PHYTOCHEMICAL SCREENING on the CONSTITUENTS of RUMEX OBTUSIFOLIUS” Ph.D Thesis

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“PHYTOCHEMICAL SCREENING ON THE CONSTITUENTS OF RUMEX OBTUSIFOLIUS” Ph.D Thesis

By ABDUL KHABIR KHAN

Institute of Chemical Sciences, Gomal University, Dera Ismail Khan, Pakistan. 2017

“PHYTOCHEMICAL SCREENING ON THE CONSTITUENTS OF RUMEX OBTUSIFOLIUS” Thesis submitted for the fulfillment of the degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY BY ABDUL KHABIR KHAN

Institute of Chemical Sciences, Gomal University, Dera Ismail Khan, Pakistan. 2017

CONTENTS

DEDICATION i

ACKNOWLEDGMENTS ii

SUMMRY iii

S.No DESCRIPTIONS Page 1 Chapter No 1: Introduction 1 1.1 Importance of medicinal plants 2 1.2 Family Polygonaceae 5 1.2.1 Botany of the family Polygonaceae 6 1.2.2 Chemistry of family Polygonaceae 6

1.2.3 Pharmacology of the family Polygonaceae 7

1.3 Genus Rumex 8

1.3.1 Distribution of genus Rumex 8

1.3.2 Morphology of genus Rumex 14

1.4 Rumex Obtusifolius 16

1.4.1 Habitat 17

1.4.2 Morphology 17

1.4.2.1 Roots 17

1.4.2.2 Stems 17

1.4.2.3 Leaves 17

1.4.2.4 Flower and Inflorescence 17

1.4.2.5 Taxonomic position 18

2 Chapter 2: Literature Review 19

2.1 Traditional uses of Rumex species 20

2.2 Traditional medicinal uses of Rumex species 21

2.3 Pharmacological and Biological screening of rumex species 23

2.4 Compound isolated from Rumex species. 29

4.2.1 Structures of the isolated compounds 33 2.5 R. Obtusifolius 46 2.6 Biosynthesis of anthraquinones 47

3 Chapter No 3: Result and Discussion 50 Preliminary phytochemical screeninig (qualitative) of crude extracts of 3.1 R.Obtusifolius 51

Biological Screening of Dichloromethane Sub-fractions of Rumex 3.2 Obtusifolius 53

3.2.1 Antibacterial screening 53

3.2.2. Antifungal screening 55

3.2.3. Cytotoxicity screening 58

3.3. Secondary metabolites from Rumex Obtusifolius 62

3.4. DCM (Dichloromethane) soluble fraction 62 3.5. Structure elucidation of compounds. 63 3.5.1. Obtusifolate A (102) 63 3.5.1.1. UV-Visible and IR of Obtusifolate A (102) 63 3.5.1.2. Mass spectrometry of Obtusifolate A (102) 64 3.5.1.3. 1H-NMR spectrum of Obtusifolate A (102) 64 3.5.1.4. 13C NMR spectrum of Obtusifolate A (102) 65 3.5.1.5. Other chemical tests for identification Obtusifolate A (102) 65 3.5.2. Obtusifolate B (103) 67 3.5.2.1 UV-Visible and IR of Obtusifolate B (103) 67 3.5.2.2 Mass spectrometry of Obtusifolate B (103) 68 3.5.2.3 1H-NMR spectrum of Obtusifolate B (103) 68 3.5.2.4 13C NMR spectrum of Obtusifolate B (103) 69 3.5.2.5 Other chemical tests for identification Obtusifolate B (103) 69 3.5.3. Obtusifolate C (104) 71 3.5.3.1 UV-Visible and IR of Obtusifolate C (104) 71 3.5.3.2 Mass spectrometry of Obtusifolate C (104) 72

3.5.3.3 1H-NMR spectrum of Obtusifolate C (104) 72 3.5.3.4 13C NMR spectrum of Obtusifolate C (104) 72 3.5.3.5 Other chemical tests for identification Obtusifolate C (104) 73 3.5.4. Obtusifolate D (105) 75 3.5.4.1 UV-Visible and IR of Obtusifolate C (104) 75 3.5.4.2 Mass spectrometry of Obtusifolate C (104) 75 3.5.4.3. 1H-NMR and 13C-NMR spectra of compound D (105) 76 3.5.4.4. Other chemical tests for identification Obtusifolate C (104) 76 3.6. Free radical scavenging activity of the isolated compounds 78 4 Chapter No 4: Experimental 80 4.1. Plant collection, Identification and Grinding 81 4.2. Extraction and Fractionations 81 4.3. Preliminary qualitative phytochemical analysis 81 4.3.1. Alkaloids 81 4.3.2. Flavonoids 81 4.3.3. Tannins 82 4.3.3. Tannins 82 4.3.4. Cardiac glycosides 82 4.3.5. Anthraquinones 82 4.3.6. Steroids 82 4.3.7. Saponins 83 4.4. Sub-fractionation of Dichloromethane soluble fraction 83 4.5. Biological Screening of Dichloromethane Sub-fractions of Rumex 85 Obtusifolius 4.5.1. Antibacterial screening 85 4.5.2. Antifungal screening 85 4.5.3. Cytotoxic screening 86 4.6. Secondary metabolites from R. Obtusifolius 87 4.6.1. Instrumentation 87 4.6.2. Chromatography 87

4.6.3. Isolation, Purification and Characterization of compounds 88 4.6.4. Obtusifolate A (102) 91 4.6.5 Obtusifolate A (103) 93 4.6.6 Obtusifolate C (104) 95 4.6.7 Obtusifolate D (105) 96 4.7. Free radical scavenging activity (RSA) of the isolated compounds 97 5 Chapter No 5: References 99

TABLES

Table No Table Name Page

1.1 Rumex species 9

2.1 Compound isolated from Rumex species. 29

Preliminary qualitative phytochemical analysis of crude extracts of 3.1. 52 R.Obtusifolius

Antibacterial screening of the sub- fractions of DCM fraction of Rumex 3.2 54 obtusifolius (in mm)

3.3 % Inhibition of fungi of sub-fractions of DCM fraction of Rumex Obtusifolius 56

Illustration of percentage mortality of brine shrimps at different 3.4. 58 concentrations of sub-fractions and respective LD50 values

3.5 1HNMR and 13CNMR data of Obtusifolate A(102) (δ=chemical shift in ppm) 66

3.6 1HNMR and 13CNMR data of Obtusifolate B(103), (δ=chemical shift in ppm) 70

3.7 1HNMR and 13CNMR data of Obtusifolate C(104), (δ=chemical shift in ppm) 74

3.8 1HNMR and 13CNMR data of Obtusifolate D(105), (δ=chemical shift in ppm) 77

Percentage of RSA of compounds 102, 103, 104 and 105 from Rumex 3.9 78 Obtusifolius

4.1 Sub-fractionation of Dichloromethane soluble fraction 84

FIGURES AND SCHEMES

Figure and scheme Page

Scheme 2.1:Polyketide pathway for anthraquinone biosynthesis 48

Scheme 2.2:Shikimate pathway for anthraquinone biosynthesis 49

Fig 3.1. % Inhibition of fungi of sub-fractions of DCM fraction of 57 Rumex obtusifolius

Fig No: 3.2. Illustration of percentage mortality of brine shrimps at 60 different concentrations of sub-fractions.

Fig 3.3. Illustration of respective LD values of brine shrimps at 50 61 different concentrations of sub-fractions

Fig 3.4 % DPPH inhibibition zone of compound the four isolated 79 compounds from Rumex Obtusifolius

Scheme 4.1 Extraction and Fraction of R. obtusifolius 89

Scheme 4.2 Chromatographic resolution on Si-gel column eluted 90 with n-hexane, DCM and EtOAs as solvent system.

Dedication Dedicated to my parents (Late) and brothers, their foresight, unconditional support and values paved the way for a privileged education.

Acknowledgements

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.

Firstly I would like to express my feelings of gratitude for the kind support and persistent encouragement of my ever smiling supervisor Dr. Shafiullah Khan. I am very thankful to him for his kind, cool calm and nice behaviour.

I am pleased to acknowledge Dr. Shafiullah Khan, Director of the Institute of Chemical Sciences for providing me with all the facilities to complete this task. Due to his tremendous efforts and dynamic effort for research facilities the Institute is now progressing day by day and the institute which was formerly Department of Chemistry got the name of institute of chemical sciences.

I would also like to acknowledge the contributions of Dr. Kamran khan, Dr. Syed Badsha, Dr. Saed Ahmad and Dr. Hidayat Ullah for their encouraging and supporting behavior.

I am extremely thankful to Dr. Afzal Shah (Chairman Chemistry Department UST Bannu) for providing lab space and Dr. Mushtaq Ahmad (Assistant Prof. UST, Bannu) for helping me in carrying out the biological activities. I am extremely thankful to Beijing University of Chemical Technology China for helping in characterization of the compounds.

I like to express my gratitude to friends especially, Dr. Farman ullah khan and Mr. Fada Khan for their nice company and kind support during the study.

Thanks to all technical and non-technical staff of the Department of Chemistry especially to Hafiz Aziz and Dr. Naqeeb Ullah for their assistance. Also, thank you to all the academic staff.

ABDUL KHABIR KHAN

Summary

The present Ph.D. thesis deals with the phytochemical screening on the constituents of Rumex

Obtusifolius.

Rumex obtusifolius is commonly known as ‘broad-leaf dock’. Itis perennial Herb that grows to a height of 50 to 130 cm. R. obtusifolius occurs along with its very close relative’s R. longifolius and R. crispus. It is widely distributed in ditches, wetlands, riparian areas, roadsides, meadows, waste grounds, disturbed damp areas and pasture fields.

This plant is very important in research point of view because of its traditional uses in medicine in several countries of South America. According to folk medicine this plant’s root has a prominent detoxifying result on the liver and is used against fever, jaundice, and as an anti- anemic tonic. The roots are also laxative. In addition, the leaves of this Rumex are used against hepatic, dermatological and eye problems. They are functional in the relief of furuncles, bruises and are also used as antiseptic and as scar healer. It is used as an antidote to nettle, astringent, depurative, tonic and laxative. It is also used for treatment of tumors, blisters, sores, burns and cancer.

The thesis is presented in following two parts.

