The 1H-NMR (Table 5) displayed four tertiary methyl signals at δ 2.01 (s, 3H, H-30), δ 0.92

(s, 3H, H-28), δ 0.88 (s, 3H, H-19) and δ 0.87 (s, 3H, H-18), while three methyls appeared as doubletsat δ 0.85(d, J = 6.2 Hz, 3H, H-26), δ 0.84(d, J = 6.2 Hz, 3H, H-27) and δ 0.81(d, J = 111

6.2 Hz, 3H, H-21). The oxymethine protons were observed at δ 3.65 (dt, J = 7.0, 4.5 Hz, 1H,

H-6) and δ 4.95 (m, 1H, H-3). A signal at δ 2.01 appeared as singlet integrating three protons indicating the presence of ester methyl in the molecule.

The broad band (BB) and DEPT (Distortionless Enhancement by Polarization Transfer)

13CNMR spectrum revealedthe presence of thirtycarbon signals, with sevenmethyl, eleven methylene, eight methineand four quaternarycarbon atoms. The downfield signal at δ 170.8 was due to acid carboxyl. Two oxymethine carbon signals were resonated at δ 71.1 and 72.3.

The EIMS and 1D NMR (1H-NMR and 13CNMR) indicated the cholestane skeleton with an additional methyl in the steroidal nucleus.

The problem was to assign the methyl and hydroxyl moieties on their specific position and was resolved by 1H-1H-COSY 45° and HMBC experiments. In 1H-1H-COSY spectrumthe signal at

δ 3.65 showedcorrelation with the two other protons at δ 1.57 and 1.34 (1H each, m, H-7) and one proton at δ 1.68 (1H, m, H-5) limiting it either at position 6 or 7. The signal at δ 1.68 further showed 1H-1H-COSY correlation with two protons at δ 1.80, 1.55 with coupling constant in range 6.5-7.1 Hz, confining the presenceof hydroxyl moiety atC-6 position in an α- configuration based on the coupling constant of carbinylic proton at δ 3.65 (1H, 9.1 and 6.5

Hz, H-6). The assignments were further confirmed by HMBC interactions. The protons at δ

3.65 showed J2 correlation withC-5 (δ 44.0) and C-7 (δ 33.2)and J3 correlationwith C-4 (δ

30.5), C-8 (δ 42.7) and C-10 (δ 34.3). Similarly a signal at δ 4.95 showed J2 correlation with

C-4 (δ 30.5) and C-2 (δ 28.8) while J3 with C-5 (δ 44.0) and C-1 (δ 35.4) allowing it at C-3 position also supported by biogenetic grounds. The stereochemistry was assigned as β and pseudo-equatorial based upon the large coupling constant of oxymethine proton (H-3, J = 9.9,

4.4 Hz). 1Dand 2D NMR (HMBC, 1H1H COSY and NOESY) data of 86 showed close 112

resemblance to the cholestane type skeleton with 14α- methyl by comparison with the literature values [71]. Hence on the basis of these grounds the compound 86 was assigned the structure as 3β-acetoxy, cholestane-6α-ol.

113

Scheme 41. Mass fragmentation pattern of Hyperinoate A (86)

114

21 H3C 22 24 CH3 20 12 19 17 23 26 11 13 25 18 CH3 16 1 9 14 27 2 O 10 8 15 H 29 3 5 28 30 4 7 H C O 3 H 6

Figure 6. Important HMBC correlation in 86

Figure 7. Important NOESY correlation in 86

115

Position No. δH δC 1 1.51 m 35.4 1.30 m 2 1.78 m 28.8 1.59 m 3 4.95 m 72.3 4 1.80 dd (7.1, 6.5) 30.5 1.55 dd (7.1, 6.5) 5 1.68 m 44.0 6 3.65 dt (7.0, 4.5) 71.1 7 1.57 m 33.2 1.34 m 8 1.39 m 42.7 9 1.18 m 48.4 10 - 34.3 11 1.49 m 19.7 1.29 m 12 1.27 t (6.4) 21.2 0.85 t (6.4) 13 - 44.8 14 - 45.9 15 1.80 t (7.7) 33.4 1.71 t (7.7) 16 1.66 m 31.6 1.41 m 17 1.46 m 50.9 18 0.87 s 15.0 19 0.88 s 15.9 20 1.13 m 35.8 21 0.81 d (6.5) 18.5 22 1.15 m 36.3 0.95 m 23 1.05 m 23.6 24 1.20 m 39.1 25 1.47 m 27.5 26 0.85 d (6.3) 22.7 27 0.84 d (6.3) 20.3 28 0.92 s 13.9 29 - 170.8 30 2.01 s 20.8

116

4.2: Hyperinoate B (87)

The compound 87 wasisolated as colorless solid from chloroformsoluble fraction of the whole plant, behaved positively against Liebermann-Burchard and Salkowski tests for steroids [69].

HREIMS provided molecularion peak at m/z 476.3862 (calcd.for C30H52O4, 476.3865) establishing the molecular formula C30H52O4, showing five degrees of unsaturation. The FT-

IR spectrum of 87 showedabsorption due to hydroxyl and estercarbonyl at 3440-3390, 1729,

1230 and 1090 cm-1respectively.

In EIMS spectrum the fragment ion at m/z 129, 170, 306 and 347 were due to the removal of side chain and ring D fission in the molecule and were characteristics of cholestane type steroids [70]. Further peaks at m/z 111, 152, 288 and 329 resulted from the loss of water molecules from these fragments, indicating the presence of one hydroxyl in side chain and one in the steroidal nucleus. It also indicated the presence of one hydroxyl in ring A or B. The loss of fragment ion at m/z 59 from the steroidal nucleus indicated the presence of acetate group

(Scheme 42). 117

The 1H-NMR (Table 6) displayed sixmethyl singlets at δ 0.84, 0.85, 0.87, 0.88, 0.92 and 2.01, while one methyl appeared as doubletat δ 0.81 (3H, J = 6.6 Hz, H-21). The downfield oxymethine protons appeared at δ 3.65 (1H, dd, 7.0, 4.2 Hz, H-6).

The 13CNMR spectral data (BB and DEPT) showed thirty carbon signals, corresponding the presence of seven methyl, elevenmethylene, seven methine andfive quaternarycarbon atoms.

