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
and My Uncle DR. MEHBOOB ALI KHAN
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
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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 120 Buddleja asiatica by GC-MS
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-Butanol
CDCl3 Deutreated Chloroform
CHCl3 Chloroform DMSO Dimethylsulphoxide EtOAc Ethyl acetate EtOH 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 Distortionless enhancement by polarization DEPT 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 High resolution electron impact mass HR-EIMS spectrum IR Infrared spectrophotometry m/z Mass to charge ratio (in mass spectrometry) NMR Nuclear magnetic resonance NOESY Nuclear overhauser effect spectroscopy Thermogravimetric and differential TG/DTA thermogravimetric analysis Thermogravimetric and differential TG/DTG 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
13 1 13 7. C- NMR (CDCl3, 75 MHz) and H/ C correlations of buddlejone (180) 56
13 1 13 C- NMR (CDCl3, 75 MHz) and H/ C correlations of dihydrobuddledin A 8 59 (181)
13 1 13 9 C- NMR (CDCl3, 75 MHz) and H/ C correlations of buddledone B (182). 62
13 1 13 10 C- NMR (CDCl3, 75 MHz) and H/ C correlations of ursolic acid (183) 65
13 1 13 C- NMR (CDCl3, 75 MHz) and H/ C correlations of 11 68 2-phenylethyl-β-D-glucoside (184)
13 1 13 C- NMR (CDCl3, 75 MHz) and H/ C correlations of 12 71 7- deoxy-8-epiloganic acid (185)
13 1 13 C- NMR (CDCl3, 75 MHz) and H/ C correlations of 13 74 scutellarin-7-O-β-D-glucopyranoside (186)
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
13 C- NMR (CDCl3, 150 MHz) chemical shifts and multiplicities of (24S)- 17 89 stigmast-5,22-diene-7β-ethoxy-3β-ol (188)
13 1 13 C- NMR (CDCl3, 150 MHz) and H/ C correlations of (24S)-stigmast-5, 22- 18 90 diene-7β-ethoxy-3β-ol (188) xii
13 C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate A 19 96 (189)
13 1 13 20 C- NMR (CDCl3, 75 MHz) and H/ C correlations of asiatoate A (189) 97
13 C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate B 21 102 (190)
13 1 13 22 C- NMR (CDCl3, 75 MHz) and H/ C correlations of asiatoate B (189) 103
13 1 13 C- NMR (CDCl3, 75 MHz) and H/ C correlations of 23 106 Chrysoeriol-7-O-β-D-glucopyranoside (191).
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
Concentration-dependent inhibitory effect of the crude extract of B. asiatica on 27 117 spontaneous contractions of isolated rabbit jejunum preparations Concentration-dependent relaxant effect of the crude extract of B. asiatica on 28 117 K+-induced contractions of isolated rabbit jejunum preparations.
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
Antibacterial activity of crude extract and its various fractions towards 10 112 S. flexneri, E.col and S.boydi Antifungal activity of crude extract and its various factions towards A. Flavus, 11 115 F. Solani and T. Longifusus Concentration-dependent inhibitory effect of the crude extract of B. asiatica on 12 spontaneous and K+- induced contractions of isolated rabbit jejunum 118 preparations. Values shown are mean ± SEM, n=3. Concentration-dependent inhibitory effect of verapamil against spontaneous 13 118 and high K+-induced contractions in isolated rabbit jejunum preparations.
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 | Page
2 | Page Introduction
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
3 | Page Introduction
(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 <2000 a.m.u) produced by an organism. Secondary metabolism concept arises due to shunt metabolism produced during idiophase, regulatory hormones or due to the overflow metabolism as a result of limitation of nutrients, etc [5]. Natural products can be from any terrestrial or marine source/plants (e.g., Paclitaxel from Taxus brevifolia), animals
(e.g., vitamins A and D from cod liver oil), or microorganisms (e.g., doxorubicin from
Streptomyces peucetius). Natural products also includes many other classes of compounds like amino acids, peptides, proteins, polyketides, carbohydrates, lipids, nucleic acids, terpenoids, and so on.
1.2 Medicinal plants
Plants have been used by men since ancient times for many purposes especially as a
need for healing and the most intrinsic, survival. They used to be the “basis of sophisticated traditional medicine systems for thousands of years and are still continuing to serve mankind as
providing new remedies”. Natural products obtained from plants have played extraordinary role in the development of medicinal chemistry. An estimation by the World Health Organization
(W.H.O.) suggests that nearly 80 % of the population depends on natural sources for their prime medical need while the rest (20 %) of the population use integrated natural sources [6].
Medicinal plants are known to be used by mankind as a “source” of medicines since ages. Information on the ancient uses of plant materials as medicines can be found in archaeological finds, old literature, history books and pharmacopoeias. In fact in the Quran and the Bible, about 20 and 125 plants are mentioned, respectively, as being used as medicinal
agents to treat various ailments [7]. “Approximately 35,000 or more different plants are known
4 | Page Introduction
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
5 | Page Introduction 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.
6 | Page Introduction
Table 1: History of natural products.
Period Type Description Ref#
Remains of Neanderthal
Before Mesopotamian record Introduced medicinal properties of plants [11] 3000 BC Ayurveda (knowledge of life) and other natural products
Asclepius
Ebers Papyrus Presented a large number of crude drugs
1550 BC Asclepius from natural sources (e.g., castor seeds [11-12]
Chinese traditional medicine and gum Arabic)
Hippocrates, ‘‘The Father of Described several plants and animals that 460–377 BC [13] Medicine’’ could be sources of medicine
Described several plants and animals that 370–287 BC Theophrastus [14] could be sources of medicine
Wrote De Materia Medica, which 60–80 AD Dioscorides [15] described more than 600 medicinal plants
Practiced botanical medicines Galen (Galenicals) and made them popular in
131–200 AD the West. [9]
A succinct outline of Roman and Greek Avicenna medicine
Kra¨uterbuch (herbals) Presented information and pictures of 15th century [16] medicinal plants
“Pen-ts as kang Mu”, describing 1898
1596 AD Li Shih- Chen herbal drugs along with 8160 [17]
prescriptions
1743 John Wesley, founder of “Primitive physic”, the popular reference [18]
7 | Page Introduction
Methodism book about various natural remedies.
Thompson’s New guide to health” was
Samuel Thompson one of the most popular publications [2, 19- 1822 (Early America) contained various natural product based 25]
methods of medicinal information
Morphine, first commercial pure natural 1826 Merck (marketing agency) [26] product introduced for therapeutic use
8 | Page Introduction
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 and as 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
9 | Page Introduction
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.
10 | Page Introduction
Table 2: Some of the medicinal plants mentioned in the Holy Quran and Ahadith.
Plant S.No Medicinal Uses References form the Holy Quran and Ahadith Specie Family
Holy Quran Verse #. 68, Surah Baqra [33]
Antidote, cholera, infection, influenza, Bukhari (Ravi: Jabir bin Abdullah) Kitabut-Tib [34] Allium cepa L 1 Alliaceae improves sperm production, hepatitis, piles, Muslim (Ravi: Jabir Bin Abdullah) Chap. Abwab Ul Attamah (Basal) menstruation and intestinal diseases. [35]
Al-Jozi (Aljawziyya), Ibn Ul Qayyim. Zadul Maad [36]
Holy Quran Verse #. 61, Surah Baqra [33] Allium sativum L. 2 Antidote, dog bite, paralysis, asthma, parkensis, Bukhari (Ravi: Hazrat Anas), Kitab ul Tamaih [34] Alliaceae cough, hysteria, tuberculosis. Muslim (Ravi: Abu Ayub) [35] (Soom) Ibne Majja (Ravi: Umer bin Alkhitab) [37]
Holy Quran Verse 15, 1, Surah Al insane [33] Cinnamomum camphora Tetanus, parkensis, hysteria, tuberculoses, Bukhari, Chapt. Kitab ul Tib [34] 3 L Lauraceae headache, liver and kidney pains, breast pain, Muslim (Ravi: Um-e-Atiya) Kitabul-Janayez [35] (Kafoor) inner wounds etc. Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad [36]
11 | Page Introduction
Remove kidney and urinary bladder stone, References from Holy Quran Verse #.1-4, Surrah Teen [33] Ficus carica L. 4 Moraceae release intestinal pain, pile, dyspepsia and Bukhari [34] (Teen) anorexia. Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad [36]
Trimzi [38] Fever, weakness, increase immunity, heart Hordeum vulgare L. Bukhari [Ravia: Hazrat Ayesha (Chap; Haiz ul Shahir [34] 5 Poaceae diseases, kidney pain, intestinal ulcer, maintain (Shair) Bukhari. Ravia: Aisha. Kitabul-Athama [34] cholesterol level, jaundice etc Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad [36]
Holy Quran Verse.# 48, Surah Younis [33] Arthritis, Maleness, Piles, lungs infection, Lagenaria siceraria Standl Bukhari, Kitab ul Tamamiah [34] 6 Cucurbitaceae common cold, kidney and liver disorder and (Yakteen, Daba) Ibn e Maja, Chap Bab ul Daba[37] heart diseases. Ibn e Maja. Ravi: Anas. Kitabul-athama [36]
Lens culinaris Medic Holy Quran Holy Quran, Verse #. 61, Surah Al Baqra [33]
(Adas) Maleness, measles, paralysis, common cold, Al-Jozi (Aljawziyya), Ibn-ul Qayyim, Tibb-e-Nabvi (Urdu Tans. 7 Papilionaceae parkensis, face clearness, eye infection etc by Hakim Azizur Rehman A’zmi and Mukhtiar Ahmad Nadvi)
[39]
Holy Quran Verse #. 12, 13, Surah Al Rahman [33] Fever, cough, eczema, baldness, arthritis, Ocimum basilicum L. Bukhri. Ravi: Abu Musa Al Asharii [34] 8 Lamiaceae antidote, pain killer, tuber closes, asthma, piles, (Rehan) Trimzi (Bab ul Tib)[38] hepatitis, malaria, heart diseases etc Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad [36]
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Holy Quran Verse #.191, Surah Alanam; verse #. 99, Surah
Strengthen body muscles, slow down aging, Alanam; verse #. 11, Surah Alnahal; verse #. 35, Surah Alnnor;
Olea europea L. clear the blood, remove the measles spot, piles, verse #. 1-4, Surah Teen [33] 9 Oleaceae (Zaiytoon) tuberculosis, eczema, baldness, kidney, Bukhri, Ravi: Khalid Bin Sahad [34]
pancreas pain, etc. Trimzi, Abwab ul Tamah [38]
Ibne Majja, Ravi; Zahid Bin Arkum[37]
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; Heart diseases, skin diseases, antidote, swelling verse #. 23, 25, Surah Mariam; verse #.148, Surah Shurah; verse Phoenix dactylifera L of kidney, intestinal pain, heart attack, wound #. 71, Surah Taha; verse #. 34, Surah Yaseen; verse #. 60, Surah 10 (Nahal, Balah, Tammar, Arecaceae healer, diarrhea, labour pain, sexual weakness, Al Qamar; verse #. 11-28, Surah Rahman; verse #. 7, surah Al Rutab, etc) stomach pain, piles, etc. Haqqa; verse #. 39, Surah Abbus [33]
Ibne Majja. Ravi-Bussar (R.A)[37]
Trimzi [38]
Bukhri. Ravi – Ans Bin Malik [34]
Stomach cough, hepatitis, muscle pain, heart Holy Quran Verse #. 99, Surah-Al Anam; verse #. 141, Surah Al Punica granatum L. and liver diseases, piles, eye diseases, 11 Punicaceae Anam; verse #. 69, Surah Rehman [33] (Rumman) dental problems, oral diseases, diarrhea and Al-Jozi (Aljawziyya), Ibn-ul Qayyim. Zadul Maad[36] dysentery.
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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.
14 | Page Introduction
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].
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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. Anticholinergic Camptothecin Indole alkaloid Camptotheca acuminatea Antineoplastic Capsaicin Phenyl arrine alkaloid Cupsicum spp. Topical analgesic Codeine, morphine Opium alkaloid Papaer somniferum Analgesic, antitussive Cocaine Cocain alkaloid Erythroxylum coca Local anesthetic Colchicines Isoquinoline alkaloid Colchicum autumnale Antigout Emetine Isoquinoline alkaloid Cephaelis ipecacuanha Antimoebic Galanthamine Isoquinoline alkaloid Leucojum aestivum Cholinesterase inhibitor Nicotine Pyrollidine alkaloid Nicotiana spp. Smoking cessation therapy Physostigmine Indole alkaloid Physostigma venenosum Cholinergic Pilocarpine Imidazole alkaloid Pilocarpus jaborandi Cholinergic Quinine Quinoline alkaloid Cinchona spp. Antimalarial Quinidine quinoline alkaloid Cinchona spp. Cardiac depressant Reserpine Indole alkaloid Rauwolfia serpentine Antipertansive, psycotropic Bisbenzyl isoquioline Chondodendron tomentosum Tubocurarine Skeletol muscles relaxant alkaloid Strychnos toxifera Vinblastine, vincristine Bis- indole alkaloid Catharantus roses Antineoplastic Aphrodisiac Yohimbine Indole alkaloid Apocynaceae, Rubiaceae spp.
16 | Page Intorduction
Terpenes and Steroids Artemisinin Sesqiterpene lactone Armetisia annua Antimalarial Diosgenin, 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 Hydroxy-anthracene Sennosides A and B. Cassia angustifoila Laxative glycosides Others Mixture of ipecac Ipecac alkaloids and other Cephaelis ipecacuanha Emetic components Podophyllotoxin Lignan Podophyllum peltatum Antineoplastic
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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].
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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.
19 | Page Introduction
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
20 | Page Introduction
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
21 | Page Introduction
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]
22 | Page Introduction
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].
23 | Page Introduction
24 | Page Introduction
25 | Page Introduction
Figure 1: Structures of the mentioned compounds
26 | Page
27 | Page Literature Review
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.
28 | Page Literature Review
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).
29 | Page Literature Review
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]
40 Buddlejoside A3 C33H42O17 710.6764 B. japonica [86]
41 Buddlejoside A4 C33H42O17 710.6764 B. japonica [86]
42 Buddlejoside A5 C33H42O17 710.6764 B. japonica [86]
43 Buddlejoside A6 C33H42O17 710.6764 B. japonica [86]
44 Buddlejoside A7 C33H42O17 710.6764 B. japonica [86]
45 Buddlejoside A8 C32H42O17 698.2422 B. japonica [86]
46 Buddlejoside A9 C34H44O18 740.7024 B. japonica [86]
47 Buddlejoside A10 C34H44O18 740.7024 B. japonica [86]
48 Buddlejoside A11 C34H44O18 740.7024 B. japonica [86]
49 Buddlejoside A12 C33H42O18 726.6758 B. japonica [86]
50 Buddlejoside A13 C46H56O24 992.9224 B. japonica [86]
51 Buddlejoside A14 C47H58O24 1006.949 B. japonica [86]
52 Buddlejoside A15 C46H56O24 992.9224 B. japonica [86]
53 Buddlejoside A16 C46H56O24 992.9224 B. japonica [86]
54 6-Vanillyajugol C23H30O12 498.4771 B. japonica [86]
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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]
59 Neolignans 2 C35H44O16 720.64 B.devidi [89]
60 Neolignans 3 C35H44O16 720.78 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- C20H28O3 316.4345 B. yunenesis [93]
abietatrien-7-one
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]
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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- C30H45O3 453.6765 B. asiatica [102]
oleanene-3-one
95 13,28-Epoxy-21β,23-dihydroxy-11- C30H46O4 470.8638 B. asiatica [102]
oleanene-3-one
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]
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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- C34H44O19 756.00 B. madagascariensis [107]
rhamnopyrunosyl)-β-D-
glucopyranosid
117 Diosmetin 7-0 (2”, 6”-di-O-α-L- C34H42O19 753.00 B. madagascariensis [107]
rhamnopyrunosyl)-β-D-
glucopyranosid
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]
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128 Jionoside D C30H38O15 638.6137 B. davidii [99]
129 2-Acetylmartynoside C33H42O16 694.6770 B. davidii [99]
130 3-Acetylmartynoside C33H42O16 694.6770 B. davidii [99]
131 4-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 C34H60O3 516.8384 B. cordata [110]
hexacosanoate
` 140 2[4 -Hydroxyphenyl]-ethyl C31H54O3 474.7587 B. cordata [110]
tricosanoate
` 141 2[4 -Hydroxyphenyl]-ethyl C33H58O3 502.8118 B. cordata [110]
pentacosanoate
` 142 2[4 -Hydroxyphenyl]-ethyl C28H48O3 432.6789 B. cordata [110]
arachidate
` 143 2[4 -Hydroxyphenyl]-ethyl C25H42O3 390.5992 B. cordata [110]
heptadecanoate
` 144 2[4 -Hydroxyphenyl]-ethyl C27H46O3 418.6523 B. cordata [110]
nonadecanoate
` 145 2[4 -Hydroxyphenyl]-ethyl behenate C30H52O3 460.7321 B. cordata [110]
` 146 2[4 -Hydroxyphenyl]-ethyl C32H56O3 488.7852 B. cordata [110]
lignocerate
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` 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- C35H59O7 591.8388 B. crispa [113]
ol-3 β-O-β-D-galactopyranoside
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]
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171 3-(4-Acetoxy-phenyl)-acrylic acid 3- C27H24O5 427.4765 B. crispa [114]
phenyl-propyl ester
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]
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Figure 2: Structures of the compounds reported from Buddleja asiatica.
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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
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2.5 Scientific classification
Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Lamiales
Family: Scrophulariaceae Genus: Buddleja Species: Buddleja asiatica Lour
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].
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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.
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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 ”.
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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.
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1 The H-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.