Part-A: Biological screeing

The present study has been carried out to investigate the antibacterial, antifungal and cytotoxic screening of sub-fractions of dichloromethane. The bioactivity screening of F1, F2, F3, F4, F5 and

F6 sub-fractions of dichloromethane of R. obtusifolius were assessed using conventional disc diffusion method, agar tube dilution method and the brine shrimp lethality assay. The most

remarkable antibacterial activities are observed with the F6having zone of inhibition (22.5mm) against ampicillin-resistant Escherichia coli. The antifungal results indicate that F3, F4 and F5 inhibited fungal growth more competently as compared to F1 F2 and F6. Maximum inhibition was recorded by F4 (69.4 %) against Aspergillus flavas. F1 does not show any prominent inhibition

(7.4 ± 2.51 to10.3 ± 2.21) against all fungal strains, b/c the probability of bioactive compounds in F1 is less, as ethanolic crude was already extracted with n-hexane in solvent extraction process. LD50 values of cytotoxicity indicate that F4 sub fraction of dichloromethane fraction is the most effective (LD50 =437.4), having 66.65 % maximum mortality of brine shrimp at 100 ppm,while least effective one is fraction of Rumex obtusifolius at early vegetative stages for seeking bioactive sub-fractions.

Part-B: Isolation and Characterization

Phytochemical studies on DCM fraction of R. obtusifolius have resulted in the isolation and characterisation of four new compounds for the first time.

New compounds

1. 1, 8-dihydroxy-5-methyl-3-(4'-(5''methylbut-4''-enoyl)-2'-(2''oxopropyl)-6-tert-pentyl-

anthraquinone-2-carboxylate (Obtusifolate A)(102)

2. 1, 8-dihydroxy-5-methyl-3-(2'methyl-4'-(2''methylbut-1''-enoyl)-6-tert-pentyl-

anthraquinone-2-carboxylate (Obtusifolate B)(103)

3. Ethyl 2-(18,18- dimethylbutanoyl)-1-methyl9,10-dioxo-5-(2-oxopropyl) anthraquinone-

7-carboxylate (Obtusifolate C)(104)

4. Ethyl 2-(16,16-dimethylbutanoyl) -4-methoxy, -1-methyl 9,10-dioxo, anthraquinone-7-

carboxylate (Obtusifolate D)(104)

19 O OH O OH

21 18 17 1 9a 9 8a 8 O 13 6' 3 6 4a 10 10a 16 1' 5 12

4' 2' O 11 14 1'' 4'' H 5'' 2'' O 3'' Obtusifolate A (102) 7'' 6'' O 8''

O OH O OH 18 1 9 8 O 17 9a 8a 13 6' 3 6 10 10a 4a 16 1' 5 12

4' O 11 14 3' 7' 1'' Obtusifolate B (103) O 2'' 4'' H 3''

5'' O 23 O 8 1 21 13 17 7 8a 9 1a O 11 18 20 12 19 O 3 5a 10 4a 5 4

O 14 O 15 Obtusifolate C(104) 16

O 21 O 8 1 19 13 8a 9 1a O 15 11 7 16 18 12 17 O 3 5a 10 4a 5 4 Obtusifolate D (105) 14 O O

Chapter No 1

Introduction

1.1. Importance of medicinal plants

Local plant derivatives play a key role in the provision of medical care for human beings all over the World. The reason which compels people to use wild plants species as foodstuff is times of famine, however on the other hand eating plant derivatives is becoming a fashion in our modern culture [1]. Plants and their derivatives are getting more importance against many diseases due to their no or less side effects and non-toxic nature. So, scientists have been constantly studying on the identification, analysis, investigation, characterization and assessment of medicinal plants [2,

3]. Approximately 0.4 billion people at present time depend on traditional herbal medicine even in the modern world. About 25 percent of standard drugs recommended by physicians originate from folk and traditional medicines still in the present era [4]. People of the developing world have turned back their interest and concentration towards herbal medicines for substitutive health care purposes because they are cheap and easily accessible as compared to synthetic medicines

[5].

Nature has been a source of therapeutic agents for thousands of years and many new drugs have been resulted from natural sources, many of these identifications and isolations were based on the uses of the agents in traditional remedy [6]. In the early 20th century botanical medicine was chief healthcare system as analgesics and antibiotics were not as yet exposed. Botanical medicine slowly lost its attractiveness among the people due to arrival of allopathic system of medicine which is based on the quick curative actions of synthetic medicine [7]. Lately there has been a shift in worldwide tendency from synthetic to botanical medicine, which may be called “Return to Nature”. Medicinal plants have been recognized for millennia and are extremely valued worldwide as a large and rich source of medicinal agents for the prevention of ailments [8]. The search for perfect health and for relieves pain, early man to explore immediate and instant natural

products. It brings about for the development of plants, animals and minerals products, as a variety curative agents [9].

Plants have been used as medicines all time during the history. In fact, investigations on wild animals confirm that they eat certain wild plants to care for themselves for some illnesses. The use of botanical medicine is extremely well established and recognized in Asia; therefore the majority of the medicinal plants species that have global recognition come from India and China.

The applications of herbal medicine are increasing rapidly, particularly for correcting imbalances caused by modern diet and lifestyle in N. America and Europe. Most people now days take medicinal plant derivatives on a daily basis, to maintain good health as much as to treat disease

[10]. The importance of traditional health systems and medicinal plants in solving the health problems in world is gaining increasing concentration. Due to this interest, the research on plants of medicinal importance is growing extraordinarily all over the world, frequently to the harm of natural habitats and mother populations in the countries of origin. Most of the developing countries have adopted traditional medicinal practice as a fundamental component of their civilization. The medicinal preparations are resulting from plants, either in the simple form of raw plant materials or in complex form of mixtures and crude extracts etc [11].

In beginning of development of modern medicines, biologically active compounds from higher plants species have showed an important role by providing medicines pain to and combat diseases e.g. In the British Pharmacopoeia in1932; more than seventy percent of organic monographs are on plant derivative products, but with the initiation of antibiotics and synthetic medicines the role of plant derived remedial products much declined especially in the developed countries. Therefore, In the British Pharmacopoeia in (1980), the share of plant-derived monographs fell to about twenty percent. In terms of new chemical entities introduced as

remedial agents over the past few decades, the share of herbal-derived medicines has been no

more than two percent [12].

This latest revival of interest and importance in botanical medicines has been spurred on by

several reasons, which are listed below [13-16].

I. The effectiveness of herbal medicines

II. Source of direct remedial agents

III. Model for new synthetic compounds

IV. The preference of patients for natural therapies, a greater interest in alternative drugs and

a commonly held belief that botanical medicines are better to artificial products.

V. The consumption, production and international trade in medicinal plants species are

growing and predicted to grow in future somewhat notably.

VI. A disappointment with the outcomes from modern synthetic products and the idea that

phytomedicines may be useful in the treatment of some diseases where usual therapies

and medicines have verified to be insufficient and poor.

VII. A movement and progress towards self-medication

VIII. The high cost of modern drugs

IX. Side effects of the most modern drugs

Study of the biological activities of plants species for the last two centuries have resulted

compounds for the development and improvement of modern synthetic organic chemistry as a

key direction for discovery of more effective novel and useful therapeutic products [9]. There are

2, 50,000 flowering plants are estimated on the earth, in which only six percent have been analyzed for biological activity and about 15 percent have been screened phytochemically, so consistent research study should be carried out to find out a probable abundance of medicinal extracts in these plant species. [17]. Plants contain biologically active agents. For their investigation and analysis, necessary methods and tools are required. These consist of suitable chemical screening procedures and biological activities. To investigate and explore for novel antimicrobial agents is necessary to observe microbial occurrence and resistance of fetal opportunistic infections [18].

Natural products have a broad diversity of functions and structures, and have traditionally provided substantial inspiration for drug development and improvement programs. Modern screening and isolation techniques have enhanced the search for new molecules and increased interest in folk medicinal plant extracts [19].

1.2. Family Polygonaceae

Polygonaceae comprises about 50 genera and 1120 species distributed worldwide, but primarily north temperate with a few species in tropical zones [20].

Secondary metabolites of the families Polygonaceae and Asteraceae species can be considered as promising starting materials for pharmaceutical discoveries, in consequence of their pharmacological potential, and in particular their remarkable anti-inflammatory and antitumour properties, which provides a basis for screening for new active constituents from these families for the treatment of cardiovascular diseases and Cancer [21].

1.2.1. Botany of the family Polygonaceae

The family was divided into two subfamilies, Polygonoideae and Eriogonoideae by Brandbyge

(1993) [22]. The largest genera include Rumex, Polygonum, Rheum, Coccoloba, Calligonum and

Persicaria. Polygonaceae is most diverse in the Northern Temperate Zone. Some species of

Persicaria, Fallopia, Rumex and Polygonum are among the most troublesome invasive species in

N. America and Europe [23]. Morphologically its plants are herbs, shrubs, or small trees, sometimes monoecious or dioecious [24].

Most of the species of Polygonaceae family are annual or perennial herbs, some are small shrubs, but trees and vines are rarely present. A distinctive feature of this family is the ocreae, a nodal sheath variously interpreted as an outgrowth of the sheathing base of the petiole, as an expanded axillary stipule, or as connate stipules. The leaves of buckwheat are nearly always alternate and penninerved, rarely whorled or opposite. In most genera the stem is characteristically swollen at the points of nodes. Flowers are generally perfect and actinomorphic with two to six uniform petaloid petals, often in two whorls of three or one whorl of five, persistent in the fruit, the ovule is generally orthotropus, the fruit is a lenticular nut or trigonous, and the seeds contain copious endosperm [22, 23, 25].

1.2.2. Chemistry of family Polygonaceae

The most important compounds isolated from species belonging in the Polygonaceae family are polysaccharides, flavonoids mainly flavonols or their O/C-glycosides (O- glycosides and C- glycosides) and phenylpropanoids particularly caffeic acid and its glycosides, sinapic acid and chlorogenic acid [22-24]. Other important phenolic compounds are anthraquinones particularly physcion, chrysophanol and emodin [25-27] and stilbenes often piceid, trans-resveratrol and its

glycoside [22, 23, 28, 29]. Characteristic components of some Polygonum plants are drimane- type norsesqui- and sesquiterpenoids and sulfated flavonoids [30-32]. It is of great interest that

Rumex plants contain 24-norursane-type triterpenoids [33].

The most of the species occurring in the Carpathian Basin have only been poorly analyzed except Fallopia species. Anthraquinones, flavonoids, stilbenes and polysaccharides have been identified from their stems, roots, flowers and leaves [23, 34, 35].