The oxymethine carbons resonated at δ 72.3 (C-3) and 71.6 (C-6). The BB spectra displayed one quaternary carbon atom at δ 70.2 representing the existence of one hydroxyl group. A signal at δ 170.1 was attributed to the ester carbonyl. The 1D (1H-NMR and 13CNMR) spectra of 87 was similar to the 86 except for the presence of one additional hydroxyl moiety in the side chainof the molecule.

The position of various substituents and their stereochemistry were further confirmed by

HMBC, 1H-1H COSY 45° and NOESY experiments. The 1H-NMR and 13CNMR signals of the side chain were different from 86 indicating the presence of hydroxyl moiety at C-25 position, confirmed finally by HMBC experiments in which a methyl at δ 0.86 showed J2 correlation with C-25 (δ 70.9) and J3 correlation with C-27 (δ 29.8) and C-24 (δ 44.3). Similarly same is the correlation observed by protons at δ 0.85 (3H, Me-27) with C-25 (δ 70.9), C-24 (δ 44.3) and C-26 (δ 30.1). The NOESY results from H-3 and H-6 were similar to the 86, allowing to assign the structure as 3β-acetoxy, cholest-6α,25-diol (Figure 9).

118

Scheme 42: Mass fragmentation pattern of Hyperinoate B (87)

119

Figure 8. Important HMBC correlation in 87

Figure 9. Important NOESY correlation in 87

120

Position No. δH δC 1 1.51 m 35.4 1.30 m 2 1.78 m 28.8 1.59 m 3 4.95 m 72.3 4 1.80 dd (7.1, 6.5) 30.5 1.55 dd (7.1, 6.5) 5 1.68 m 44.0 6 3.65 dt (7.0, 4.5) 71.1 7 1.57 m 33.2 1.34 m 8 1.39 m 42.7 9 1.18 m 48.4 10 - 34.3 11 1.49 m 19.7 1.29 m 12 1.27 t (6.4) 21.2 0.85 t (6.4) 13 - 44.8 14 - 45.9 15 1.80 t (7.7) 33.4 1.71 t (7.7) 16 1.66 m 31.6 1.41 m 17 1.43 m 50.9 18 0.87 s 15.0 19 0.88 s 15.9 20 1.13 m 35.8 21 0.81 d (6.5) 18.5 22 1.16 m 36.3 0.96 m 23 1.05 m 20.5 24 1.20 dt (6.7, 4.4) 43.8 25 - 70.2 26 0.85 s 29.7 27 0.84 s 27.3 28 0.92 s 13.9 29 - 170.8 30 2.01 s 20.8

121

4.3: Hyperinone (88)

Compound 88 was isolatedas colorless needles from the chloroform solublefraction of H. oblongifolium, behaved positively against Liebermann-Burchard test for triterpenes [69]. The

IR spectrumshowed absorption bands at 2950 cm1 (CH- stretching), 1755 cm1 (γ-lactone)

1 1 1695 cm (C=O), and 1650 cm (terminal double bond). The molecularformula C30H44O3 was determined by HREIMS, which produced the molecular ion peak [M]+ atm/z 452.3265

1 (calcd. for C30H44O3 452.3290), with nine degrees of unsaturation. Inspection of the H-NMR indicated signals of six methyls; among them five appeared separately as singlets at δ 0.76,

0.94, 0.95, 0.97 and 1.04 (3H each) and one as doublet at δ 1.10 (3H, J = 6.9). Two characteristic signals at δ 4.66 (1H, s) and δ 5.10 (1H, s) were observed, which indicatedthe presence of terminal olefinic moiety inthe molecule [72]. The BB and DEPT 13C-NMR spectra displayed thirty carbon signals out of which six were methyl, eleven methylene, four methine and nine quaternary carbons. A signal at δ 216.5 depicted the presence of keto group while a signal at δ 178.4 indicatedthe presence of γ-lactone moiety inthe molecule. The olefinic carbons resonated at δ 151.9 and δ 106.8 and an oxygenated carbon appeared at δ 89.9. All 122

above spectral data revealed that it was a tarxastane type triterpene having a lactone, a ketone group and a terminal olefinic moiety. Further confirmation of the structure was done by the inspection of HMQC and HMBC experiments. The proton of C-18 position resonated at δ 2.22

(1H, J = 4.6 Hz) and the lower magnitude of coupling constant pointed towards the β configuration of this proton. It also confirmed the existence of equatorial and β configuration of neighboring proton at C-19 (δ 2.02), hence proved the axial and α configuration of the secondary methyl group attached to C-19 [73]. In HMBC correlations (Figure 10) H-23 (δ

1.04) and H-24 (δ 0.76) showed J2 correlation with C-4 (δ 40.6), and J3 correlations with C-3

(δ 216.5) and C-5 (53.0) allowing to place the oxo moiety at C-3, which also supported by the biogenetic analogy. The carbonyl carbon of γ-lactone at δ 178.4 showed correlations with H-

16 (δ 1.30, 2.02) and H-22 (δ 1.68, 1.58) while carbon bearing lactone oxygen at δ 89.9 correlated with H-15 (δ 1.87, 2.11), H-18 (δ 2.22), and H-27 (δ 0.99). This data indicated that the carbonyl and tertiary oxygen bond of a lactone were connected at C-17 and C-13 position, respectively, in the taraxastene terpenoid skeleton. The olefinic protons δ 106.8 (H-30) showed

J2 correlation with C-20 (δ 151.9) and J3 correlations with C-19 (δ 32.8) and C-21 (δ 30.6) establishing the presence of terminal double bond at C-20. On thebasis of these evidences and comparisonwith the spectral data of already reported compounds in the literature, [73-74] the compound was considered as a new taraxastene type triterpene and hence the structure of 88 was assigned as 3-oxo-20(30)-taraxastene-28,13β-olide.

