prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/369/1/1781s.pdfiii acknowledgments first of...

PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF THE CONSTITUENTS OF

BUDDLEJA ASIATICA LOUR

By

FARMAN ALI KHAN

DEPARTMENT OF CHEMISTRY, GOMAL UNIVERSITY,

DERA ISMAIL KHAN, KPK, (PAKISTAN)

2013

PHYTOCHEMICAL STUDIES AND BIOLOGICAL ACTIVITIES OF THE CONSTITUENTS OF

BUDDLEJA ASIATICA LOUR

A THESIS SUBMITTED

FOR THE PARTIAL FULFILMENT OF THE DEGREE

OF

DOCTOR OF PHILOSOPHY IN

CHEMISTRY BY

FARMAN ALI KHAN

(062-000111-PS3-223)

DEPARTMENT OF CHEMISTRY, GOMAL UNIVERSITY,

DERA ISMAIL KHAN, KPK, (PAKISTAN)

2013

ii

DEDICATED TO

My Father

SHABAB ALI KHAN

DR. MEHBOOB ALI KHAN

and My Uncle

iii

ACKNOWLEDGMENTS

First of all I bow down my head to the Omnipotent, the most Merciful, the

Compassionate, and the Omniscient Al-Mighty ALLAH, whose clemency resulted into my

success. I wish to pay homage to the most perfect personality of the world

Hazrat Muhammad (PBUH), who enlightened our minds to recognize our Creator.

Completing my Ph. D was never going to be easy, but the support that I have received from

the people around me has made it an incredible experience that I will cherish for the rest of my

life.

Firstly, I would like to thank my supervisor, Prof. Dr. Irshad Ali, whose infectious

enthusiasm for natural product chemistry had hovered over me for the entire period of my

studies. I am very thankful for his excellent supervision, advice, constant encouragement and

precise attention during write up of my Ph.D thesis. It was indeed a pleasure working for him.

I am also thankful to Prof. Dr. Azim Khan Khattak (Chairman, Department of

Chemistry) for providing me with all the facilities to complete this task. I am somewhat

overwhelmed to express my feelings because the extremely dedicated services given by him

are beyond the limits of words of acknowledgment. I also pay my sincerest gratitude to

Prof. Dr. Musa Kaleem Baloch (Dean, Faculty of Sciences, Gomal University) for his

intermittent suggestions, instrumental and moral support at crucial times during my course of

studies. This work is carried out and completed with the partial but vital guidance of

Dr. Shafi Ullah Khan (Asst. Prof., Department of Chemistry) who helped me a lot in laboratory

work as well as structure elucidation. I am also indebted to all all the technical and admin staff

in Department of Chemistry.

I would also like to acknowledge Higher Education Commission, Pakistan for

providing me financial support during my Ph.D under HEC Indigenous 5000 PhD Fellowship

Program Batch III and International Research Support Initiative Program (IRSIP).

I would like to thank my colleagues, lab fellows and “partners in crime”, Masood Afzal,

Dilfaraz khan, Abdul Samad and Hazrat Ali (Ph.D Scholars) who have travelled through this

journey with me. Thanks for helping me re-word my sentences and always have a suggestion

when I felt like I had hit a wall. From travelling in search of unexplored medicinal plants, to

conferences and dinner-trips as well as in the organic lab. I have utterly enjoyed all that we

have done together and am privileged to call them dear friends.

Thanks to Dr. Hidayat Ullah (Assistant Prof., USTB, Bannu) for assisting and helping me

a lot in conducting my experiments and Dr. Arif Ullah khan (Assistant Prof., KUST, Kohat) for

helping me in carrying out the biological screening at Department of Biological and

iv

Biomedical Sciences, Aga Khan University Medical College, Karachi under the kind supervision

of Prof. Dr. Anwarul Hassan Gilani.

Above all else I wish to express my deepest love and gratitude to my parents and

other family members for the encouragement and support and their constant prayers for my

success. Especially to my Father and first ever teacher, Shabab Ali Khan Shabab. I would not

be at this point without you and I would never be able to step out without knowing you are

behind me. I am also thankful to my mother, Aapa and my sisters for their moral support

throughout this research work. Much gratitude to my brothers, Qaiser Ali Khan (Gold

medallist and Lecturer in English), Nayab Ali Khan (S.E.T) and Seemab Ali Khan (MSc, Botany)

for supporting me in every manner during my Ph.D. I will specially thank my Dear Uncle,

Dr. Mehboob Ali Khan for his extreme affection and kindness from my childhood to till.

May God bless you all.

Farman Ali Khan

v

LIST OF CONTENTS

CERTIFICATE i

DEDICATION ii

ACKNOWLEDGMENTS iii

LIST OF CONTENTS v

LIST OF ABBREVIATIONS ix

LIST OF TABLES xi

LIST OF FIGURES xiii

SUMMARY xv

1.0 CHAPTER 1: INTRODUCTION 01

1.1 Natural products 02

1.2 Medicinal plants 03

1.3 Natural products from plants as medicine 04

1.4 Medicinal plants and Islamic literature 08

1.5 Plants as traditional medicine and drug discovery 13

1.6 Natural products as anticancer drugs 17

1.7 Modern drug discovery and natural products 21

2.0 CHAPTER 2: LITERATURE REVIEW 26

2.1 Family Scrophulariaceae 27

2.2 The genus Buddleja 27

2.3 Medicinal importance of genus Buddleja 27

2.4 Buddleja asiatica Lour 48

vi

2.5 Scientific classification 49

2.6 Pharmacological significance of Buddleja asiatica 49

3.0 CHAPTER 3: RESULTS AND DISCUSSION 50

PART A

3.1 Secondary metabolites from Buddleja asiatica 51

3.2 Extraction and isolation 51

3.3 Chloroform soluble fraction 52

3.4 Structure elucidation of compounds 53

3.4.1 Buddlejone (180) 53

3.4.2 Dihydrobuddledin-A (181) 57

3.4.3 Buddledone-B (182) 60

3.4.4 Ursolic acid (183) 63

3.4.5 2-Phenylethyl-β-D-glucoside (184) 66

3.4.6 7-Deoxy-8-epiloganic acid (185) 69

3.4.7 Secutellarin-7-O- β-D-glucopyranoside (186) 72

3.4.8 Antimicrobial activities of compounds 180-186. 75

3.5 Ethyl acetate soluble fraction 79

3.6 Structure elucidation of compounds 80

3.6.1 Lignoceric Acid (187) 80

3.6.2 (24S)-Stigmast-5, 22-diene-7β-ethoxy-3β-ol (188) 83

3.6.3 Asiatoate- A (189) 92

3.6.4 Asiatoate- B (190) 98

3.6.5 Chrysoeriol-7-O- β-D-glucopyranoside (191) 104

PART B

3.7 Biological screening of the crude extract and fractions 107

3.7.1 Brine shrimp lethality assay 108

vii

3.7.2 Antibacterial activity 110

3.7.3 Antifungal activity 113

3.7.4 Antispasmodic activity and Ca ++ antagonist action 116

PART C

3.8 Qualitative and quantitative assessment of fatty acids of Buddleja asiatica by GC-MS

120

3.8.1 Fatty acid profile of non-volatile oil 121

3.8.2 Fatty acid profile of fat 125

3.8.3 Discussion 129

3.8.4 Thermal stability measurement of non-volatile oil 132

3.8.5 GC/MS analysis of essential oils from leaves of B. asiatica 134

4.0 CHAPTER 4: EXPERIMENTAL 138

PART A

4.1 Secondary metabolites from Buddleja asiatica 139

4.2 General notes 139

4.3 Plant material 142

4.4 Extraction and fractionation 142

4.5 Isolation and characterization from chloroform soluble fraction 144

4.5.1 Buddlejone (180) 147

4.5.2 Dihydrobuddledin-A (181) 148

4.5.3 Buddledone-B (182) 149

4.5.4 Ursolic acid (183) 150

4.5.5 2-phenylethyl-β-D-glucoside (184) 151

4.5.6 7-deoxy-8-epiloganic acid (185) 152

4.5.7 Secutellarin-7-O- β-d-glucopyranoside (186) 153

4.6 Isolation and characterization from ethyl acetate soluble fraction 154

4.6.1 Lignoceric acid (187) 157

viii

4.6.2 (24S)-Stigmast-5, 22-diene-7β-ethoxy-3β-ol (188) 158

4.6.3 Asiatoate A (189) 160

4.6.4 Asiatoate B (190) 161

4.6.5 Chrysoeriol7-O- β-d-glucopyranoside (191) 162

PART B

4.7 Biological screening of the crude extract and fractions of B. asiatica 163

4.7.1 Brine shrimp lethality assay 163

4.7.2 Antibacterial activity 163

4.7.3 Antifungal activity 164

4.7.4 Antispasmodic activity 165

4.7.5 Determination of Ca++ antagonist action 165

4.7.6 Statistical analysis 165

4.7.7 Bioassay of the compounds 165

PART C

4.8 GC/MS Analysis of oil from Buddleja asiatica 167

4.8.1 Volatile oil extraction 167

4.8.2 Non-volatile oil extraction 167

4.8.3 Instrumentation 167

4.8.4 Thermal stability measurements 168

5.0 CHAPTER 5: BIBLIOGRAPHY 169

ix

LIST OF ABBRIVIATIONS

Reagents and Solvents

BuOH n-ButanolCDCl3 Deutreated Chloroform CHCl3 ChloroformDMSO DimethylsulphoxideEtOAc Ethyl acetateEtOH Ethanol

Hex n- Hexane

MeOH Methanol

Me2CO Acetone

KBr Potassium bromide

BSTFA N,O-Bis (trimethylsilyl) trifloroacetamide

TMS Trimethylsilylene

x

Techniques

BB Broad (decoupled) band CC Column chromatography COSY Correlated spectroscopy

DEPT Distortionless enhancement by polarization

transfer EI-MS Electron impact mass spectrum FAB-MS Fast atom bombardment mass spectrometry

GC/MS Gas chromatography mass spectrometry

HMBC Heteronuclear multiple bond connectivity

HMQC Heteronuclear multiple quantum coherence

HR-EIMS High resolution electron impact mass

spectrum

IR Infrared spectrophotometry

m/z Mass to charge ratio (in mass spectrometry)