1.2.3. Pharmacology of the family Polygonaceae

Many plants of Polygonaceae are rich sources of bioactive constituents which have a wide range of medicinal properties. Anti-inflammatory, antioxidant, antimicrobial, antitumour, antiulcerogenic and antileukaemic properties and aldose reductase, α-glycosidase, lipid peroxidation and platelet aggregation-inhibitory effects were reported, previously.

The polysaccharides have been found to exhibit significant radical-scavenging properties, representing their potential application as novel natural antioxidants [34]. Stilbene derivatives such as resveratrol and piceid with antifungal and antibacterial activities have also been isolated from many Polygonaceae plants [36]. Chalcones and flavonoids have various biological activities. Their strong antioxidant properties in particular play significant roles against radical oxidative stress causing pathological processes i.e. cancer or arteriosclerosis [37]. Quercetin, isolated and elucidated from many species of this family has the ability to induce apoptosis in human leukaemic cells [38].

A quercetin (quercetin-3-O-β-D-glucuronopyranoside) was identified and isolated in large amount from Rumex aquaticus and has been investigated in many experimental models. It confirmed to inhibit neutrophil infiltration into the gastric mucosa, pro-inflammatory cytokine

(IL-1β and TNF-α) production [39], the production of intracellular ERK½ and ROS activation

[40]. It also decreased the area injuries of gastric lesion sizes, the gastric pH and acid output [41].

Its anti-inflammatory and antioxidative properties were evaluated on cultured feline oesophageal epithelial cells [42].

Sesquiterpenes extracted and isolated from P. hydropiper possessed interesting biological properties, i.e. antitumour-promoting, antifungal and lens aldose reductase-inhibitory effects.

[32, 43]. The same species also has tyrosinase-inhibitory, oestrogenic, antimutagenic and antinociceptive properties [44-46].

R.palmatum, among the well-known species of the Polygonaceae, produces anthranoids as the most characteristic compounds. Their pharmacological effects have been studied in many assays.

Aloe emodin induces the apoptosis of human being nasopharyngeal carcinoma cells [47].

Emodin has antitumour and antidiabetic properties [48-50]. Additionally, emodin has been evaluated for its neuroprotective and lipid-lowering activities in rat cortical neurons [51, 52].

1.3. Genus Rumex

The name Rumex derived from the Latin words for dart, alluding to the shapes of the leaves. It is the largest genus of family Polygonaceae [53].

1.3.1. Distribution of genus Rumex

This genus includes more than 250 species distributed worldwide [54]. These species are listed in Table No 1.1.

Rumex plants can constitute a considerable part of the biomass (about 70 % in the mentioned studies) and the yield reduction is directly proportional to the area of ground covered by dock

species. The loss in grass growth is caused by competition from the dock plants, mostly by shading but also by below ground competition for nutrients and water [55]. Several investigations confirm that Rumex species can cause reduced grass yields [56, 55].

The population dynamics of Rumex pants in undamaged grassland is an example of the so-called

‘phalanx’ strategy (The strategy of a phalanx species is to remain in a relatively fixed position as long as possible and to gradually colonise neighboring fields) [57].

Table No 1.1. Rumex species

1. Rumex acetosa 121. Rumex acetosella

2. Rumex balcanicus 122. Rumex brownie

3. Rumex densiflorus 123. Rumex dentatus

4. Rumex lorentzianus 124. Rumex x lousleyi

5. Rumex x acutus 125. Rumex albescens

6. Rumex brownie 126. Rumex bucephalophorus

7. Rumex diclinis 127. Rumex digynus

8. Rumex ludovicianus 128. Rumex lugdunensis

9. Rumex x alexidis 129. Rumex alpestris

10. Rumex chrysocarpus 130. Rumex confertus Willd.

11. Rumex dimidiatus 131. Rumex dimorphophyllus

12. Rumex lunaria 132. Rumex luxurians

13. Rumex altissimus 133. Rumex alpinus

14. Rumex conglormeratus 134. Rumex confuses

15. Rumex dobrogensis 135. Rumex dissimilis

16. Rumex maderensis 136. Rumex lycheanus

17. Rumex aquaticus 137. Rumex angiocarpus

18. Rumex crispus 138. Rumex costaricensis

19. Rumex dregeanus 139. Rumex dolosus

20. Rumex maritimus 140. Rumex magellanicus

21. Rumex azoricus 141. Rumex aquitanicus

22. Rumex crystallinus 142. Rumex cristatus

23. Rumex drummondii 143. Rumex drobovii

24. Rumex polygamous 144. Rumex polycarpus

25. Rumex dumosiformis 145. Rumex dufftii

26. Rumex flexuosiformis 146. Rumex flexicaulis

27. Rumex gusuleacii 147. Rumex gussonii

28. Rumex promiscuous 148. Rumex polyklonos

29. Rumex ellenbeckii 149. Rumex dumosus

30. Rumex frutescens 150. Rumex foliosus

31. Rumex hayekii 151. Rumex hadmocarpus

32. Rumex pulcher 152. Rumex Propinquus

33. Rumex engelmanni 153. Rumex dumulosus

34. Rumex fueginus 154. Rumex fontanopaludosus

35. Rumex hazslinszkyanus 155. Rumex halophilus

36. Rumex quarrei 156. Rumex protractus

37. Rumex ephedroides 157. Rumex durispissimus

38. Rumex gamsii 158. Rumex foveolatus

39. Rumex heimerlii 159. Rumex hararensis

40. Rumex raulini 160. Rumex pseudonatronatus

41. Rumex erosus 161. Rumex ecklonianus

42. Rumex gangotrianus 162. Rumex franktonis

43. Rumex hellenicus 163. Rumex hasslerianus

44. Rumex rechingerianus 164. Rumex pseudopulcher

45. Rumex erubescens 165. Rumex ecuadoriensis

46. Rumex gieshueblensis 166. Rumex fraternus

47. Rumex henrardi 167. Rumex hastatulus

48. Rumex rectinervius 168. Rumex pseudoscutatus

49. Rumex erythrocarpus 169. Rumex elbrusensis

50. Rumex giganteus 170. Rumex fringillimontanus

51. Rumex hesperius 171. Rumex hastatus

52. Rumex recurvatus 172. Rumex pseudoxyria

53. Rumex hymenosepalus 173. Rumex esquirolii

54. Rumex interruptus 174. Rumex ginii

55. Rumex kaschgaricus 175. Rumex heteranthos

56. Rumex ruwenzoriensis 176. Rumex rhaeticus

57. Rumex x impurus 177. Rumex euxinus

58. Rumex inundates 178. Rumex gmelini

59. Rumex kaschmirianus 179. Rumex heterophylus

60. Rumex sagittatus 180. Rumex rhodesius

61. Rumex inconspicuous 181. Rumex evenkiensis

62. Rumex iseriensis 182. Rumex gombae

63. Rumex kerneri 183. Rumex hexagynus

64. Rumex sagorski 184. Rumex x romanicus

65. Rumex integer 185. Rumex exspectatus

66. Rumex jacutensis 186. Rumex gracilescens

67. Rumex khekii 187. Rumex hippiatricus

68. Rumex salicetorum 188. Rumex romassa

69. Rumex integrifolia 189. Rumex fallacinus

70. Rumex japonicas 190. Rumex gracilipes

71. Rumex khorasanicus 191. Rumex hirsutus

72. Rumex salicifolius 192. Rumex rosemurphyae

73. Rumex intercedens 193. Rumex fascicularis

74. Rumex johannis-moorei 194. Rumex graminifolius

75. Rumex knafii 195. Rumex horizontalis

76. Rumex salinus 196. Rumex roseus

77. Rumex intermedius 197. Rumex fascilobus

78. Rumex kamtshadalus 198. Rumex granulosus

79. Rumex komarovii 199. Rumex hoschedei

80. Rumex samuelssoni 200. Rumex rossicus

81. Rumex krausei 201. Rumex fimbriatus

82. Rumex marschallianus 202. Rumex griffithii

83. Rumex obovatus 203. Rumex hostilis

84. Rumex sanguineus 204. Rumex rothschildianus

85. Rumex lachanus 205. Rumex finitimus

86. Rumex maximus 206. Rumex x grintzescui

87. Rumex obtusifolius 207. Rumex hultenii

88. Rumex sanninensis 208. Rumex rugosus

89. Rumex lacustris 209. Rumexhungaricus

90. Rumex megalophyllus 210. Rumex hybridus

91. Rumex occidentalis 211. Rumexhydrolapathum

92. Rumex suzukianus 212. Rumex rupestris

93. Rumex leptocaulis 213. Rumex lanceolatus

94. Rumex monistrolensis 214. Rumex meyeri

95. Rumex oxysepalus 215. Rumex occultans

96. Rumex nankingensis 216. Rumex vesceritensis

97. Rumex leptophyllus 217. Rumex langloisii

98. Rumex montanus 218. Rumex mezei

99. Rumex pakistanicus 219. Rumex ochotensis

100. Rumex natalensis 220. Rumex vesicarius

101. Rumex limoniastrum 221. Rumex lanuginosus

102. Rumex monticola 222. Rumex microcarpus

103. Rumex pallidus 223. Rumex orbiculatus

104. Rumex neglectus 224. Rumex violascens

105. Rumex linearis 225. Rumex lapponicus

106. Rumex muelleri 226. Rumex microdon

107. Rumex palustris 227. Rumex orientalis

108. Rumex nematopodus 228. Rumex wachteri

109. Rumex lingulatus 229. Rumex lanuginosus

110. Rumex munshii 230. Rumex mirabilis

111. Rumex palustroides 231. Rumex orthoneurus

112. Rumex nemorosus 232. Rumex weberi

113. Rumex litoralis 233. Rumex latifolius

114. Rumex muretii 234. Rumex mixtus

115. Rumex pamiricus 235. Rumex oryzetorum

116. Rumex nepalensis 236. Rumex longifolius

117. Rumex lonaczewskii 237. Rumex lativalvis

118. Rumex muricatus 238. Rumex moedlingensis

119. Rumex pannonicus 239. Rumex osswaldii

120. Rumex nervosus 240. Rumex longisetus

1.3.2. Morphology of genus Rumex

Rumex plants belong to the hemicryptophytes. Characterizing for this group, which includes many rosette plants and grasses, is that buds are positioned at the soil surface, protected by leaf and stem bases [58].