123

Scheme 43: Mass fragmentation pattern of Hyperinone (88)

124

Figure 10. Important HMBC correlation in 88

125

Figure 11. Important NOESY correlation in 88

126

Position 1H-NMR 13C-NMR 1 1.52 m 36.1 1.30 m 2 2.10 m 29.4 3 - 216.5 4 - 40.6 5 1.01 m 53.0 6 1.67 m 16.5 1.58 m 7 1.75 m 57.8 1.18 m 8 - 42.7 9 1.30 dd (11.6, 4.8) 51.3 10 - 33.9 11 1.27 m 15.7 1.09 m 12 1.84 m 29.6 1.68 m 13 - 89.9 14 - 40.6 15 2.11 m 23.4 1.87 m 16 2.14 m 19.4 1.30 m 17 - 42.1 18 2.22 d (4.6) 53.2 19 2.02 dq (6.9, 4.4) 32.8 20 - 151.9 21 2.15 m 30.6 22 1.68 m 28.1 1.58 m 23 1.03 s 22.3 24 0.76 s 20.7 25 0.94 s 14.2 26 0.95 s 16.2 27 0.97 s 17.2 28 - 178.4 29 1.10 d (6.9) 14.4 30 5.10 s 106.8 4.66 s

127

4.1.4: Hyperinoic acid (89)

The compound 89 was isolated as colorless solid, gave positive Libermann Buchard test for triterpenes [69]. HREIMS gave molecularion peak at m/z 472.3549 establishing the molecular formulaC30H48O4 with seven degrees of unsaturation. FT-IR spectrum showed peaks at 3450,

3051, 1697, 1634, 810 indicating the existence of hydroxyl, carboxyl and olefinic moieties in the molecule. EI-Massspectrum gave molecular ion peak atm/z 472, while daughter ions are at m/z 264, 251, 221, 219, 208 and 133. The peaks at m/z 264 and 208 were formed due to RDA

(Retro Diels Alder) fragmentation revealing the presence of one additional hydroxyl and carboxyl in the ring AB or DE (Scheme 44) [75].

The 1H-NMR spectrum of hyperinoic acid provided seven methyl signals, five singlet at δ 0.90,

0.93, 1.03, 1.15 and 1.20, while two methyls appeared as doublet at δ 0.89 (J = 6.5) and 0.96

(J = 7.0). Two downfield signals of hydroxymethine protons at δ 3.27 (1H, dd, J = 9.1, 6.3) and 3.81 (1H, dt, J = 9.8, 8.7, 7.4) depicted the presence of two hydroxyl moieties in the molecule. A characteristic signal at δ 5.37 (1H, t J = 3.5) represented the existence of 128

trisubstituted double bond in the structure. This indicated that 89 is a Δ12-ursene type triterpene with one additional hydroxyl moiety.

The BB and DEPT 13CNMR spectra displayed thirtycarbon signals, sevenmethyl, eight methylene, eight methineand seven were quaternarycarbon atoms. The observation of carbon signals at δ 77.3 and 70.2 (methine carbons) also proved the existence of two hydroxyl groups in the structure. Two olefinic carbons resonated at δ 124.6 and 137.8, while an acidic carboxyl was appeared at δ 180.1 [76].

The position of various substituents were finally confirmed by HMBC (Heteronuclear multiple bond connectivity) and 1H-1H COSY 45° experiments. The signals at δ 3.81 (H-21) showed

1H-1H COSY correlation with H-22 (δ 1.96 and 1.76) which further showed J3 correlation with

C-28 (δ 180.1) and C-18 (δ 52.5) allowed us to assign the hydroxyl at C-21 position. The magnitude of coupling constant of H-21 (J = 9.8, 8.7) allowed us to assign β and equatorial orientation to the hydroxyl group which could also be confirmed through NOESY experiments in which H-21 showed interactions with H-30 and H-22 α, respectively. In HMBC experiments

H-21 showed J2 correlation with C-20 (δ 48.1) and C-22 (δ 40.4) and J3 correlationswith C-30

(δ 14.0), C-19 (δ 36.5) and C-17 (δ 45.6). The other important HMBC and NOESY correlations showed in Figure 12 and 13.

On thebasis of these evidencesthe structure of 89 was assignedas 21β-hydroxy-12-ene-ursane-

28-oic acid. 129

Scheme 44: Mass fragmentation pattern of Hyperinoic acid (89) 130

Figure 12. Important HMBC correlation in 89

131

Figure 13. Important NOESY correlation in 89

132

Position No. δH δC 1 1.79 m 38.0 1.36 m 2 1.83 m 26.5 1.50 m 3 3.27 dd (9.1, 6.3) 77.3 4 - 39.2 5 0.70 t (8.9) 54.3 6 1.54 m 17.8 1.41 m 7 1.57 m 32.3 1.18 m 8 - 38.9 9 1.56 t (5.4) 46.8 10 - 36.2 11 2.42 m 22.7 2.23 m 12 5.37 t (3.5) 124.6 13 - 137.8 14 - 41.9 15 1.98 m 27.4 1.80 m 16 2.41 m 25.5 2.31 m 17 - 45.6 18 2.15 d (8.8) 52.5 19 2.01 m 36.5 20 1.77 m 48.1 21 3.81 dt (9.8, 8.7, 7.4) 70.2 22 1.96 m 40.4 1.76 m 23 1.20 s 22.3 24 1.15 s 20.2 25 0.93 s 15.1 26 0.90 s 16.2 27 1.03 s 23.4 28 - 180.1 29 0.96 d (7.0) 17.3 30 0.89 d (6.5) 14.0

133

4.5: 4,4-Dimethyl cholesterol (90)

The compound 90 was isolated in the form of colorless crystalline solid showing melting point

20 109-110 °C and [α]D + 20.5 (c 0.50, CHCl3). The HREIMS of this compound provided the molecular ion peak at m/z 414.3845 along with a stronger [M-1]+ peak at m/z 413 resulting the molecular formula C29H50O. The IR spectrum exposed the existence of OH moiety and an

-1 olefinic bond in the molecule by providing absorption bands at ʋmax 3437 cm and 1610, 1485,

1038 and 812 cm-1. The EIMS spectrum of compound 90 showed diagnostic peaks at m/z 414,

317, 303, 301, 260, 113, 111 and 97 which are characteristics for steroids with mono-saturation and dimethyls at C-4 [77].

The 1HNMR spectrum provided the signals for seven methyls in the compound among these methyl four appeared as singlet (δ 0.74, 1.34 and 1.55) while three as doublet (δ 1.01 (J = 6.5

Hz, Me-21), 1.67 (J = 6.9 Hz, Me-26, 27)). This spectrum also showed carbinylic proton signal at δ 3.69 (dd, J = 7.1, 2.9 Hz). The olefinicproton resonated at δ 5.38 asa doublet (J = 5.1 Hz) which is characteristic of Δ5,6 cholesterol derivatives [78].