13 The C- 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
1 13 respectively. “The chemical shifts of the various carbons and H/ C connectivities ” are shown in
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Table - 7. The comparison of spectral data with those reported in the literature [97] established the identity of the compound as buddlejone (180).
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13 1 13 Table 7: C- NMR (CDCl3, 75 MHz) and H/ C correlations of buddlejone (180).
Multipilicity C. No Chemical shift () 1H/13C Connectivity (= Hz) DEPT
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)
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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,
-1 -1 1738 cm and 2955 cm 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 %).
1 The H-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.
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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.
13 C-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).
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13 1 13 Table 8: C- NMR (CDCl3, 75 MHz) and H/ C correlations of dihydrobuddledin A (181).
Multipilicity C.No 13C- NMR () 1H/13C Connectivity (= Hz) DEPT
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)
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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].
1 The H-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.
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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.
13 C-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).
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13 1 13 Table 9: C- NMR (CDCl3, 75 MHz) and H/ C correlations of buddledone B (182).
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
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)
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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
12 pattern, a characteristic of Δ - 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.
1 “The H-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 -
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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
1 13 carbon atoms and H/ C 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)”.
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13 1 13 Table 10: C- NMR (CDCl3, 75 MHz) and H/ C correlations of ursolic acid (183)
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
1 CH2 38.4 0.96 (1Hα, m), 1.54 (1Hβ, m)
2 CH2 27.4 1.02 (1Hα, m), 1.83 (2H, m)
3 CH 79.1 2.99 (1Hα, dd, J = 10.20 Hz, 4.5 Hz)
5 CH 52.4 0.85 (1Hα, m)
6 CH2 18.1 1.35 (1Hβ, m), 1.56 (1Hα, m)
7 CH2 32.6 1.36 (1Hα, m), 1.55 (1Hβ, m)
9 CH 48.4 1.62 (1H, m)
11 CH2 23.7 1.90 (1Hα, m) , 1.95 (1Hβ, m)
12 CH 124.8 5.10 (1H, m)
15 CH2 28.4 1.20 (1Hα, m), 2.32 (1Hβ, m)
16 CH2 22.5 2.11 (1Hα, m), 2.00 (1Hβ, m)
18 CH 54.2 2.63 (1Hβ, d, J = 11.3 Hz)
19 CH 30.2 1.46 (1Hα, m)
20 CH 30.1 0.99 (1Hβ, m)
21 CH2 27.6 1.36 (1Hα, m), 1.45 (1Hβ, m)
22 CH2 36.6 1.97 (2H, m)
23 CH3 24.0 1.05 (3H, s)
24 CH3 14.7 0.84 (3H,s)
25 CH3 15.6 0.96 (3H, s)
26 CH3 16.0 1.06 (3H, s)
27 CH3 24.3 1.20 (3H, s)
29 CH3 21.4 0.79 (3H, d, J = 6.8 Hz)
30 CH3 24.0 0.90 (3H, d, J = 6.6 Hz)
66 | Page Results and Discussion
3.4.5 2-Phenylethyl-β-D-glucoside (184)
Further elution of the same column with CHCl3 - MeOH (9 : 1) afforded several fractions which showed a major spot with minor impurities. In order to purify, it was subjected to preparative TLC (silica gel) in n-hexane - EtOAc - acetone (6.5 : 2 : 1.5) furnishing a pure compound 184 as white crystals having m.p. 168-170 oC. Its molecular ion peak in mass spectrum (HR-EIMS) appeared at m/z: 284.2190 (calcd. for C14H20O6, 284.1840) with other major peaks at m/z 284 (32 %), 185 (27 %), 143 (100 %) and 157 (26 %). The IR (KBr) spectrum exhibited strong absorption at 1466 cm-1 which indicated aromatic group while absorption at 3010 cm-1 was due to the aliphatic C - H stretching vibration.
1 The H-NMR spectrum (300 MHz) in CDCl3 showed multiplets for five aromatic protons in the region between δ 7.16 - 7.28 (H – 2 to H - 6). The C - 7 methylenic protons appeared as triplet at 2.85 (2H, t, J = 6.5 Hz) and the other C - 8 methlyenic protons Hα and Hβ resonated as multiplets centered at 3.64 and 3.93 respectively. A methine proton of Glc - 1΄ resonated as doublet at 4.17 (1H, d, J = 8.0 Hz) while the other protons of sugar moiety resonated as multiplets, centered at 3.30 - 3.50 (6H, m, H - 2΄ to H - 6΄).
67 | Page Results and Discussion
13 The C-NMR (75 MHz, CDCl3) spectrum of this compound showed the presence fourteen carbon atoms. The C - 7 and C - 8 methylenic carbons showed resonance at 36.35 and
70.16 while the methine carbon of (C - 1΄) was found at 103.55. The six aromatic carbon signals resonated at 139.42 (C - 1), 128.91 (C - 2, C - 6), 129.60 (C - 3, C - 5) and 126.75
(C - 4) respectively. Other signals were at 61.79 (C - 6΄), 70.80 (C - 4΄), 74.13 (C - 2΄), 77.48
(C - 5΄) and 77.60 (C - 3΄). 1H/13C correlation were determined by using HMQC technique [128].
Table – 11 enlists the chemical shifts of various carbons as well as 1H/13C connectivities.
All the spectral data coincided with the reported data [131] and established the identity of the compound as 2-phenylethyl-β-D-glucoside (184).
68 | Page Results and Discussion
13 1 13 Table 11: C- NMR (CDCl3, 75 MHz) and H/ C correlations of 2-phenylethyl-β-D-glucoside (184).
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
2 CH 128.91
3 CH 129.60
4 CH 126.75 7.16 -7.28 (5H, m)
5 CH 129.60
6 CH 128.91
7 CH2 36.35 2.85 (2H, t, J = 6.5 Hz),
8 CH2 70.16 3.64 (1H, m), 3.93 (1H, m),
1΄ CH 103.55 4.17 (1H, d, J = 8.0 Hz, Glc-1΄ )
2΄ CH 74.13
3΄ CH 77.60
4΄ CH 70.80 3.30 - 3.50 (6H, m)
5΄ CH 77.48
6΄ CH2 61.79
69 | Page Results and Discussion
3.4.6 7- Deoxy-8-epiloganic acid (185)
“Further elution of the same column with CHCl3 - MeOH (7.5 : 2.5) yielded several
fractions. The first few fractions showed similar spots on TLC, were mixed and concentrated. It
was re-chromatographed over silica gel eluting with the mixtures of CHCl3 - MeOH in
increasing order of polarity. The fraction obtained from CHCl3 - MeOH (7.8 : 2.2) was found to
contain a major with some minor spots on TLC was further purified by preparative TLC on
silica plates in n-hexane - EtOH - diethylamin (6.5 : 3.5 : 2 drops) afforded a pure compound
185, as white amorphous powder, showed m.p. 118 - 120 oC. The mass spectrum of the
compound exhibited molecular ion peak in HR-EIMS at m/z: 374.3854 which agreed with the
mass calculated for the formula C16H22O10 (374.3734) and other major peaks at m/z 346
(32 %), 343 (24 %), 330 (52 %), 211 (100 %) and 179 (69 %). The IR (KBr) spectrum showed
prominent absorption at 3455 cm-1 for hydroxyl group, 1687 cm-1 indicated the presence of
-1 enolic group while the absorption at 1633 cm showed the presence of vinylic group”.
1 The H-NMR (300 MHz) in CDCl3 showed the presence of hemiacetal proton (C - 1) as a doublet at 5.15 (1H, d, J = 9.6). It was found to be coupled with C - 9 proton, which in turn
70 | Page Results and Discussion revealed a double doublet at 2.18 (1H, dd, J = 7.4 Hz and 9.6 Hz). A broad singlet was observed at 7.22 which was attributed to the olefinic proton (C - 3) while a multiplet centered for methine proton (C - 5) was observed at 2.81. A downfield multiplet centred at 3.29 was assigned to
C - 7 proton (1H, m). The C - 6 methylenic protons (Hα, β) resonated as double doublets for each at
1.41 (dd, J = 10.3 Hz, 13.8 Hz, Hα) and 2.63 (dd, J = 7.5 Hz, 13.8 Hz, Hβ) while a singlet was assigned at 1.63 for C - 10 methyl protons. In glycone part, one proton doublet at 4.81 (1H, d,
J= 7.9 Hz) was assigned to the C - 1΄ proton, while four protons resonated as complex multiplets in the region between 3.19 - 3.44 (4H, m, H - 2΄ and H - 5΄). The C-6΄ hydroxyl methylene protons (Hα, β) resonated as double doublets at 3.60 (1H, dd, J = 5.8 Hz, 11.9 Hz, Hα - 6΄) and
3.93 ((1H, dd, J = 5.8 Hz, 11.9 Hz, H β - 6΄).
13 C-NMR (75 MHz) in CDCl3 spectrum exhibited signals for all 16 carbons. The six glycone carbons resonated at 99.9 (C - 1΄), 75.6 (C - 2΄), 78.3 (C - 3΄), 72.1 (C - 4΄), 78.7
(C - 5΄), and 63.2 (C - 6΄) while the aglycone carbons were assigned as one methyl at 18.5
(C - 10), one methylenic at 37.1 (C - 6), five methine at 32.8 (C - 5), 45.6 (C - 9), 64.0 (C - 7),
95.9 (C - 1), 148.2 (C - 3), one quaternary at 115.7 (C - 4) and one carboxylic at 174.1. The chemical shifts of various carbon atoms and 1H/13C correlations are presented in Table - 12. All the spectral data showed complete resemblance with those reported in the literature [132] established the identity of 7-deoxy-8-epiloganic acid (185).
71 | Page Results and Discussion
13 1 13 Table 12: C- NMR (CDCl3, 75 MHz) and H/ C correlations of 7- deoxy-8-epiloganic acid (185)
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
1 CH 95.9 5.15 ( 1H, d, J= 9.6)
3 CH 148.2 7.22 ( 1H, s)
5 CH 32.8 2.81 ( 1H, m)
1.41 (1Hα , dd, J = 10.3, 13.8 Hz) 6 CH2 37.1 2.63 (1Hβ, dd, J =7.5, 13.8 Hz)
7 CH 64.0 3.29 (1H, m)
9 CH 45.6 2.18 (1H, dd, J = 7.4, 9.6 Hz)
10 CH3 18.5 1.63 (1H, s)
1΄ CH 99.9 4.81 (1H, d, J = 7.9 Hz) 2΄ CH 75.6
3΄ CH 78.3 3.19 - 3.44 ( 4H, m) 4΄ CH 72.1
5΄ CH 78.7
3.66 ( 1Hα, dd, J = 5.8, 11.9 Hz) 6΄ CH2 63.2 3.93 ( 1Hβ, dd, J = 1.9, 11.9 Hz)
72 | Page Results and Discussion
3.4.7 Scutellarin-7-O-β-D-glucopyranoside (186)
The fraction FD obtained from the chloroform soluble fraction was concentrated and subjected on a silica gel column and eluted with increasing polarities of CHCl3 - MeOH. The eluates obtained from CHCl3 - MeOH (9.0 : 1.0) were combined, concentrated and subjected to column chromatography over silica gel (TLC grade). Elution was carried out in increasing polarity of CHCl3 - MeOH mixtures which yielded several fractions. The fractions obtained on elution with CHCl3 - MeOH (9.5 : 0.5) showed similar spots on TLC, were combined, concentrated and subjected to preparative TLC in n-hexane – EtOAc - diethylamin (5.5 : 4.5 : 2 drops) to obtain 186 as yellow powder which showed a sharp melting point 184-185 oC. The molecular ion peak in mass spectrum of the compound appeared at m/z 448.3264 which corresponded to the composition C21H20O11 (448.3322). Other major peaks appeared at m/z 417
(100 %), 404 (23 %), 285 (64 %), 331 (25 %), 179 (55 %) and 118 (38 %). The IR (KBr) spectrum revealed strong absorption at 3250 and 1648 cm-1, indicated the presence of hydroxyl and carbonyl groups, respectively while the UV spectrum (MeOH, λmax) displayed two characteristic flavone bands at 381 and 265 nm [133-134]. The acid hydrolysis of the compound yielded D-glucose, confirmed the flavonoid glucoside.
73 | Page Results and Discussion
1 The H-NMR (300 MHz, CDCl3) spectrum showed the presence of four aromatic protons
resonated as multiplets centered at 7.12 (H - 2΄ & H - 6΄) and 7.64 (H - 3΄ & H - 5΄).
The C - 3 methine proton appeared as a singlet at 6.58 (1H, s). The C - 8 methine proton
resonated as doublet at 6.70 (1H, d, J= 3.0 Hz) while the methine proton of anomeric carbon
(C - 1΄΄) of glycone part resonated as doublet at 5.4 (1H, d, J = 7.5 Hz) and other four methine
protons of glycone unit resonated as multiplets at 3.86 (H - 2΄΄), 3.59 (H - 3΄΄), 4.52 (H - 4΄΄)
and 3.38 (H - 5΄΄) respectively. The C - 6΄΄ hydroxyl methylene protons (Hα, β) resonated as
double doublets at 3.78 (1H, J = 5.6 Hz, 11.7 Hz, Hα - 6΄΄) and 3.89 (1H, J = 2.1 Hz, 11.8 Hz,
Hβ - 6΄΄).
13C-NMR and DEPT spectra showed 21 signals, 6 signals were assigned for sugar moiety resonated at 62.6 (C - 6΄΄), 70.6 (C - 5΄΄), 74.6 (C - 4΄΄), 77.8 (C - 3΄΄), 78.2 (C - 2΄΄) and 102.3
(C - 1΄΄). On the other hand, 15 signals were attributed to the flavone part. The 6 carbon signals of phenyl ring were assigned at 122.4 (C - 1΄), 129.44 (C - 2΄& C - 6΄), 118.0 (C - 3΄ & C - 5΄) and at 163.36 (C - 4΄) respectively. The carbons of benzo-pyran moiety resonated at 164.8
(C - 2), 104.5 (C - 3), 146.5 (C - 5), 132.7 (C - 6), 152.8 (C-7), 96.6 (C - 8), 150.6 (C - 9) and
108.2 (C - 10). The downfield signal for carbonyl carbon resonated at 185.2 (C - 4). The chemical shifts of the various carbon atoms and 1H/13C correlations are presented in Table - 13.
The spectral data was in a good agreement with the reported data in literature [107] which established the identity of the compound as secutellarin-7-O-β-D-glucopyranoside (186).
74 | Page Results and Discussion
13 1 13 Table 13: C- NMR (CDCl3, 75 MHz) and H/ C correlations of scutellarin-7-O-β-D-glucopyranoside (186)
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
3 CH 104.5 6.58 (1H, s)
8 CH 95.6 6.70 ( 1H, d, J = 3.0 Hz)
2΄ CH 129.44 7.12 (2H, d, J = 9.0 Hz)
3΄ CH 118.0 7.64 (2H, d, 9.0 Hz)
5΄ CH 118.0 7.64 (2H, d, 9.0 Hz)
6΄ CH 129.44 7.12 (2H, d, J = 9.0 Hz)
1΄΄ CH 102.3 5.04 (1H, d, J = 7.5)
2΄΄ CH 74.6 3.86 (1H, m)
3΄΄ CH 77.8 3.59 (1H, m)
4΄΄ CH 70.6 4.52 ( 1H, m)
5΄΄ CH 78.2 3.38 ( 1H, m)
6΄΄ CH2 62.6 3.78 (2H, m)
75 | Page Results and Discussion
3.4.8 Antimicrobial activities of the compounds 180-186
Antibacterial activity:
The antibacterial study of compounds 180 - 186 was tested against six bacteria viz.,
Proteus vulgaris, Klebsiella pneumonia, Salmonella typhi, Escherichia coli, Streptococcus pyogenes and Staphylococcus aureus according to the literature protocol [105, 135]. Tetracycline was used as standard drug in this bioassay and the results were compared with those of tetracycline. It was indicated from the data (Table - 14) that compounds 184 - 186 were almost same and showed high activity whereas compounds 180 - 183 exhibited low activity in killing the
P. vulgaris, S. typhi and E. coli. It was further observed that compounds 184 - 186 were found to possess low activity against K. pneumonia, S. pyogenes, and S. aureus while compounds 180 - 183 revealed weak or inactive.
76 | Page Results and Discussion
Table 14: Antibacterial bioassay of compounds 180-186 from Buddleja asiatica.
Compounds a and their zone of Microorganism Standard Drug inhibition b Bacteria 180 181 182 183 184 185 186 Tetracycline
Proteus vulgaris 08 05 06 09 19 21 20 32
Klebsiella pneumoniae 00 00 00 00 08 10 09 29
Salmonella typhi 09 06 05 010 19 20 19 30
Escherichia coli 08 06 07 08 17 19 18 29
Streptococcus pyogenes 00 00 00 00 08 10 09 28
Staphylococcus aureus 04 00 00 03 09 11 11 30
Note: a = Concentration used 100 µL, b = Zone of inhibition in mm.
Incubation period = 8 hour, 37 oC; Colony Forming Unit = 104- 106; Size of Well = 5 mm radius.
77 | Page Results and Discussion
Antifungal activity:
Compounds 180 - 186 were tested for their antifungal activity against six fungi according to the standard procedure [136]. The results obtained were evaluated to those obtained for the reference drug, miconazole. The data indicated (Table - 15) that the compounds 184 - 186 were the most active whereas compounds 180 - 183 exhibited moderate to low activity against T. longifusus, C. albicans, M. canis, and C. glaberata. In case of A. flavus and F. solani, moderate activity was showed by compounds 184 – 186. The compounds
180, 183 showed low activity while compounds 181 and 182 were found to be inactive against these fungi.
78 | Page Results and Discussion
Table 15. Antifungal activity of compounds 180 - 186 from B. asiatica.