Plants of Rumex are perennials, biennials or annuals, mostly herbs, rarely shrubs. Leaves are usually alternate, occasionally sagittate or hastate. They have long fleshy roots, rarely the roots

are rhizomatous. Flowers are generally unisexual or hermaphrodite arranged in whorls on branched or simple in florescences. In most members of Rumex the flowers are green, however in some species like Rumex acetosella the stems and flowers are brick-red. Typically fruits are trigonous nuts [59].

1.4. Rumex obtusifolius

https://en.wikipedia.org/wiki/Rumex_obtusifolius and picture taken from my own mobile Q-LT700

1.4.1. Habitat

It is commonly known as ‘broad-leaf dock’ [60]. Rumex is a pliny name for sorrel while the meaning of Obtusifolius is ‘obtuse leaved’ [61].

Ditches, wetlands, riparian areas, roadsides, meadows, waste grounds, disturbed damp areas and pasture fields. [62] In Sweden R. obtusifolius occurs along with its very close relative’s R. longifolius and R. crispus. [63]

1.4.2 Morphology

Rumex obtusifolius is perennial Herb that grows to a height of 50 to 130 cm. Its morphology is listed below [54, 64].

1.4.2.1 Roots

Brown -black, vertical, large, up to 2.5 cm in diameter.

1.4.2.2 Stems

Erect, 30-60 cm, branched, grooved. It is branched above middle or in upper 2/3 and glabrous.

1.4.2.3 Leaves

Basal leaves; leaf blade oblong, 5-20 × 3-8cm, petiole 3-4 cm, both surfaces glabrous, midvein prominent abaxially, base subcordate or rounded , apex acute or obtuse, margin somewhat undulate. Cauline leaves small; shortly petiolate, ocrea fugacious.

1.4.2.4 Flowers and Inflorescence

Flowers are bisexual and dense. Inflorescence is broadly paniculate and large.

1.4.2.5 Taxonomic position

Kingdom: Plantae

Unranked: Angiosperms

Unranked: Eudicots

Unranked: Core eudicots

Order: Caryophyllales

Family: Polygonaceae

Genus: Rumex

Species: Rumex Obtusifolius

Chapter No 2

Literature Review

Many species of genus Rumex have been used as vegetables and for their medicinal importance.

Based on the traditional data and knowledge, different pharmacological and phytochemical studies have been at the focus of researchers. The aim of this chapter is to collect information of the recent state of knowledge of local and traditional medicinal values, pharmacological properties and chemical constituents, in order to identify the therapeutic potential of Rumex species and further directions and guidelines of research study.

A numerous ethnopharmacological and ethnobotanical literature reports regarding to the traditional uses and occurrence of Rumex species is present. [65-67].

2.1. Traditional uses of Rumex species

Rumex species are used traditionally as edible plants in many parts of the World. In some regions of world the leaves of Rumex species for example Rumex vasicarius, Rumex crispus, Rumex acetosella, Rumex abyssinicus, Rumex singuineus, Rumex acetosa Rumex tuberosus and Rumex thirsiflorus are used as foods mostly in the forms of salads and sauces [68-75]. The airborne portions of many plants e.g. Rumex acetosella, Rumex acetosa, Rumex crispus, Rumex pseudonatronatus & Rumex patientia are gathered mostly in the spring season and used as vegetables [69, 76, 77]. In majority cases, the roots are used for treatment, while the other parts, such as the fruits, seeds and leaves are also applied. Leaves are used for soups, sauces and dressed with olive oil. Sometime leaves are mixed with boiled potatoes to moderate their PH [70,

72, 74, 76, 78]. Some members of Rumex are consumed fried in olive oil or lard or sautéed with butter or are used as filling for pie (Rumex alpinas, Rumex acetosa, Rumex nepalansis and

Rumex acetosella) [79-82]. In N. America R. hymenosepalus’s roots are used as chewing gum

[79, 83], While the stems of R. alpinus and R. acetosa are used as raw snacks [72, 79, 84]. In

some parts of India nearly all parts of R. crispus are consumed as medicine and also as food. The extremely young leaves of this species are cooked as a potherb or added to salad and soup.

Sometime stems are peeled and the inner parts are eaten. The flour which is made from dry seeds of the said plant can be used as for making pancakes [85].

In many parts of alpine, R. alpinus was used in past for different purposes, e.g. stem was peeled and applied instead of rhubarb, put into cakes, puddings and biscuits or eaten fresh [86]. The leaves of some Rumex species for example that of R. acetosa is used for making sarma i.e. a traditional south-Eastern and Middle Eastern food [87]. The tuberous roots of R. abyssinicus are

applied to purify butter s and give it a yellow colour [88]. Furthermore, rhizomes the of some species (R. hymenosepalus, R. abyssinicus) have been used in North America & Kenya as a source of a yellow stain which renders cellulose fibers red-brown when applied in the presence of sodium carbonate (Na2CO3) [79, 89]. Several species of genus Rumex are cultivated like

Rumex vasicarius & Rumex acetosa [90].

2.2. Traditional medicinal uses of Rumex species

The airborne parts, roots and leaves of the plant species are used in traditional medicine for the cure of several diseases e.g. infections, constipation, mild diabetes, diarrhoea, jaundice, oedema, inflammation and as an analgesic antihypertensive and diuretic. These parts are also used for the treatment of skin, liver and gallbladder diseases. The genus Rumex has attracted the concentration of many scientists and investigators due to its medicinal importance and phytoconstituents. The various crude extracts of this genus species, and compounds extracted and isolated from them, have been verified to possess various pharmacological properties, such as antioxidant, antiinflammatory, antibacterial, antitumour, antifungal properties and antiviral in

vivo and in vitro [28, 29,39, 91-95]. Local and traditional names for many species used as food reveal their gustatory properties, aroma. Due to gentle laxative effect of the roots of many plants of Rumex, they have been used in medicines from ancient time. R. acetosa is present in the

Korean Food Code (Korea Foods and Drugs Administration) as one of the key food resource and has been used in folk medicine as a mild purgative & also for the treatment of cutaneous diseases

[96].

For medicinal purposes mostly infusions or decoctions are prepared from the various parts of rumex plant e.g. the young leaves of R. nepalensis forms tinging nettles by rubbing over the affected parts after injury [94].

There is an old verse about R. obtusifolius. “Nettle in, dock out. Dock rub nettle out!” there is no objective proof which supports this rhyme aside from the reality that firm rubbing (by itself) was found to create a short-lived reduction of the pain inflicted by nettle. It is also possible that the

effort times and spent on finding a dock (R. Obtusifolius) leaf is enough for distracting the victim s from the itching caused by nettle rash [97, 98].

Various parts of Rumex alpinus, Rumex acetosella, Rumex acetosa, Rumex confertus, Rumex obtusifolius and Rumex crispus are mainly used in Europe to treat of various diseases. These species are also used for the treatment of diarrhoea, constipation, swellings, sores, kidney disorders and rashes and as an astringent in Romania and Hungary [76, 99].

In Ireland and England R. acetosa is applied for warts, scurvy, wounds, bruises, sore throat and jaundice .Furthermore, R. hydrolapathum, R. palustris and R. conglomeratus are also used for the treatment of scurvy, as “blood purifier”, for sunburn. They are also used for cancer therapy and bathing rashes. The decoctions of the seeds of R. obtusifolius are applied against all kinds of

coughs, bronchitis and colds [100]. In Turkey Rumex alpinus is used to treat diarrhoea, constipation and eczema and in Bulgaria and Ukraine this plant is used for the treatment stomach diseases and as a laxative [86].

Rumex alpinus roots and leaves have been used in traditional medicine in Austria and to treat viral diseases [101]. R. nervosus is used as a medicinal species for the treatment of acne, as an ophthalmic antiseptic agent and as a hypoglycaemic agent. It is also applied for the cure of eczema, wounds and rabies [102].

In Pakistan R. crispus is used for the treat of cough, constipation, Skin problems, rheumatism and tonic [103].

2.3. Pharmacological and Biological screening of Rumex species

A number of crude extracts and isolated compounds have been screened for their antioxidant, antibacterial and antitumor properties. The antifungal, antiviral, antiulcerogen, hepatoprotective, antidiabetic, purgative, anthelminthic, anti-inflammatory, antiplasmodial and antifertility, properties have also been evaluated. A detail of the biological activities performed on various species of Rumex is listed below.

The antioxidant properties of thirty medicinal species of Rumex that used in Mexico were evaluated. The powder plant materials were extracted with water by heating (85°C) for ten minutes. Two-stage Trolox-based assay analysis showed that the extracts of the stem of R. hymenosepalus had significant activity [104]. The antioxidant activities of traditional medicinal species of Cameroon were evaluated by hemoglobin ascorbate peroxidase activity inhibition

(HAPX), the Trolox equivalent antioxidant capacity and DPPH bleaching methods. The result confirmed that R.abyssinicus has the greatest activities in all the assays. In DPPH method, the

area under the kinetic curve was 10. Gallic acid was added as standard instead of trolox in TEAC assay. Gallic acid equivalent antioxidant capacity (GEAC) was 50 μg/cm3 for R. abyssinicus. In

HAPX assay the inhibition of the ascorbic acid consumption of R. abyssinicus was 100% [105].

Lone etal in 2007 evaluated the antioxidant potential of an ethanol extract of R. patientia revealed its strong activity. The extract scavenged DPPH radicals significantly and dose-

3 3 3 dependently {O2 (IC50 =29 μg/cm ), NO (IC50 =33 μg/cm ) and OH radicals (IC50=63 μg/cm )}.

Its total polyphenols content calculated in gallic acid equivalent was 315 mg/g [106]. The antioxidant study of orcinol and flavans (anthraquinones) isolated from R. patientia showed that just catechin and 6-chlor-ocatechin have strong DPPH radical scavenging activity. In this study

TLC plate was sprayed with a 0.2% DPPH solution in MeOH after developing and drying.

Active compounds appeared as yellow spots on a purple background [92].