NMR Nuclear magnetic resonance

NOESY Nuclear overhauser effect spectroscopy

TG/DTA Thermogravimetric and differential

thermogravimetric analysis

TG/DTG Thermogravimetric and differential

thermogravimetry

TLC Thin layer chromatography

UV Ultraviolet

xi

LIST OF TABLES

Table Title Page

1. History of natural products 06

2. Medicinal plants mentioned in the Holy Quran and Ahadith 10

3. Some important pharmaceutical innovations 14

4. Some of the most important drugs derived from natural products 15

5. Anticancer compounds derived from plants 19

6. Isolated compounds from the genus Buddleja 29

7. 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of buddlejone (180) 56

8 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of dihydrobuddledin A

(181) 59

9 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of buddledone B (182). 62

10 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of ursolic acid (183) 65

11 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of

2-phenylethyl-β-D-glucoside (184) 68


7- deoxy-8-epiloganic acid (185) 71


scutellarin-7-O-β-D-glucopyranoside (186) 74

14 Antibacterial bioassay of compounds 180-186 from Buddleja asiatica 76

15 Antifungal activity of compounds 180-186 from B. asiatica 78

16 13C-NMR (150 MHz) and 1H/13C correlations of lignoceric acid (187) 82

17 13 C- NMR (CDCl3, 150 MHz) chemical shifts and multiplicities of (24S)-

stigmast-5,22-diene-7β-ethoxy-3β-ol (188) 89

18 13C- NMR (CDCl3, 150 MHz) and 1H/13C correlations of (24S)-stigmast-5, 22-

diene-7β-ethoxy-3β-ol (188) 90

xii

19 13 C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate A

(189) 96

20 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of asiatoate A (189) 97

21 13 C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate B

(190) 102

22 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of asiatoate B (189) 103


Chrysoeriol-7-O-β-D-glucopyranoside (191). 106

24 Brine shrimp bioassay of different fractions of Buddleja asiatica 109

25 Antibacterial activity of crude extracts and various fractions of B. asiatica. 111

26 Antifungal activity of crude extract and various fractions of B. asiatica 114

27 Concentration-dependent inhibitory effect of the crude extract of B. asiatica on

spontaneous contractions of isolated rabbit jejunum preparations 117

28 Concentration-dependent relaxant effect of the crude extract of B. asiatica on

K+-induced contractions of isolated rabbit jejunum preparations. 117

29 Fatty acids composition of B. asiatica non-volatile oil. 123

30 Fatty acids composition of B. asiatica fat. 127

31 Comparison between various fatty acids found in both oil and fat of B. asiatica. 131

32 GC/MS analysis of essential oil from leaves of Buddleja asiatica. 136

xiii

LIST OF FIGURES

Figure Title Page

1. Structures of the mentioned compounds 23

2. Structures of the compounds reported from Buddleja asiatica. 36

3. Buddleja asiatica Lour 48

4. Important mass spectral peaks in HR-EIMS of 188 87

5. Important HMBC and COSY correlations of 188 87

6. Mass fragmentation pattern of 189 95

7. Important HMBC correlations of 189 95

8 Mass fragmentation pattern of 190 101

9 Important HMBC correlations of 190 101

10 Antibacterial activity of crude extract and its various fractions towards

S. flexneri, E.col and S.boydi 112

11 Antifungal activity of crude extract and its various factions towards A. Flavus,

F. Solani and T. Longifusus 115

12

Concentration-dependent inhibitory effect of the crude extract of B. asiatica on

spontaneous and K+- induced contractions of isolated rabbit jejunum

preparations. Values shown are mean ± SEM, n=3.

118

13 Concentration-dependent inhibitory effect of verapamil against spontaneous

and high K+-induced contractions in isolated rabbit jejunum preparations. 118

14 BSTFA derivatised GC/MS - TIC of B. asiatica oil. 122

15 Ratio between different fatty acids in B. asiatica non volatile oil 124

16 BSTFA derivatised GC/MS - TIC of B. asiatica fat 126

17 Ratio between different fatty acids in B. asiatica fat 128

xiv

18 Comparison amongst fatty acids found in both fat and oil of B. asiatica. 131

19 TG/DTA thermogram of B. asiatica oil 133

20 Total Ion Chromatogram of leaves essential oil 135

xv

Summary

The present thesis comprises three parts, A, B and C.

Part A describes the isolation and structure elucidation of compounds from the

chloroform and ethyl acetate soluble fractions of Buddleja asiatica Lour. Three new and nine

known compounds have been isolated for the first time from this species.

New compounds.

1. (24S)-Stigmast-5, 22-diene-7β-ethoxy-3β-ol (188)

2. Asiatoate A (189)

3. Asiatoate B (190)

Known compounds isolated for the first time.

1. Buddlejone (180)

2. Dihydrobuddledin-A (181)

3. Buddledone-B (182)

4. Ursolic acid (183)

5. 2-Phenylethyl-β-D-glucoside (184)

6. 7- Deoxy-8-epiloganic acid (185)

7. Secutellarin-7-O-β-D-glucopyranoside (186)

8. Lignoceric acid (187)

9. Chrysoeriol-7-O-β-D-glucopyranoside (191)

Part B deals with the antibacterial, antifungal, antispasmodic and Ca++ antagonist

activities of the crude extract, chloroform (F2), ethyl acetate (F3) and n-butanol (F4) soluble

fractions of Buddleja asiatica Lour. The antibacterial activity of these fractions was performed

against eleven bacteria, in which the crude extract, F3 and F4 exhibited significant activity while

F2 showed low activity against Shigella flexenari, Sternostoma boydi and Escherichia coli. The

xvi

fungicidal activity was performed against six fungi; the crude extract, F2 and F3 displayed

significant activity against Fusarium solani, while F4 exhibited high activity against

Microsporum canis. The crude extract of B. asiatica caused concentration-dependent relaxation

of spontaneous and high K+ (80 mM) - induced contractions in isolated rabbit jejunum

preparations.

In addition to these, anti-microbial activity of the isolated compounds 180-187 was

carried out against twelve pathogens. The compounds 185-187 showed significant activity

against Proteus vulgaris, Salmonella typhi, Escherichia coli, Trichophyton longifusus, Candida

albicans, Microsporum canis, Candida glabrata, Fusarium solani and Aspergillus flavus while

other compounds showed weak to moderate activity.

Part C describes the GC/MS studies of non-volatile oil, fat and essential oils obtained

from various parts of B. asiatica. The non-volatile oil and fat obtained from the whole plant

were analyzed by GC/MS. The non-volatile oil contained 59 % fatty acids and 41 % other

constituents. The palmatic acid (46.75 %), linoleic acid (37.80 %) and stearic acid (15.98 %)

were found in large quantities while lignoceric acid (1.22 %), archidic acid (2.0 %) and

margaric acid (1.22 %) were present in small quantities (< 3 %).

The fat was found to contain 83.33 % saturated fatty acids; lignoceric acid (24:0) was

found to be in the highest quantity (43.12 %), while behenic acid (22:0) was the second highest

(26.39 %) of all FAs. The trycosylic acid (23:0) was found in small amount (4.83 %) and was

the only fatty acid, which showed odd carbon number chain. The archidic acid (9.29 %), stearic

acid (5.58 %), montanic acid (4.46 %) and cerotic acid (4.09 %) were also found in very small

quantities, while melissic acid and palmatic acid were present in traces (2.6 %, 1.86 %).

The non-volatile oil showed a low thermal stability, when subjected to TG/TDA

analysis, probably due to the absence of phenolic contents and PUFA (poly unsaturated fatty

acids).

xvii

The analysis of essential oil from the leaves of B. asiatica, seventeen compounds were

detected by GC/MS, in which fourteen were identified as four monoterpenes hydrocarbons, four

oxygenated monoterpenes, one sesquiterpene hydrocarbon and five oxygenated sesquiterpene.

They are listed as:

1. α –Pinene (4.95 %)

2. α - Phellandrene (5.79 %)

3. α –Thujene (3.37 %)

4. Limonene oxide (38.11 %)

5. Furan, 2-(1-pentenyl) - (E) (1.79 %)

6. Terpinen-4-ol (1.89 %)

7. Bicyclo[5.1.0]octane, 8-(1-methylethylidene) (1.0 %)

8. Eugenol (1.53 %)

9. Kushimone (1.0 %)

10. α – Cubebene (1.42 %)

11. 12-Nor-preziza-7(15)-en-2-one (1.84 %)

12. Sesquichamaenol (1, 10-seco-1-hydroxycalamenen-10-one) (10.21 %)

13. Isospathulenol (0.95 %)

14. β-Sinensal (11.84 %)

Mass spectral data and retention indices of the constituents were analyzed by the data

system library and were confirmed by comparison of their mass spectra using NIST Mass

Spectral Search Program or Kovat’s retentions indices.

1 | P a g e

2 | P a g e I n t r o d u c t i o n

1.1 Natural products

Humans have relied on nature throughout history for their basic needs like clothing,

shelter, food, flavours, fertilizers, means of transportation and not the least, medicine [1]. The

traditional medicine and remedies played a key role in human societies throughout ages.

Natural products find their interest in the earliest points of history by providing curatives for

pain, palliatives for an array of maladies or recreational use. Mankind has always been

interested in the exploration and understanding of certain plants, animals and fungi which

contain substances that have an effect on human bodies in specific ways. Natural products have

been an integral part of mankind’s history by finding uses in the improvement of health and

enhancing quality of life.

The term, “natural products”, refers to “hurbs, herbal concoctions, dietary supplements,

traditional medicine or alternative medicine in common” [2]. A natural product can be defined

as “A chemical substance produced by a living organism; a term used commonly in reference to

chemical substances found in nature that have distinctive effects. Such a substance is

considered as natural product even if it can be prepared by total synthesis” [3]. It can also be

described as “compounds isolated from plants, animals, marine organisms, microbes and fungi

or naturally occurring compounds that are end products of secondary metabolism”. They are

frequently unique for particular organism or classes of organisms.