Compounds a and their zone of Microorganism Standard Drug inhibition b Fungi 180 181 182 183 184 185 186 Miconazole
Trichophyton longifusus 16 6 8 14 24 22 25 30
Candida albicans 14 7 7 13 25 23 23 30
Aspergillus flavus 08 2 3 7 18 17 17 25
Microsporum canis 14 6 7 15 24 23 24 30
Fusarium solani 7 0 2 6 18 17 18 25
Candida glaberata 12 7 6 11 21 20 20 25
Note: a = Concentration used 100 µL, b = Zone of inhibition in mm,
Incubation period = 8 hour, 37 oC; Colony Forming Unit = 104- 106; Size of Well = 5 mm radius.
79 | Page Results and Discussion
3.5 Ethyl acetate soluble fraction
The ethyl acetate soluble fraction (F3) was re – chromatographed over silica gel and the elution was carried out with mixtures of n-hexane - EtOAc and EtOAc - EtOH in increasing polarity to obtain ten fractions (FA - FJ). These fractions were again loaded to a series of column chromatographic separations, which resulted in the isolation of five compounds, including a fatty acid (lignoceric acid, 187), a novel sterol ((24S)-stigmast-5, 22-diene-7β-ethoxy-3β-ol,
188), two benzoic acid derivatives (asiatoate A, 189) and asiatoate B, 190) and one flavonoid glycoside (chrysoeriol-7-O-β-D-glucopyranoside, 191) were isolated and characterized for the first time form this plant.
80 | Page Results and Discussion
3.6 Structural elucidation of compounds
3.6.1 Lignoceric acid (187)
The fraction FB obtained from n-hexane - EtOAc (6 : 4) was re-chromatographed over silica gel, elution being carried out in increasing polarity of n-hexane - EtOAc and EtOAc - EtOH mixtures. The fractions afforded from n-hexane - EtOAc (3 : 7) showed a major spot on TLC. It was purified in solvent mixture n-hexane - EtOAc (3.6 : 6.4) by preparative TLC on silica plates to afford white amorphous powder as lignoceric acid (187) showed m.p. 81-83 0C. The mass
+ spectrum of this compound showed [M - H] at m/z: 367.3 (calcd. for C24H48O2, 368.07) with other major fragmentation pattern which bore a distinct resemblance to that reported for mass spectra of typical fatty acids [137]. The IR spectrum revealed strong absorption at 3308.65 cm–1 (OH), 2955 cm–1 (C - H starch) and 1699 cm–1 (carbonyl).
1 H-NMR spectrum (600 MHz, CDCl3) exhibited a three proton triplet at 0.88
(3H, J = 8.3 Hz, H - 24) was assigned to the methyl group, coupled to the methlyenic protons
(C - 23). A two proton triplet 2.34 (2H, J = 4.5 Hz, H - 2) was assigned for two methylenic protons while two protons multiplets for the straight chain methlyenic protons were observed in the region from 1.24 to 2.34. The correlations were strongly supported by 1H-1H-COSY spectrum.
81 | Page Results and Discussion
13 C-NMR (150 MHz) spectrum in CDCl3 of this compound exhibited 24 signals which indicated one methyl carbon at 24.8, twenty two methylenic carbon atoms were found to occur in the region between of 33.9 to 29.5 and a downfield signal of the carbonyl carbon resonated at
178.7 (C=O).
The chemical shifts of the various carbons and 1H/13C connectivities are listed in
Table - 16. All these spectral data showed complete agreement with those reported in the literature
[138-139], the compound was identified as lignoceric acid (187)
82 | Page Results and Discussion
Table 16: 13C-NMR (150 MHz) and 1H/13C correlations of lignoceric acid (187)
Multipilicity 13C- NMR () 1H/13C Connectivity C. No DEPT ( = Hz)
2 CH2 33.9 2.34 (2H, t, J = 4.5 Hz)
3 CH2 24.7 1.63 (2H, m)
CH2 4 29.8 1.24 (2H, m)
5-17 CH2 29.8 (13) 1.24 (26H, m)
18 CH2 29.1 1.24 (2H, m)
19 CH2 29.3 1.24 (2H, m)
20 CH2 29.4 1.24 (2H, m)
21 CH2 29.5 1.24 (2H, m)
22 CH2 24.8 1.24 (2H, m)
23 CH2 22.8 1.31 (2H, m)
24 CH3 14.26 0.88 (3H, t, J = 8.3 Hz) - - - 11.0 ( OH, s)
83 | Page Results and Discussion
3.6.2 (24S)-stigmast-5, 22-diene-7β-ethoxy-3β-ol (188)
Further elution of the same column with mixtures of n-hexane - EtOAc and EtOAc -
EtOH yielded several fractions. The fractions obtained with EtOAc - EtOH (9.5 : 0.5, 9.0 : 1.0) showed same results on TLC, were combined and again subjected to column chromatography on TLC grade silica gel. The repeated elution of the column with gradually increasing polarity with EtOAc – EtOH, the first few fractions obtained from (9.4 : 0.6) showed a major spot with some impurities. These were combined and were subjected to preparative TLC in EtOAc - diethylamine (100 %: 2 drops) as a solvent system yielded 188 as amorphous powder showed m.p. 144 – 145 0C. It exhibited positive “Liebermann-Burchard test" as identification of sterols
[140]. The mass spectrum in HR-EIMS afforded the molecular ion at m/z 456.4065 consistent with the formula C31H52O2 (calcd. 456.4124). Hence compound 188 C31H52O2 possessed six degrees of unsaturation. Five of these were eventually accounted for the tetracyclic α β unsaturated skeleton and one for the olefinic double bond in the chain. Other major peaks were
+ + observed at “m/z 438 (10 %) [M - H2O] , 410 (100 %) [M - CH3CH2OH] , 315 (13 %) [M -
+ + + C10H21] , 269 (9 %) [M - C10H21 - CH3CH2OH] and 141 (11 %) [M - C21H31O2] ” (Figure - 4).
84 | Page Results and Discussion
The ion at m/z 438 and 410 indicated a hydroxyl and ethoxy group in the molecule. The ion at m/z 315 (C21H31O2) indicated stigmastane skeleton as tetracyclic α β unsaturated structure, which possessed the presence of five unsaturation, was exactly the same to that of
“(24S)-stigmast-5-ene-7β- ethoxy-3β-ol)” (192) [141]. The ion at m/z 141 (C10H21) could arise by the cleavage of C-20/ C-17 bond, was in accordance to that reported in literature for stigmasterol [142]. It was also closely resembled to the known ion at m/z 143 (C10H23) obtained from (192) but the main difference was the lack of 2H and addition of double bond at C-22/C-
23 in compound 188.
-1 The IR spectrum (CHCl3) showed intense absorption at 3480 cm , indicating the presence of hydroxyl group while the absorption at 3046 and 1693 cm-1 consistent with the presence of aliphatic CH stretching and olifenic bond in the structure.
The 1H and 13C-NMR pattern of signals was very similar to that provided by 192 [141], a part from the absence of signal in 188 that corresponded to those of C - 22 and C - 23 in 192.
However compound 188 showed two extra methine carbons (C - 22, C - 23) at 138.33 and
131.62 compared with 192. It proved that the compound 188 considered analogues to “(24S)- stigmast-5-ene-7β- ethoxy-3β-ol” by addition of a double bond between C-22 and C-23. The presence of an additional double bond in the chain confirmed the sixth unsaturation in the molecule 188.
1 Other signals in H-NMR (600 MHz, CDCl3) displayed two mutually coupled olefinic protons (H - 22 and H - 23) in 188 resonated as two double doublets at 5.16 (1H, J22, 23= 9.4
Hz, J22, 20 = 3.4 Hz) and 5.02 (1H, J23, 22 = 10.2 Hz, J23, 24 = 3.4 Hz) while these shifts value are shown up field in 192.
The H - 22 and H - 23 protons showed vicinal coupling in 1H-1H COSY spectrum in which H - 22 methine proton coupled with H - 23 ( 5.02) and H - 20 ( 2.33) protons while
H - 23 methine proton showed coupling with H - 24 ( 2.15) and H - 22 ( 5.16) protons.
85 | Page Results and Discussion
The methyl protons of the C - ethoxyl side chain appeared as a triplet at 1.18
(3H, t, J = 6.8 Hz) while the methylene protons of C - ethoxyl group resonated as two multiplets centered at 2.87 and 2.66 respectively.
The C - 1α proton resonated at 1.6 as a multiplet and was observed to be coupled with
C - 1β proton at 2.64, C - 2α proton at 1.84 and C - 2β proton at 1.76. The C - 1β proton also resonated as a multiplet at 2.64 and was coupled with C - 1α, C - 2α and C - 2β protons respectively.
A methine proton at C - 3 resonated downfield as multiplet at 3.45, showed coupling with C - 2 α & β protons ( 1.84 and 1.76) and C - 4 α & β ( 2.02, 2.23) while methlyenic protons
(4α & 4β) exhibited geminal and vicinal coupling resonated as two double doublets at 2.02
(1H, J 4α, β = 12.6, J 4α, 3 = 3.50 Hz) and 2.23 (1H, J 4β, α =12.6, J 4 β, 3 = 3.22 Hz). The C - 7 methine proton resonated as a double doublet at 3.30 (1H, J 7, 6 = 10.2 Hz, J 7, 8 = 3.4 Hz) showed vicinal coupling with C - 6 and C - 8 proton. A doublet at 5.32 (1H, J 6, 7 = 10.6 Hz) was assigned to the olefinic proton at C - 6 and was seen to be coupled with C - 7 proton while another methine proton at C - 8 exhibited a multiplet at 2.86. The methlyenic protons at C - 11
α & β ( 1.27 & 1.22), C - 12 α & β ( 1.45 & 1.46), C - 15 α & β ( 1.6 & 1.7) and C - 16 α & β ( 2.1
& 1.9) also resonated as multiplets.
The two methyl group protons resonated as singlets at 1.08 (3H, s, H - 18) and 1.28
(3H, s, H - 19) while four methyl group protons resonated as doublets at 0.91
(3H, d, J = 7.7 Hz, H - 21), 0.82 (3H, d, J = 6.6 Hz, H - 26), 0.84 (3H, d, J = 6.6 Hz, H - 27) and 0.90 (3H, d, J = 7.2 Hz, H - 29) respectively.
13 The C - NMR spectrum in CDCl3 (broad band and DEPT) [143] showed signals for all thirty one carbons including seven methyl, nine methylenic, twelve methine and three quaternary carbons (Table - 17). All the assignments of 13C resonance signals were made by comparison with 13C-NMR spectrum of 192 [141] and stigmasterol [142]. These signals
86 | Page Results and Discussion showed close similarities to that reported. A notable feature was the appearance of downfield signals of the C - 22 and C - 23 which substantiated the presence of the double bond in the chain part. The olefinic C - 22 resonated at 138.33 while the C - 23 showed a signal at
131.62.
The methyl and methylene carbons of ethoxyl side chain resonated at 15.9 and 64.2 while the six methyl carbons resonated at 12.17 (C - 18), 19.53 (C - 19), 19.1 (C - 21), 20.2
(C - 26), 20.2 (C - 27) and 12.2 (C - 29) respectively. The four vinyllic carbons resonated at
140.86 (C - 5), 121.84 (C - 6), 138.33 (C - 22) and 131.62 (C - 23) respectively. A downfield methine carbon signal resonated at 71.94 was assigned to the hydroxy carbon (C - 3) as supported by HMQC correlation H - 3 ( 3.45). The one bond HMQC correlations (1H/13C) are shown in Table - 18. Further more in HMBC spectrum, the location of ethoxy group at C - 7
3 was confirmed as the methlyenic protons of ethoxy group (H = 3.29) showed a J correlation to
C – 7 (C = 82.5). Other important HMBC interactions include correlations of proton at 3.45
(H - 3) to the carbons at 29.06 (C - 1) and 140.86 (C - 5) while the proton at 5.32 (H - 6) demonstrated correlation to the carbons at 37.36 (C - 4), 51.56 (C - 8) and 34 (C -10), confirming the A, B ring connectivity. The proton at 5.16 (H - 22) illustrated correlation to the carbons at 56.5 (C - 17), 19.1 (C - 21) and 42.33 (C - 24) which established the connectivity of side chain to the cyclopentane ring of sterol. The proton at 5.02 (H - 23) showed correlation with carbons at 36.2 (C - 20), 30.6 (C - 25), 25.4 (C - 28) while it also showed 1H-1H-COSY relationship with protons at 1.86 (H - 25), and 5.16 (H - 22)
(Figure - 5). On the basis of spectroscopic data and comparison evidence, the structure of 188 was established as (24S)-stigmast-5, 22-diene-7β-ethoxy-3β-ol.
87 | Page Results and Discussion
= 315
= 141
HO OCH CH
= 438 = 410
Important mss spectral peaks in HR-EIMS of
88 | Page Results and Discussion
89 | Page Results and Discussion
13 Table 17: C- NMR (CDCl3, 150 MHz) chemical shifts and multiplicities of (24S)-stigmast-5,22-diene-7β-ethoxy-3β-ol (188)
Multipilicity Multipilicity C. No 13C- NMR () C. No 13C- NMR () DEPT DEPT
1 CH2 29.06 16 CH2 28.6
2 CH2 32.00 17 CH 56.5
3 CH 71.94 18 CH3 12.17
4 CH2 37.36 19 CH3 19.53
5 C 140.86 20 CH 36.2
6 CH 121.84 21 CH3 19.1
7 CH 82.5 22 CH 138.33
8 CH 51.36 23 CH 131.62
9 CH 50.15 24 CH 42.33
10 C 34.00 25 CH 30.6
11 CH2 21.2 26 CH3 20.2
12 CH2 39.7 27 CH3 20.2
13 C 42.33 28 CH2 25.4
14 CH 55.0 29 CH3 12.2
15 CH2 25.8 7-O-CH2-CH3 CH2 64.2
7-O-CH2-CH3 CH3 15.9
90 | Page Results and Discussion
13 1 13 Table 18: C- NMR (CDCl3, 150 MHz) and H/ C correlations of (24S)-stigmast-5, 22-diene-7β-ethoxy-3β-ol (188)
Multipilicity C. No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
1 CH2 29.06 1.6 (1Hα, m), 2.64 (1Hβ, m)
2 CH2 32.00 1.84 (1Hα, m), 1.76 (1Hβ, m)
3 CH 71.94 3.45 (1H, m)
2.02 (1Hα, dd, J = 12.6, 3.50 Hz) 4 CH2 37.36 2.33 (1Hβ,dd, J = 12.6, 3.22 Hz)
6 CH 121.84 5.32 (1H, d, J = 3.4 Hz)
7 CH 82.5 3.30 (1H, dd, J = 10.2, 3.4 Hz)
8 CH 51.36 2.86 (1H, m)
9 CH 50.15 1.44 (1H, m)
11 CH2 21.2 1.27 (Hα, m), 1.22 (Hβ, m)
12 CH2 39.7 1.45 (1Hα, m), 1.46 (1H β, m)
14 CH 55.0 1.40 (1H, m)
15 CH2 25.8 1.6 (1H α, m), 1.7 (1H β ,m)
16 CH2 28.6 2.1 (1H α , m), 1.9 (1H β , m)
17 CH 56.5 1.51 (1H, m)
18 CH3 12.17 1.08 (3H, s)
19 CH3 19.53 1.28 (3H, s)
20 CH 36.2 2.33 (1H, m)
21 CH3 19.1 0.91 (3H, d, J = 7.7 Hz)
22 CH 138.33 5.16 (1H, dd, J = 15.12, 8.59 Hz)
23 CH 131.62 5.02 (1H, dd, J = 15.12, 8.59 Hz)
24 CH 42.33 2.15 (1H, m)
25 CH 30.6 1.86 (1H, m)
91 | Page Results and Discussion
26 CH3 20.2 0.82 (3H, d, J = 6.6 Hz)
27 CH3 20.2 0.84 (3H, d, J = 6.6 Hz)
28 CH2 25.4 1.44 (H α, m), 1.46 (1H β ,m)
29 CH3 12.2 0.90 (3H, t, J = 7.2 Hz)
7-O-CH2-CH3 CH2 64.2 3.29 (1H, q, J= 6.8 Hz)
7-O-CH2-CH3 CH3 15.9 1.18 (3H, t, J = 6.8 Hz)
92 | Page Results and Discussion
3.6.3 Asiatoate A (189)
The fraction FD obtained from the ethyl acetate soluble fraction with n-hexane - EtOAc
(2 : 8) was “subjected to column chromatography over silica gel. Elution was carried out with mixtures of EtOAc and EtOH in increasing order of polarity. The fractions obtained from
EtOAc - EtOH (8 : 2) showed a major spot with some impurities on TLC. They were mixed, concentrated and subjected to preparative TLC on silica plates in n-hexane – EtOAc – EtOH
(2 : 6 : 2) as a solvent system afforded a pure compound 189 as white crystals showed m.p.
157-158 0C. The IR (KBr) spectrum displayed absorption at 3051 and 1460 cm-1 indicated aromatic functionality, absorption at 1720 cm-1 was due to carbonyl group while strong
-1 absorption at 1250 cm showed methoxy group in the molecule”.
The mass spectrum and DEPT proved highly instructive in the determination of the structure 189. The HR-EIMS exhibited molecular ion peak at m/z 436.2871 which corresponded closely with the mass calculated for the molecular formula of C25H40O6 (436.5815) with other important major peaks at m/z 421 (12 %), 393 (21 %), 323 (23 %), 253 (15 %), 237 (100 %),
199 (16 %), 156 (14 %), 113 (12 %), 98 (10 %) and 84 (6 %). The molecular formula C25H40O6
93 | Page Results and Discussion indicating six degree of unsaturation. Four of them were eventually accounted for by the penta substituted benzene ring and two were due to double bonds, one for ketonic and other for ester carbonyl.