The total phenolic content, reducing power and antioxidant activity and of hexane, EtOH,

CHCl3, EtOA and aqueous extracts of the stems, leaves and seeds of R.japonicus were studied by

DPPH assay, using superoxide radical and β-carotene bleaching method. The ethyl acetate fraction has strong antioxidant activity (Superoxide radicals scavenging activity=16.4±1.44ppm

3 3 and DPPH radical scavenging activity=86.0±0.20 μg/cm , EC50=0.04±10.0001 μg/cm ), which linked with the high content of phenolic compounds, especially pyrocatechins and pyrogallols

[107]. An investigation was performed with R. pulcher and R.papillaris and recognized that the

3 3 radical scavenging activity of the said plants were EC50=02.45 mg/cm and 03.31mg/cm , the reducing power developed as 0.60 mg/cm3 and 0.80 mg/cm3and the β-carotene bleaching inhibition were 0.30 mg/cm3 and 0.34 mg/cm3 respectively [108]. R. vesicarius extracts (MeOH acetone and EtOA and) were tested on different methods i.e. lipid per oxidation and DNA-sugar damage inhibitory activity, hydrogen peroxide scavenging effect and DPPH radical. In each case

the methanol extract of the showed the maximum inhibitions, 33.80%, 66.30% and 96.60% lipid per oxidation, DNA–sugar damage, and DPPH radical scavenging respectively [109]. The antioxidant properties of crude extracts (MeOH, hexane, EtOH, CHCl3, BuOH and H2O) of R. hastatus were examined, and the total flavonoid and phenolic contents were also investigated.

The BuOH and MeOH extracts showed the highest activities in all except H2O2 radical scavenging assay, where the CHCl3 extract showed strong activity[110].

An antioxidant study of water and acetone extracts of R. hastatus was performed with different assays like OH-radical scavenging, DPPH, ferric ion, total antioxidant capacity and reducing power. The results proved that both extract had moderated activities [111]. Ethanol, ether, and hot aqueous extracts of the seeds and leaves of R. crispus were investigated pharmacologically, the water extracts showed the higher antioxidant activities than the others, while total phenolic contents was highest in the EtOH extract of the seeds (220.0 μg/500 μg extract). In case of DPPH scavenging activity and reducing power, the EtOH extract of the seeds showed potential activities [112]. The singlet oxygen quenching abilities and the protective properties of n-hexane, chloroform, ethyl acetate and butanol extracts of R. crispus seeds against photodynamic damage were studied in biological system. High levels of total phenol contents were seen for the EtOA

3 3 and BuOH (QC50=82 μg/cm for EtOA and QC50=116 μg/cm for BuOH) which were somewhat

3 similar to that of the positive control ascorbic acid (QC50 =86 μg/cm [113].

The MeOH extract of R. crispus roots showed strong DPPH radical scavenging property

3 +3 (IC50=42.86 μg/cm ). Therefore, it has a significant ability to guard against H2O2/Fe /ascorbic acid-induced protein damage [114]. The free radical DPPH scavenging activity EtOH extract of

R. dentatus showed a higher activity (96%) than MeOH extract (73%). The ascorbic acid

(positive control) displayed 95% scavenging potential. The in vitro inhibition of LP was 86%

(ethanol extract) and 78% (methanol extract) [115]. The antioxidant potential of acetone and water extracts of R. dentatus was examined with different methods like DPPH, Fe+3 reducing power, OH radical scavenging, and total antioxidant activity. The results showed that both of the extracts have moderate antioxidant property [111]. A lyophilized extract of R. induratus leaves

3 showed a concentration-dependant antioxidant potntial (IC50=149.90 μg/cm ). Furthermore, this

3 extract exhibited inhibitory effects on XO (IC25 =708.80 μg/cm ) [116]. Another investigation proved that a lyophilized aqueous extract of R. induratus leaves had a strong concentration-

3 dependant antioxidant property with IC50 value of 106.50 μg/cm . It also showed significant

3 scavenging effects against NO free redical (IC50=92.70 μg/cm ) [78].

The cytotoxic activities of EtOH extracts of the roots, leaves and fruits of Rumex acetosella,

Rumex acetosa, Rumex confertus, Rumex crispus , Rumex hydrolapathum and Rumex obtusifolius

were studied against the 2 humans leukaemic cell lines 1301(human Tlymphoblastic cells) and

EOL-1(humans eosinophilic leukaemia) and the normal H9 (a clonal derivative of the T cell line).

3 The IC50 values exposed that R. confertus showed the highest activity 0.220 mg/cm (1301) and

3 0.230 mg/cm (EOL-1)] for roots. The leaves extracts of R. obtusifolius IC50 values were 0.47mg/ cm3 (1301) and 0.44 mg/ cm3 (EOL-1), while for R. hydrolapathum were 0.420 mg/cm3 (1301) and 0.170 mg/cm3 (EOL-1)] for fruits extracts [117].

The antiproliferative properties of water and various organic solvent extracts of twenty seven

plants species belonging to Polygonaceae (Rumex) were investigated against humans tumour cell lines (MCF-7, A-431 and HeLa) by performing the MTT assay. The chloroform extract of R. aquiticus (60.90% at30 μg/cm3 on HeLa cells and 69.30% at30 μg/cm3 on MCF7cells), R. acetosa (77.70% and 97.00% at10 and 30 μg/cm3 on HeLa cells), R. scuitatus (51.20% at 30

μg/cm3 on HeLa cells and 56.20% at 30 μg/cm3 on MCF7cells) and R. thirsiflorus (96.20% at 30

μg/cm3 on A431cells and 88.55% at 30 μg/cm3 on MCF7cells) showed considerable cell growth inhibitory effects against one or more cell lines [118]. In a previous research, Rumex patientia

3 was proved to have strong cytotoxic property against brines hrimp(LC50=1.3 mg/cm ) [119]. The

cytotoxic property of R. obtusifolius extract was screened by brine shrimps lethality assays. The

3 3 LD50 values of DCM (1.00 mg/cm ) and MeOH (41.00 mg/cm ) extracts revealed that this species has comparative low cytotoxic effects with the positive control podophyllotoxin [120].

Demirezer etal performed cytotoxicity screening on human breast carcinoma (MCF), humanmelanoma (HM02) and human epidermoid carcinoma (HEPG2) cell lines. In this study, anthraquinones (compounds No 1-3), flavans (51, 53) andorcinol (85) isolated from R. patientia

were examined. The result showed that none of the isolated compounds inhibited the growths of the cell lines [92]. Whereas anthraquinone aglycones (1–4) from R.scutatus proved to possess potent cytotoxic activities [LC50=0.05 mg/cm3 (1); 0.06 mg/cm3 (2); 0.15 mg/cm3 (3) and 0.01 mg/ cm3 (4)]. Catechin (51) and anthraquinone glycosides were inactive [92].

The antiproliferative properties of torachrysone-8-glucosides, nepodin-8-glucoside and chrysophanol were tested on MCF-7, 7901(gastriccancer), SKOV-3(oophoroma) and

A375(melanoma) tumour cell lines, the results indicated that compound No 2 showed higher activity(IC50=20.4 μM on MCF7,513 μM on7901,83.1 μM on A375 and 5.62 μM on SKOV-

3cells), than the naphthalene compounds [121]. Emodin (1) is proved to be as a atyrosine kinase inhibitor [122]. The tumour inhibitory property of Emodin (1) is based on the mammalian cells cycle modulation in definite oncogene-overexpressing cell [123].

A cytotoxic activity of emodin, emodin-8-O-β-D-glucopyranosides, emodin-8-O-β-D-(6′-O- acetyl)gluco-pyranoside, chrysophanol, physcion , citreoresin, , chrysophanol-8-O-β-D-(6′O-

acetyls) glucopyranoside, chrysophanol-8-O-β-D-glucopyranosides, aloesin, nepodin-8-O-β-D- glucopyranoside, nepalensides A and B, orcinol glucoside, hastatuside A, orientaloside,

torachrysone, torachrysone-8-O-β-D-glucopyranosides, rumexneposides A and B, hastatuside B,

(–)-epicatechin, (–)-epicatechin-3-O-gallate, lyoniresinol3α-O-β-D-A. glucopyranoside, and

(3,5-dimethoxy-4-hydroxyphenols)-1- O-β-D-(6-O-galloyl)-glucoses, isolated from R. hastatus and R. nepalensis was investigated against H522 (lungcancer), A549, MCF-7, MCF-10A and

SKBR3 cancer cell lines by performing the MTT method, with cisplatin as positive control.

Among these compounds chrysophanol-8-O-β-D-(6′O-acetyl)glucopyranoside, orientaloside and rumexneposides A, and exhibited marked activities [95].

2.4. Compound isolated from Rumex species.

The record of previously isolated compounds from Rumex species is listed in table No 2.1.

Table No 2.1. Compound isolated from Rumex species.

S. Name Reference No Basic skeleton No 1 Emodin 2 Chrysophanol 89,28,92,94,124,2 3 Physcion 6,27, 125, 126 4 aloe-emodin 5 Citreoresin 6 Rhein 121 7 Endocrocin 95

8 emodin-6-O-β-D-glucopyranosides 127

9 emodin-8-O-β- D-glucopyranosides 128 10 Ziganein 129, 130 11 Przewalsquinone 12 Barbaloin 127 Antraquinones 13 Rumexone 130 14 rumejaposide A(10R) 131,132,133 15 rumejaposide B(10S) 16 rumejaposide C(10R) 17 rumejaposide D(10S) 18 rumejaposide E(10R) 19 rumejaposide F(10S) 20 patientoside A(10S) 21 patientoside A(10S) 22 Dianthrones of chrysophanol 26, 127 23 Dianthrones of physcion

24 Heterodianthrone of chrysophanol and physcion 25 (R+) senno- side A 26 (mezo)sen- noside B 27 Aloesin 128 28 nepalenside A 128 29 nepalenside B 30 3-acetyl-2-methyl-1,5-dihydroxy-2,3- 134

epoxynaphthoquinols 31 3-Acetyl-2-methyl-1,4,5-trihy-droxy-2,3- 131

epoxynaphthoquinols 32 Nepodin 135; 94, 26 , 33 nepodin monoglucoside 42,136,121, 137, 34 patientoside A 138,128, 134 35 patientoside B Naphthalenes 36 Rumexoside 37 Orientaloside 38 Torachrysone 39 torachrysone-8-O-β-D-glucopyranoside 40 2-methoxystypandrone 136 41 Rumexneposide A 95 42 Rumexneposide 95 43 hastatuside B 135 44 Isovitexin 139

45 Apigenin 53

46 Vitexin 110, 139, 140

47 Luteolin 110

48 luteolin-7-O-glucoside 139, 126,53

49 Orientin Flavonoids 50 Isoorientin

51 Catechin 92, 141 52 Gallocatechin 142 53 6-chlorocatechin 92 54 catechin-6-C-glucoside 139 55 Epicatechin 28, 141, 142 56 Epigallocatechin