13CNMR spectrum (BB and DEPT) displayed twenty nine carbons, seven methyl, ten methylene, eight methine and four quaternary carbons. It showed the olefinic carbon signals at 134

δ 121.6 and 140.7. This information together with the molecular formula of the compound showed that the compound is a tetracyclic triterpenoid with a Δ5,6 tri-substituted double bond.

All the physical and spectral data of compound 90 coincided with the values reported in literature for 4,4-dimethyl cholesterol [79].

135

4.6: β – Sitosterol (91)

The compound91 was isolated as colorless crystals. The molecularformula wasestablished

+ with thehelp of HREIMS, showed [M] atm/z 414.3851 (calcd.for C29H50O, 414.3861) indicating five degreesof unsaturation. The IR spectrumshowed a broad peak of OH moiety at

-1 -1 ʋmax 3450 cm , while olefinic bond showed absorption at ʋmax 1660 cm

Major peaks shown by 1H-NMR indicate the presence of six methyl groups. Among of them two methyl groups resonated as singlet at δ 0.68 (3H, s, Me-18) and 1.03 (3H, s, Me-19). Three methyl groups appeared as doublet at δ 0.81(3H, d, J = 6.5 Hz, Me-26), 0.84(3H, d, J = 6.5

Hz, Me-27)andδ 0.92(3H, d, J = 6.2 Hz, Me-21). While one methylgroup provide a triplet at δ

0.86 (3H, t, J = 7.0 Hz, Me-29). The carbinylic proton at C-3 showed a downfield signal at δ

3.36 (1H, m, H-3) due to presence of β and equatorial OH group. Peak at δ 5.14(1H, m, H-6) is a characteristic signal of Δ5 sterols [78]. 13C-NMR spectrum (BB and DEPT) confirmedthe presence oftwenty nine carbons, six methyl, eleven methylene, nine methine and three quaternarycarbon. EIMS spectrum indicated peaks at m/z 399, 381, 329 275, 255 which are characteristics of Δ5 steroidal molecules [80]. All the physical and spectral data of compound

91 was consistence with β-sitosterol [81].

136

4.7: Lupeol (92)

20 Compound 92 was isolated as colorless needles, melting point 215-216°C and [α]D +26.9 (c

1.0, CHCl3). HREIMS of 92 showedmolecular ion peak at m/z 426.3835, (calculated 426.3861) consistentwith the molecularformula C30H50O. The IR spectrum showeda broadpeak at ʋmax

3475 cm-1 which is the indication of the presence of a hydroxyl moiety in the molecule. The

-1 other bands are at ʋmax 3081 (C-H), 1645 (terminal double bond) and 873 (C-H bending) cm .

EIMS of 92 gave molecularion peak at m/z 426 [M+]. Other fragments produced by mass spectrum appeared at m/z 411 (M-CH3),408 (M-H2O), 393 (M-H2O-CH3), 385 (M-C3H5), 220

(M-C3H5), 218 (M-C14H24O), 207 (M-C16H27), 189 (M-C16H29O), 139 (M-C20H34O).

The inspection of 1H-NMR spectrum showed the existence of seven methyls, appeared as singlets at δ 0.75, 0.78, 0.83, 0.94, 0.96, 1.03, 1.36. Two protons of terminal olefinic bond appeared as singlet at δ 4.57 and 4.69. The carbinylic proton resonated at δ 3.22 asdouble doublet ( Jax, ax = 9.9 Hz, Jax, eq = 4.5 Hz), showing the equatorial and βconfiguration of hydroxyl moiety atC-3. 137

13C-NMR confirmed the presence of thirty carbon atoms among them seven are methyl, eleven methylene, six methine and six are quaternarycarbons. 13C-NMR dataalso correspond to the presenceof lupeol skeleton [82].

The physicaland spectral dataof compound 92 corroborated to thoseof lupeol reportedin literature [83].

138

4.8: Taraxerol (93)

20 The compound 93 was isolated as white crystals, melting point 275-277°C and [α]D + 0.72

(c 1.15, CHCl3). HREIMS produced molecularion peak at m/z 426.3826 (calcd.for C30H50O,

426.7261) establishing the molecular formula C30H50O with six degrees of unsaturation. The

-1 peaks in IR spectrum at ʋmax cm 3584, 3052, 1638 and 818 depicted the presence of OH and olefinic moieties in the molecules respectively.

1H-NMR produced five singlet signals for eight methylgroups at δ 0.82 (3H, s, Me-24), δ 0.85

(3H, s, Me-28), δ 0.89 (3H, s, Me-25), δ 0.93 (3H, s, Me-30), δ 0.95 (3H, s, Me-23), δ 0.97

(3H, s, Me-29), δ 1.13 (3H, s, Me-26) and δ 1.19 (3H, s, Me-27). The proton of olefinic moiety appeared as double doublet at δ 5.54 (J = 7.1, 4.2) and the carbinylic carbon gave a triplet peak at δ 3.38 with large coupling constant (J = 0.82 Hz) indicating the OH at equatorial and β configuration. 13CNMR corroborated with the existence of seven quaternary, five tertiary, ten secondary and eight primary carbon atoms. It also confirmed the presence of olefinic moiety

(δ 158.3, 117.3) and OH group (δ 79.3). 139

The mass spectrum of the compound 93 showed peaks at m/z 426, 408, 393, 363, 348 correspond with that reported for taraxerane type skeleton of triterpenes. All the physicaland spectraldata was in consistence with that reportedin literature for taraxerol [84].

140

4.9: 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94)

23 The compound 94 was obtained as colorless powder, [α]D -15.2° (c 2.8, MeOH). The molecularformula C30H48O3 was establishedwith the helpof HREIMS which provide the molecularion peak at m/z456.360345 (calcd. forC30H48O3, 456.7062) which was further confirmed by 1H-NMR and 13CNMR. The UV spectrum of the compound depicted an absorptionband at 247 nm pointing towards the presenceof hetroannular diene system, which

-1 was alsoconfirmed by IR spectrumwhich gave peak for conjugated alkene at ʋmax 1631 cm .