Natural products include:

(a) an entire organism (e.g., a plant, an animal, or microorganism) that has not been subjected

to any kind of processing or treatment other than a simple process of preservation (e.g., drying),

(b) part of an organism (e.g., leaves or flowers of a plant, an isolated animal organ),

(c) an extract of an organism or part of an organism, and exudates, and


(d) pure compounds (e.g., alkaloids, flavonoids, glycosides, lignans, steroids, sugars,

terpenoids, etc.) isolated from plants, animals, or micro-organisms [4].

In many cases the same term, natural products, refers to secondary metabolites that are

small molecules (mol wt


to be used in various human cultures around the world for medical purposes” [8]. However, the

number could be much higher as knowledge of indigenous uses of plants mainly passed on

verbally from one generation to another and has largely remained undocumented. Amongst

250,000 higher plants species, around 5-15 % have been scrutinized for their natural products

[9]. Similarly a little of the marine organisms has been explored which are abundant in the

oceans. A recent survey suggests that the currently known bacterial species are less than 1 %

while that of fungi are less than 5 % [10]. Hence it is very important that natural product

chemistry continues to investigate for new natural products.

1.3 Natural products from plants as medicine

The medicinal usage of natural products predates recorded history. Remains of old

civilizations contain leftovers of many medicinal herbs. The earliest known records date back to

Mesopotamia around 2600 B.C. and were written on clay tablets in cuneiform script, the

earliest known form of written expression. “Nei Ching” which dates back to 13th century B.C.,

is amongst one of the “earliest health science compilations” ever produced. Cedar oils (Cedrus

species), cypress (Cupressus sempervirens), licor (Glycyrrhiza glabra), myrrh (Commiphora

spss.) and poppy juice (Papaver somniferum) have been accounted to be used extensively in

that times and still used today in treatment of various illness like coughs, colds and parasitic

infections. One of the most famous written records include the, dating from about 1500 B.C. in

Egypt, which documents nearly 1000 different formulations and substances, mostly, plant based

medicines have been documented in the famous book "Ebers Papyrus” [11]. It, for example,

specify use of willow leaves as an antipyretic and early English herbalists also recommended

the use of teas made from willow bark for treatment of fever.

Asclepius (1500 B.C.) was a Greek physician, have used many plants in medicine. The

Chinese “Meteria Medica” (1100 B.C.) has been repeatedly documented and some of its early


texts describe various plants that have been sources of very important modern medicines. The

Indian traditional healing system, “Ayurvedic Hymns” (1000 B.C.) describes the use of number

of different plants used as medicine. Theophrastus (300 B.C.) wrote about medicinal qualities

of herbs in his history of plants. The very famous and well known European document “De

Material Medica”, written by Pedanious Dioscorides a Greek botanist (100 A.D.) illustrates

about the use of plants in medicine.

The Arabs maintained the knowledge by documentation of Greek and Roman

knowledge of natural products. The work entitled “Canon Medicine” of Avicenna, a Persian

philosopher and physician, is regarded as a succinct outline of Roman and Greek medicine [9].

“Primitive physic” and “Thompson’s New guide to health” were amongst some of the most

popular publications written, which contained various natural product based methods of

medicinal information. In fact, due to several reasons, the curiosity in natural products

continues to this very day. A brief summary of natural products in historical prospective has

been illustrated in Table 1.


Table 1: History of natural products.

Period Type Description Ref#

Before

3000 BC

Remains of Neanderthal

Mesopotamian record

Ayurveda (knowledge of life)

Asclepius

Introduced medicinal properties of plants

and other natural products [11]

1550 BC

Ebers Papyrus

Asclepius

Chinese traditional medicine

Presented a large number of crude drugs

from natural sources (e.g., castor seeds

and gum Arabic)

[11-12]

460–377 BC Hippocrates, ‘‘The Father of

Medicine’’

Described several plants and animals that

could be sources of medicine [13]

370–287 BC Theophrastus Described several plants and animals that

could be sources of medicine [14]

60–80 AD Dioscorides Wrote De Materia Medica, which

described more than 600 medicinal plants [15]

131–200 AD

Galen

Avicenna

Practiced botanical medicines

(Galenicals) and made them popular in

the West.

A succinct outline of Roman and Greek

medicine

[9]

15th century Kra¨uterbuch (herbals)

Presented information and pictures of

medicinal plants [16]

1596 AD Li Shih- Chen

“Pen-ts as kang Mu”, describing 1898

herbal drugs along with 8160

prescriptions

[17]

1743 John Wesley, founder of “Primitive physic”, the popular reference [18]


Methodism book about various natural remedies.

1822 Samuel Thompson

(Early America)

Thompson’s New guide to health” was

one of the most popular publications

contained various natural product based

methods of medicinal information

[2, 19-

25]

1826 Merck (marketing agency) Morphine, first commercial pure natural

product introduced for therapeutic use [26]


1.4 Medicinal plants and Islamic literature

In Islamic teachings, medicinal plants have always been a silent feature throughout. The

so called “Islamic medicine” started from “Hazrat Adam (A.S) and completed at Hazrat

Muhammad (Sallallaho Alaihe Wasallam)” but still continued to explore after his death.

The Holy Quran is a religious book more the 1400 years old with a total of 6600 verses

dealing with many aspect of regular life, about 1000 of those verses are of scientific nature.

There are 900 verses in the Holy Quran that signify new scientific discoveries [27]. Al-Quran

illustrates the significance of plants in various verses in different chapters.

Twenty two identifiable plants belong to seventeen plant families are cited in the Holy

Quran [28]. They include Phoenix dactylifera, Olea europea, Ficus carica, Vitis vinifera,

Punica granatum, Ocimum basilicum , Zingiber officinale, brassica nigra, salvadora Persia,

Tamarix, Zizyphus spina-christi, Citrulus colocynthis, Cucurbita pepo, Cucumis sativus, Allium

sativum, Allium cepa, Lens esculents, Musa sapientum, Hordeum vilgare, Triticum vulgare and

trifolium.

In Islamic terms, the plant based medicines are known as Tibb. Tibb is concerned

with the use of medicinal plants related to human health and personal hygiene. Fig, olive, date

palm and pomegranates have been mentioned distinctly in the Holy Quran. The other plants

mentioned include onion, garlic, lentils, zinger, basil, camphor and colocynth [28]. The Holy

Prophet (Sallallaho Alaihe Wasallaam) used various herbs and recommended certain

medicinal plants that has many references in Holy Quran as remedy of m a n y common

diseases an d a s food [29]. The medicinal plants mentioned by our Holy Prophet in various

Ahadith are being compiled and explained by different people afterwards, regarding as Islamic

medicine.

The history of Islamic medicine started form second century of Hijra and some of the

famous books include “Tib-e-Nabvi, Kanzulamal Fee Sanan Walakwal and Haddi Kabeer


Kamal-ul-Sannat” [30-31]. The medicinal plants mentioned in those books were extensively

used in the treatment of many ailments as a key source of drugs.

A recent survey into medicinal plants mentioned in the Holy Quran, Ahadith and Islamic

literature confined thirty two medicinal plants of different families in plant kingdom [32] . A

check list of some medicinal plants mentioned in the Holy Quran and in the Ahadith with their

scientific names, Arabic names, families, medicinal uses and specific references from Islamic

literature is provided in Table 2.


Table 2: Some of the medicinal plants mentioned in the Holy Quran and Ahadith.

S.No Plant

Medicinal Uses References form the Holy Quran and Ahadith Specie Family

1 Allium cepa L

(Basal) Alliaceae

Antidote, cholera, infection, influenza,

improves sperm production, hepatitis, piles,

menstruation and intestinal diseases.

Holy Quran Verse #. 68, Surah Baqra [33]

Bukhari (Ravi: Jabir bin Abdullah) Kitabut-Tib [34]

Muslim (Ravi: Jabir Bin Abdullah) Chap. Abwab Ul Attamah

[35]

Al-Jozi (Aljawziyya), Ibn Ul Qayyim. Zadul Maad [36]

2

Allium sativum L.

(Soom)

Alliaceae Antidote, dog bite, paralysis, asthma, parkensis,

cough, hysteria, tuberculosis.

Holy Quran Verse #. 61, Surah Baqra [33]

Bukhari (Ravi: Hazrat Anas), Kitab ul Tamaih [34]

Muslim (Ravi: Abu Ayub) [35]

Ibne Majja (Ravi: Umer bin Alkhitab) [37]

3

Cinnamomum camphora

L

(Kafoor)

Lauraceae

Tetanus, parkensis, hysteria, tuberculoses,

headache, liver and kidney pains, breast pain,

inner wounds etc.

Holy Quran Verse 15, 1, Surah Al insane [33]

Bukhari, Chapt. Kitab ul Tib [34]

Muslim (Ravi: Um-e-Atiya) Kitabul-Janayez [35]

Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad [36]


4 Ficus carica L.

(Teen) Moraceae

Remove kidney and urinary bladder stone,

release intestinal pain, pile, dyspepsia and

anorexia.

References from Holy Quran Verse #.1-4, Surrah Teen [33]

Bukhari [34]


5 Hordeum vulgare L.

(Shair) Poaceae

Fever, weakness, increase immunity, heart

diseases, kidney pain, intestinal ulcer, maintain

cholesterol level, jaundice etc

Trimzi [38]

Bukhari [Ravia: Hazrat Ayesha (Chap; Haiz ul Shahir [34]

Bukhari. Ravia: Aisha. Kitabul-Athama [34]


6 Lagenaria siceraria Standl

(Yakteen, Daba) Cucurbitaceae

Arthritis, Maleness, Piles, lungs infection,

common cold, kidney and liver disorder and

heart diseases.