57 epicatechin-3-O-gallates 128, 142

58 epigallocatechin-3-O-gallates 59 procyanidin A2 143

60 (2R,2’R,3R,3’S,4R)- procyanidins B1 142, 143

61 (2R,2’R,3R,3’R,4R)-procyanidins B2

62 (2R,2’R,3S,3’S,4S)-procyanidins B3

63 (2R,2’R,3S,3’R,4S)- procyanidinsB4

64 (2R,2’R,3R,3’R,4S)- procyanidinsB5 143

65 (2R,2’R,3R,3’S,4S)- procyanidins B7

66 B2-3,3′ -O-digallate 142 67 epiafzelechin-(4β -8)-epicatechin 142 68 trans-resveratrol 28,144, 29 69 Pinostilbene

70 deoxyrhapontigenin 71 4-{(E)-2-(3,5-dihydroxyphenyl)ethenyl}-

1,2-benzenediols 72 4-{(E)-2-(3,5- Stilbenoids dihydroxyphenyl)ethenyl}phenyl-

hexopyranosides 73 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]-2-

hydroxyphenyl-hexopyranosides 74 Piceid 75 (2-methoxy-2,4-dihydroxyphenol)-1-O-β- 128 Tannins

D-(6-O-galloyl)glucose 76 (3,5-dimethoxy-4-hydroxyphenol)-1-O-β- 143 D-(6-O-galloyl)glucose

77 2α,3α,19α-trihydroxys-24-norurs-4(23),12- 33

dien-28-oicacids

78 23-epoxy-2α,3α,19α-trihydroxys-24-norurs- 33 Triterpenes

12-en-28-oicacids 79 tormentic acid 33 80 myrianthic acid 33 81 Lutein 90,145 82 Anhydrolutein I 145 Carotenoids 83 Anhydrolutein II 145 84 β-carotene 90 85 Orcinol 92 86 2-acetylorcinol 26 87 Sakakin 128 , 26 88 2-acetylorcinol-3-O-glucoside 89 Rumexin 146 90 paraben-acid 131 91 vanillic acid 147 92 2,6-dihydroxybenzoic acid 131 Other 93 2,6-dimethoxy-4-hydroxybenzoic acid compounds 94 caffeic acid 146 95 1-methylcaffeic acid 148,147 96 1-O-caffeoyl-β-D-glucopyranoside 97 sinapic acid 98 Neochlorogenic acid 149 99 Hastatuside A 95 100 Lyoniresinol glucoside 128

2.4.1. Structures of the compounds

OH O OH OH O OH

HO 2 1 O O

OH O OH OH O OH

O 3 4 O O OH

OH O OH OH O OH

HO 5 6 O OH O O

OH O OH O OH O OH

OH OH

HO O O 7 8 HO O O

O OH glc O O OH

10 HO OH O 9 O

OH O OH OH O OH

OH O 11 12 OH O glc

O OH OH OH O OH O OH

10 13 14 OH OH O glc

OH O OH OH O OH O O OH OH

10 10 HO

16 OH 15 OH glc glc

OH O OH OH O OH O OH

10 10 HO HO 18 17 OH OH glc glc

OH O OH OH O OH

10 10 HO O 19 OH 20 OH glc glc

OH O OH OH O OH

10 O 10 21 OH glc 22

OH O OH

10 OH O OH O 23 O

OH O OH

OH O OH Oglc O OH

O 10 10 24 OH O 25 OH

OH O OH O glc O OH

Oglc O OH

O glc O 10 HO O O OH 26 OH 27

OH O

O glc O OH

O glc O OH O glc O OH OH O

HO HO HO O 28 HO O 29

OH OH

O O

O O 30 31 OH O OH OH

OH OH O glc O OH O

32 33 glc glc O OH O OH O

35 34 Cl Cl glc glc

O OH O glc O OH O

COOH 36 37

glc O OH O OH OH O

O O 39 38 O

O O O OH O O OH O

OH HO

O 40 O 41 O

O O

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

O 42 43

OH OH

HO O HO O

glc 44 45

OH O OH O

OH OH glc

HO O HO O OH

47 46 OH O OH O OH OH glc glc

HO O O O OH OH

49 48

OH O OH O

HO O OH

glc 50

OH O

OH OH

HO O HO O OH OH

OH OH 52 51 OH OH

OH OH

HO O HO O OH OH

OH glc OH Cl 54 53 OH OH OH

OH OH

HO O HO O OH OH

OH OH 56 55 OH OH OH

OH HO O OH

OH O

57 OH O OH

OH OH

HO OH OH

OH HO O OH OH O O OH

58 OH O OH OH OH OH O HO O OH 59 O OH OH OH OH OH OH O HO OH OH

OH OH O OH 60,61,62,63 OH OH OH O OH OH

OH 65 OH OH

OH OH HO O OH

OH 64 OH O OH

OH OH OH OH OH HO O OH

O 66 OH OH O

OH OH O

OH OH 68 OH OH HO O

OH 67 OH O

OH HO

O 70 69

OH Oglc

OH HO

HO OH 72

71 OH

OH OH Oglc glcO

HO OH 74

73 OH

O O

O HO O O O O

O O HO HO O O O OH HO H HO O OH HO OH HO OH HO O 76 75

HO HO

COOH COOH HO HO

HO 78 HO 77 O

HO HO

COOH COOH HO HO

HO 80 HO 79 HO OH

O O

HO OH Oglc HO OH HO HO Oglc

86 85 87 88

O COOH COOH COOH

HO OH

OH O 90 89 91 92 Oglc OH OH OH

COOH O O

O O HO HO OH O

94 95 HO HO 93 OH

O O

O HO Oglc OH

96 97 HO HO

HO COOH

O HO O O

O OH

98 OH HO 99 Oglc OH

O OH O OH

Oglc HO

O 100 HO OH 101 O

O O

2.5. R. Obtusifolius

Rumex obtusifolius (Polygonaceae) has long been used in traditional medicines. It is used as an depurative, astringent, antidote to nettle, laxative and tonic. It is also used to treat sores, tumors, blisters, cancer and burns [150].

Among the isolated compounds from this plant previously, we can find a glycopyranoside: 6-O- malonyl-β methylglucopyranoside [152]. Another effort has precise direction of the isolation and identification of amino acid plastocyanin [153]. Compound 101 (An anthraquinone) was isolated from R. Obtusifolius by Sandra L. etal in 2099 [154]. Compounds 4, 60, 61, 62, 65 have also been isolated from R. Obtusifolius [155, 127].

2.6. Biosynthesis of anthraquinones

In plants, anthraquinones are found in many species, especially in the plant families

Rhamnaceae, Polygonaceae, and Rubiaceae [156].

Anthraquinones are structurally made from an anthracene ring (tricyclic aromatic) with a keto group each on carbon atom nine and ten. In plants, two main biosynthetic pathways for the biosynthesis of anthraquinones have been described:

I. The shikimate or chorismate/o-succinylbenzoic acid pathway is used to synthesize

anthraquinones with only one hydroxylated ring like 1,2-dihydroxylated anthraquinone

(alizarin)

II. the polyketide pathway producing anthraquinones by folding of a polyketide chain with

both rings hydroxylated(emodin)[156, 157 and references therein]

Anthraquinones and their precursors, the anthrones, are widespread substances in many different organisms ranging from fungi, bacteria, plants and some animals [158, 159, 156].

O O O CO2 O

O O O HO SCoA SCoA 2 1 SCoA O O O 3

O R1

R8 R2

CO2

R7 R3

R6 O R4 4

Polyketide pathway for anthraquinone biosynthesis; 1 acetyl-CoA, 2 malonyl-CoA, 3 octa-â-ketoacyl chain, 4 Basic structure of Anthraquinone

OH O OH

Emodin(1,8-dihydroxylated anthraquinone) from polyketide pathway

Scheme 2.1 Polyketide pathway for anthraquinone biosynthesis

HO COOH COOH COOH COOH O

HO A B C COOH OH O

COOH O R1 OH

D E OH O R3 OH

Shikimate pathway for anthraquinone biosynthesis; A shikimic acid, B o-succinoylbenzoic acid, 6 á- ketoglutaric acid,C mevalonic acid, D anthraquinone.

O OH

O Alizarin(1,2-dihydroxylated anthraquinone) from shikimate pathway

Scheme 2.2 Shikimate pathway for anthraquinone biosynthesis

Chapter No 3

Result and Discussion

3.1. Preliminary phytochemical screeninig (qualitative) of crude extracts of R.Obtusifolius

The preliminary phytochemical analysis (qualitative) of the various extracts of R.Obtusifolius was done to evaluate the existence of bioactive components. The presence of alkaloids

(Dragendroff, Wagner, Mayer), tannins, flavonoids, anthraquinones, steroids, cardiac glycosides and saponins was analyzed (Table 3.1).

Results of different experiments performed for phytochemical screening revealed that alkaloids are absent in n-hexane and aqueous extracts. Alkaloids are present in lower amount (+) with

Dragondroff test and Wagner test in DCM extract, while considerable amount is present with

Wagner test in ethyl acetate extract. Tannins are only present in considerable amount in DCM and ethyl acetate extracts. Cardiac glycosides and Steroids are present in all extracts except n- hexane. DCM and ethyl acetate extracts contain considerable amount of Anthraquinones. The table 3.1 results show that flavonoids are present in all the tested extracts.

Table 3.1. Preliminary qualitative phytochemical analysis of crude extracts of R.Obtusifolius

Alkaloids S.No Extracts TN CG ST FL SP AN DR MR WR

1 n-hexane ------+ - -

2 DCM + - + ++ + ++ ++ + ++

3 Ethyl acetate + + ++ ++ +++ ++ ++ +++ ++

4 Aqueous - - - - + + + - -

MR= Mayer’s reagent; DR= Dragondroff’s reagent; WR= Wagner’s reagent; TN=Tannins;

FL=Flavonoids; ST=Steroids; SP=Saponins; CG=Cardiac glycosides; AN=Anthraquinones and -

= Absent; + = present; ++= present considerable; +++ = present very considerable.

3.2. Biological Screening of Dichloromethane Sub fractions of Rumex Obtusifolius

3.2.1. Antibacterial screening

A disc diffusion method was performed to assess the antibacterial screening of sub-fractions of

Dichloromethane fraction of Rumex obtusifolius. The results are presented in table No 3.2. The

F1 and F2 do not show any antibacterial property. The F6 is active against all bacteria, while F3 is active only against E. coli. The most significant antibacterial properties are seen for F6. Highest zone of inhibition (22.5 mm) is showed for F6 against Ampicillin-resistant Escherichia coli.