-1 IR spectrum also provided peaks for terminal alkene (ʋmax 3039 & 1631 cm ). Another

-1 absorption band in IR spectrum was appeared at ʋmax 3472 cm for hydroxyl moiety. EIMS of the compound 94 provide signals at m/z 456, 438, 420, 397, 394, 379, 369, 327, 301, 300, 273 and 271 which were in accordance with that provided by steroids.

1HNMR yield four singlets at δ 0.82, 0.86, 1.01 and 1.03 representing four methyl groups attach with tertiary carbon atoms. It also provide three doublets for methyl groups attach with secondary carbons (δ 1.04, 1.07 and 1.16). Two carbinylic protons exhibited double doublets at δ 3.19 and 3.94 (J = 10.0 & 10.5 Hz respectively) with β orientation. It also gave broad singlet at δ 4.72 approving the terminal alkene moiety. A triplet at δ 5.44 (J = 6.5 Hz) 141

corroborated the existence of a trisubstituted double bond. 13CNMR provide the evidence for the presence of thirty carbon atoms. Among these seven were primary carbons, nine secondary, six tertiary and eight were quaternary carbon atoms.

All the physicaland spectral data of compound 94 was corroborated for that already present in literature for 4,4-dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol [85].

142

4.10: Oleanolic acid (95)

The compound 95 was obtained as colorless needles from methanol showing melting point

20 305-306°C and [α]D +78.9° (c 1.07, CHCl3). The molecularformula C30H48O3 was established by HREIMS which provide molecularion peak at m/z 456.3603 (calcd.for C30H48O3,

-1 456.3610). The peaks in IR spectrum (ʋmax 3420-3285, 1707, 1637 and 815 cm ) revealed hydroxyl, carboxyl and olefinic moiety in the molecule.

1H-NNMR spectrum provide seven singlet signals (δ 0.82, 0.92, 0.93, 0.96, 0.99, 1.01, 1.10) for seven methyl indicating pentacyclic triterpene skeleton. The downfield shift of carbinylic proton (3.62, dd, J = 9.9, 4.1 Hz) proved the existence of hydroxyl moiety at C-3 and its J value proved the β configuration of hydroxyl group, also compared from literature values [86].

1H-NNMR spectra also showed a triplet for olefinic proton at δ 5.23 (J = 3.4 Hz).

13CNMR spectrum confirmed the thirty carbon skeleton for the compound with seven methyl, ten methylene, five methine and eight quaternary carbon atoms. Inspection of BB and DEPT confirmed the presence of hydroxyl and olefinic moieties in the molecules. It also provide clue for the existence of a carboxyl group at C-17 by observing signals at δ 183.7. 13CNMR 143

spectrum produced a spectrum similar to oleanane type pentacyclic triterpenes, which was further confirmed by the mass fragmentation pattern appeared in EIMS ([M]+ 456, 248, 208,

203, 133).

All the physical and spectral data of compound 95 was corroborated with oleanolic acid as reported in literature [87].

144

4.11: Erectasteroid D (96)

The compound 96 has been isolated as gummy solid. The HREIMS spectrum provided molecular ion peak at m/z 458.3396 establishing the molecular formula as C29H46O4 with seven degrees of unsaturation. The IR spectrum indicated the existence of hydroxyl moiety (ʋmax

-1 -1 3414 cm ), an ester moiety (ʋmax 1725, 1037 cm ) and olefinic moiety (ʋmax 1665, 1454, 754 cm-1) inthemolecule. EIMS gave molecular ion peakat m/z 458. After loss of acetyl group the daughter ionpeak was observed at m/z 399 whichproduce further fragments by losing H2O molecules, side chain (381, 363 and 252) [88].

The 1HNMR spectrum exhibited four signals due to five methyl groups among of these two methyl groups appeared as singlet at δ 0.75 (Me-19) and 2.02 (Me-29) while others appeared as doublet at δ 0.84 (6H, d, J = 6.1 Hz, Me-26, 27) and 1.02 (3H, d, J = 6.5 Hz, Me-21), respectively. This spectrum also showed the presence of two oxymethines and one oxymethylene by providing downfield signals at δ 3.67 (1H, m, H-3), 3.89 (2H, d, J = 11.5 Hz,

H-18) and 4.94 (1H, brd, J = 8.5 Hz, H-7). The protons of disubstituted olefinic bond resonated at δ 5.21(1H, dd, J = 15.0, 7.8 Hz, H-22),5.23(1H, dt, J = 15.0, 7.8 Hz, H-23) while single 145

proton of trisubstituted double bond resonated at δ 5.58 (1H, brs, H-6). The 1HNMR spectrum followed the pattern shown in literature by Δ5,22 steroids [89].

The presence of the oxo groups was also corroborated by 13CNMR spectrum which produce signals for these groups at δ 70.9 (CH), 75.6 (CH), 62.7 (CH2). Beside the other signals

13CNMR spectrum produced signals for ten methylenes, five methines and two quaternary carbon atoms.

Further confirmation was made by 2D NMR (HMBC, HMQC, 1H-1H COSY 45° and NOESY) experiments. By comparing the values with that of literature, compound 96 was considered as

Erectasteroid D [90].

146

4.12: (S)-4', 5-Dihydroxy-7-methoxyflavanone (97)

20 The compound 97 was isolated as colorless needles, m.p 152-154°C and [α]D -8.0° (c 1.0,

MeOH). The molecular formula of the compound was deduced with the help of HREIMS which gave molecular ion peakat m/z 286.0980 (calcd for C16H14O5, 286.0961). The UV spectrum showed broad absorptionbands at λmax 286 and 329 nm revealed the flavone skeleton of the compound. On additionof AlCl3 andAlCl3/HCl a red shiftof 40 nmwas observed indicating thepresence of achelated hydroxyl group atC-5 in the flavonoid skeleton. IR

-1 spectrum gave peaks at ʋmax 3434, 1685, 1705 cm which provide evidence about the existence of OH moiety, cyclic ketone and cyclic ether moiety in the molecule. EIMS spectrum gave several peaks along with characteristic RDA fragmentation in ring at m/z 286, 194, 166, 178,

166, 148, 120 and 94 give confirmation of one hydroxyl and methoxyl group in ring A and one hydroxyl group in ring B.