Holy Quran Verse.# 48, Surah Younis [33]

Bukhari, Kitab ul Tamamiah [34]

Ibn e Maja, Chap Bab ul Daba[37]

Ibn e Maja. Ravi: Anas. Kitabul-athama [36]

7

Lens culinaris Medic

(Adas)

Papilionaceae Maleness, measles, paralysis, common cold,

parkensis, face clearness, eye infection etc

Holy Quran Holy Quran, Verse #. 61, Surah Al Baqra [33]

Al-Jozi (Aljawziyya), Ibn-ul Qayyim, Tibb-e-Nabvi (Urdu Tans.

by Hakim Azizur Rehman A’zmi and Mukhtiar Ahmad Nadvi)

[39]

8 Ocimum basilicum L.

(Rehan) Lamiaceae

Fever, cough, eczema, baldness, arthritis,

antidote, pain killer, tuber closes, asthma, piles,

hepatitis, malaria, heart diseases etc

Holy Quran Verse #. 12, 13, Surah Al Rahman [33]

Bukhri. Ravi: Abu Musa Al Asharii [34]

Trimzi (Bab ul Tib)[38]



9 Olea europea L.

(Zaiytoon) Oleaceae

Strengthen body muscles, slow down aging,

clear the blood, remove the measles spot, piles,

tuberculosis, eczema, baldness, kidney,

pancreas pain, etc.

Holy Quran Verse #.191, Surah Alanam; verse #. 99, Surah

Alanam; verse #. 11, Surah Alnahal; verse #. 35, Surah Alnnor;

verse #. 1-4, Surah Teen [33]

Bukhri, Ravi: Khalid Bin Sahad [34]

Trimzi, Abwab ul Tamah [38]

Ibne Majja, Ravi; Zahid Bin Arkum[37]

10

Phoenix dactylifera L

(Nahal, Balah, Tammar,

Rutab, etc)

Arecaceae

Heart diseases, skin diseases, antidote, swelling

of kidney, intestinal pain, heart attack, wound

healer, diarrhea, labour pain, sexual weakness,

stomach pain, piles, etc.

Holy Quran Verse #.6, Surah Baqra; verse #. 99, Surah Al

Anam; verse #. 4, Surah Al Rahad; verse #. 11, 27, Surah Al

Nahal; verse #. 91, Surrah Al Israa; verse #.36, Surah Al Kahaf;

verse #. 23, 25, Surah Mariam; verse #.148, Surah Shurah; verse

#. 71, Surah Taha; verse #. 34, Surah Yaseen; verse #. 60, Surah

Al Qamar; verse #. 11-28, Surah Rahman; verse #. 7, surah Al

Haqqa; verse #. 39, Surah Abbus [33]

Ibne Majja. Ravi-Bussar (R.A)[37]

Trimzi [38]

Bukhri. Ravi – Ans Bin Malik [34]

11 Punica granatum L.

(Rumman) Punicaceae

Stomach cough, hepatitis, muscle pain, heart

and liver diseases, piles, eye diseases,

dental problems, oral diseases, diarrhea and

dysentery.

Holy Quran Verse #. 99, Surah-Al Anam; verse #. 141, Surah Al

Anam; verse #. 69, Surah Rehman [33]

Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad[36]


21.5 Plants as traditional medicine and drug discovery

Plants based traditional medicine systems used to be exercised in China and India for

many years [40]. Their uses have also been documented in various “Traditional medicine

systems of other culture”. These “plant-based systems” still continue to play a vital role in

health care, as it has been estimated by W.H.O. During 1959-1980, at least 119 chemical

substances, considered as important drugs were derived from 90 plant species and are still in

use [41]. 74% amongst these were discovered through bioassay directed isolation from plants

used in traditional medicine. A review from Newman and Cragg suggest that “from 1940s to

2007, 73 % of 155 small molecules are non-synthetic with 47 % being either natural products

or natural product derivatives”. In 1990, synthetic medicinal chemistry caused the proportion of

natural products drugs to drop to 50 %. Between 2005 and 2007, 13 “new natural product

derived drugs” were approved in the U.S.A, five amongst those were first members of new

classes [42].

In 1820, “quinine (1) an anti-malarial drug, was isolated from the bark of cinchona

species by French pharmacists, Caventou and Pеllеtierе”. The bark was introduced into Europe

in early 1600 as a cure of malaria and has long been used for the treatment of several kinds of

fevers. Chloroquine (2) and Mefloquine (3) are the derivatives of quinine. Artimisnin (4) has

been isolated from Artemisia annua, a Chinese traditional medicine, which was an effective

plant in ailment of fevers. The derivatives of (4), artemeter (5) and artether (6), are reported to

be effective against resistant strains. Morphine (7), isolated in 1816 was used in ancient

Mesopotania and is used as an analgesic. This discovery lead to the basis of alkaloid chemistry

[43]. Digoxin (8) was isolated from Digitalis purpurea in 1785 and so on. Some important

pharmaceutical innovations/discoveries are listed in Table – 3.


Table 3: Some important pharmaceutical innovations.

Year Drug Drug type Therapeutic

1785 Digitoxin Cardioglycoside Inotropic agent

1803 Morphine Narcotic Analgesic

1867 Carbolic acid Phenol Antiseptic

1884 Phenzone Alkaloid Analgesic

1910 Salversan Arsenical Antisyphillitic

1935 Sulfamidchrysolidine Sulfonamides Bactericide

1942 Penicillin Antibiotic Bactericide

1987 Humulin Hormone Recombinant DNA

Some major drugs which developed from medicinal plants (traditional) include

“reserpine (9) (anti-hypertensive), isolated from Rauwolfiа serpentinа used in Ayurvedic

medicine to cure snakebite and other ailments. Ephedrine (10) was isolated from Ephedra

sinicа (Ma-Huang), used in trаditional Chinеse medicine. Salbutamol (11) and sаlmetrol (12)

are anti-asthаmic agents while tubocurarine (13) is used for the arrow poison, isolated from

Chondrodendron and Curаrea species” [40, 43].

15 | P a g e I n t o r d u c t i o n

Table 4: Some of the most important drugs derived from natural products.

Name Type Source Therapeutic uses

Alkaloids Atropine , hyocyamine, scopolamine Tropane alkaloid Solanaceae spp. AnticholinergicCamptothecin Indole alkaloid Camptotheca acuminatea AntineoplasticCapsaicin Phenyl arrine alkaloid Cupsicum spp. Topical analgesicCodeine, morphine Opium alkaloid Papaer somniferum Analgesic, antitussiveCocaine Cocain alkaloid Erythroxylum coca Local anestheticColchicines Isoquinoline alkaloid Colchicum autumnale AntigoutEmetine Isoquinoline alkaloid Cephaelis ipecacuanha AntimoebicGalanthamine Isoquinoline alkaloid Leucojum aestivum Cholinesterase inhibitorNicotine Pyrollidine alkaloid Nicotiana spp. Smoking cessation therapyPhysostigmine Indole alkaloid Physostigma venenosum CholinergicPilocarpine Imidazole alkaloid Pilocarpus jaborandi CholinergicQuinine Quinoline alkaloid Cinchona spp. AntimalarialQuinidine quinoline alkaloid Cinchona spp. Cardiac depressant Reserpine Indole alkaloid Rauwolfia serpentine Antipertansive, psycotropic

Tubocurarine Bisbenzyl isoquioline

alkaloid Chondodendron tomentosum

Strychnos toxifera Skeletol muscles relaxant

Vinblastine, vincristine Bis- indole alkaloid Catharantus roses Antineoplastic

Yohimbine Indole alkaloid Apocynaceae, Rubiaceae spp. Aphrodisiac

16 | P a g e I n t o r d u c t i o n

Terpenes and Steroids Artemisinin Sesqiterpene lactone Armetisia annua AntimalarialDiosgenin, hecogenin, stigmaterol Steroids Dioscorea spp Oral contraceptives and hormonal d

Taxol and other taxoids Diterpens Taxus brevifoila Antineoplastic

Glycosides Digoxine, digitoxin Steroidal glycosides Digitalis spp Cardiotonic

Sennosides A and B. Hydroxy-anthracene

glycosides Cassia angustifoila Laxative

Others

Ipecac Mixture of ipecac

alkaloids and other

components Cephaelis ipecacuanha Emetic

Podophyllotoxin Lignan Podophyllum peltatum Antineoplastic

17 | P a g e

1.6 Natural products as anticancer drugs

Cancer is one of the most deadly threats to humans and is considered to be the second

leading cause of deaths worldwide. Over ten million people around the world are diagnosed

with cancer per year [44]. In U.S.A, approximately, one million cancer patients are registered

per year and deaths from cancer continued to increase from 1973-1990. In 1900, about 510,000

Americans were reported to be dead due to cancer [45]. Modulation of single targets is amongst

one of the ongoing anticancer therapies but due to high cost and less safety, these therapies

have encouraged alternative approaches e.g. natural product compounds along with their

derived prototypes, they are now being used in cancer therapy as chemo preventive compounds.

Much improvement has been made in the war against cancer in the past twenty years.

The modern advancement in molecular and cellular biology aided a lot in comprehending

different mechanisms of this disease which led to the development of different anticancer drugs

and vaccines. Natural products continued to contribute in the improvement of anticancer drugs

significantly. In a recent review, from 1981-2006, “there were total 100 NCEs anticancer drugs

in which; the number of non-biologicals was 81. Using 81 as 100%, 22 % of the total anticancer

drugs were classified into S (synthetic) category. Expressed as a proportion of the non-

biologicals/vaccines, 63 of 81 (77.8%) were either natural products or were based

pharmacophore originated from natural compounds” [46].

Despite major progress in combinatorial chemistry, natural product derived drugs are still

making a good contribution in drug discovery today [47-49]. The search for effective anticancer

drugs has prompt researchers to investigate the efficiency of natural products in the treatment of

cancer. Laboratory trials have proven that there are hundreds of different compounds that

possess anticancer activities [50]. Some other new classes have also been isolated from marine

organisms which have been shown to bear cytotoxic activities against multiple tumor types

[51-52].


Amongst the class of natural alkaloids, Vinblastine (14) and vincristine (15), isolated

from Catharanthus roseus are used to treat “lymphoma and leukemia” [53]. One example is

vindesine (16) possess less neurotoxicity and causes “complete remission in adult non-

lymphocytic leukemia and acute lymphatic childhood leukaemia” [54].

Camptothecin (17), another natural alkaloid that has been modified through several

structural modification, has been isolated from Camptotheca acuminate [55] and is used in the

treatment of “gastric, rectal, colon and bladder cancers”. 10-hydroxycamptothecine (18) [55], 9-

aminocamptothecin (19), topotecan (20) and irinotecan (21) are “potent antitumor and DNA

Topol inhibitory agents” [56] [57] [58].