Linked scan measurements on the molecular ion exhibited that the ions at m/z 421, 393,
+ 323, 253, 237, 199 arose directly from it. These are resulted from the loss of M - CH3,
+ + + + + M - CH3CO, M - C8H17, M - C13H27, M - C13H27O and M - C12H13O5 while peaks at m/z
156, 113, 98 and 84 arose from C13H27O – C2H3O, C13H27O – C5H10O, C8H17 – CH3 and
C8H17 – C2H5 (side chain). (Figure - 6)
1 The H-NMR (300 MHz) spectrum in CDCl3 showed one proton singlet at 6.68 was assigned to the proton of aromatic region (1H, s, H - 5) showing a pentasubstituted benzene ring. A three proton singlet at 3.28 (3H, s) was ascribed to the methyl protons for each of the three methoxy groups on the ring resonated as nine proton singlet at 3.28 (9H, s) while three protons singlet at 2.50 (3H, s) was assigned to the methyl protons of acetyl group. On the basis of high chemicals shifts value and HMBC correlations, the acetyl group was at C - 4 and three methoxy at C - 2, 3 and 6 respectively.
The methine proton of C-1 resonated at 1.32 (3H, d, J=6.8 Hz) and was found to be coupled with the methine proton at C-2 which showed a quartet at 4.12 (1H, q, J = 6.8 Hz).
A triplet for three protons was observed at 0.81 (3H, t, J = 6.8 Hz) for terminal methyl group
(C - 11΄) which was coupled with methlyenic protons (C - 10΄) as multiplet at 1.29 (2H, m).
Other six methlyenic protons resonated as multiplets between the range at 1.25 - 1.29 for
C - 4΄, C - 5΄, C - 6΄, C - 7΄, C - 8΄and C - 9΄ respectively. The appearance of a peak at m/z 237
(M+ - 199) in the mass spectrum attributed to the loss of dimethylundecanyl side chain.
The 13C-NMR (75 MHz) spectrum of compound 189 showed the presence of twenty five carbon atoms while their multiplicity was determined by DEPT which indicated eight methyl, seven methylene, two methine and eight quaternary carbons. The methyl and carbonyl
94 | Page Results and Discussion carbons of the acetyl group resonated at 26.6 and 182.8 while three methoxy carbons at C - 2,
3 and 6 were found to occur at 60.8, 61.3 and 60.1 respectively. The carbonyl carbon (C - 7) resonated at 169.9 where as the side chain terminal methyl carbon (C - 11) resonated at
14.7. The two methyl carbons (C - 3) resonated at 13.5 and 13.6 while the methyl carbon
(C - 1) resonated at 14.9. The methine carbon (C - 2) was found to occur at 75.8. The aromatic carbons resonated at 120.8 (C - 1), 153.6 (C - 2), 152.0 (C - 3), 124.2 (C - 4), 93.2
(C - 5) and 152.5 (C - 6) respectively. All the 13C- NMR spectral assignments are shown in
Table - 19.
3 In HMBC spectrum, the C - 5 aromatic proton (H = 6.68) showed J H-C coupling with
C - 1 (C = 120.8), C - 3 (C = 152.0) and acetyl carbon (C = O) (C = 182.8). The methoxy
3 protons (H = 3.28) showed J H-C correlations with benzene ring carbon (C - 2, 153.6) hence showed the exact attachment point in the ring. The other methoxy protons (H = 3.28) showed
3 J H-C correlation with C - 3 and C - 6 in the ring while the C - 2 methine proton (H = 4.12)
3 showed J H-C relationship with the two methyl substituents (C - 3, C = 13.5, 13.6) as well as with the carbonyl carbon atom at position 7 (C = 169.9). The Important HMBC correlations are listed in Figure - 7. One bond 1H/13C correlations were determined by HMQC technique [144].
The carbons and protons (1H/13C) connectivity are shown in Table - 20. On the basis of these evidences the structure of asiatoate A (189) was assigned as 3´,3´-dimethylundecan-2´-yl 4- acetyl-2,3,6-trimethoxybenzoate.
95 | Page Results and Discussion
96 | Page Results and Discussion
13 Table 19: C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate A (189)
C. No. Multiplicity 13C-NMR C. No Multiplicity 13C-NMR (DEPT) () (DEPT) ()
1 -C- 120.8 7 CH2 23.8
2 -C- 153.6 8 CH2 23.9
3 -C- 152.0 9 CH2 22.8
4 -C- 124.2 10 CH2 22.4
5 CH 93.2 11 CH3 14.7
6 -C- 152.5 2 - methoxy CH3 60.8
7 C=O 169.9 3 - methoxy CH3 61.3
1 CH3 14.9 6 - methoxy CH3 60.1
2 CH 75.8 4 - C=O -C- 182.8
3 -C- 36.5 4 – CH3-C=O CH3 26.6
4 CH2 28.3 C - 3 - methyl CH3 13.5
5 CH2 25.4 C - 3 - methyl CH3 13.6
6 CH2 24.2
97 | Page Results and Discussion
13 1 13 Table 20: C- NMR (CDCl3, 75 MHz) and H/ C correlations of asiatoate A (189)
Multipilicity 1H-NMR Connectivity C. No Chemical shifts () DEPT ( = Hz)
5 CH 93.2 6.68 (1H, s)
1΄ CH3 14.9 1.3 (3H, d, J = 6.8 Hz) 2΄ CH 75.8 4.12 ( 1H, q, J = 6.8 Hz)
4΄ CH2 28.3 1.25 (2H, m)
5΄ CH2 25.4 1.25 (2H, m)
6΄ CH2 24.2 1.25 (2H, m)
7΄ CH2 23.8 1.25 (2H, m)
8΄ CH2 23.9 1.29 (2H, m)
9΄ CH2 22.8 1.29 (2H, m)
10΄ CH2 22.4 1.29 (2H, m)
11΄ CH3 14.7 0.81 (3H, t, J = 6.8 Hz)
2 - methoxy -OCH3 60.8 3.28 (3H, s)
3 - methoxy -OCH3 61.3 3.28 (3H, s)
6 - methoxy -OCH3 60.1 3.28 (3H, s)
4 – CH3 – C=O CH3 26.6 2.50 (3H, s)
C -3 - methyl CH3 13.5 1.2 (3H, s)
C - 3 - methyl CH3 13.6 1.2 ( 3H, s)
98 | Page Results and Discussion
3.6.4 Asiatoate B (190)
Further elution of the same column with mixture of EtOAc – EtOH in increasing polarity afforded several fractions. The fractions obtained with EtOAc – EtOH (7.5: 2.5) exhibited a major with several minor spots on TLC. They were mixed, concentrated and loaded to preparative TLC on silica gel plates in n-hexane – EtOAc – EtOH (2.5 : 5.0 : 2.5) as solvent system to yield crystalline compound 190, showed m.p 160-161 0C.
The IR (KBr) spectrum, of 190 showed absorption at 3403, 3060 and 1705 cm-1 which were tentatively assigned to phenolic group, aromatic functionality and carbonyl group. The spectrum also possessed an absorption at 1247 cm-1 for methoxy group.
High resolution mass measurement on the molecular ion afforded the exact mass to be
+ m/z 422.2732 (calcd. for C24H38O6, 422.5548), other major peaks at m/z 407 (9 %, M - CH3),
+ + + 379 (20 %, M - CH3CO), 309 (22 %, M - C8H17), 266 (12 %, M - C11H24), 223 (100 %,
+ + M - C13H27O), 199 (15 %, M - C11H11O5), 156 (11 %, C13H27O – C2H3O), 113 (13 %,
C13H27O – C2H3O - C3H7), 98 (12 %, C8H17 – CH3) and 84 (7 %, C8H17 – C2H5) from side chain. The overall fragmentation pattern was very similar to that of 189. The ion fragmentation
99 | Page Results and Discussion of the side chain peaks were same as 189 except the penta substituted benzene containing ion fragments at m/z 407, 379, 309, 266 and 223 which arose by the loss of 14 mass unit. The IR spectrum and 1H-NMR showed the presence of phenolic group (OH). The losses of 14 a.m.u and the presence of OH group (IR) suggested the attachment of OH group instead of methoxy at
C - 6. (Figure - 8)
1 The H-NMR (300 MHz) spectrum in CDCl3 showed only one proton singlet in the aromatic region at 6.68 (1H, s, H - 5) rendering the benzene to be highly substituted. A broad singlet at 10.30 was assigned for the hydroxyl group at C - 6. A three methyl proton for each methoxy group (C - 2, C - 3) appeared as a singlet at 3.28 (3H, s) while another singlet at
2.50 (3H, s) was assigned to the methyl protons of acetyl group.
C-2 proton exhibited a quartet at 4.12 (1H, q, J= 6.8 Hz), coupled with the methyl protons (C-1) which resonated as a doublet at 1.3 (3H, d, J = 6.8 Hz). A triplet was observed at 0.81 (3H, t, J = 1.6 Hz) for terminal methyl protons at C - 11΄ which was in turn coupled to the methlyenic protons (C - 10΄) showed multiplets at 1.29 (2H, m). Other multiplets were observed in the region between 1.25 - 1.29 for methlyenic protons of C - 4΄, C - 4΄, C - 5΄, C -
6΄, C - 7΄ and C - 9΄ respectively while a three proton singlet at 1.2 (3H, s) was assigned for each of the two methyl groups proton (C – 3).
The 13C-NMR (75 MHz) of this compound afforded twenty four signals which were confirmed by employing “DEPT pulse sequences” to confirm the presence of seven methyl, seven methylene, two methine and eight quaternary carbon atoms. The most downfield signal at
183.4 was assigned to the carbonyl carbon of acetyl group (C = O) while its methyl carbon atom resonated at 26.6. The two methoxy carbons of penta substituted benzene ring (C - 2, 3) resonated at 61.3 and 61.5 respectively. The carbonyl carbon (C - 7) was found to occur at
171.3 while the aromatic carbons resonated at 121.7 (C - 1), 154.5 (C - 2), 152.4 (C - 3),
124.3 (C - 4), 92.2 (C - 5) and 156.6 (C - 6) respectively.
100 | Page Results and Discussion
The side chain methyl carbons resonated at 14.1 (C - 11), 17.2 (C - 1), 13.8 and 13.8
(3 substituents) while the methlyenic carbons (C - 4 to 10) were found to occur in the region between 22.7 to 27.5. The methine carbon resonated at 76.6 (C - 2). The assignments are presented in Table - 21.
3 In HMBC spectrum, the C - 5 aromatic proton (H = 6.68) showed J H-C coupling with
C - 1 (C = 121.7), C - 3 (C = 152.4) and acetyl carbon at C - 4 (C = 183.4). The methoxy
3 protons (C - 2) (H = 3.28) showed J H-C correlations with the aromatic carbon (C - 2, 154.5)
3 while the C - 2 methine proton (H = 4.12) showed J H-C relationship with the two methyl substituents (C - 3, C = 17.2, 13.8) as well as with the carbonyl carbon (C - 7, C = 171.3). The important HMBC correlations have been provided in Figure - 9. One bond 1H/13C correlations were determined by heteronuclear multiple quantum correlation (HMQC) [144]. The chemical shifts of the various carbons and protons (1H/13C) connectivity are listed in Table - 22. On the basis of these evidences the structure of asiatoate B (190) was assigned as 3´,3´- dimethylundecan-2´-yl 4-acetyl-6-hydroxy-2,3-dimethoxybenzoate.
101 | Page Results and Discussion
102 | Page Results and Discussion
13 Table 21: C- NMR (CDCl3, 75 MHz) chemical shifts and multiplicities of asiatoate B (190)
C. No. Multiplicity 13C-NMR C. No Multiplicity 13C-NMR (DEPT) () (DEPT) ()
1 -C- 121.7 6 CH2 22.7
2 -C- 154.5 7 CH2 23.6
3 -C- 152.4 8 CH2 24.4
4 -C- 124.3 9 CH2 22.8
5 CH 92.2 10 CH2 22.8
6 -C- 156.6 11 CH3 14.1
7 -C- 171.3 2 - methoxy CH3 61.3
C - 1- CH3 17.2 3 - methoxy CH3 61.5 methyl 2 CH 76.6 4 – C =O -C- 183.4
3 -C- 38.8 4 – CH3 - C=O CH3 26.6
4 CH2 27.5 C - 3- methyl CH3 13.8
5 CH2 25.4 C - 3- methyl CH3 13.8
103 | Page Results and Discussion
13 1 13 Table 22: C- NMR (CDCl3, 75 MHz) and H/ C correlations of asiatoate B (190)
Multipilicity 1H-NMR Connectivity ( = C. No 13C- NMR () DEPT Hz)
5 CH 92.2 6.68 (1H, s)
1 CH3 17.2 1.3 (3H, d, J = 6.8 Hz)
2΄ CH 76.6 4.12 ( 1H, q, J = 6.8Hz)
4΄ CH2 27.5 1.25 (2H, m)
5΄ CH2 25.4 1.25 (2H, m)
6΄ CH2 22.7 1.25 (2H, m)
7΄ CH2 23.6 1.25 (2H, m)
8΄ CH2 24.4 1.29 (2H, m)
9΄ CH2 22.8 1.29 (2H, m)
10΄ CH2 22.8 1.29 (2H, m)
11΄ CH3 14.1 0.81 (3H, t, J = 6.8 Hz)
2 - methoxy -OCH3 61.3 3.28 (3H, s)
3 - methoxy -OCH3 61.5 3.28 (3H, s)
4 – CH3–C=O CH3 26.6 2.50 (3H, s)
C - 2- methyl CH3 13.8 1.2 (3H, s)
C - 2- methyl CH3 13.8 1.2 ( 3H, s)
OH - - 10.30 (1H, br s)
104 | Page Results and Discussion
3.6.5 Chrysoeriol-7-O-β-D-glucopyranoside (191)
The fraction FE obtained from EtOAc (100 %) was further chromatographed over silica gel and elution was carried out with mixture of EtOAc and EtOH in increasing polarity, yielded several fractions. The fractions obtained from EtOAc - EtOH (6.5 : 3.5) were, concentrated and re- chromatographed over TLC grade silica gel. Eluting was carried out with the mixtures of
EtOAc - EtOH in increasing polarity. The eluates obtained from EtOAc - EtOH (6.8 : 3.2) were mixed and loaded to pre preparative TLC in EtOAc - EtOH - (CH3)2CO (7 : 2 : 1) afforded compound 191 as yellow powder, mp 173-174 0C. The compound showed molecular ion peak at m/z 462.1233 (calcd. C22H22O11, 462.1240) and other major peaks at m/z 431(100 %), 418 (16 %),
299 (34 %), 345 (52 %), 179 (61 %) and 148 (10 %). The compound exhibited typical flavonoids
UV spectrum with absorption at 381 and 265 nm (MeOH, λmax) [133-134]. The IR (KBr) showed the presence of hydroxyl group at 3452 cm-1 and carbonyl group at 1648 cm-1. Upon acid hydrolysis, the compound yielded D-glucose, it was also confirmed by 1H-NMR chemical shifts.
1 “The H-NMR (300 MHz) spectrum in CDCl3 showed a singlet at 6.68 (1H, s,) for C – 3 methine proton while the other C - 8 and C - 6 methine proton resonated as a double for each at
6.49 (1H, d, J = 1.6 Hz) and at 6.84 (1H, J = 1.6 Hz). A Three protons singlet at 3.83 was attributed to the methoxy protons.
105 | Page Results and Discussion
The C - 2΄ and C - 5΄ methine protons appeared as a doublet for each at 7.74 (1H, d, J=
2.9 Hz) and 6.93 (1H, d, J = 7.6 Hz) while the other C - 6΄ methine proton resonated as a double doublet centered at 7.59 (1H, dd, J= 8.0, 2.2 Hz).
The methine proton of anomeric carbon (C - 1΄΄) of glycone part resonated as doublet at
5.4 (1H, d, J = 7.5 Hz) while other four methine protons of glycone unit resonated as multiplets at
3.86 (H - 2΄΄), 3.59 (H - 3΄΄), 4.52 (H - 4΄΄) and 3.38 (H - 5΄΄) respectively. The C - 6΄΄ hydroxyl methylene protons (Hα, β) resonated as double doublets at 3.78 (1H, J= 5.6 Hz, 11.7 Hz, Hα - 6΄΄) and 3.89 (1H, J = 2.1 Hz, 11.8 Hz, Hβ - 6΄΄).
13C-NMR and DEPT spectra revealed 22 signals, 6 were assigned for sugar moiety, 15 for flavone part and one methoxy carbon. The downfield signal for carbonyl carbon in flavone part was observed at 185.4, the phenyl ring carbons resonated at 134.3 (C - 1΄), 125.6 (C - 3΄),
117.5 (C - 2΄), 149.9 (C - 4΄) and 141.8 (C - 5΄) and 112.44 (C - 6΄).
The chemical shifts of various carbon atoms and 1H/13C correlations are presented in
Table - 23. Comparison of these spectral data with those reported in the literature [145] established
the identity of the compound as chrysoeriol-7-O-β-D-glucopyranoside (191)”.
106 | Page Results and Discussion
13 1 13 Table 23: C- NMR (CDCl3, 75 MHz) and H/ C correlations of Chrysoeriol-7-O-β-D-glucopyranoside (191).