Table No 3.2 shows that generally activity increases with polarity. The antibacterial effects of

DCM fraction of Rumex obtusifolius is mostly due to average polarity compounds, e.g.

(flavonoids, phenolics) present in medium polar fractions F4, F5 and F6. It is also cleared that the active compounds accountable for antibacterial effects is more soluble in polar organic solvent.

Antibacterial screening of all the six fractions is also shown in figure No 3.1.

Table No3.2. Antibacterial activities of the sub- fractions of DCM fraction of R. obtusifolius (in mm)

Sub-fractions 1 2 3 4 5 6 7 8

F1 R R r r r R r R

F2 R R r r r R r R

F3 R R r r r R r R

F4 13 9 14 19 8 12.5 9.5 R

F5 14 11 17.5 11 9 13 10.5 R

F6 15 14 16 22.5 9 14.5 11 10

Cefotaxime 32 31 30 30 30 30 30.5 30

̽1 = Bacillus cereuss , 2 = Bacillus subtilis , 3 = Escherichia coli , 4 = Ampicillin-resistant

Escherichia coli , 5 = Staphylococcus aureuss and 6 = Salmonella typhii , 7= Pseudomonas

aeruginosas ,8= Streptococcus pneumonia. r=Resistant=Show no activity

3.2.2. Antifungal screening

The results of table No 3.3 indicate that F3, F4 and F5 inhibited fungal growth more proficiently as compared to F1, F2 and F6. Maximum inhibition was recorded by F4 is 69.4% against Aspergillus flavas. F4 and F5 show significant inhibition ranging (35.1±2.21 to 69.4±3.18) except F4 against

Fusarium moniliformes.F1does not show any prominent antifungal activity (7.4±2.51 to10.3±2.21) against all fungi, b/c the probability of bioactive agents in F1 is less, as ethanolic crude was already extracted with n-haxane in solvent extraction procedure. Shortly, F4 is the most active and F1 least active among the 6 fractions. The results confirm previous study in which fungi toxic surface flavonoids (Isoflavonoid) have been isolated and characterized are reported to be extracted with low polar solvents and non polar [160], like n-hexane and DCM mixture. Percentage inhibition of fungi has been shown in figure No 3.1.

Table 3.3. % Inhibition of fungi of DCM sub-fractions of R. obtusifolius

Fractions A.flavass F.solani M.species A.nigers A.fumigatus A.alterata F.moniliformes

F1 9.4±1.39 10.3±2.1 8.2±1.4 9.4±2.85 8.4 ± 2.53 9.8±1.39 7.4 ± 2.51

F2 30.2±1.3 15.3±1.3 23.6±1.32 14.2±2.1 21.3±1.43 37.4±2.2 23.8±2.13

F3 25.4±2.53 15.8±1.39 14.1 ± 1.51 31.7±2.49 31.7 ± 2.49 31.7±2.49 34.2±1.25

F4 69.4±3.18 36.4±1.89 38.0 ± 1.34 36.0±1.68 56.2 ± 2.77 37.6±1.32 30.1±1.31

F5 66.2±1.41 36.2±2.14 41.3±1.34 49.1±1.23 50.2±2.41 42.4±2.51 35.1±2.21

F6 22.6±2.32 27.0±2.56 43.2 ± 1.80 51.3±1.67 15.5 ± 2.34 40.5±1.07 30.4±1.34

Terbinafin 87.6±2.70 84.5±2.13 81.78±2.12 88.4±3.16 87.4 ± 3.17 84.4±3.13 89.2±1.23

DMSO N N N N N N N

Mean ± SE and N = Nil

100%

90%

80%

70%

60% Terbinafin F6 50% F5

40% F4 F3 30% F2 F1 20%

10%

Fig 3.1. percentage inhibition of fungi of DCM sub-fractions of R. obtusifolius

3.2.3. Cytotoxicity screening

Brine shrimp assay is recommended to be a suitable tool for the pharmacological activities in plant analysis [161]. Cytotoxicity results are presented in Table No 3.4. The results indicate that the F4 is the most active (LD50 437.4), having maximum mortality of brine shrimp (66.65%) at

100ppm, while F1 is the least active fraction. Our results showed that none of the fractions are found highly effective (P > 0.05), however at high concentration, fractions were effective at probability level 0.05. Other fractions do not any show prominent results, as higher the value of

LD50 lower will be percentage mortality of brine shrimp. It can be thought that presence of a wide range of bioactive compounds with different structures and their synergistic effect may put in to the overall activity of F4. F1, F2, F4 has shown lowest % mortality of brine shrimp at 10 ppm, while it has highest value at 1000 ppm (66.6%). Figure No 3.2 Illustrated percentage mortality of brine shrimps at different concentrations of sub-fractions, while respective LD50 values of brine shrimps at different concentrations have been shown in figure No 3.3.

Table 3.4. Illustration of percentage mortality of brine shrimps at different concentrations of sub-fractions and respective LD50 values

Concentration F1 F2 F3 F4 F5 F6

10ppm 13.3 % 23.3% 13.3 % 13.3 % 20.0 % 16.6%

100ppm 23.3% 30.0% 16.6% 20.0% 26.6% 20.0%

1000ppm 30.0% 43.3% 56.6% 66.6% 50.0% 50.0%

LD50 values 53970.9 4753.8 867.8 437.4 1371.8 1552.1

70.00%

60.00%

50.00%

40.00% 10ppm 100ppm 30.00% 1000ppm

20.00%

10.00%

0.00% F1 F2 F3 F4 F5 F6

Fig No: 3.2. Illustration of percentage mortality of brine shrimps at different concentrations of sub-fractions.

60000

50000

40000

30000 LD50 values

20000

10000

0 F1 F2 F3 F4 F5 F6

Fig 3.3. Illustration of respective LD50 values of brine shrimps at different concentrations of sub-fractions

3.3. Secondary metabolites from Rumex Obtusifolius

In search of bioactive secondary metabolites from medicinal plants, Rumex Obtusifolius belonging to the genus rumex was investigated. The ethno-pharmacological and chemotaxonomic importance of the genus prompts us to start evaluation and screening on this plant. As a result, four compounds were isolated from DCM soluble fraction of Rumex

Obtusifolius. (Scheme -1, 2)

Structures of the all isolated compounds were elucidated using spectral as well as published data in literature. In this section of my project, the compounds are discussed briefly.

3.4. DCM soluble fraction

The DCM soluble fraction was concentrated and subjected to column chromatography over silica gel for preliminary fractionation. Elution was carried out with n-hexane 100 % (F1), n- hexane75% - DCM 25% ( F2), n-hexane 50% - DCM50% (F3), n-hexane25% - DCM75%(F4),

DCM100%(F5), EtOAc100%(F6), in increasing order of polarities.

It has been that F4, F5 and F6 sub-fractions of DCM fraction of Rumex obtusifolius appeared as an important source for the discovery of new cytotoxic, antibacterial and antifungal agents in biological screening, therefore these fractions were further loaded to a series of column chromatography to obtain four anthraquinone compounds, Obtusifolate A(102), Obtusifolate

B(103), Obtusifolate C(104) and Obtusifolate D(105).

All the compounds were characterized using latest spectroscopic techniques and chemical methods.

3.5. Structure elucidation of compounds.

3.5.1. Obtusifolate A (102)

19 O OH O OH

21 18 17 1 9a 9 8a 8 O 13 6' 3 6 4a 10 10a 16 1' 5 12

4' 2' O 11 14 1'' 4'' H 5'' 2'' O 3'' Obtusifolate A (102) O 7'' 6'' 8''

The fraction F4 obtained with n-hexane - DCM (25%: 75%) was subjected to column chromatography over silica gel eluting with n-hexane – DCM mixtures in increasing order of polarities. The compound obtained as yellow amorphous solid from n-hexane - DCM (1:9) was identified as Obtusifolate A (102). The Compound was elucidated through UV-VISIBLE, IR,

NMR and MS studies as an anthraquinone derivate.

3.5.1.1. UV-Visible and IR of Obtusifolate A (102)

The absorptions at (3441,3425), (1701–1728), (1590) and (1619) in cm-1 of the IR spectrum showed the presence of chelated hydroxyl groups, ester groups, conjugated carbonyl groups of anthraquinone nucleus and benzene ring respectively. Absorption at 1715 cm-1 is due to non

conjugated carbonyl group. The UV spectra of compound (102) showed maximum absorptions at

211, 239, 252, 275, and 366 nm, attributed a highly conjugated system [162].

3.5.1.2. Mass spectrometry of Obtusifolate A (102)

The molecular formula was determined through HREI-MS as C39H42O8 (m/z =638.2870; calcd.

638.2880), which shows 15 degrees of unsaturation. Eight of them are eventually accounted for anthraquinone ring, 3 for the tri-substituted benzene ring; one was due to double bonds, two for ketonic and last one for ester carbonyls group.

3.5.1.3. 1H-NMR spectrum of Obtusifolate A (102)

The 1H-NMR spectrum of compound (102) confirm alkene moiety (double bond) with a proton at (6.69ppm, s) together with two different methyl groups at (1.65ppm, s), (1.70ppm, s) attached to the same double bonded carbon. Two singlet’s at (12.13ppm, HO-1) and (12.31ppm, HO-8) revealed the presence of two peri-positioned chelated hydroxyl groups [163, 164].

Two peri-positioned chelated hydroxyl groups together with two conjugated carbonyl groups(C-

9, δ190.50; C-10; δ181.00)ppm confirmed the presence of anthraquinone nucleus. A pair of singlet appearing at (7.02ppm, H-4) and (7.12ppm, H-7) indicated a tetra-substituted 1,8- dihydroxy anthraquinone. A mutiplet at 4.20ppm corresponding to H-18 was attributed to the oxymethylene protons. Signals at (7.66d, H-3') due to meta coupling having small J (3Hz) value;

(7.72dd, H-5' J=3&8.8Hz) corresponding to ortho and meta coupling; (7.56d, H-6' J=8.8) for ortho coupling) showing another benzene ring. Signals at (2.38s, H-11 &2.13s, H-3'') having same height indicated methyl groups directly attached to aromatic ring and to carbonyl group respectively. A singlet at (3.62ppm, H-1'') was due to methylene group present b/w carbonyl and aromatic ring. A highest singlet in up field region (1.28s, H-13 and H-14) confirmed two methyl

groups in same chemically environment. The up field signals (0.85—1.70ppm) were due to protons of various chains of alkyl groups listed in table No 3.5.