1 H-NMR spectrum showed a D2O exchangeable downfield signalat δ12.55 due to the chelated hydroxylgroup at C-5. The AMX pattern of 1H-NMRspectrum showed signals at δ 5.54 (1H, dd, J = 13.5, 2.8 Hz), 3.21(1H, dd, J = 17.5, 13.5 Hz) and 2.86(1H, dd, J = 17.5, 2.8) was the characteristic signal of H-2, H-3ex, and 3eq of the flavanone moiety. The spectrum further showed two meta coupled proton signals at δ 6.41 (1H, J =2.1 Hz) and6.66 (1H, J = 2.2 Hz) wereassigned to the H-6 andH-8 of ring A of flavanone structure. Furthermore two signals 147

were resonated as A2B2 system at δ 7.27 and 7.69, each showing 2H integration with dd (J =

8.2, 2.3 Hz, H2', H3', and H5', H6') establishing the presence of a 3' mono oxygenated ring B.

One methoxyl groupwas also observedat δ 3.67 (3H, s).

13C-NMRspectrum (BB and DEPT) provided information that molecule has sixteen carbon atoms. Among these sixteen carbons one was primary, one was secondary, seven were tertiary and seven were quaternary carbon atoms.

Applications of HMQC, HMBC, and 1H-1H COSY experiments give full assignments of 1H and 13C- NMR chemical shifts of 97. The absolute stereochemistry at C-2 was established as

S on the basis positive Cotton effect at 333 and negative Cotton effect at 298 nm in circular dichroism spectrum [91-92].

On the basis of comparisonof physical and spectraldata in literature thecompound 97 was named as (S)-4', 5-Dihydroxy-7-methoxyflavanone [93].

148

4.13: 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98)

8 1 HO O 7 9 2

3 2' 6 4 1' 3' OCH3 10 5 4' OCH3 O 6' OH 5'

The compound 98 was obtained in colorless crystal form showing melting point 283-285°C.

The HREIMS gave molecularion peak at m/z 314.0780 (calcd. for C17H14O6, 314.0790) establishing the molecular formula C17H14O6. The UV spectrum of the compound produce signal at 258 and 285, 372 indicating the existence of flavonoid skeleton. TheIR spectrum presented a peak at 1665 cm-1 indicating the presence of conjugated ketones. IR spectrum also provide evidence for OH moiety by rising a broad band at 3483-3345 cm-1.

The 1H-NMR and13C-NMR spectrum of compound 98 exhibited resonances due to the aromatic systems. In the 1H-NMR the aromatic region exhibited an ABX system atδ 7.69 (1H, d, J = 2.1 Hz, H-2'),δ 7. 55 (1H, J = 7.2 and 2.1 Hz, H-6') and δ 6.79 (1H, J = 7.2 Hz, H-5') due to the 3', 4', di-substitution of ring B. The signalsat δ 6.30 (1H, d, J = 2.2)and 6.15 (1H, d,

J = 2.2) were due tothemeta coupled H-6 and H-8,respectively. Twomethoxyl groups were observed at δ 3.77 and 3.64 as singlet, and further sported by the EI mass spectrum which indicate one hydroxyl with ring A and other with ring B. (experimental).

13C-NMR spectrum confirmed the existence of isoflavone skeleton. It showed peaks for seventeen carbon atoms, out of which six were methine, nine were quaternary, and two signals were for methoxy carbon atoms. The 13C-NMR signals were assigned by the HMQC 149

experiment. Applications of HMQC, HMBC, and 1H-1H COSY experiments give full assignments of 1H and 13C- NMR chemical shifts of 98.

On thebasis of physical and spectraldata thecompound 98 was namedas 7, 4'-dihydroxy-5, 3'- dimethoxyisoflavone, also compared with data reported in literature [94].

150

4.14: α-D-Glucopyranosyl-6'-O-hexadecanoate (99)

6' O 3 1 5 7 5' O 2 9 4 1' 11 4' OH 6 8 13 2' 15 HO OH 10 3' 12 OH 14 16

18.6 The compound 99 was isolated as white powder, with m.p 106-107 °C and [α]D +66.7(c 1.3, pyridine). The negative ion HRFABMS of the molecule gave a qausi molecular ion peak at

-1 417.2847 [M-H] suggesting molecular formula C22H42O7 with two degrees of unsaturation.

The IR spectrum of the molecule provided peaks at 3453, 1735, 1172 cm-1 revealing the presenceof hydroxyl andester moiety.

The 1H-NMR depicted a broad singlet of 24 protons at δ 1.22 indicating the existence of long chain ester. It also showed a signal of anomeric carbon with α configuration at δ 5.88 (1H d, J

= 3.6 Hz). The other oxymethine signals appeared in 1H-NMR spectrum (δ 4.12-4.93) were assigned to the protons of glycoside moiety.

The BB and DEPT 13CNMR spectrum confirmed the presence of an anomeric carbon at δ 94.0, ester carbonyl at δ 173.5 and oxymethylene carbon at δ 64.9. The other signals in this spectrum revealed the presenceof one methyl, fifteen methylene, five methineand one quaternarycarbon atoms.

Acid hydrolysis of 99 with KOH in MeOH yielded a free glycoside and methyl palmitate. The glycoside was confirmed as D-glucose through Co-TLCwith an authenticsample and sign of its opticalrotation. ([α]D + 52.9°). The methyl palmitate was also confirmed by comparative

TLC with an authentic samaple, by melting and boiling point comparison (mp = 34.5 °C and 151

bp = 184.5-185.5 °C) and by the comparison of its IR and 1H-NMR spectra with those reported in the literature [95-97]. All the physical and spectral data of compound 99 corroborated with that present in literature for α-D-glucopyranosyl-6'-O-hexadecanoate [98].

152

4.15: β -Sitosterol-3-O-β-D-glucopyranoside (100)

21 28 29 22 19 12 20 24 11 23 27 18 13 17 25 16 1 9 14 2 26 10 8 15 CH2OH 3 O 7 O 5 4 6

HO OH

25 The compound 100 was obtained as white solid, melting point 279-280°C and [α]D -14.5° (c

-1 1.3, CHCl3). The IR spectra gave bands at ʋmax cm 3442, 3038, 1649 depicting the existence of hydroxyl and olefinic moiety respectively. The molecularformula C35H60O6 was established withthehelp of HRFABMS (positive mode) which demonstrated the molecular ion peak at m/z

576.4386(calcd. forC35H60O6 =576.4389).