Podophyllotoxin (22) is a biologically active compound which is isolated from

Podophyllum species. It is a “mitotic inhibitor which reversibly bind to tubulin and also inhibit

microtubule assembly” [59]. Etoposide (23) and teniposide (24) are the “structural mimic” of

22. Rabdosiin (25) , is natural lignan which is also equipotent to 23 and DNA Topo II inhibitor,

has been isolated from Arnebia euchroma [60].

According to the latest findings from the plants, some novel antitumor compounds are:

Bryostatin 1 (26):- Potent inhibitor of “protein kinase C (PKC), which is involved in the

phosphorylation of serine and threonine residues, and actually counteracts tumor promotion

induced by phorbol esters”.

Dolastatins (27):- Mechanistically, strongly inhibit microtubule assembly, tubulin-dependent

Guanosine triphosphate hydrolysis.

Auristatin (28): - Acts by the inhibition and disruption of the microtubule assembly, auristatin

has a dual action in blocking blood supply to tumor vasculature.

Combretastatin A4 (29):- is a low molecular weight vascular disruptive agent (VDA), a new

class of cancer chemotherapy designed to induce rapid and selective vascular shutdown in

tumors [61]. Some anticancer compounds derived from plants are shown in Table – 5.


Table 5: Anticancer compounds derived from plants.

S No Compound Source Family

1 Allamandin Allamanda cathartica Apocynaceae

2 4-ipomeanol Ipomoea batatas Convolvulaceae

3 Penstimide Penstemon deutus Scrophulariaceae

4 Baccharin Baccharis megapotamica Compositae

5 Helenalin Helenium autumnale Compositae

6 Liatrin Liatris chapmanii Compositae

7 Phyllanthoside Phyllanthus acuminatus Euphorbiaceae

8 Vernolepin Vernonia hymenolepis Compositae

9 Gnidin Gnidia lamprantha Thymelaeaceae

10 Jatrophone Jatropha gossypiifolia Euphorbiaceae

11 Taxol Taxus brevifolia Taxaceae

12 Tripdiolide Tripterygium wilfordii Celastraceae

13 Bruceantin Brucea antidysenterica Simaroubaceae

14 Glaucarubinone Simarouba glauca Simaroubaceae

15 Holacanthone Holacantha emoryi Simaroubaceae

16 Cucurbitacin Marah oreganus Cucurbitaceae

17 Acer saponin P Acernegundo Aceraceae

18 Hellebrigenin Bersama abyssinica Melianthaceae

19 Withaferin A Acnistus arborescens Solanaceae

20 Combretastin A- 4 Combretum caffrum Combretaceae

21 α-and β-Peltatin Podophyllum peltatum berberidaceae

22 Podophyllotoxin Podophyllu hexandrum berberidaceae

23 Steganacin Steganotaenia araliaaceae umbelliferae

24 Jacaranone Jacaranda caucana bignoniaceae

25 Lapachol Stereospermum sauveolens bignoniaceae

26 Monocrotaline Crotalaria spectabilis leguminosae

27 Indicine-N-oxide Heliotropium indicum boraginaceae


28 Emetine Cephaelis acuminata rubiaceae

29 Tetrandrine Cyclea peltata menispermaceae

30 Thalicarpine Thalictrum dasycarpum ranunculaceae

31 Nitidine Fagara zanthoxyloides rutaceae

32 Nitidine Fagara macrophylla rutaceae

33 Tylocrebine Tylophora crebiflora asclepiadaceae

34 Acronycine Acronychia baueri rutaceae

35 Ellipticine Ochrosia elliptica apocynaceae

36 9-Methoxyellipticine Ochrosia maculata apocynaceae

37 Camptothecin Camptotheca acuminata nyssaceae

38 Harringtonine Cephalotaxus harringtonia cephalotaxaceae

39 Homoharringtonine Cephalotaxus harringtonia cephalotaxaceae

40 Leurosine Catharanthus lanceus apocynaceae

41 Vinblastine Cephalotaxus roseus apocynaceae

42 Vincristine Cephalotaxus.roseus apocynaceae

43 Maytanacine Maytenus buchananii celastraceae

44 Maytansine Maytenus buchananii celastraceae

45 Maytanvaline Maytenus buchananii celastraceae

46 Colchicine Colchicum speciosum liliaceae

47 Bouvardin Bouvardia ternifolia rubiaceae

48 Deoxybouvardin Bouvardia ternifolia rubiaceae


1.7 Modern drug discovery and natural products

In the 1990s and early 2000s, many pharmaceutical companies moved away from

natural product discovery, following the early successes in drug discovery. Natural products

struggled to provide a good number of compounds desired for High Throughput Screening

(HTS). It put a strain on natural product programs. Besides this, the introduction of

combinatorial chemistry as a better approach to creating large sets of “drug-like” compounds

which ultimately, led to diminish the numbers of natural product discovery programs in the

pharmaceutical industry [62].

The “chemical space” “occupied by natural products is now considered both more

versatile and more drug-like than that of combinatorial chemical collection”. Natural products, a

part from being most productive source of leads, are currently off fashion from the

pharmaceutical industry, which continue to favour combinatorial techniques [63]. Interestingly,

combinatorial chemistry has not proved very fruitful so far, with only one de novo new

compound, Sorafenib (Nexavar, 30), an anti-tumor compound produced by Bayer and was

approved by FDA in 2005 [46].

The authors of a recently published statistical comparison between three classes of

compounds, marketed drugs, combinatorial compounds, and natural products, suggested that

“by mimicking certain distribution properties of natural compounds, combinatorial products

might be made that are substantially more diverse and have greater biological relevance” [64].

This statement was based on the assumption that most natural products have a function, and the

biosynthetic routes which generate these metabolites have coevolved with the specific receptor

systems which they target. It is therefore thought that combinatorial chemistry must also evolve

beyond “synthetic feasibility” to focus on the creation of compounds with desired biological

function. [64]


Despite the trends in the pharmaceutical industry away from natural product discovery,

the field has continued to deliver new drugs and drug leads. In the 25 years from January 1981

to June 2006, nearly two third of the new drugs approved world-wide were natural products

(N), natural product derivatives (ND), synthetic mimics of natural product action (NM), or

derivatives from natural product pharmacophores (S*).

Newman and Cragg et al. have published several reviews on “natural products as

sources of new drugs” [46, 65]. In their most recent review, they summarise their findings by

stating “we strongly advocate expanding, not decreasing, the exploration of nature as a source

of novel active agents that may serve as the leads and scaffolds for elaboration into desperately

needed efficacious drugs for a multitude of disease indications” [46].


Figure 1: Structures of the mentioned compounds

26 | P a g e

27 | P a g e L i t e r a t u r e R e v i e w

2.1 Family Scrophulariaceae Family Scrophulariaceae is also called the figwort family. About 275 genera and 5000

species belonging to this family are found all over the world. The plants are annual or perennial

herbs. The flowers are either aygomorphic (bilateral) or actinomorphic (radial) [66]. The name

of family is due to its genus, Scropularia [67]. Members of this family are distributed in

temperate and tropical mountain area.

2.2 The genus Buddleja

Buddleja (Buddleia) is a genus of flowering plants [68]. In the past, it was classified in

either Loganiaceae or Buddlejaceae [69] but now it is included in family Scrophulariaceae

[70-71].

The genus Buddleja comprises of 100 species. Most of them are shrubs including some

as trees. They are widely distributed throughout the world from Southern United States to

Chile, Africa and Asia. In Pakistan only four species are found including B. asiatica Lour, B.

crispa Benth, B. davidii Franch and B. lindleyana [72].

2.3 Medicinal importance of genus Buddleja

Plants of this genus have been used as a remedy against various diseases and in

treatment of several health problems. The ethnopharmacology of Buddleja species summarise

major traditional uses such as wound healing and related conditions, treatment of liver diseases,

bronchial complaints, diuretic activity, sedative effects, antirheumatic and analgesic activities

and many other medicinal uses [73]. Various species of Buddleja have been used in the

treatment of variety of complaints like skin ailments, ulcer, clustered nebulae and conjunctival

congestion.


Plants of this genus posses anti-inflammatory, analgesic, antipyretic, anticataratic,

antihepatotoxic, hypotencive, hypoglycaemic, neuroprotective, antimicrobial, molluscicidal and

amoebocidal activities [74]. Buddleja specie have also been used in treatment of cancer [75].

Several parts of B. asiatica have been used traditionally to cure, articular rheumatism and

diarrhea in the Chinese traditional medicine [76]. The plant of B. saligna is used as treatment of

coughs, colds, and purgatives [77]. The leaves of B. globosa were used by indigenous

“Mapuche” in wound healing [78] and ulcers [79]. Leaves of B. globosa are effective in

wounds healing, thus showing strong anti-oxidant activities [80].The flowers and flower buds

of B. officinalis are used as a cure for hepatitis [81], as an antispasmodic, cholagogue,

ophthalmic and various eye problems [82]. Linarin, isolated from B. davidii, is a strong

inhibitor of acetylcholinesterase enzyme [83]. The leaves of B. madagascariensis have been

traditionally used against asthma, coughs and bronchitis and a soap substitute [84].

Buddleja species contain typical chemical characteristics (iridoids and phenylethanoids)

of the group of dicotyledons having flowers with fused petals [73]. Phytochemical studies of

the genus Buddleja resulted in the isolation of various compounds such as iridoids, lignan-

iridoids, lignans, neolignans, phenylethanoids, phenylpropanoids, terpenes (sesquiterpenes,

diterpenes, triterpenes and their glycosides), flavonoids (glycosides and flavones), sterols, aryl

esters, phenolic fatty acid esters and saponins (Table – 6).


Table 6: Isolated compounds from the genus Buddleja.