Multipilicity C No 13C- NMR () 1H/13C Connectivity ( = Hz) DEPT
3 CH 104.5 6.68 (1H, s)
8 CH 94.3 6.84 ( 1H, d, J = 1.6 Hz)
2΄ CH 125.6 6.49 (1H, d, J = 1.6 Hz)
3΄ CH 117.5 7.74(1H, d, J = 1.3 Hz)
5΄ CH 141.8 6.93 (1H, d, J = 8.3 Hz)
6΄ CH 112.44 7.59 (1H, d, J = 8.5, 1.3 Hz)
1΄΄ CH 102.6 5.65 (1H, d, J = 7.5 Hz)
2΄΄ CH 74.4 3.86 (1H, m)
3΄΄ CH 77.2 3.59 (1H, m)
4΄΄ CH 70.4 4.52 ( 1H, m)
5΄΄ CH 78.8 3.38 ( 1H, m)
6΄΄ CH2 62.2 3.78 (2H, m)
OCH3 56.1 3.83 (3H, s)
107 | Page Results and Discussion
PART B
3.7 Activities on crude extract and fractions
The main principle of biological screening is to imply chances of utilization of the
samples (synthetic or natural) in future, for the welfare of humanity. This is the primary step
towards drug formulation. The plant B. asiatica was investigated chemically as a result, crude
extract, various semi pure fractions and pure compounds were gated. The following
bio-assays were conducted.
Brine shrimp lethality assay
Antibacterial activity
Antifungal activity
++ Antispasmodic and Ca antagonist effect
108 | Page Results and Discussion
3.7.1 Brine shrimp lethality assay
In preliminary assessment of toxicity, the brine shrimp lethality assay is considered to be one of the most rapid and useful tool. This assay was put forwarded by Michael et al [146] and was further modified later by Vanhaecke et al [147] and Sleet & Brendal [148].
The basis of this assay relies upon the ability to kill the laboratory cultured Artemia naupili brine shrimp. This assay is very useful in the detecting toxicity [149] and is also used to detect fungal and cyanobacteria toxins [150-151].
The crude methanolic extract was fractionated into n-hexane (F1), chloroform (F2), ethyl acetate (F3) and n-butanol (F4). F1 - F4 were tested for their cytotoxicity. ED50 (effective dose) measurements of these fractions were carried out against brine shrimp larvae according to the standard method [152]. The ED50 valve of extracts F2 and F3 fall in a very narrow range of
-1 12.044 - 7.086 µg ml revealed highest effect against larvae. The extract F1 showed optimum
-1 lethality with ED50 102.44 µg ml while extract F4 exhibited low lethality with ED50 416.78 µg ml-1 (Table - 24). Etoposide was used as a positive control which exhibited high lethality with
-1 ED50 6.632 µg ml . Due to least inhibitory effect, F1 fraction was not considered to be used for further biological screening.
109 | Page Results and Discussion
Table 24: Brine shrimp bioassay of different fractions of Buddleja asiatica.
% Deaths at doses ED50
Fractions 1000 g ml-1 100 g ml-1 10 g ml-1 g ml- Results
F1 (n-hexane) 80 60 50 102.44 ++
F2 (chloroform) 100 100 90 12.044 ++++
F3 (ethyl acetate) 100 90 90 7.086 ++++
F4 (n-butanol) 70 30 10 416.78 +
Etoposide 90 80 60 6.632 ++++ (positive control)
Key: ++++ = significant activity, +++ = high activity, ++ = optimum activity and + = low activity
110 | Page Results and Discussion
3.7.2 Antibacterial Activity
Antibacterial activity of crude extract and three fractions viz. chloroform (F2), ethyl acetate (F3) and n-butanol (F4) soluble fractions of B. asiatica was performed against eleven human pathogens including Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella boydi, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae,
Klebsiella pneumoniae, Pseudomonas maltophilia and Mycobacterium leprae. The crude extract displayed high to low activity in killing the S. boydi (56%), E. coli (44%), S. flexneri
(36%), S. aureus (25%), V. cholera, (25%), B. subtilis (17%), M. leprae (13%) and P. aeruginosa (10%) but no activity was seen against the S. typhi, K. pneumoniae and P. maltophilia. F2 was most effective against S. flexenari (23 %) and S. boydi (16 %) while less effective in case of inhibiting growth of P. aerigenosa (12 %) and E. coli. F2 was totally ineffective against the rest of bacteria. F3 exhibited significant activity against S. boydi (67%) and E. coli (44%), while low activity was seen in killing the S. flexneri (29%), P. aeruginosa
(21%), S. aurous (18%), B. subtilis (10%) and V. cholera (7%) but no activity was observed in the rest of bacteria. The fraction F4 revealed moderate to low activity in killing the S. flexneri
(58%) and S. boydi (32%), V. cholera (29%), E. coli (27%), P. aeruginosa (17%), S. aurous
(11%) and did not show any activity against the others. These results were compared with standard drug, imepinem (Table - 25, Figure - 10).
111 | Page Results and Discussion
Table 25: Antibacterial activity of crude extracts and various fractions of B. asiatica.
Z.I of Standard drug (Imipenem) Crude Extract F2 F3 F4 Bacteria Z.I Inh Z.I Inh Z.I Inh Z.I Z.I
E .coli 34 15 44 5 9 15 44 9 27
B. subitiltis 30 5 17 ------3 10 ------
S. aurous 28 7 25 2 7 5 18 3 11
S. boydii 25 14 56 4 16 16 67 8 32
S. flexenari 31 11 36 7 23 9 29 18 58
P. aerigenosa 30 3 10 4 12 2 21 5 17
S. typhi 29 ------
V. cholera 24 6 25 ------3 7 7 29
K. pneumoniae 25 ------
P. maltophilia 28 ------
M. leprae 31 4 13 3 10 ------2 7
Z.I = Zone of Inhibition in mm, Inh = Inhibition in %. The plates were inaculcated at a concentration mg/ml of DMSO
112 | Page Results and Discussion
80 70 E. Coli
S. Boydii 60 (%) S. flexenari 50 40 inhibition
30
Zoneof 20 10 0 CrudeF2F3F4 Test samples
Figure 10: Antibacterial activity of crude extract and its various factions towards E.col, S.boydi and S. flexneri,
113 | Page Results and Discussion
3.7.3 Antifungal Activity:
The fungicidal activity of the crude extract and different fractions F2 - F4 of B. asiatica was evaluated against six fungi including Aspergillus flavus, Fusarium solani, Candida albicans,
Ttichophyton longifusus, Microsporum canis and Candida glaberata. The results (Table - 26) indicated that the crude extract and fractions F2, F4 displayed significant activity while fraction F3 showed moderate activity in killing the F. solani. In case of A. flavus, and T. longifusus, the area of inhibition was almost same and exhibited very high activity in crude extract while in fractions
F2-F4 showed optimum activity. It was further observed that the crude extract and fraction F4 showed high activity and fractions F2, F3 revealed weak activity against the M. canis. The crude extract and fractions F2 - F4 remained totally ineffective in killing the C. albicans and
C. glaberata (Figure - 11).
114 | Page Results and Discussion
Table 26. Antifungal activity of crude extract and various fractions of B. asiatica.
Crude extract F2 F3 F4 Standard drugs Fungi Control L.G L.G Inh L.G Inh L.G Inh L.G Inh Name MIC µg/ml
A. flavus 100 25 71 60 40 80 20 65 35 Amphotericin-B 30
F. solani 100 70 66 35 65 54 46 41 59 Miconazole 105
C. albicans 100 100 ----- 100 ----- 100 ----- 100 ----- Miconazole 20
T. longifusus 100 50 59 44 46 65 35 55 4 Miconazole 88
M .canis 100 40 60 80 20 75 25 35 65 Miconazole 94
C. glaberata 100 100 ----- 100 ----- 100 ----- 100 ----- Miconazole 103
L.G = Linear growth in mm, Inh= Inhibition in %, The plates were inculcated at a concentration of mg/ml of DMS
115 | Page Results and Discussion
100 90 80 70 60 A. flavus 50 F. solani 40 T. longisus 30 20 10 0 Crude F2 F3 F4
Figure 11: Antifungal activity of crude extract and its various factions towards A. Flavus, F.
Solani and T. Longifusus.
116 | Page Results and Discussion
3.7.4 Antispasmodic and Ca++ antagonist effect
“The crude extract of B. asiatica inhibited the spontaneous contractions concentrations of
0.03, 0.1, 0.3 and 1.0 mg/ml by 5.7 ± 4.8, 11.7 ± 9.3, 38 ± 8.5 and 100% (mean ± SEM, n=3) respectively (Table - 27, Figure - 12) thus showing antispasmodic effect. The contraction of smooth muscle preparations including rabbit jejunum is dependent upon an increase in the cytoplasmic free (Ca++), which activates the contractile elements [153-154]. The increase in intracellular Ca++ is due to either influx via voltage dependant L-type Ca++ channels (VDCs) or to release from intracellular stores in the sarcoplasmic reticulum. Periodic depolarization regulates the spontaneous movements of intestine and at the height of depolarization the action potential appears as a rapid influx of Ca++ via VDCs [155]. The inhibitory effect of the plant extract on spontaneous movements of jejunum may be due to interference either with the Ca++ release or with the Ca++ influx through VDCs. In our earlier studies, we have observed that the spasmolytic effect of the medicinal plants is usually mediated through Ca++ channel blockade
[156]. To see whether the spasmolytic effect of this plant is also mediated via the same mechanism, the extract was tested on high K+ (80 mM)-induced contraction, which was relaxed by B. asiatica extract at 0.1, 0.3 and 1.0 mg/ml by 6.7 ± 4.4, 32.7 ± 9.8 and 100% (mean ±
SEM, n=3) respectively (Table – 28, Figure - 13). At high concentration (> 30 mM), K+ is known to cause smooth muscle contractions through opening of VDCs, thus allowing influx of extra cellular Ca++ causing a contractile effect [157] and a substance causing inhibition of high
+ ++ K -induced contraction is considered an inhibitor of Ca influx [158-159]”.
117 | Page Results and Discussion
Table 27: Concentration-dependent inhibitory effect of the crude extract of B. asiatica on spontaneous contractions of isolated rabbit jejunum preparations.
Concentration S.No Inhibition (%) (mg/ml)
1 0.03 5.7 ± 4.8
2 11.7 ± 9.3 0.1
3 38 ± 8.5 0.3
4 100 1.0
Values shown are mean ± SEM, n=3.
Table 28: Concentration-dependent relaxant effect of the crude extract of B. asiatica on K+-induced contractions of isolated rabbit jejunum preparations.
Concentration S.No Relaxation (%) (mg/ml)
1 0.1 6.7 ± 4.4
2 32.7 ± 9.8 0.3
3 100 1.0
Values shown are mean ± SEM, n=3.
118 | Page Results and Discussion
100
75
50 A
% of Control Spontaneous 25
K+ (80 mM)
0 0.01 0.1 1
[Buddleja asiatica extract] mg/ml
Figure 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.
B 100
75
50
% of Control 25 Spontaneous
K+ (80 mM) 0 0.003 0.03 0.3 [Verapamil] M
Figure 13. Concentration-dependent inhibitory effect of verapamil against spontaneous and
+ high K -induced contractions in isolated rabbit jejunum preparations..
119 | Page Results and Discussion
Preliminary analysis of the plant extract for various phytochemical classes was carried out following the reported methods [160]. Plant material treated with petroleum ether and subsequently extracted with chloroform was noted for green to pink or pink to purple colour after reaction with acetic anhydride and hydrochloric acid in succession to detect sterols and terpenes, respectively. Appearance of yellow colour with aluminium chloride reagent detected the presence of flavonoids.
In the phytochemical studies, the B. asiatica fractions were screened for various natural products. It was proven that plant extract contain terpenes, flavonoids, tannins, sterols and alkaloids, whilst the result was not good for the remaining types of natural product. The extract contains 101 ± 1.8 mg of quercetin equivalent/g of the total flavonoid concentration. It was concluded that antispasmodic and antimicrobial activities of the B. asiatica extracts perhaps will be displayed by flavonoids, usually it was determined that flavonoids are mostly identified to display these actions [161]. But, the function of several more components cannot be neglected. In the end, in our recent research findings, the antifungal, antispasmodic (probably reconciled via Ca++ antagonist effect) and antibacterial activities of the plant, B. asiatica has been reported.
120 | Page Results and Discussion
PART C
3.8 Qualitative and quantitative assessment of fatty acids of
Buddleja asiatica by GC-MS
GC-MS is most common technique used to determine the lipid composition of various biologically important components of plants such as fatty acids, flavonoids, alkaloids, terpenoids and various amino acids. BSTFA (N,O-Bis(trimethylsilyl) trifloroacetamide) is an effective TMS (trimethylsilyl) donor for derivatization of polar compounds producing volatile and thermally stable derivatives of the parent compound for GC-MS [162-163] .
Analysis of fatty acid profiles has become increasingly important due to the fact that people are more curious about their diet, health and nutritional implications. Fatty acids determination is carried out worldwide in order to obtain information regarding fat composition of various food matrices, such as vegetable oils, animal oils and sea food etc. Many of the lipids and fats of various plants have been extensively investigated for their fatty acid profile [164-
165]. Several studies have shown the dietary importance of fatty acid composition of lipids.
Recently, it was proven by clinical evidence that fatty acids, especially unsaturated Fatty acids
(UFAs) are able to alleviate symptoms of certain diseases such as coronary heart disease, stroke and rheumatoid arthritis [166]. Omega 6 family has been considered to have very important role during fatal and infant growth, in particular in the formation of the central nervous system and retina [167]. Investigators have described the essentiality of FAs in human health, the role of
FAs in health promotion and disease prevention [168].
Because of the noticeable importance of UFAs/PUFAs in human health and nutrition, different means are used to increase the human consumption of PUFAs from different food sources such as direct intake as food additives and nutraceuticals [169]. Fatty acid composition
121 | Page Results and Discussion is usually controlled as an index of quality of food and their distribution provides a unique fingerprint for a given food [170].
Current research in nutritional medicine indicates that fatty acids are essential components of the human diet and the most important omega-6 fatty acid is gamma-linolenic acid [171]. It was shown that administration of γ-linolenic acids (GLA) from natural sources can correct both the biochemical abnormality and the clinical disorders [172].
Main objective of this study is to identify and quantify the major fatty acid components found in B. asiatica. In this study, oil and fat obtained from the leaves of B. asiatica were hydrolyzed, derivatised with BSTFA and analyzed with GC-MS. All fatty acids were detected and characterized for the first time from this plant. The oil showed a low thermal stability when subjected to TG/TDA (Thermo gravimetric / Differential Thermal analysis) due to the absence of phenolic contents and PUFA (Poly unsaturated fatty acids).
3.8.1 Fatty acid profile of non-volatile oil
The oil was found to contain 59 % fatty acids, 41 % other constituents including hydrocarbons. Palmatic acid (46.75 %), linoleic acid (37.80 %) and stearic acid (15.98 %) were found abundantly. Lignoceric acid (1.22 %), Archidic acid (2.0 %) and margaric acid (1.22 %) were found in minor quantity (< 3 %). Saqualene (< 1 %) was also detected. (Figure – 14 & 15,
Table - 29).
122 | Page Results and Discussion
Abundance TIC: FAeoil.D\data.ms
750000
700000
650000
600000
550000
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time--> Figure 14: BSTFA derivatised GC/MS - TIC of B. asiatica oil.
123 | Page Results and Discussion
Table 29. Fatty acids composition of B. asiatica non-volatile oil.
Retention Time Common Name IUPAC Name Abundance (%) (in minutes)
Palmitic acid Hexadecanoic acid (16:0) 8.5 46.75 Margaric acid Heptadecanoic acid (17:0) 8.95 1.22
Linoleic acid Octadecadienoic acid (18:2) 9.2 37.80
Stearic acid Octadecanoic acid (18:0) 9.35 10.98
Arachidic acid Eicosanoic acid (20:0) 10.1 2.03
Lignoceric acid Tetracosanoic acid (24:0) 11.9 1.22
124 | Page Results and Discussion
46.00 41.00 36.00 31.00 26.00 21.00 16.00 11.00 6.00 1.00 ‐4.00 Palmitic acid Margaric Linoleic acid Stearic acid Arachidic Lignoceric acid acid acid
Figure 15: Ratio between different fatty acids in B. asiatica non volatile Oil.
125 | Page Results and Discussion
3.8.2 Fatty acid profile of fat
Fat was found to contain 83.33 % fatty acids. All detected FAs were saturated.
Lignoceric acid (24:0) was found to be in the highest quantity, 43.12 % while behenic acid
(22:0) was second highest of (26.39 %) all FAs. Trycosylic acid (23:0) was found in less amount 4.83 % and was the only fatty acid which detected odd carbon number carbon chain.
Other fatty acids, archidic acid (9.29 %), stearic acid (5.58 %), montanic acid (4.46 %) and cerotic acid (4.09 %) were also found in minor quantities while melissic acid and palmatic acid were found in traces (2.6 %, 1.86 %). (Figure – 16 & 17, Table - 30).
126 | Page Results and Discussion
Abundance TIC: FAM3C1r.D\ data.ms 150000
140000
130000
120000
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time-->
Figure 16: BSTFA derivatised GC/MS – TIC of B. asiatica fat
127 | Page Results and Discussion
Table 30. Fatty acids composition of B. asiatica fat.
Retention Time Abundance Common Name IUPAC Name (in minutes) (%)
Palmitic acid Hexadecanoic acid (16:0) 9 1.86
Stearic acid Octadecanoic acid (18:0) 10 5.58
Arachidic acid Eicosanoic acid (20:0) 10.9 9.29
Behenic acid Docosanoic acid (22:0) 11.5 26.39
Tricosylic acid Tricosanic acid (23:0) 11.85 4.83
Lignoceric acid Tetracosanoic acid (24:0) 12 43.12
Cerotic acid Hexacosanic acid (26:0) 12.8 4.09
Montanic acid Octacosanic acid (28:0) 13.5 4.46
Melissic acid Triacontanoic Acid (30:0) 14 2.60
128 | Page Results and Discussion
B. asiatica Fat
40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00
Figure 17: Ratio between different fatty acids in B. asiatica fat
129 | Page Results and Discussion
3.8.3 Discussion
Although the FAs values seems low compared to other plants oils [173-174], but it encourage the use of this plant as a source of oil due the increase of the demand of new oils.