The 1H-NMR spectra of 100 was in accordance with the data of β-sitosterol except additional resonance at δ 3.84-4.46 due to protons of sugar moiety and peaks at δ 5.15 (1H, dd, J = 5.4

Hz) & 5.34 (1H, br d, J = 7.2 Hz) for protons of C-6 and anomeric proton respectively. The

13C-NMR spectrum was also completely corresponded with the published data of β-sitosterol except additional peaks for sugar moiety.

The mass spectrum corroborated with the specific pattern of Δ5 sterols. EIMS produced M- glycoside and M-glycoside-H2O peaks at m/z 414 and 396, respectively. Other peaks appeared in EIMS spectrum at m/z 381, 329, 256, 179 were also similar to the mass fragmentation pattern of β-sitosterol. 153

On the basis of above data, co TLC of sugar moiety and comparative m.p withan authentic samplethe compound 100 was confirmed as β-sitosterol-3-O-β-D-glucopyranoside [99].

154

4.16: Quercetin-3'-O-β-D-glucopyranoside (101)

25 The compound 101 wasisolatedas an amorphous powder with [α]D -20° (c 1.01 methanol).

The molecular formula of the compound was determined as C21H21O12 by HR-FAB-MS positive mode. The UV spectrum of the compound produce signal at 255 and 279, 364 indicating the existence of flavono-glycoside skeleton. A board peak in the IR spectrum at

3478-3395 cm-1 confirmed the existence of hydroxyl moieties inthe molecule. IRspectrum of

- 101 further showedabsorption bandsat ʋmax 1630 1736 cm 1 gave the idea about the presence of a cyclic ketone and an ether moiety in the molecule.

The 1H-NMR and 13 C-NMRspectrum of compound 101 exhibited resonances due to the aromatic and glycoside systems. In the 1H-NMR the aromatic region exhibited an ABX system at δ 7.98 (1H, d, J = 2.2 Hz, H-2'), δ 7. 74 (1H, J = 7.8 and 2.2 Hz, H-6') and δ 6.77 (1H, J =

7.8 Hz, H-5') due to the 3', 4', di-substitution of ring B. The signals at δ 6.38 (1H, d, J = 2.1) and 6.22 (1H, d, J = 2.1) were due to the metacoupled H-6andH-8,respectively The anomeric proton signal appearedat δ 5. 02 as doublet (J = 7.4 Hz, H-1'') and the signals in the region of

3.61-4.40 (6H, m, H-2'', H-3'', H-4'', H-5'', H-6'') together with the corresponding carbons signals assigned from HMQC spectrum indicated the presence of β-glycoside moiety. 155

13C-NMR (BB and DEPT) spectrum confirmed peaks for twenty one carbon atoms, one methylene, ten for methine, ten for quaternary carbon atoms. The 13C-NMR signals were assigned by the HMQC experiment. Applications of HMQC, HMBC, and 1H-1H COSY experiments give full assignments of 1H and 13C-NMR chemical shifts of 101. In the HMBC spectrum, cross peaks between C-1'' and C-3' setup the linkage between sugar and quercetin.

The 13C-NMR signals were assigned by the HMQC experiment. Applications of HMQC,

HMBC, and 1H-1H COSY experiments give full assignments of 1H and 13C-NMR chemical shifts of 101.

The physical and spectral data of the compound 101 was in consistent with that already reported quercetin-3'-O-β-D-glucopyranoside [100].

156

4.17: Biological Screening

4.17.1: Lipoxygenase inhibitory activity

4.17.1.1: Lipoxygenases

Lipoxygenases is a class of enzymes that contain non-heam iron withdioxygenases that catalyze the stereospecific additionof molecular oxygento PUFAs (polyunsaturated fatty acids) containinga cis, cis-1,4-pentadieneto their hydroperoxide derivatives. These enzymes are present in the cytosol of the lungs, platelets and leukocytes. They are mostly found in animals and plants where they produce signaling molecules and bring structural and metabolic changes in cells. Therefore they are targets for control by inhibition. In mammals, they play a vital role in number ofdisorders such asasthma and inflammation [101]. These enzymes are classified into several subcategories, most common are 5-, 12-, and 15- lipoxygenases with the number indicating at which carbon the oxygen is inserted.

The 5-LOX pathway is the main focus study because it leads to 5,6-epoxyleukotrienes that are involved ina variety of inflammatoryresponses whereas the 12-LOX and 15-LOX pathways play a potent role in the development of diseases such as cancer, psoriasis and atherosclerosis

[102].

4.17.1.2: The 5-Lipoxygenase Pathway

Lipoxygenase are present in the cytosol of cell as soluble enzymes where cellular stimulation takes place. 5-LOX and cPLA2 (cytolsolic phospholipase A2) transfer to the nucleus and liberate arachidonic acid which is the main substrate for 5-LOX enzyme and a twenty carbon fatty acid [Figure 14].

157

The arachidonic acid is converted to 5-HpETE (5-hydroperoxy-eicostetraeoic acid) by 5-LOX and as a result of this conversion following products are produced

 5-HETE (5-hydroxy-eicostetraeoic acid) and then 5-oxo-ETE (5-oxo-eicosatetraenoic

acid) by glutathione peroxidase

 LTA4 (leukotriene A4) by the action of dehydrase enzyme

Leukotriene A4 is then converted to further leukotrienes (Scheme 45). 158

 LTB4 (leukotriene B4) formed by the action of hydrolase enzyme mainly in neutrophils

 LTC4 (leukotriene C4) formed by the action of glutathione transferase enzyme which is

further converted to LTD4 (leukotriene D4) and LTE4 (leukotriene E4) respectively by

amino acid substitution.

Leukotrienes have been recognized as intermediates for a variety of inflammatory and allergic ailments including inflammatory bowel disease, arthritis, atopic dermatitis and rhinitis [103-104]. The 5- LOX pathway has also recently been related to the progression of osteoporosis and certain type of cancers [105].