S. # Compound Name Mol. Formula Mol. Weight Source Ref.

31 Acubin C16H22O9 358.56 B. globosa [85]

32 Buddlejoside A2 C33H42O16 694.6770 B. japonica [86]

33 Buddlejoside A C22H25O11 465.4243 B. crispa [87]

34 Buddlejoside B C22H25O12 481.4267 B. crispa [87]

35 Buddlejoside C C20H29O10 429.4383 B. crispa [87]

36 Methyl Catalpol C16H24O11 392.00 B.asiatica [74]

37 Benzoyl Catalpol C22H26O13 498.77 B.dividi [88]

38 p-methoxycinnamoyl Catalpol C25H32O13 540.44 B.dividi [89]

39 Dimethoxycinnamoyl Catalpol C26H34O14 570.32 B.dividi [89]















54 6-Vanillyajugol C23H30O12 498.4771 B. japonica [86]


55 6-Feruloyl-ajugol C25H32O12 524.5144 B. japonica [86]

56 Buddlejoside A1 C30H48O15 648.6931 B. japonica

57 Buddlin C9H14O5 202.2045 B. asiatica [90]

58 Neolignans 1 C35H42O15 702.55 B.devidi [89]



61 Buddledin A C17H24O3 276.3707 B. davidii [91]

62 Buddledin B C15H22O2 234.3419 B. davidii [91]

63 Buddledin C C15H22O 218.3346 B. davidii [92]

64 Buddledin D C15H22O 218.3346 B. davidii [92]

65 Buddledin E C15H24O 220.3505 B. davidii [92]

66 Dihydroxy buddledin A C17H20O3 272.3389 B. globosa [93]

67 Zerumbone C15H22O 218.3346 B. globosa [93]

68 Buddledone A C15H24O 220.3505 B. globosa [93]

69 Buddledone B C15H22O2 234.3340 B. globosa [93]

70 Cycloclorinone C15H22O 218.33 B. cordata [94]

71 1-Hydroxy cycloclorinone C15H22O2 234.33 B. sessiliflora [94]

72 Buddlejone II C16H16O3 256.3006 B. crispa [95]

73 Buddlindeterpene A C15H22O2 234.1619 B. lindleyana [96]

74 Buddlindeterpene B C15H22O 218.1670 B. lindleyana [96]

75 Buddlejone C20H28O2 300.4351 B. albiflora [97]

76 Deoxy Buddlejone C20H28O 284.4357 B. globosa [98]

77 11,14-Dihydroxy-8,11,13-

abietatrien-7-one

C20H28O3 316.4345 B. yunenesis [93]

78 Maytenone C40H60O4 604.9020 B. globosa [98]

79 Crocetin-gentiobiose ester C30H42O14 626.26 B. officinalis [93]

80 Buddlindeterpene C C20H30O3 318.2195 B. lindleyana [96]


81 Saikosaponin A C42H68O13 780.9815 B. japonica [99]

82 Buddlejasaponin i C54H88O22 1089.2633 B. japonica [99]

83 Buddlejasaponin ii C53H86O22 1075.2367 B. japonica [99]

84 Buddlejasaponin iii C47H76O17 913.0961 B. japonica [99]

85 Buddlejasaponin iv C48H78O18 943.1221 B. japonica [99]

86 Mimengoside A C54H88O21 1072 B. officinalis [100]

87 Mimengoside B C55H92O22 1104 B. officinalis [100]

88 Mimengoside C C54H88O22 1088 B. officinalis [101]

89 Mimengoside D C47H76O17 912 B. officinalis [101]

90 Mimengoside E C54H88O22 1088 B. officinalis [101]

91 Mimengoside F C47H76O17 912 B. officinalis [101]

92 Mimengoside G C54H88O21 1072 B. officinalis [101]

93 Songaroside A C54H88O21 1073.263 B. officinalis [101]

94 13,28-Epoxy-23-dihydroxy-11-

oleanene-3-one

C30H45O3 453.6765 B. asiatica [102]

95 13,28-Epoxy-21β,23-dihydroxy-11-

oleanene-3-one

C30H46O4 470.8638 B. asiatica [102]

96 11-Keto-β-amyrin C30H48O2 440.7009 B. madagascariensis [69]

97 α-Amyrin C30H50O 426.7174 B.madagascariensis [69]

98 β-Amyrone C30H49O 425.7095 B. officinalis [7]

99 β-Amyrin C30H50O 426.7174 B. globosa [93]

100 β-Amyrin acetate C32H52O2 468.7541 B. globosa [93]

101 Glutinol C30H50O 426 B. globosa [103]

102 Lupeol C30H50O 426.50 B. globosa [103]

103 Luteolin C15H10O6 286 B. officinalis [104]

104 Luteolin glucopyranoside C21H20O11 448 B. officinalis [104]

105 Apigenin C15H10O5 270 B. officinalis [104]


106 Apigenin-7-O-glucoside C21H22O10 434.3934 B. globosa [104]

107 Acacetin C16H12O5 284.2635 B. officinalis [104]

108 Acacetin-7-O-rutenoside C28H32O14 592.5453 B. davidii [83]

109 Quercitin C15H10O7 302.55 B. davidii [83]

110 Dosmin C28H32O15 608.25 B. globosa [98]

111 6-Hydroxyluteolin C15H10O7 302.12 B. globosa [98]

112 Isorhoifolin C15H10O7 302.66 B. officinalis [105]

113 Eriodictyol C15H12O6 288.88 B. perviflora [106]

114 Glucohesperetin C22H22O12 358.77 B. perviflora [106]

115 Pyracanthoside C21H22O11 406.66 B. perviflora [106]

115 Hesperetin C15H14O6 290.76 B. madagascariensis [107]

116 Hesperetin 7-0(2”,6”-di-O-α-L-

rhamnopyrunosyl)-β-D-

glucopyranosid

C34H44O19 756.00 B. madagascariensis [107]

117 Diosmetin 7-0 (2”, 6”-di-O-α-L-

rhamnopyrunosyl)-β-D-

glucopyranosid

C34H42O19 753.00 B. madagascariensis [107]

118 Secutellarein 7-O-glucoside C21H20O11 448.76 B. globosa [108]

119 Calceolarioside A C23H26O11 478.4459 B. officinalis [93]

120 Campneoside II C29H36O16 640.5865 B. officinalis [93]

121 Pliumoside C35H46O19 770.7283 B. officinalis [93]

122 Echinacoside C35H46O20 786.7277 B. officinalis [93]

123 Forsythoside B C34H44O19 756.7018 B. officinalis [93]

124 Angoroside A C34H44O19 756.7018 B. officinalis [93]

125 Angoroside C C36H48O19 784.7549 B. davidii [99]

126 Verbascoside A C31H40O16 686.6397 B. japonica [86]

127 Plantainoside C C30H38O15 638.6137 B. davidii [99]


128 Jionoside D C30H38O15 638.6137 B. davidii [99]

129 2-Acetylmartynoside C33H42O16 694.6770 B. davidii [99]



132 Martynoside C31H41O15 653.6482 B. davidii [109]

133 Isomartynoside C31H40O15 652.6403 B. davidii [99]

134 Leucosceptoside A C30H38O15 638.6137 B. davidii [99]

135 Leucosceptoside B C36H48O19 784.7549 B. davidii [99]

136 Phenylethyl glycoside C14H20O6 284.1840 B. officinalis [93]

137 Bioside C20H32O12 480.45 B. officinalis [93]

138 Salidroside C14H20O7 300.44 B. officinalis [93]

139 2[4`-Hydroxyphenyl]-ethyl

hexacosanoate

C34H60O3 516.8384 B. cordata [110]


tricosanoate

C31H54O3 474.7587 B. cordata [110]


pentacosanoate

C33H58O3 502.8118 B. cordata [110]


arachidate

C28H48O3 432.6789 B. cordata [110]


heptadecanoate

C25H42O3 390.5992 B. cordata [110]


nonadecanoate

C27H46O3 418.6523 B. cordata

[110]

145 2[4`-Hydroxyphenyl]-ethyl behenate C30H52O3 460.7321 B. cordata

[110]


lignocerate

C32H56O3 488.7852 B. cordata

[110]


147 2[4`-Hydroxyphenyl]-ethyl palmitate C24H40O3 376.5726 B. cordata

[110]

148 2[4`-Hydroxyphenyl]-ethyl stearate C26H44O3 404.6258 B. cordata [110]

149 Asiatiside A C18H22O8 366.3625 B. asiatica [111]

150 Asiatiside B C18H22O9 382.3619 B. asiatica [111]

151 Asiatiside C C19H24O9 396.3885 B. asiatica [111]

152 Asiatiside D C18H22O8 366.3625 B. asiatica [111]

153 Buddlenol A C31H34O11 582.5951 B. davidii [112]

154 Buddlenol B C31H36O11 584.6109 B. davidii [112]

155 Buddlenol C C31H38O12 602.6262 B. davidii [112]

156 Buddlenol D C33H40O13 644.6629 B. davidii [112]

157 Buddlenol E C31H36O11 584.6109 B. davidii [112]

158 Buddlenol F C32H38O12 614.6369 B. davidii [112]

159 Balanophonin C29H48O15 356.3692 B. davidii [112]

160 Syringaresinol C29H48O15 418.4370 B. davidii [112]

161 Glutinol C30H50O 426.7174 B. globosa [98]

162 Chondrillasterol C29H48O 412.6908 B. globosa [98]

163 β-sitosterol C29H50O 414.7067 B. yunenesis [93]

164 Stigmasterol C29H48O 412.3705 B. mad. [69]

165 β-sitosterol-O-glucoside 593.24 B. asiatica [74]

166 (22R)-Stigmasta-7,9(11)-dien-22α-

ol-3 β-O-β-D-galactopyranoside

C35H59O7 591.8388 B. crispa [113]

167 Sucrose C12H22O11 342.2965 B. yunenesis [93]

168 Hexyl p-hydroxy-cinamate C15H20O3 248.3175 B. crispa [113]

169 Ferulic acid methyl ester C11H12O4 208.2106 B. globosa [108]

170 p-Coumeric acid methyl ester C10H10O3 178.1846 B. globosa [98]


171 3-(4-Acetoxy-phenyl)-acrylic acid 3-

phenyl-propyl ester

C27H24O5 427.4765 B. crispa [114]

172 Nonyl benzoate C16H24O2 248.3606 B. crispa [113]

173 Methyl β-orcinolcarboxylate C10H12O4 196.1999 B. cordata [110]

174 β-orcinolcarboxylate C9H10O4 182.1733 B. cordata [110]

175 Coniferaldehyde C10H10O3 178.1846 B. davidii [112]

176 Buddamin C10H15NO5 229.2298 B. davidii [112]

177 Crispin A C40H74NO9 712.5364 B. crispa [115]

178 Crispin B C51H84NO12 902.44 B. crispa [115]

179 BDL-H3 (Aryl ester) C27H24N5 428.48 B. crispa [114]


Figure 2: Structures of the compounds reported from Buddleja asiatica.