Those results show that leaves of B. asiatica are rich in fatty acids in comparison with some other leafy vegetables [175-176].
The oil of B. asiatica was found to be as a good source of various fatty acids. Majority of the fatty acids were found saturated except linoleic acid. Its fatty acid profile evinces that palmitic acid (46.75 %) was the dominating fatty acid, followed by linoleic acid (37.80 %) and stearic acid (15.98 %). Linoleic acid belongs to essential n-6 fatty acids class and found abundantly in many vegetable oils. It is shown to be useful in various health related problems such as diabetes, dermatitis [177], cystic fibrosis [178], anti-inflammatory, acne reductive, and moisture retentive [179] and is also an essential ingredient in various industries. Lack of LA in the diet causes dry hair, hair loss [180], and poor wound healing [181].
Table - 31 shows ratio between four common fatty acids which are found in both oil and fat. A comparative study suggests that palmatic acid (16:0) was found to be in ratio (25:1), stearic acid (2.86:1), arachidic acid (1:4.5) and lignoceric acid (1: 35.3) in oil and fat respectively. The results indicate that short chain fatty acids are found in the oil while long chain fatty acids are obtained from the fat (Figure -18).
Palmatic acid and stearic acid were found as major constituents in oil. They are saturated FAs. In clinical studies, stearic acid associates with lowered low-density lipoprotein cholesterol in comparison with other saturated fatty acids .These findings indicate that stearic acid is less unhealthy than other saturated fatty acids [182]. Generally, both act as energy generators, when activated in the body [183].
130 | Page Results and Discussion
Arachidic acid has been used in the production of detergents, photographic materials and lubricants [184] and Lignoceric is considered to inhibit estradiol from binding to estrogens receptors α and β, thus stimulating estrogens inducible genes [185].
Due to the low thermal stability (compared TG/DTA curve with that of standard olive oil) and absence of PUFA and other poly phenols, the oil lacks properties of edible oils but may be suitable to be used for other chemical/pharmaceutical purposes.
131 | Page Results and Discussion
45 40 35 30 25 FAT 20 OIL 15 10 5 0 Palmitic acid Stearic acid Arachidic acid Lignoceric acid
Figure 18: Comparison amongst fatty acids found in both fat and oil of B. asiatica.
Table 31. Comparison between various fatty acids found in both oil and fat of B. asiatica.
Fatty Acid Oil (%) Fat (%)
Palmitic acid 46.75 1.86
Stearic acid 15.98 5.58
Archidic acid 2.03 9.29
Lignoceric acid 1.22 43.12
132 | Page Results and Discussion
3.8.4 Thermal stability measurements of non-volatile oil
The thermal stability of non-volatile oil was measured from its Thermo gravimetric /
Differential Thermal analysis. The thermogravimetric curve (Figure – 19) indicated the thermal decomposition of this oil, which occurred between 72 oC and 400 oC, with no residue remaining after thermal treatment up to 540 oC. According to DTG curve, the thermal decomposition of the oil occurred in two steps, related to the decomposition of unsaturated and saturated fatty
o acids respectively. The thermal stability of the oil was found to be very low (Tonset=72 C ) and weight of the oil at Tonset was 2.001 mg. The maximum degradation took place at temperature
o o Tmax = 201.1 C with sample weight 1.051 mg. At 400 C all the oil sample got degraded and the weight of the oil at the end of degradation was 0.01 mg. In spite of being highly saturated
(Table - 29), the low thermal stability of the oil is attributed to the absence of natural antioxidants, such as tocopherols, ferulic acid, polyphenols etc [186].
133 | Page Results and Discussion
1.400
2.000 40.00 1.300 0.017min 3.367min 39.8Cel 72.3Cel 2.020mg 2.001mg 17.38min 1.500 1.200 35.00 201.1Cel 1.051mg
1.000 1.100 30.00
1.000 39.57min 0.500 56.47min 25.00 393.0Cel 0.012mg 539.8Cel -0.028mg 0.900 0.000 20.00 0.800 -0.500
15.00 17.38min 0.700 201.1Cel 10.43uV 39.57min -1.000
3.367min TG mg DTA uV 10.00 393.0Cel
DTG mg/min 0.600 72.3Cel 56.47min -192J/g 8.13uV 6.26uV -170J/g 539.8Cel -1.500 7.39uV 0.017min -1120J/g 0.500 5.00 39.8Cel 4.73uV -2.000 0.400 0.00 -2.500 0.300
-5.00 17.38min 201.1Cel 0.200 -3.000 0.153mg/min -10.00 3.367min 0.100 39.57min -3.500 72.3Cel 393.0Cel 56.47min 0.017min 0.002mg/min 0.002mg/min 539.8Cel -15.00 39.8Cel 3.9ug/min 0.000 0.005mg/min -4.000
-50.0 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0 Temp Cel
Figure 19: TG/DTA thermogram of B. asiatica oil
134 | Page Results and Discussion
3.8.5 GC/MS analysis of B. asiatica leaves essential oil
The essential oil was obtained from the leaves of B. asiatica by standard method and was subjected to Infra red (IR) and Gas Chromatography/Mass Spectrometry (GC/MS) analysis.
-1 -1 The IR spectrum in CHCl3 showed major absorption at 3410 cm and 1077 cm which indicated hydroxyl group. The absorption appeared at 3008 cm-1, 886 cm-1 and 741 cm-1 were assigned for aromatic functional group. The absorption at 2924 cm-1 was indicative of aliphatic while absorption at 1681 cm- 1 was due to the presence of carbonyl functionalities.
The GC chromatogram obtained for leaves essential oil showed total of 17 peaks which appeared at varies retention indices/times (Figure – 20). Both combined GC and MS studies resulted in the identification of 14 compounds. The scan numbers, retention time, compound names and their percentage abundances /quantities as well as other related results have been provided in Table - 32. The mass spectral data and retention times of the constituents were analyzed by the data system library and confirmed by comparison of their mass spectra using
NIST mass spectral search program or Kovat’s Retention Index (RI).
The results showed that both monoterpenes and sesquiterpenes were detected in the leaves essential oil. They included four monoterpenes hydrocarbons, four oxygenated monoterpenes, one hydrocarbon sesquiterpenes and five oxygenated sesquiterpene. The major constituent being found was Limonene oxide (38.11 %) whiles α-phellandrene, α-pinene and α- thujene were found to be 5.79 %, 4.95 % and 3.37 %. Furan, 2-(1-pentenyl)-(E), terpinen-4-ol,
Bicyclo[5.1.0]octane, 8-(1-methylethylidene) and Eugenol were found in traces. Because of the high content of monoterpenes, especially, limonene oxide, the oil may be classified as a
“medicinal type” [187]. Amongst sesquiterpenes, β-sinensal amounted 11.84 % while isospathulenol, kushimone, 12-nor-Preziza-7(15)-en-2-one, α-cubebene, isospathulenol and germacrene-B were found to be in traces.
135 | Page Results and Discussion
Figure 20: Total Ion Chromatogram of leaves essential oil.
136 | Page Results and Discussion
Table 32: GC/MS analysis of essential oil from leaves of Buddleja asiatica.
Peak No GC/MS R.I Compound identified M. F [M]+ % age m/z (%)a Scan Number Abundance 1 139 3.12 α –Pinene C10H16 136 4.95 136, 121, 105, 93, 77, 65, 53, 41
2 172 3.83 α-Phellandrene C10H16 136 5.79 136, 121, 107, 93, 77, 67, 55, 43
3 197 4.37 α –Thujene C10H16 136 3.37 136, 121, 105, 93, 77, 65, 51, 41
4 228 5.3 Limonene oxide C10H18O 154 38.11 154, 139, 107, 96, 93, 81, 71, 68, 59, 43
5 287 6.32 Furan, 2-(1-pentenyl)- (E) C9H12O 136 1.79 136, 121, 107, 93, 79, 65, 43
6 411-416 9.06 Terpinen-4-ol C10H18O 154 1.89 154, 136, 111, 93, 86, 77, 71, 55, 43
7 439 9.62 Bicyclo[5.1.0]octane, 8-(1- C11H18 150 1.00 150, 139, 135, 121, 107, 96, 93, 81, methylethylidene) 71, 67, 59, 55, 43
8 687 14.98 Eugenol C10H12O2 164 1.53 164, 149, 137, 121, 103, 91, 77, 55
9 778 16.95 Kushimone C14H20O 204 1.00 204, 189, 161, 147, 133, 121, 105, 91, 79, 67, 55
10 809 17.63 α –Cubebene C15H23 204 1.42 204, 189, 174, 161, 147, 133, 119, 105, 91, 81, 67, 55
11 860 18.73 12-Nor-Preziza-7(15)-en-2-one C14H20O 204 1.84 204, 189, 173, 161, 136, 121, 107, 93, 81, 67, 55, 43
12 1009 21.95 Sesquichamaenol (1,10-seco-1- C14H20O2 220 10.21 220, 202, 187, 177, 159, 147, 133,
137 | Page Results and Discussion
hydroxycalamenen-10-one) 119, 107, 95, 81, 69, 55, 43
13 1015 22.08 C16H14O 222 4.05 222, 204, 189, 175, 161, 147, 133, Unidentified alocohol 121, 107, 95, 81, 69, 55, 43
14 1074 23.37 Isospathulenol C15H24O 220 0.95 220, 205, 187, 177, 162, 147, 134, 119, 105, 93, 79, 69, 55, 43
15 1093 23.78 Unknowen C15H10O2 222 0.89 222, 204, 189, 177, 161, 147, 133, Diphenyl, Di-ketone 121, 109, 95, 79, 69, 55, 43
16 1197 26.03 β-Sinensal C15H22O 218 11.84 218, 203, 189, 175, 161, 147, 133, 121, 107, 91, 79, 69, 55, 43
17 2034 44.17 Unidentified C18H17O2N 279 9.42 279, 248, 167, 149, 131, 113, 104, 97, 83, 71, 57, 43
138 | Page Experimental
139 | Page Experimental
PART A
4.1 Secondary metabolites from Buddleja asiatica
4.2 General notes
“Physical Constants: Melting points were measured by using Gallenkamp and Kofler hot-stage apparatus. Glass capillaries were used and melting points are uncorrected. Optical rotations were measured on JASCО DIP-360 digital polarimeter.
Spectroscopy: Ultra Violet (UV) spectra were recorded in MeOH on Shimadzu UV 240 spectrophotometer (Shimadzu, Japan). Infra Red (IR) spectra were recorded on а Jаsco-320-A spectrophotometer in KBr and Perkin-Elmer (Spectrum 100 FT-IR Spectrometer). The Mass spectra (MS) were determined using Micromаss ZMD and Varian MAT 312 double focusing mass spectrometer connected to DEC–PDP 11 / 34 computer system. Nuclear Magnetic
Resonance (NMR) spectra were recorded in CDCl3 on a Bruker AM–300 NMR spectrometer and JEOL ECA 600 MHz NMR spectrometer (75 MHz and 150 MHz for 13C–NMR respectively). Carbon atom types were determined by employing a combination of 13C-NMR broad-band proton decoupling spectra, and DEPT experiments. Assignments were established by employing a combination of 1D and 2D NMR experiments. 2D NMR spectra were processed by JEOL Delta and ACD Lab software.1H - 1H correlations were established by
Double Quantum-Filtered COSY. 1H-13C NMR correlations were assigned by using HMQC,
H2BC and HMBC pulse sequences.
Chromatography: Column chromatography (CC) was carried out on silica gel (70–230 mesh,
Merck, Darmstadt, Germany) as stationery phase while organic solvents as mobile phase. Thin layer chromatography (TLC) was carried out using precoated silica gel GF254 plates and TLC cards (20 × 20, 0.5 mm thick, Merck) along with preparative TLC plates (20 × 20 and 120 × 20) for thin layer chromatography, while Vacuum Liquid Chromatography (VLC) was carried out
140 | Page Experimental
using silica gel Si 60 (Merck)”.
Visualization reagents:
Ceric sulphate reagent: A mixture of ceric sulphate (0.1 g) and trichloroacetic acid (1 g) was added into 5 ml distilled water. The solution was boiled with drop wise addition of conc. sulfuric acid, until the disappearance of turbidity.
Vanillin sulfuric acid reagent: Vanillin reagent was prepаred by mixing 1 g vanillin in
100 ml ethanol. Sulfuric acid (5 ml, conc.) was added drop wise with constant shaking to keep the solution colourless. The plates were sprayed and heated at 100 OC for 10 minutes in an oven.
p-Anisaldehyde reagent: p-anisaldehyde (0.5 ml) was mixed with 50 ml glacial acetic acid. Conc. sulfuric acid (1 ml) was added drop wise to the solution. The plates were placed in an oven at 110 ̊C for 10 minutes until visualization.
Iodine reagent: Small amount of iodine crystals were placed in a TLC tank and warmed for a few minutes on a water bath (40 oC). TLC plates were placed immediately after warming and the spots were visualized.
Characterization of glucose:
“Acid hydrolysis: 0.1 g of the compound was dissolved in 1N H2SO4 (5 ml). The solution was refluxed for 6 hours resulting in formation of black degradation product which was filtered off. The solution was neutralised with Ba(OH)2 saturated solution. The obtained suspension was then filtered, evaporated and chromatographed over silica gel (70-230 mesh) in solvent system CHCl3 - MeOH (7:3) to yield D-glucose. It was identified by comparison with an authentic sample (Rf, 1H-NMR (J =8.0 Hz of anomeric proton) and was also confirmed as β-D-
25 Glucose from its optical rotation [α] D +46.3°(c 0.02, H2O) [188] ”
141 | Page Experimental
Characterization of steroids:
Libermann-burchardt reaction: Small amount of compound was dissolved in chloroform, added a few drops of conc. sulfuric acid and followed by addition of 3 drops of acetic anhydride, the solution becomes violet and finally green [140].
142 | Page Experimental
4.3 Plant material
Buddleja asiatica (whole plant) was collected from Banda Piran, Siran valley (34° N/73°
E, 2900 meters above the sea level), District Mansehra, in October, 2007. It was identified by
Professor Dr. Manzoor Ahmad, Plant Taxonomist, Department of Botany, Government Degree
College, Abbotabad, Pakistan, where a voucher specimen has been deposited in the herbarium
(accession no. B - 0015).
4.4 Extraction and fractionation
The shade-dried plant of Buddleja asiatica (21 kg) was ground and extracted with
MeOH (65 L) at room temperature for a period of seven days (3 X 65 L). The combined methanolic extract was evaporated to obtain a greenish gummy residue (522 g) which was divided into n-hexane (F1, 63 g), chloroform (F2, 101 g), ethyl acetate (F3, 59 g) and n-butanol
(F4, 48 g) (scheme 1).
143 | Page Experimental
Extraction and fractionation of Buddleja asiatica
Buddleja asiatica Powdered 21 Kg
Extracted with methanol (3 × 65 L)
Methanolic Extract Concentrated on rotary (Crude) 522 g evaporator (- MeOH)
Extracted with n-hexane + H2O
n- Hexane soluble Aqueous layer fraction (F1, 63 g)
Extracted with chloroform
Chloroform soluble fraction Aqueous layer (F2, 101 g)
Extracted with ethyl acetate
Ethyl acetate soluble Aqueous layer fraction (F3, 59 g) Extracted with n-butanol
n-Butanol soluble Aqueous layer fraction (F4, 48 g)
Scheme 1
144 | Page Experimental
4.5 Isolation and characterization of the compounds from chloroform
soluble fraction (F2)
The chloroform soluble fraction (F2, 101 g) was subjected to column chromatography over silica gel eluting successively 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). (Scheme 2)
The fraction FB (16 g) was subjected to column chromatography over silica gel eluting with mixtures of n-hexane - CHCl3 and CHCl3 - MeOH in increasing order of polarity. The fractions which eluted with n-hexane - CHCl3 (6 : 4) were combined, concentrated and re- chromatographed over silica gel using n-hexane - CHCl3 (6.5 : 3.5) as eluent to provide buddlejone (180, 23 mg) and dihydrobuddledin-A (181, 20 mg) from the top and tail fractions, respectively. The fractions which eluted with n-hexane - CHCl3 (4 : 6) showed a major spot on
TLC. It was purified in n-hexane - acetone (6 : 4) to yield pure compound, buddledone-B (182,
19 mg).
The fraction FC (19 g) was subjected to column chromatography over silica gel eluting with mixtures of n-hexane - CHCl3 and CHCl3 - MeOH in increasing order of polarity, yielded several fractions. The fractions obtained from n-hexane - CHCl3 (2 : 8) showed a major spot on
TLC and subsequent preparative TLC using n-hexane - EtOAc (7 : 3) as solvent system afforded ursolic acid (183, 31 mg). Further elution of the same column with CHCl3 : MeOH
(9 : 1) yielded several fractions which were combined, concentrated and subjected to preparative TLC using n-hexane – EtOAc - acetone (6.5 : 2 : 1.5) as solvent system to furnish
2-phenylethyl-β-D-glucoside (184, 21 mg). Elution of the same column with CHCl3 - MeOH
(7.5 : 2.5) yielded several fractions. The last six fractions showed similar spots on TLC plate
(silica gel) were combined and again subjected to column chromatography on silica gel. The column was eluted with the mixtures of CHCl3 - MeOH (7.8 : 2.2). First few fractions were
145 | Page Experimental found to contain a semi pure compound. It was purified in n-hexane - EtOH - diethylamin
(6.5 : 3.5 : 2 drops) as solvent system to afford 7-deoxy-8-epiloganic acid (185, 25 mg).