159

4.17.1.3: Lipoxygenase inhibitory activity of compounds 86-101

All the compounds showed moderate to good inhibition of lipoxygenase. Among new compounds 86, 87 and 89 showed good inhibition, while 89 was moderately active against lipoxygenase. The testing of known compounds against lipoxygenase inhibition indicated that flavonoids 97, 98 and 101 have good inhibitory activity, followed by compounds 90, 91, 94,

96 and 100. The remaining compounds showed moderate lipoxygenase inhibition. 160

Compound (IC50 ± SMEa) [µM] 86 41.7 ± 0.15

87 42.3 ± 0.20

88 65.7 ± 0.10

89 49.8 ± 0.25

90 43.5 ± 0.10

91 38.4 ± 0.23

92 53.5 ± 0.10

93 57.0 ± 0.10

94 45.0 ± 0.10

95 58.5 ± 0.10

96 31.0 ± 0.10

97 31.8 ± 0.10

98 33.7 ± 0.25

99 41.3 ± 0.30

100 39.3 ± 0.10

101 30.1 ± 0.20

Baicaleinb 22.0 ± 0.04

161

4.17.2: Antimicrobial activity

4.17.2.1: Introduction

The microorganism are a vital part of our ecosystem and have been found very beneficial for humans. In both terrestrial and aquatic ecosystems microorganisms play an important role in carbon, sulphur, oxygen and nitrogen cycles. However these microorganisms found to be harmful for human beings in many ways because they have the ability grow on or in other organisms and form colonies which may cause disability and death. So, it is very necessary to inhibit the growth of these organisms using effective agents or chemicals. Those chemicals that have the capacity to kill or to stop the growth of microorganisms are known as antimicrobial agents. Furthermore these agents are classified on the basis of their spectrum of activity and application into micro biostatic and germicides agents. Germicides may be acting as bacteriocides (bacteria killing), fungicide (fungi killing), algaecides (algae killing) and viricides (virus killing) [106].

4.17.2.2: Antibacterial activity

The antimicrobial drugs have a specialty and that is its selective toxicity, it means that these drugs targets the bacteria selectively and cause less or no harm to the human host. These are prokaryotes so they provide a many unique targets for their selective toxicity in comparison to viruses and fungi. Every class of the antibacterial drug has a unique mode of action which is shown in Figure 15. 162

4.17.2.3: Antifungal activity

Fungi are eukaryotes so it seems to be challenging to treat fungal infections because these are more common in human beings as compared to prokaryotic bacteria. So, it is difficult to find specific targets for drug. Currently the drugs are designed to target the specific fungal pathways to kill the fungi rather than the human host. The fungal cell wall and cell membrane is different from humans because it is composed of β-1,3-glucan, squalene, ergosterol, β-1,6-glucan and chitin. At present the drugs available for treatment of fungal infections target cell membrane and cell wall. Depending upon their mode of action these drugs are classified into four major classes such as polyenes, allylamines, echinocandins and azoles, [107]. A schematic preview

(Scheme 46) showed the fungal mode of action.

163

164

4.17.2.4: Antimicrobial activities of compounds 86-101

All the compounds showed moderate to good antimicrobial activity. Among new compounds

86, 87 and 89 showed good activity, while 89 gave moderate activity. The testing of known compounds for antimicrobial potential indicated that flavonoids 97, 98 and 101 have good antimicrobial activity, followed by compounds 90, 91, 94, 96 and 100. The remaining compounds showed moderate antimicrobial activity.

165

Micoorganism Zone of inhibition diameter (mm) Bacteria 86 87 88 89 90 91 92 93 Standard drugs Imipenem Gram positive Staphylococcus avreus 27 28 13 29 27 25 26 24 33 Bacillus subtilis 26 27 12 28 26 27 25 23 33 Shigella flexneri 21 22 09 23 22 21 21 23 27 Gram negative Salomella typhi 19 20 08 21 20 19 18 19 25 Escherichia coli 24 25 12 26 23 24 25 24 30 Pseudomonas aeruginosa 19 20 07 21 19 17 18 19 24 Fungi Miconazole Microsporum canis 92 93 41 94 89 90 89 88 98.4 Candida albicans 103 104 39 105 102 103 101 99 110.8 Fusarium solani 66 67 32 68 63 62 60 61 73.3 Tricophyton longifusus 65 66 29 67 60 58 59 57 70 Candida glabrata 104 105 42 106 101 98 99 96 110.8 Aspergillus flavus 14 15 05 16 11 12 13 11 20

166

Micoorganism Zone of inhibition diameter (mm) Bacteria 94 95 96 97 98 99 100 101 Standard drugs Imipenem Gram positive Staphylococcus avreus 26 29 26 28 27 29 30 29 33 Bacillus subtilis 24 28 27 27 26 28 29 30 33 Shigella flexneri 20 24 21 22 21 23 25 24 27 Gram negative Salomella typhi 21 20 18 19 18 21 22 21 25 Escherichia coli 22 25 22 24 23 26 27 26 30 Pseudomonas aeruginosa 17 19 18 18 17 20 21 22 24 Fungi Miconazole Microsporum canis 87 93 89 92 91 94 95 96 98.4 Candida albicans 102 105 99 104 101 106 105 107 110.8 Fusarium solani 63 68 62 66 65 69 69 70 73.3 Tricophyton longifusus 56 65 58 63 62 66 67 68 70 Candida glabrata 95 104 97 102 100 105 106 107 110.8 Aspergillus flavus 10 15 09 14 13 16 16 17 20

Conclusion

167

Conclusion

Hypericum oblongifolium was investigated for its bioactive constituents using column chromatography over silica gel with n-hexane-chloroform and chloroform-methanol solvent system. This isolation was successfully led to sixteen compounds. The structures of all the compounds were elucidated with the help of spectrometry (1D and 2D NMR and Mass spectrum) and some chemical and physical experiments. All the compounds were tested for their pharmacological potential. Lipoxygenase inhibition and antimicrobial (antifungal and antibacterial) activity was checked for isolated compounds. All the compounds showed moderate to strong antimicrobial activity. The new compounds 86, 87 and 89, showed strong lipoxygenase inhibition as well as good antifungal and antibacterial activity, comparable to standards. While 88 was moderately active against all biological activities. Similarly compounds 90, 91, 96, 94, 97, 98, 100 and 101 showed strong antifungal and antibacterial activity, comparable to standards. These compound were also highly active against lipoxygenase inhibition when compared with Baicalein. Extension of this work to biotransformation of isolated compounds is included in our future plans. The compounds showing good biological potential can be used for drug designing, for several other biological activities like anti-oxidant, anti-proliferative, anti-depressant etc and enzyme inhibition activities (chymotrypsin inhibition, urease inhibition, cholinesterase inhibition).

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