2.4 Buddleja asiatica Lour B. asiatica Lour is a woody shrub up to 15 feet high. It is native to South Asia and East

Indies. In Pakistan, it is found in Siren Valley, Mansehra, KPK and in District Kotli, Azad

Jammu & Kashmir. It is locally known as Bui, Banna or Batti [116].

Leaves of this plant are almost entire dark green and glabrous above while lighter and

tomentose below. The flowers are borne in three-five flowered cymes arranged in a slender

pendulous panicle 10-30 cm. long. The corolla is white, without glabrous and yellow pubescent

five-six mm long. The calyx is campanulate and is two-three mm long. It has lobes of one mm

each, triangular, acute and tomentulose. The stamen insertion is median while the ovary is

ovoid. The flowers have a very attractive sweet odour. This species blooms profusely from

December to February [117].

Figure 3: Buddleja asiatica Lour


2.5 Scientific classification

2.6 Pharmacological significance of B. asiatica

This plant has been used medicinally in different regions in past and present. It has been

used as an abortifacient [118] and in skin complaints [119]. A paste of its roots is used as a

tonic when mixed with rice water [120]. This plant is also used as a medicine for skin disease, a

cure for loss of weight and in cases of abortion [121]. Roots and leaves of this plant are

employed to treat head tumour [122]. An infusion of roots is used to treat malaria [123], while

its leaves cause hypotensive effect on cats and dogs [124]. The essential oil of the leaves has

been reported to posses in vitro antifungal activities [125]. The flowers have been used in the

treatment of cystitis, cold [81] and to treat oedema [126]. The extracts of B. asiatica also

showed strong cyclo-oxygenase (COX) inhibitory activities using elicited rat peritoneal

leukocytes [93].

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Lamiales

Family: Scrophulariaceae

Genus: Buddleja

Species: Buddleja asiatica Lour

50 | P a g e

51 | P a g e R e s u l t s a n d D i s c u s s i o n

PART A

3.1 Secondary metabolites from Buddleja asiatica

In search of bioactive secondary metabolites from medicinal plants, Buddleja asiatica

belonging to the family Scrophulariaceae was investigated. It occurs abundantly in Pakistan and

the genus Buddleja is represented by four species [72].

The ethno-pharmacological and chemotaxonomic importance of the genus prompted us to

start investigation on this plant. As a result, twelve compounds were isolated from chloroform

and ethyl acetate soluble parts of Buddleja asiatica.

“Structures of all the isolated compounds were established using spectral as well as

published data in literature. In this part, the compounds are discussed briefly”.

3.2 Extraction and isolation

The methanolic crude extract of air-dried plant of B. asiatica was concentrated to a gum.

The gummy material was divided into four fractions, e.g., n-hexane (F1), chloroform (F2), ethyl

acetate (F3) and n-butanol (F4) soluble fractions.

The chloroform (F2) and ethyl acetate (F3) soluble fractions (F2 and F3) showed high

toxicity in brine shrimp lethality test (as discussed in part B). They were subjected to series of

column chromatographic techniques to yield twelve compounds (180-191). All the compounds

were characterized using latest spectroscopic techniques.


3.3 Chloroform soluble fraction

“The chloroform soluble fraction (F2) was concentrated and subjected to column

chromatography over silica gel for preliminary fractionation. Elution was carried out with

n-hexane (100 %, FA), n-hexane - CHCl3 (1:1, FB), CHCl3 (100 %, FC), CHCl3 - EtOAc (1:1,

FD), EtOAc (100 %, FE), EtOAc - MeOH (1:1, FF) and MeOH (100 %, FG) in increasing order

of polarities. These fractions were further loaded to a series of column chromatography to

obtain seven compounds, a diterpene (Buddlejone, 180), two sesquiterpenes

(dihydrobuddledin-A, 181 and buddledone-B, 182), a triterpene (ursolic acid, 183), a phenyl

ethyl glycoside (2-phenylethyl-β-D-glucoside, 184), an iridoid glycoside (7-deoxy-8-epiloganic

acid, 185) and one flavonoid glycoside (secutellarin-7-O-β-D-glucopyranoside, 186)

respectively”.


3.4 Structure elucidation of compounds.

3.4.1 Buddlejone (180)

The fraction FB obtained with n-hexane - CHCl3 (1 : 1) was subjected to column

chromatography over silica gel eluting with n-hexane - CHCl3 in increasing order of polarities.

The eluates obtained with n-hexane - CHCl3 (6 : 4) showed two major with some minor spots

on TLC were combined and re-chromatographed with mixtures of n-hexane - CHCl3 in

increasing polarities over silica gel. The eluates obtained from n-hexane - CHCl3 (6.5 : 3.5)

showed two major spots on TLC, were purified by preparative TLC on silica gel in

n-hexane - Me2CO (8.0 : 2.0). The faster moving orange colour oil compound was identified as

buddlejone (180) which showed molecular ion peak in HR-EIMS at m/z 300.2053 (calcd. for

C20H28O2, 300.2089), other major peaks obtained at m/z 300 (97 %), 285 (100 %), 257 (82 %),

215 (17 %), 177 (24 %), 149 (30 %), 129 (23 %) and 113 (20 %). The IR (KBr) spectrum

showed prominent absorption at 3425 cm–1, which indicated the presence of hydroxyl (OH)

group while the absorption at 1731 cm–1 showed the presence of carbonyl group.


The 1H-NMR spectrum (CDCl3) showed a three protons singlet at 1.18 and was

assigned to the uncoupled methyl group (3H, H - 20). Two geminal methyl groups showed

distinct singlet at 1.01 (3H, H - 18) and 0.95 (3H, H - 19) respectively. Two three protons

doublets were observed at 1.25 (3H, J = 4.1 Hz, H - 16) and 1.26 (3H, J = 4.1 Hz, H - 17),

which were coupled to a methine proton of C - 15, appeared as septet at 3.43 (1H, J=6.8 Hz),

exhibited vicinal coupling characteristic of an isopropyl side chain group.

The methylenic protons of C - 1 showed multiplets resonated at 2.14 (1Hα) and 1.52

(1Hβ) due to the coupling with two different methylenic protons of C - 2 which in turn also

showed multiplets for each of the two protons at 1.77 (1Hα) and 1.61 (1Hβ). The two

methylenic protons of C - 3 resonated as two multiplets at 1.50 (1Hα) and 1.26 (1Hβ) while

two other methlyenic protons belonging to C - 6 resonated as a singlet at 2.65 (1Hα) and a

doublet at 2.66 (1Hβ, J = 2.8 Hz).

A methine proton of C - 5 resonated as a multiplet centered at 1.86 and showed vicinal

coupling with methylenic protons (2H, H - 6). The two aromatic methine protons resonated as

two doublets at 6.20 (1H, J =4.1 Hz, H - 11) and 7.31 (1H, J = 4.1 Hz, H - 12) respectively.

The 13C- NMR spectrum (75 MHz) in CDCl3 showed 20 signals. The five methyl

carbons resonated at 20.9, 20.7, 32.9, 21.6 and 21.8 while four methylene carbons were found

to resonate at 36.7, 18.6, 41.4 and 31.8 respectively. The olefinic carbons (C - 12 and C - 13)

resonated at 137.0 and 120.2 whereas other olefinic carbons (C - 9 and C - 11) resonated at

167.8 and 116.7 respectively. The signals of the two methine carbons (C - 5, C - 15) were found

to occur at 52.5 and 32.9, while the two quaternary carbon atoms resonated down field at

188.8 and 192.3 and were attributed to hydroxy and carboxy carbons (C - 7, C - 14). Three

quaternary carbons (C - 10, C - 4 and C - 8) were found to occur at 33.1, 36.7 and 120.2

respectively. “The chemical shifts of the various carbons and 1H/13C connectivities” are shown in


Table - 7. The comparison of spectral data with those reported in the literature [97] established

the identity of the compound as buddlejone (180).


Table 7: 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of buddlejone (180).

C. No Multipilicity

DEPT Chemical shift () 1H/13C Connectivity (= Hz)

1 CH2 36.7 2.14 (1Hα, m), 1.52 (1Hβ, m)

2 CH2 18.6 1.77 (1Hα, m), 1.61 (1Hβ, m)

3 CH2 41.4 1.50 (1Hα, m), 1.26 (1Hβ, m)

5 CH 52.5 1.86 (1H, m)

6 CH2 31.8 2.65 (1H, s), 2.66 (1H, d, J = 2.8 Hz)

11 CH 116.7 6.20 (1H, d, J = 4.1 Hz)

12 CH 137.0 7.31 (1H, d, J = 4.1 Hz)

15 CH 32.9 3.43 (1H, septet, J = 6.8 Hz)

16 CH3 20.9 1.25 (3H, d, J = 4.1 Hz)

17 CH3 20.7 1.26 (3H, d, J = 4.1 Hz)

18 CH3 32.9 1.01 (3H, s)

19 CH3 21.6 0.95 (3H, s)

20 CH3 21.8 1.18 (3H, s)


3.4.2 Dihydrobuddledin A (181)

The fractions obtained from n-hexane - CHCl3 (6.5 : 3.5) were combined and subjected

to preparative TLC on silica plates in n-hexane - Me2CO (8.0 : 2.0) afforded faster and slower

moving compounds. The slower moving compound 181 isolated by the same preparative TLC

procedure as compound 180 was obtained as colourless oil which was identified as

dihydrobuddledin A (181). The IR (KBr) spectrum revealed strong absorption at 1700 cm-1,

1738 cm-1 and 2955 cm-1 indicated ketonic, ester carbonyl and vinyllic groups. Its molecular ion

peak in HR-EIMS appeared at m/z 278.1945 which agreed with the formula mass calculated for

C17H26O3 (278.1960), while other major peaks were observed at m/z 278 (17 %) 263 (100 %),

236 (35 %), 252 (9 %) and 235 (12 %).