The fraction FD (21 g) was chromatographed over silica gel eluting with mixtures of
CHCl3 - MeOH in increasing order of polarity yielded several fractions. The fractions obtained from CHCl3 - MeOH (9 : 1) were combined and re-chromatographed over TLC grade silica gel eluting with mixtures of CHCl3 - MeOH in increasing polarity. The fractions obtained from
CHCl3 - MeOH (9.5 : 0.5) were subjected to preparative TLC in solvent system n-hexane - EtOAc - diethylamin (5.5 : 4.5 : 2 drops) to obtain secutellarin-7-O-β-D- glucopyranoside (186, 27 mg).
146 | Page Experimental
147 | Page Experimental
4.5.1 Buddlejone (180)
Physical Data
Orange oil (23mg)
25 o [α]D = - 28.6 (c 0.32, CHCl3)
–1 IR (KBr): νmax cm = 3425, 2924, 1731, 1559, 1430, and 1374
+ HR-EIMS: m/z = 300.2053 [M] (calcd. for C20H28O2, 300.2089)
1 H-NMR (300 MHz, CDCl3): δ = 7.31 (1H, d, J = 4.1 Hz, H-12), 6.20 (1H, d, J = 4.1 Hz, H-11),
3.43 (1H, septet, J = 6.8 Hz, H-15), 2.66 (1H, d, J = 2.8 Hz, Hβ-6), 2.65 (1H, s, Hα-6), 2.14 (1H, m, Hα-1), 1.86 (1H, m, H - 5), 1.77 (1H, m, Hα-2), 1.61 (1H, m, Hβ -2), 1.52 (1H, m, Hβ-1), 1.50
(1H, m, Hβ-3), 1.26 (1H, m, Hβ-3), 1.26 (3H, d, J = 4.1 Hz, CH3-17), 1.25 (3H, d, J= 4.1 Hz,
CH3-16), 1.18 (3H, s, CH3-20), 1.01 (3H, s, CH3-18), 0.95 (3H, s, CH3-19)
13 C-NMR (75 MHz, CDCl3): δ = 36.7 (C-1), 18.6 (C-2), 41.4 (C-3), 36.7 (C-4), 52.5 (C-5), 31.8
(C-6), 188.8 (C-7), 120.2 (C-8), 167.8 (C-9), 33.1 (C-10), 116.7 (C-11), 137.0 (C-12), 120.2
(C-13), 192.3 (C-14), 32.9 (C-15), 20.9 (C-16), 20.7 (C-17), 132.9 (C-18), 21.6 (C-19), 21.8
(C-20).
148 | Page Experimental
4.5.2 Dihydrobuddledin A (181)
Physical Data
Colourless oil (20 mg)
25 o [α]D = - 64.8 (c 0.51, CHCl3)
–1 IR (KBr): νmax cm = 2955, 1738, 1455, 1020, 890
+ HR-EIMS: m/z = 279.1945 [M] (calcd. for C17H27O3, 279.1960)
1 H-NMR (300 MHz, CDCl3): δ = 5.12 (1H, d, J = 11.4 Hz, H-2), 4.75 (1H, s, H-15), 4.62 (1H, t,
J =1.8 Hz, H-15), 3.03 (1H, m, H-9), 2.96 (1H, m, H-4), 2.08 (3H, s, OAc), 2.07 (1H, dd, J =
11.4 Hz,10.0 Hz, H-1), 2.01-1.53 (8H, m, CH2-5, CH2-6, CH2-7, CH2-10), 1.14 (3H, s, H-12),
1.12 (3H, s, H-13), 1.11 (3H, d, J = 6.8 Hz, H-14)
13 C-NMR (75 MHz, CDCl3): δ = 213.4 (C-3), 171.0 (2-OOCCH3), 153.2 (C-8), 111.4 (C-15),
79.6 (C-2), 50.5 (C-1), 44.4 (C-4), 39.4 (C-10), 38.2 (C-9), 34.5 (C-11), 32.9 (C-7), 31.6 (C-12),
30.7 (C-5), 26.4 (C-6), 23.0 (C-13), 15.8 (C-14), 21.0 (2-OOCCH3).
149 | Page Experimental
4.5.3 Buddledone B (182)
Physical Data
Colourless oil (19 mg)
25 o [α]D = +58.2 (c 0.12, CHCl3)
–1 IR (KBr): νmax cm = 2955, 2935, 1680, 1675, 1455, 1371, 1244, 1020
+ HR-EIMS: m/z = 235.1706 [M] (calcd. for C15H23O2, 235.1699)
1 H-NMR (300 MHz, CDCl3): δ = 6.47 (1H, d, J = 16.5 Hz, H-10), 6.01 (1H, d, J = 16.5 Hz,
H-9), 5.07 (1H, m, H-1), 2.76 (1H, m, H-7), 1.93-2.29 (2H, m, H-4), 1.61 (3H, d, J = 2.7 Hz, H-
12), 1.43-1.60 (2H, m, H-5), 1.29-1.68 (2H, m, H-6), 1.25 (3H, s, H-14), 1.20 (3H, s, H-15),
1.10 (3H, d, J=6.6 Hz, H-13)
13 C-NMR (75 MHz, CDCl3): δ = 100.8 (C-1), 101.7 (C-2), 203.7 (C-3), 32.2 (C-4), 24.6 (C-5),
33.2 (C-6), 43.2 (C-7), 207.9 (C-8), 124.9 (C-9), 153.9 (C-10), 39.0 (C-11), 19.0 (C-12), 15.6
(C-13), 26.3 (C-14), 28.3 (C-15).
CH
H C H
H C
CH
150 | Page Experimental
4.5.4 Ursolic acid (183)
Physical Data
Amorphous powder (31 mg)
Melting Point = 290-291 0C
25 o [α]D = +66.2 (c 0.01, CHCl3)
–1 IR (KBr): νmax cm = 3480, 3046, 1693, 1632
+ HR-EIMS (m/z) = 456.3603 [M] (calcd. for C30H48O3, 456.3595)
1 H-NMR (300 MHz, CDCl3): δ = 5.10 (1H, m, H-12), 2.99 (1H, dd, J = 10.20 Hz, 4.5 Hz, Hα-3),
1.20 (3H, s, CH3-27),1.05 (3H, s, CH3-23), 0.96 (3H, s, CH3-25), 0.90 (3H, d, J = 6.6 Hz,
CH3-30), 0.84 (3H, s, CH3-24), 0.79 (3H, d, J = 6.8 Hz, CH3-29)
13 C-NMR (75 MHz, CDCl3): δ = 38.4 (C-1), 27.4 (C-2), 79.1 (C-3), 38.8 (C-4), 52.4 (C-5), 18.1
(C-6), 32.6 (C-7), 38.8 (C-8), 48.4 (C-9), 36.8 (C-10), 23.7 (C-11), 124.8 (C-12), 137.6 (C-13),
40.0 (C-14), 28.4 (C-15), 22.5 (C-16), 46.9 (C-17), 54.2 (C-18), 30.2 (C-19), 30.1 (C-20), 27.6
(C-21), 36.6 (C-22), 24.0 (C-23), 14.7 (C-24), 15.6 (C-25), 16.0 (C-26), 24.3 (C-27), 176.2
(C-28), 21.4 (C-29), 24.0 (C-30).
151 | Page Experimental
4.5.5 2-phenylethyl-β-D-glucoside (184)
Physical Data
White crystals (21mg) from acetone
Melting Point = 168-170 0C
–1 IR (KBr): νmax cm = 3400, 2930, 1500, 1450, 1375, 1050.
+ HR-EIMS: m/z = 284.2190 [M] (calcd. for C14H20O6, 284.1840)
1 H-NMR (300 MHz, CDCl3): δ = 7.16 -7.28 (5H, m, H-2, H-3, H-4, H-5, H-6), 2.85 (2H, t, J =
6.5 Hz, H-7), 3.64 (1H, m, H-8), 3.93 (1H, m, H-8 ), 4.17 (1H, d, J = 8.0 Hz, Glc-1΄), 3.30-3.50
(sugar protons).
13 C-NMR (75 MHz, CDCl3): δ =139.42 (C-1), 128.91 (C-2, C-6), 129.60 (C-3, C-5), 126.75
(C - 4), 36.35 (C-7), 70.16 (C-8), 103.55 (C-1΄), 74.13 (C-2΄), 77.60 (C-3΄), 70.80 (C-4΄),
77.48 (C-5΄), 61.79 (C - 6΄).
152 | Page Experimental
4.5.6 7- deoxy-8-epiloganic acid (185)
Physical Data
White amorphous powder (26 mg)
Melting point= 118 - 120 0C
25 o [α]D = - 28.6 (c 0.32, CHCl3)
–1 IR (KBr): νmax cm = 3455, 1687, 1633
+ HR-EIMS: m/z = 374.3854 [M] (calcd. for C16H22O10 374.3734)
1 H-NMR (300 MHz, CDCl3): δ = 7.22 ( 1H, br s, H-3), 5.15 (1H, J= 9.6, H-1), 3.29 (1H, m,
H-7), 2.81 (1H, m, H-5), 1.41 (1H, dd, J= 10.3 Hz, 13.8 Hz, Hα-6), 2.63 (1H, dd, J =7.5 Hz,
13.8 Hz, Hβ-6), 2.18 (1H, dd, J = 7.4 Hz, 9.6 Hz, H-9), 1.63 (3H, s, H-10), 4.81 (1H, d, J = 7.9,
H-1΄), 3.19 - 3.44 (4H, m, H-2΄, H-3΄, H-4΄ and H-5΄), 3.66 ( 1H, dd, J = 5.8, 11.9, Hα-6΄), 3.93
( 1H, dd, J = 1.9 Hz, 11.9 Hz, Hβ-6΄)
13 C-NMR (75 MHz, CDCl3): δ = 95.9 ( C-1), 148.2 ( C-3), 115.7 (C- 4), 32.8 ( C-5), 37.1
(C- 6), 64.0 (C-7), 65.4 ( C-8), 45.6 (C-9), 18.5 (CH3-10 ), 174.1 (COOH), 99.9 (C-1΄), 75.6
(C-2΄), 78.3 (C-3΄), 72.1 (C-4΄), 78.7 (C-5΄), 63.2 (C-6΄).
153 | Page Experimental
4.5.7 Scutellarin 7-O-β-D-glucopyranoside (186)
Physical Data
Yellow powder (27 mg)
Melting point= 184 - 185 0C
25 o [α]D = - 83.2 (c 0.11, CHCl3)
–1 IR (KBr): νmax cm = 3250 and 1648
+ HR-EIMS: m/z = 448.3264 [M] (calcd. for C21H20O11, 448.3322)
1 H-NMR (300 MHz, CDCl3): δ = 7.64 (2H, d, 9.0 Hz, H-3΄ and H-5΄), 7.12 (2H, d, J = 9.0 Hz,
H-2΄ and H-6΄), 6.70 ( 1H, d, J = 3.0 Hz, H-8), 6.58 (1H, s, H-3), 5.04 (1H, d, J = 7.5, H-1΄΄),
4.52 ( m, H-4΄΄), 3.89 (1H, dd, J = 2.1 Hz, 11.8 Hz, Hβ-6΄΄), 3.86 (1H, m, H-2΄΄), 3.78 (1H, dd,
J = 5.6 Hz, 11.7 Hz, Hα-6΄΄), 3.59 (m, H-3΄΄), 3.38 ( m, H-5΄΄)
13 C-NMR (75 MHz, CDCl3): δ = 164.8 (C-2), 104.5 (C- 3), 185.2 (C-4), 146.5 ( C-5), 132.7
(C-6), 152.8 (C-7), 95.6 (C-8), 150.6 (C-9), 108.2 (C-10), 122.4 (C-1΄), 129.44 × 2 (C - 2΄and
C - 6΄), 118.0 × 2 (C - 3΄ and C - 5΄), 163.36 (C - 4΄), 102.3 ( C-1΄΄), 77.0 (C-3΄΄), 74.6 (C- 2΄΄),
74.6 (C- 2΄΄), 77.8 ( C-3΄΄), 70.6 ( C- 4΄΄), 78.2 (C- 5΄΄), 62.6 (C-6΄΄).
154 | Page Experimental
4.6 Isolation and characterization of the compounds from ethyl acetate soluble fraction (F3)
The ethyl acetate soluble fraction (F3, 59 g) was subjected to column chromatography over silica gel. Elution with mixtures of n-hexane, EtOAc and EtOH in increasing order of polarity yielded ten fractions (FA-FJ).
The fractions FB (5.1 g), obtained from n-hexane - EtOAc (6 : 4) were concentrated and further subjected to column chromatography over silica gel eluting with mixture of n-hexane-
EtOAc and EtOAc - EtOH in increasing polarity. The eluates obtained with n-hexane - EtOAc
(3 : 7) showed a major spot on TLC and subsequent preparative TLC using n-hexane - EtOAc
(3.6 : 6.4) as a solvent system afforded white amorphous powder as lignoceric acid (187, 15 mg). The fractions which obtained from the same column with EtOAc - EtOH (9.5 : 0.5 and 9.0
: 1.0) showed same results on TLC, were combined and re-chromatographed over silica gel column eluting with mixture of EtOAc - EtOH in increasing order of polarity. The fractions obtained with EtOAc - EtOH (9.4 : 0.6) were subjected to preparative TLC in solvent system
EtOAc - diethylamine (100 % : 2 drops) to obtain (24S)-stigmast-5,22-diene-7β-ethoxy-3β-ol
188 (20 mg).
The fractions FD (5.2 g) obtained with n-hexane - EtOAc (2 : 8) were concentrated and subjected to column chromatography over silica gel eluting with mixtures of EtOAc and EtOH in increasing polarity. The fractions obtained with EtOAc - EtOH (8 : 2) showed a major spot with some impurities. They were concentrated and subjected to preparative TLC on silica plates in n-hexane – EtOAc - EtOH (2 :6 :2) as solvent system to obtain asiatoate A (189, 30 mg).
The fractions of the same column obtained from EtOAc - EtOH (7.5 : 2.5) also showed a major along with some miner spots on TLC. The major spot was purified in n-hexane - EtOAc - EtOH (2.5 : 5 :2.5) as solvent system to obtain asiatoate B (190, 28 mg).
155 | Page Experimental
The fractions FE (6 g), obtained with EtOAc (100 %) were concentrated and subjected to column chromatography on silica gel. Elution was carried out with mixtures of EtOAc and
EtOH in increasing order of polarity. The fractions obtained from EtOAc - EtOH (6.5 : 3.5) were concentrated and again loaded to column chromatography over silica gel eluting with mixtures of EtOAc-EtOH in increasing order of polarity. The fractions obtained from
EtOAc - EtOH (6.8 : 3.2) were combined and subjected to preparative TLC in EtOAc – EtOH -
(CH3)2CO (7 : 2 : 1) as solvent system to afford chrysoeriol-7-O-β-D-glucopyranoside (191, 20 mg).
156 | Page Experimental
157 | Page Experimental
4.6.1 Lignoceric acid (187)
Physical Data
White solid (15 mg)
Melting point = 81-83 0C
–1 IR (KBr): νmax cm = 3308.65, 2955, 2916, 1699, 1462 and 1052
+ HR-ESMS: m/z = 367.3 [M-H] (calcd. for C24H48O2, 368.07)
1 H-NMR (300 MHz, CDCl3): = 11.0 ( OH, s), 2.34 (2H, t, J = 4.5 Hz, H-2), 1.63 (2H, m, H-
3), 1.24 (2H, m, H-4), 1.24 (38H, m, H-4 to H-22), 1.31 (2H, m), 0.88 (3H, t, J =8.3 Hz, H-24)
13 C-NMR (75 MHz, CDCl3): = 178.7 (C-1), 33.9 (C-2), 24.7 (C-3), 29.8 (C-4), 29.8 X 13
(C-5 to C-17), 29.1 (C-18), 29.3 (C-19), 29.4 (C-20), 29.5 (C-21), 24.8 (C-22), 22.8 (C-23),
14.26 (C-24).
158 | Page Experimental
4.6.2 (24S)-stigmast-5,22-diene-7β-ethoxy-3β-ol (188)
Physical Data
Amorphous powder (20 mg)
Melting point= 144-145 0C
–1 IR (CHCl3): νmax cm =3480, 3046, 1693, 1632, 1462, 1381 and 1314
+ HR-EIMS: m/z = 456.4065 M (calcd. for C31H52O2, 456.4124)
1 H-NMR (600 MHz, CDCl3): = 5.32 (1H, d, J = 9.8 Hz, H-6), 5.16 (1H, dd, J = 9.4, 3.4 Hz,
H-22), 5.02 (1H,dd, J = 10.2, 3.4 Hz, H-23), 3.45 (1H, m, H-3), 3.30 (1H, dd, J = 10.2, 3.4 Hz,
H -7), 2.86 (1H, m, H-8), 2.64 (1H, m, Hβ - 1), 2.33 (1H, dd, J = 12.6, 3.22 Hz, Hβ - 4), 2.33
(1H, m, H-20), 2.15 (1H, m, H-24), 2.1 (1H, m, Hα-16), 2.02 (1H, dd, J = 12.6, 3.5 Hz, Hα - 4),
1.9 (1H, m, Hβ -16), 1.86 (1H, m, H-25), 1.84 (1H, m, Hα - 2), 1.76 (1H, m, Hβ - 2), 1.7 (1H, m,
Hβ - 15), 1.6 (1H, m, Hα - 1), 1.6 (1H, m, Hα - 15), 1.51 (1H, m, H-17), 1.46 (1H, m, Hβ - 28),
1.44 (1H, m, Hα - 28), 1.28 (3H, s, H-19), 1.08 (3H, s, H-18), 0.91 (3H, d, J = 7.7 Hz, H-21),
0.90 (3H, t, 7.2 Hz, H-29), 0.84 (3H, d, J = 6.6 Hz, H-27), 0.82 (3H, d, J = 6.6 Hz, H-26), 2.87
(1H, m, 7-O-CH2-CH3), 2.66 (1H, m, 7-O-CH2-CH3), 1.18 (3H, t, J = 6.8 Hz, 7-O-CH2-CH3)
13 C-NMR (150 MHz, CDCl3): = 29.06 (C-1), 32.0 (C-2), 71.94 (C-3), 37.36 (C-4), 140.86
(C-5), 121.84 (C-6), 82.5 (C-7), 51.36 (C-8), 50.15 (C-9), 34.0 (C-10), 21.2 (C-11), 39.7
(C-12), 42.33 (C-13), 55.0 (C-14), 25.8 (C-15), 28.6 (C-16), 56.5 (C-17), 12.17 (C-18), 19.53
(C-19), 36.2 (C-20), 19.1 (C-21), 138.33 (C-22), 131.62 (C-23), 42.33 (C-24), 30.6 (C-25), 20.2
(C-26), 20.2 (C-27), 25.4 (C-28), 12.2 (C-29), 64.2 (7-O-CH2-CH3), 15.9 (7-O-CH2-CH3)
159 | Page Experimental
160 | Page Experimental
4.6.3 Asiatoate-A (189)
Physical Data
White Amorphous powder (30 mg)
Melting point= 157-158 0C
–1 IR (KBr): νmax cm = 3051, 2923, 2853, 1720, 1460, 1250, 741
+ HR-EIMS: m/z = 436.2871 [M] (calcd. for C25H40O6, 436.5815)
1 H-NMR (300 MHz, CDCl3): = 6.68 (1H, s, H - 5), 4.12 ( 1H, q, J = 6.8 Hz, H - 1΄), 1.3 (3H, d, J = 6.8 Hz, H-1΄), 1.25 (8H, m, H-4΄, H-5΄, H-6΄, H-7΄), 1.29 (6H, m, , H-8΄, H-9΄, H-10΄),
0.81 (3H, t, J = 6.8 Hz, H-11΄), 3.28 (9H, s, 3 × OCH3), 2.50 (3H, s, CH3-C=O), 1.2 (6H, s, 2 ×
CH3 at C-3΄).
13 C-NMR (75 MHz, CDCl3): = 120.8 (C-1), 153.6 (C-2), 152.0 (C-3), 124.2 (C-4), 93.2
(C-5), 152.5 (C-6), 169.9 (C-7), 14.9 (C-1), 75.8 (C-2), 36.5 (C-3), 28.3 (C-4), 25.4 (C-5),
24.2 (C-6), 23.8 (C-7), 23.9 (C-8), 22.8 (C-9), 22.4 (C-10), 14.7 (C-11), 182.8 (CH3-C=O),
26.6 (CH3-C=O), 60.8 (OCH3 at C-2), 61.3 (OCH3 at C-3), 60.1 (OCH3 at C-6), 14.9 (CH3 at C-
1), 13.5 (CH3 at C-3),13.6 (CH3 at C-3) .
161 | Page Experimental
4.6.4 Asiatoate-B (190)
Physical Data
White Amorphous powder (28 mg)
Melting point= 160-161 0C
–1 IR (KBr): νmax cm = 3403, 3060, 2923, 2853, 1705, 1460, 1247, 741
+ HR-EIMS: m/z = 422.27 [M] (calcd. for C24H38O6, 422.5548)
1 H-NMR (300 MHz, CDCl3): = 6.68 (1H, s, H-5), 4.12 ( 1H, q, J = 6.8 Hz, H-1΄), 1.3 (3H, d,
J = 6.8 Hz, H-1΄),1.25 (8H, m, H-4΄, H-4΄, H-5΄, H-7΄), 1.29 (6H, m, H-8΄, H-9΄, H-10΄), 0.81
(3H, t, J = 6.8 Hz, H-11΄), 3.28 (6H, s, 2 × OCH3), 2.50 (3H, s, CH3-C=O), 1.2 (6H, s, 2 × CH3
(C-3΄), 10.30 (1H, s, OH)
13 C-NMR (75 MHz, CDCl3): = 121.7 (C-1), 154.5 (C-2), 152.4 (C-3), 124.3 (C-4), 92.2
(C-5), 156.6 (C-6), 171.3 (C-7), 17.2 (C-1), 76.6 (C-2), 38.8 (C-3), 27.5 (C-4), 25.4 (C-5),
22.7 (C-6), 23.6 (C-7), 24.4 (C-8), 22.8 (C-9), 22.8 (C-10), 14.1 (C-11), 61.3 (OCH3 at C-
2), 61.5 (OCH3 at C-3), 183.4 (CH3-C=O at C-4), 26.6 (CH3-C=O at C-4), 13.8 (CH3 at
C-3), 13.8 (CH3 at C-3).
162 | Page Experimental
4.6.5 Chrysoeriol 7-O-β-glucopyranoside (191)
Physical Data
Yellow Amorphous powder (20 mg)
Melting point= 173-174 0C
–1 IR (KBr): νmax cm = 3452, 3044, 1648
+ HR-EIMS: m/z = 462.1233 [M] (calcd. for C22H22O11, 462.1240)
1 H-NMR (300 MHz, CDCl3): δ = 7.74 (1H, d, J = 2.9 Hz, H-2΄), 7.59 (1H, dd, J=8.0, 2.2 Hz,
H-6΄), 6.93 (1H, d, J=7.6 Hz, H=5΄), 6.84 ( 1H, d, J = 1.6 Hz, H-6), 6.68 (1H, s, H-3), 6.49
(1H, d, J= 1.6 Hz, H-8), (Sugar protons) 5.65 (1H, d, J = 7.5, H-1΄΄), 3.86 (1H, m, H-2΄΄), 3.59
(m, H-3΄΄), 4.52 ( m, H-4΄΄), 3.89 (1H, J= 2.1 Hz, 11.8 Hz, Hβ-6΄΄), 3.78 (1H, J= 5.6 Hz, 11.7
Hz, Hα-6΄΄), 3.38 ( m, H-5΄΄).
13 C-NMR (75 MHz, CDCl3): δ =164.8 (C-2), 104.5 (C- 3), 185.2 (C-4), 146.5 ( C-5), 132.7
( C-6 ), 152.8 (C-7), 95.6 (C-8), 150.6 (C-9), 108.2 (C-10), 134.3 (C-1΄), 117.5 (C- 2΄), 125.6
(C-3΄), 149.9 (C-4΄ ), 141.8 (C-5΄), 112.44 (C-6΄), 102.3 ( C-1΄΄), 77.0 (C-3΄΄), 74.6 (C- 2΄΄),
74.6 (C- 2΄΄), 77.8 ( C-3΄΄), 70.6 ( C- 4΄΄), 78.2 (C- 5΄΄), 62.6 (C-6΄΄).
163 | Page Experimental
PART B
4.7 Biological screening of the crude extract and fractions
4.7.1 Brine shrimp lethality assay:
It is a simple and superb initial technique to find out the cytotoxicity of crude plant extract and pure compounds. In this method [189], artificial sea water was prepared by dissolving 3.8 g sea salt per litre of double distilled water and filtered. Sea water was placed in a small tank; added brine-shrimp eggs (Artemia salina, 1 mg) and was darkened by covering with aluminium foil. It was allowed to stand for 24 hours at 25oC which provided a large number of larvae. Twenty milligrams of the concentrated sample was dissolved in 2 ml CHCl3
(20 mg/2ml) and transferred to 500, 50 and 5 l vials corresponding to 1000, 100 and 10 g per ml, respectively. Then three replicates were prepared for each concentration making a total of nine vials. The vials containing material was concentrated, dissolved in DMSO (50l) and 5ml
“sea water” added to each. Then were 10 shrimps added per vial, allowed to stand for 24 hours, shrimps were counted and recorded the number of surviving shrimps. The data was analyzed with a Finney computer program to determine the LD50 values.
4.7.2 Antibacterial activity
The antibacterial activity was checked by the agar–well diffusion method [135]. In this method one loop full of 24 hours old culture containing approximately 104-106 CFU was spread on the surface of Mueller-Hinton Agar plates. Wells were dug in the medium with the help of sterile metallic cork borer. Stock solutions of the test samples (crude extract, fractions (F2-F4) in the concentration of 1 mg/ml were prepared in dimethyle sulfoxide (DMSO) and 100 μl
164 | Page Experimental dilutions were added in their respective wells. The antibacterial activity of crude extract and fractions (F2-F4) was compared with standard drug, imepinem which served as positive control.
4.7.3 Antifungal activity
The antifungal activity was determined by the Agar Well Diffusion Method [135]. In this method amphotericin and miconazole were used as the Standard drug. The crude extract and fractions (F2-F4) were dissolved in DMSO (50 mg / 5ml). Sterile sabouraud’s dextrose agar medium (5 ml) was placed in a test tube and inoculated with the sample solutions (400 µg /ml) kept in slanting position at room temperature overnight. The fungal culture was then inoculated on the slant. The samples were incubated for 7 days at 29 oC and growth inhibition was observed.
4.7.4 Antispasmodic activity
The spasmolytic activity of the plant material was studied by using isolated rabbit jejunum method [190]. Respective segments of 2-cm length were suspended in a 10 ml of
Tyrode’s solution and bubbled with carbogen gas at 37 C. Composition of Tyrode’s solution in mM was: KCl 2.68, NaCl 136.9, MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, CaCl2 1.8 and glucose 5.55. A resting tension of 1 g was applied to each tissue and kept constant throughout the experiment. Intestinal responses were recorded isotonically using a Bioscience transducer and Harvard oscillograph. Each tissue was allowed to equilibrate for at least 30 min before addition of any drug, then stabilized with sub-maximal concentration of acetylcholine (0.3 µM) and bath fluid was subsequently replaced with normal Tyrode’s solution before starting experiment. Under these experimental conditions, jejunum exhibits spontaneous rhythmic contractions, allowing testing relaxant (spasmolytic) effect directly, without use of any agonist.
165 | Page Experimental
4.7.5 Determination of Ca++ antagonist action
To assess whether the antispasmodic effect of the plant materials is mediated through
calcium channel blockade, high K+ (80 mM) was used to depolarize the preparations as [191].
Addition of high K+ to the tissue bath produced a sustained contraction. Relaxation of intestinal
preparations by the plant material, pre-contracted with K+, was expressed as percent of the
control response mediated by K+.
4.7.6 Statistical analysis
“Data expressed are mean standard error of mean (SEM, n = number of experiments)
and the median effective concentrations (EC50 values) with 95% confidence intervals (CI).
Concentration-response curves (CRCs) were analyzed by non-linear regression using Graph
Pad Program (Graph PAD, San Diego, CA, USA)”.
4.7.7 Biological assay of the compounds
Antibacterial assay:
“Compounds 180-186 were screened against the pathogenic microbes using
agar – well diffusion method [105, 135]. In this method one loop full of 24 hours old culture
containing approximately 104-106 colonies forming units (CFU /ml) was spread on the surface
of Mueller-Hinton Agar plates. Wells were dug in the media with the help of sterile metallic
cork borer. Stock solutions of the test samples (180-186) in the concentration of 1 mg/ml were
prepared in dimethyle sulfoxide (DMSO Merck) and 100 µl dilutions were added in their
respective wells. The antibacterial activity of compounds was compared with tetracycline used
as standard drug.
166 | Page Experimental
Fungicidal assay:
Agar Well diffusion method was used to determine the antifungal assay [192] using miconazole as the standard drug. The compounds 180-186 were dissolved in DMSO (50 mg /
5ml). Sterile sabouraud’s dextrose agar medium (5 ml) was placed in a test tube which was then inoculated with the sample solution (400 µg /ml) by keeping in slanting position at room temperature overnight. The fungal culture was then inoculated on the slant. The samples were
o incubated for 7 days at 29 C and growth inhibition was observed”.
167 | Page Experimental
PART C
4.8 GC/MS Analysis of oil from Buddleja asiatica
4.8.1 Volatile oil extraction
Fresh leaves (100 g) of B. asiatica were air dried and subjected to hydro-distillation for
3-4 hours using a Clevenger-type apparatus for essential oil extraction. The extracted oil (2 %) was separated from water by extraction thrice with EtOAc (300 ml) and was dried by filtration over anhydrous sodium sulphate.
4.8.2 Non-volatile oil extraction
Extraction of Oil
Dried leaves powder (100 g) of B. asiatica was extracted with n-hexane using Soxhlet apparatus to obtain the lipid contents. The solvent was evaporated under reduced pressure on a rotary evaporator and the produced oil (5 g) was dried in an oven at 105 oC to a constant weight
[193].
Extraction of Fat
The above oil was treated with acetone (100 ml), yielded a precipitate (fat). The fat was re-crystallized with CHCl3 - CH3OCH3 (2 : 3) system at room temperature to yield needle shaped crystals. It was subjected to TLC on precoated silica gel TLC card using n-hexane - EtOAc (9 : 1) as a solvent system. CoCl2 was used as spray reagent which showed many spots with different Rf values on TLC.
4.8.3 Instrumentation
Sample Preparation for non-volatile oil and fat:
1 mg of each sample was dissolved in 1 ml DCM. 1 ml of BSTFA was added to this solution, mixed well and allowed to stand for 10 minutes. Alongside the samples, a method
168 | Page Experimental blank was also prepared and analyzed.
GC/MS:
GC/MS Analysis was carried out by combined gas chromatography-mass spectrometry using an Agilent 7890A Series GC connected to an 5975C Inert XL mass selective detector.
The splitless injector and interface were maintained at 300°C and 340°C respectively. Helium was the carrier gas at constant inlet pressure. The temperature of the oven was programmed from 50 °C (2 min) to 350 °C (10 min) at 10 °C/min. The GC was fitted with a 15 m X 0.25 mm, 0.25 µm HP-5MS 5% phenyl methyl siloxane phase fused silica column. The column was directly inserted into the ion source where electron impact (EI) spectra were obtained at 70 eV with full scan from m/z 50 to 800.
4.8.4 Thermal stability measurement
Diamond Thermo gravimetric / Differential Thermal analyzer (PerkinElmer, USA) was used. 2 mg of non-volatile oil was taken in an aluminium pan and weighed on an analytical balance. The pan was then placed in the room temperature furnace of the instrument, and the exact sample weight was determined by microbalance. The experiment was performed at a heating rate of 10 oC / min; whereas, the temperature was varied from room temperature 35 oC
o to 600 C. The analysis was carried out under Nitrogen (N2) atmosphere at a flow rate of 100 ml
/ min. The continuous records of weight loss and temperature were obtained from the TG/DTG curves, i.e., thermogram. The thermal stability of the oil sample was measured as a function of initial temperature of thermal decomposition (Tonset).
169 | Page
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182 | Page Publications
PUBLISHED PAPERS
1. “Two new cholinesterase inhibitors asiatoates A and B from Buddleja asiatica”.
Farman Ali, Hidayat Ullah Khan, Masood Afzal, Abdul Samad, Shafiullah Khan and Irshad
Ali. (Journal of Asian Natural Products Research, UK, Published, DOI =
10.1080/10286020.2013.794417 (2013).
2. “Antimicrobial constituents from Buddleja asiatica”. Farman Ali, Muhammad Iqbal,
Rubina Naz, Abdul Malik and Irshad Ali, Journal of The Chemical Society of Pakistan, 33
(1): 90-94 (2011)
3. “Studies on antibacterial, antifungal, antispasmodic and Ca++ antagonist activities of
Buddleja asiatica”. Farman Ali, Irshad Ali, Hidayat Ullah Khan, Arif Ullah Khan and
Anwarul Hassan Gilani, African Journal of Biotechnology, 10 (39), 7679-7683 (2011)
4. “Qualitative and Quantitative Assessment of Fatty Acids of Buddleja asiatica by GC-MS”.
Farman Ali, Irshad Ali, Hafsa Bibi, Abdul Malik, Ben Stern & Derek James Maitland.
(Journal of the Chemical Society of Pakistan, 2013) (In press)
5. “Gut Modulatory effects of Daphne oleoides are mediated through cholinergic and Ca++
antagonist mechanisms” Arif–ullah Khan, Farman Ali, Dilfaraz Khan and Anwarul Hassan
Gilani. Pharmaceutical Biology, 49 (8): 821-825, 2011
6. “Phytochemical study of the constituents from Cirsium arvense“. Zia Ul Haq Khan,
Farman Ali, Dilfaraz Khan, Shafi Ullah Khan and Irshad Ali. Mediterranean Journal of
Chemistry, 2, 64-69, 2011.
7. “Phytochemical, antioxidant and antifungal studies on the constituents of Lonicera
quinquelocularis”. Irshad Ali, Dilfaraz Khan, Farman Ali, Hafsa Bibi and Abdul Malik,
Journal of The Chemical Society of Pakistan, 35 (1), 139-143, 2013
183 | Page Publications
8. “Biological screening and chemical constituents of Vibernum grandiflorum”.
Zaher Shah, Farman Ali, Dilfaraz Khan, Shafiullah Khan, Hidayat Ullah, Rasool Khan and
Irshad Ali, Journal of the Chemical Society of Pakistan, 2013) (In press)
9. “Bioactive constituents from Buddleja species”. (Review). Farman Ali (Submitted to
Chemistry and Biodiversity, 2013).
10. “Farmenol, A novel α-chymotrypsin inhibiter from Buddleja asiatica”,
Farman Ali, Dilfaraz Khan, Hidayat Ullah Khan, Irshad Ali and Derek James Maitland. (In
process)
11. “GC/MS analysis of Buddleja asiatica leaves essential and its antimicrobial activities”.
Farman Ali, Ben Stern, Hidayat Ullah and Irshad Ali. (In process).