The 1H-NMR spectrum (CDCl3) showed two singlets for methyl protons at 1.14 (3H,

H - 12) and 1.12 (3H, H - 13). A three proton doublet at 1.11 was assigned to one secondary

methyl protons of C-14 (3H, J = 6.8 Hz) while the C - 4 methine proton appeared as a multiplet

centred at 2.94. Another three proton singlet at 2.08 was assigned to the methyl protons of

acetyl group.


A methine proton belonging to C - 1 appeared as double doublets at 2.07 (1H, dd, J =

11.4, 10.0 Hz). “The methine proton of C - 2 resonated as a doublet at 5.12 (1H, d, J = 11.4

Hz)” while the other methine proton (C - 9) appeared as a multiplet centered at 3.03. One of

the two olefinic protons of C - 15 appeared as a singlet at 4.75 while the other olefinic proton

resonated as a triplet centred at 4.62 (1H, t, J =1.8 Hz). Four methylenic protons resonated as

multiplets in the region of 2.01-1.5 (8H, m, CH2 - 5, CH2 - 6, CH2 - 7, CH2 - 10) respectively.

13C-NMR spectrum (75 MHz) in CDCl3 showed the presence of four methyl, five

methylene, four methine and four quaternary carbon atoms. The methyl and carbonyl carbons

of ester group resonated at 21.0 and 171.0 while the olefinic carbons (C - 8 and C - 15) were

found to produce signals at 153.2 and 111.4. The two methyl carbons of the C - 11 side chain

resonated at 31.6 (C - 12) and 13.23 (C - 13) while the ketonic carbon was found to resonate

at 213.4. The methyl (C - 14) and methylenic (C - 10) carbons were found to resonate at

15.8 and 39.4 respectively. The chemical shifts of various carbons and 1H/13C connectivities are

listed in Table-8.

The spectral data was in good agreement with those reported in the literature [93] in the

identity of the compound as dihydrobuddledin A (181).


Table 8: 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of dihydrobuddledin A (181).

C.No Multipilicity DEPT 13C- NMR () 1H/13C Connectivity (= Hz)

1 CH 50.5 2.07 (1H, dd, J = 11.4,10.0 Hz)

2 CH 79.6 5.12 (1H, d, J = 11.4 Hz)

4 CH 44.4 2.96 (1H, m)

5 CH2 30.7 1.66 (1H, m), 1.44 (1H, m)

6 CH2 26.4 1.38 (1H, m), 1.27 (1H, m)

7 CH2 32.9 2.1(1H, m), 1.91(1H, m)

9 CH 38.2 3.03 (1H, m, H-9)

10 CH2 39.4 2.00 (1H, m), 1.75(1H, m)

12 CH3 31.6 1.14 (3H, s)

13 CH3 23.0 1.12 (3H, s)

14 CH3 15.8 1.11 (3H, d, J = 6.8 Hz)

15 CH2 111.4 4.75 (1H, s), 4.62 (1H, t, J = 1.8 Hz)

17 CH3COOH 21.0 2.08 (3H, s)


3.4.3 Buddledone B (182)

Further solution of the same column with n-hexane - CHCl3 (4.0 : 6.0) afforded a major

spot on TLC with some impurities and was subjected to preparative TLC in n-hexane - acetone

(6.0 : 4.0) as solvent system to afford compound 182 as colourless oil. The molecular ion peak

appeared at m/z 235.1706 in its mass spectrum which resulted in obtaining the molecular formula

C15H23O2 (calcd. 235.1699). The mass spectrum also exhibited peaks at m/z 235 (28 %), 220 (12

%), 209 (25 %) and 207 (35 %). The IR (KBr) spectrum showed absorption at 2935, 1675 and

1455 cm-1, pointed out the existence of olefinic group while the intense absorption at 1680 cm-1

which indicated ketonic carbonyl group. A strong absorption at 973 cm-1 suggested that the

compound 182 contains a humulene skeleton [127].

The 1H-NMR (300 MHz, CDCl3) spectrum of compound 182 showed a three proton

singlet at 1.25 due to methyl group at C - 14. Another singlet appeared at 1.20 was due to

methyl protons at C - 15. A secondary methyl group (C - 12) showed doublet at

1.61 (J= 2.7 Hz) due to long range coupling to the neighbouring olefinic methine proton of

C - 1. A series of multiplets were attributed to the three consecutive methylenic protons (H - 4 to

H - 6) as their chemical shifts were observed in the range of 1.29 to 2.29.


A singlet was observed at 5.07 for C - 1 olefinic proton while two other olefinic protons

(C - 9 & C - 10) appeared as doublets, each at 6.01 and 6.47 having J value of 16.5 Hz

respectively. A methine proton at C - 7 showed a multiplet centered at 2.76.

13C-NMR spectrum (75 MHz, CDCl3) corroborated the existence of 15 signals which

were identified as four methyl, three methylene, four methine and three quaternary carbons. The

four methyl carbon atoms resonated at 19.0 (C - 12), 15.6 (C - 13), 26.3 (C - 14) and

28.3 (C - 15). The four olefinic carbons showed resonance at 100.8 (C - 1), 101.7 (C - 2),

124.9 (C - 9) and 153.9 (C - 10) respectively. The three methlyenic carbons were detected at

32.2 (C – 4), 24.6 (C - 5) and 33.2 (C - 6) while carbonyl carbons signals were found to occur

down field at 203.7 (C - 8) and 207 (C - 3). The two quaternary carbons resonated at 101.7

(C - 2) and 39.0 (C - 11). One bond 1H/13C correlations were verified through HMQC techniques

[128]. The chemical shifts of the various carbons along with 1H/13C connectivities are listed in

Table - 9.

The physical and spectral data in the literature [93] was in complete agreement with those

of the compound buddledone B (182).


Table 9: 13 C- NMR (CDCl3, 75 MHz) and 1H/13C correlations of buddledone B (182).

C. No Multipilicity DEPT 13C- NMR () 1H/13C Connectivity ( = Hz)

1 CH 100.8 5.07 (1H, m)

4 CH2 32.2 1.93-2.29 (2H, m)

5 CH2 24.6 1.43-1.60 (2H, m)

6 CH2 33.2 1.29- 1.68 (2H, m)

7 CH 43.2 2.76 (1H, m)

9 CH 124.9 6.01 (1H, d, J = 16.5 Hz)

10 CH 153.9 6.47 (1H, d, J = 16.5 Hz)

12 CH3 19.0 1.61 (3H, d, J = 2.7 Hz)

13 CH3 15.6 1.10 (3H, d, J =6.6 Hz)

14 CH3 26.3 1.25 (3H, s)

15 CH3 28.3 1.20 (3H, s)


3.4.4 Ursolic acid (183)

The fraction FC which was obtained from CHCl3 (100 %) was subjected to column

chromatography over silica gel using n-hexane, CHCl3 and MeOH as eluent mixtures with

gradual increase in polarity to yield several fractions. The fractions obtained from n-hexane -

CHCl3 (2.0 : 8.0) were concentrated and subjected to precoated TLC plates (silica gel) using

n-hexane - EtOAc (7.0 : 3.0) as solvent system to obtain ursolic acid (183) as amorphous

powder. The molecular ion peak appeared at m/z 456.3603 in the HR-EIMS which was

consistent with the molecular formula, C30H48O3 (calcd. 456.3595). The mass spectrum showed

the base peak at m/z 248.1822, which was attributed to Retro-Diels-Alder fragmentation

pattern, a characteristic of Δ12- ursane type triterpenes with a COOH group at C - 17 [129] while

another prominent peak appeared at m/z 411.3862 (M+ - COOH). The IR (KBr) spectrum

showed strong absorption for hydroxyl group at 3480 cm-1 while the absorption at 1693 cm-1

indicated the presence of carbonyl group. The presence olefinic group was supported by the

absorption at 1632 cm-1.

“The 1H-NMR spectrum (300 MHz) in CDCl3 of 183 displayed five singlets for the five

tertiary methyl groups at 1.05 (3H, s, CH3 - 23), 0.84 (3H, s, CH3 - 24), 0.96 (3H, s, 2 CH3 -


25), 0.87 (3H, s, CH3 - 26) and 1.20 (3H, s, CH3 - 27) while the two secondary methyl protons

showed doublets for each at 0.79 (3H, d, J = 6.8 Hz, H - 29) and 0.90 (3H, d, J = 6.6 Hz, H -

30) which indicated the ursane type triterpene skeleton”.

A multiplet centered at 5.10 (1H, m) was assigned to the olefinic proton of C - 12

while a methine proton of hydroxyl carbon (C – 3) resonated as double doublet at 2.99 with

coupling constant J= 10.2 Hz and 4.5 Hz, which was confirmed to be at α and axial

configuration by its J values.

Two mutual coupled methlyenic protons signals resonated as multiplets centered at

0.96 (1H , Hα - 1), 1.54 (1H, Hβ - 1), 1.02 (1H, Hα - 2) and 1.83 (1H, Hβ - 2) were attributed to

the C - 1 and C - 2 methylenic protons while the other six methylenic protons (2H - 6, 7, 15, 16,

21, 22) resonated as multiplets in the region 0.90-2.32. The C-18 methine proton resonated as

a doublet at 2.63 (1H, d, J = 11.3 Hz).

13C-NMR (75 MHz) of compound 183 showed the presence of thirty carbons while their

multiplicity was determined by DEPT which indicated seven methyl, nine methylene, seven

methine, six quaternary and one carboxyl carbon atom. The olefinic carbons resonated at

137.6 (C - 12) and 124. 8 (C -13) while all the methyl carbons were found to resonate at 14.7

- 24.3. The methine carbon of hydroxyl group was found to occur at 79.1. A downfield signal

at 176.2 was assigned to carbonyl carbon of the ester group. The chemical shifts of various

carbon atoms and 1H/13C connectivities are listed in Table -10. “By comparison of these spectral

data with those reported in the literature [130], the compound was identified as ursolic acid

(183)”.

65 | P a g e R e s u l

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