3.5.1.4. 13C NMR spectrum of Obtusifolate A (102)

13C NMR spectrum determined through a DEPT (Distortionless Enhancement by Polarization

Transfer) experiment shows 38 signals for 39 carbon atoms, in which 20 are for olefinic , four for carbonyl, and the remaining are for SP3 carbons of methane, methylene and methyl groups.

The most downfield signal at (206.01ppm) is assigned to non-conjugated carbonyl carbon. Other downfield signals at (190.50, 181.00)ppm and (168.12)ppm are assigned to the conjugated carbonyls of anthraquinone ring and ester carbonyl respectively. The enone portion C=O resonated at (192.10ppm, C-4''). The carbon signal appearing at 74.00ppm is attributed to the oxymethine carbon. The most important signals, which appears at (165.42, 159.00)ppm are assigned for C-1 and C-8 to which chelated hydroxyl groups are attached. Another up field signal at 29.60ppm is due to C-13 and C-14, which confirmed two methyl groups carbons in almost same chemically environment (table No 3.5).

3.5.1.5. Other chemical tests for identification Obtusifolate A (102)

Compound (102) decolorized KMnO4 solution, which showed non-aromatic multiple bonds

(double or triple bond).

The stereochemistry at the chiral center C-18 was determined by the alkaline hydrolysis of A that yielded an alcohol that could be identified as (R)-1-methyl-1-propanol through the sign of its optical rotation.

Table No 3.5 1HNMR and 13CNMR data of Obtusifolate A(102) (δ=chemical shift in ppm) Obtusifolate A (102) Position δ (H) δ(C) 1-OH 12.31 165.42 2 ---- 117.00 3 ---- 138.90 4 7.02s 124.50 5 ---- 144.00 6 ---- 150.13 7 7.12s 144.40 8-OH 12.13s 159.00 9, ----- 190.50, 10 ----- 181.00 4a ----- 134.04 8a ----- 114.20 9a ----- 113.10 10a ----- 133.15 11 2.38s 20.01 12 ---- 34.50 13 1.28s 29.60 14 1.28s 29.60 15 1.20q 37.6 16 0.85d 9.00 17 ----- 168.12 18 4.20m 74.00 19 1.35d 22.02 20 1.75m 29.80 21 0.92 t 8.01 1' ----- 138.70 2' ----- 132.30 3' 7.66 d 129.40 4' ----- 136.60 5' 7.72 dd 127.80 6' 7.56d 127.50 1'' 3.62s 48.4 2'' ----- 206.01 3'' 2.13s 31.30 4'' ----- 192.10 5'' 6.69s 120.00 6'' ----- 133.10 7'' 1.65s 18.50 8'' 1.70s 25.50

3.5.2. Obtusifolate B (103)

O OH O OH 18 1 9a 9 8a 8 O 17 13 6' 3 6 10 10a 4a 16 1' 5 12 4' O 11 14 3' 7' 1'' Obtusifolate B (103) O 2'' 4'' H 3''

5''

The eluate obtained with 100% DCM of F4 showed one major with some minor spots on TLC were and re-chromatographed with mixtures of EtOAc - DCM in increasing polarities over silica gel. A fraction with EtOAc – DCM (1:9) contained the compound corresponding to the major spots. It was concentrated and was elucidated through UV-Visible, IR, NMR and MS studies as an anthraquinone derivate.

3.5.2.1. UV-Visible and IR of B (103)

The absorptions at (3441, 3425), (1701–1728), (1590) and (1619) in cm-1 of the IR spectrum showed the presence of chelated hydroxyl groups, ester groups, conjugated carbonyl groups of anthraquinone nucleus and benzene ring respectively.

The UV spectra of 103 showed maximum absorptions at 211, 252, 275, and 366 nm, attributed a highly conjugated system [162].

3.5.2.2. Mass spectrometry of B (103)

Through HREI-MS the molecular formula of compound 103 was determined as, C34H34O7 molecular ion peak (m/z =554.2296; calcd. 554.2305), which shows 14 degrees of unsaturation.

Eight of them are accounted for anthraquinone ring, 3 for the trisubstituted benzene ring; one was due to double bonds, one for ketonic and last one for ester carbonyls group.

3.5.2.3. 1HNMR spectrum of B (103)

The 1HNMR and 13CNMR of (103) is similar to compound (102) with little differences as shown in table No 3.5. Two singlets (12.14ppm, HO-1, and 12.33ppm, HO-8) revealed the presence of two peri-positioned chelated hydroxyl groups [163, 164]. Two peri-positioned chelated hydroxyl groups together with two conjugated carbonyl groups(C-9, 190.50ppm; C-10,181.00ppm) also confirmed the presence of anthraquinone nucleus in compound (103).

A pair of singlet appearing at (7.02, H-4) and (7.12, H-7) indicated a tetra-substituted 1,8- dihydroxy anthraquinone. A singlet at 4.23ppm corresponding to H-18 was attributed to the oxymethylene protons. Signals at {7.66d H-3' due to meta coupling having small J values; (7.72-

7.74 dd H-5') corresponding to ortho and meta coupling; (7.56d H-6') for ortho coupling} showing another benzene ring as in compound (103). Signals at (2.38s H-11 and 2.47s H-3'') having same height indicated Me groups directly attached to aromatic rings. A highest singlet in up field region (1.28ppm, H-13 &H-14) also confirmed two methyl groups in same chemically environment in compound (103). The up field signals (0.85—1.70ppm) were due to protons of various chains of alkyl groups listed in table 3.6.

3.5.2.4. 13C NMR spectrum of B (103)

The 13C NMR spectrum determined through a DEPT experiment shows 33 signals for 34 carbon atoms, in which 16 are for olefinic , three for carbonyl, and the remaining are for SP3 carbons of various methine, methylene and methyl groups. The most downfield signals at (190.50ppm,

181.00ppm) and (166.10ppm) are assigned to the conjugated carbonyls of anthraquinone ring and ester carbonyl respectively. The enone portion C=O resonated at (192.10, C-1''). The carbon signal appearing at (51.60ppm) is attributed to the oxymethyl carbon. The most important signals, which appears at (165.00, 159.00)ppm are assigned for C-1 and C-8 to which chelated hydroxyl groups are attached. Another up field signal at 29.60ppm is due to C-13 and C-14, which confirmed two methyl groups carbons in almost same chemically environment. The different olefinic carbons resonated at (113.00—150.00) which are present in various chemical environments are listed in table No 3.6.

3.5.2.5. Other chemical tests for identification Obtusifolate B (103)

Compound Obtusifolate B (103) also decolorized KMnO4 solution, which showed non-aromatic multiple bonds (double or triple bond).

Table No 3.6 1HNMR and 13CNMR data of Obtusifolate B(103), (δ=chemical shift in ppm) Obtusifolate B (103) Position δ (H) δ(C) 1-OH 12.33 165.00 2 ---- 116.01 3 ---- 138.60 4 7.02s 124.55 5 ---- 144.00 6 ---- 150.14 7 7.12s 144.40 8-OH 12.14 159.00 9 ----- 190.50 10 ----- 181.00 4a ------134.04 8a ----- 114.20 9a ----- 113.10 10a ----- 133.15 11 2.38s 20.00 12 ---- 34.50 13,14 1.28s 29.60 15 1.20q 37.6 16 0.85d 9.00 17 ---- 166.10 18 4.23s 51.60 1' ---- 139.50 2' ---- 136.70 3' 7.66 d 130.00 4' ---- 136.60 5' 7.72dd 127.80 6' 7.56d 127.50 7' 2.47s 22.50 1'' ---- 192.10 2'' 6.69s 120.00 3'' ---- 133.10 4'' 1.65s 18.50 5'' 1.70s 25.50

3.5.3. Obtusifolate C (104)

O 23 O 8 1 21 13 17 7 8a 9 1a O 11 18 20 12 19 O 3 5a 10 4a 5 4

O 14 O 15 Obtusifolate C(104)

F6 fraction was subjected to column chromatography over silica gel, eluting with n-hexane, n- hexane/DCM, DCM/AcOEt, AcOEt and MeOH in increasing order of polarity. Eluting with the solvent system AcOEt /DCM 2:1 to afford Obtusifolate C (104). It was then purified by pencil column chromatography and concentrated as an orange gummy solid.

3.5.3.1. UV and IR spectra of Obtusifolate C (104)

In UV spectra intense benzenoid absorption at 240-260 nm, medium absorption at 320-330 nm a strong quinonoid electron transfer band at 270-290nm accompanied by a weak quinonoid absorption band at 405 nm showed anthraquinones skeleton. The absorptions at (1701–1728) and

(1590) in cm-1 of the IR spectrum showed the presence of ester group and conjugated carbonyl groups of anthraquinone nucleus. Absorption at 1715cm-1 is due to non conjugated carbonyl group (C-15).

3.5.3.2. Mass spectrometry of Obtusifolate C (104)

The molecular formula was determined through HREI-MS as C27H28O6 (m/z=448.1876; calcd.

448.1886), which shows 11 degrees of unsaturation. Eight of them are eventually accounted for anthraquinone ring, two for ketonic and last one for ester carbonyls group.

3.5.3.3. 1HNMR spectrum of Obtusifolate C (104)

Two pair of doublets appearing at (7.55ppm, H-4, J=8Hz & 7.57ppm, H-3, J=8Hz) and

(7.72ppm, H-6, J=3Hz & 7.74ppm, H-8, J=3Hz) due ortho and meta coupling respectively indicated a tetra-substituted anthraquinone. Signals at (2.39s H-23 &2.13s H-16) having same height indicated methyl groups directly attached to aromatic ring and to carbonyl group respectively. A singlet at (3.91ppm, H-14) was due to methylene group present between carbonyl and anthraquinone ring. A highest singlet in up field region (1.35ppm, H-19 and H-20) confirmed two methyl groups in same chemically environment. The various up field signals at

(0.96-1.28) ppm were due to protons of various chains of alkyl groups listed in Table No 3.7.

3.5.3.4. 13C NMR spectrum of Obtusifolate C (104)

13C NMR spectrum shows 26 signals for 27 carbon atoms, in which 12 are for olefinic, four for carbonyl, one for ester and the remaining are for SP3 carbons of methine, methylene and methyl groups.

The most downfield signals at 209.00ppm and 206.01ppm are assigned to C-17 and C-15 carbonyl carbons. Other downfield peaks at (181.00, 182.50) ppm and (168.12) ppm are assigned to the carbonyls carbons of anthraquinone ring and ester carbonyl carbon respectively.

The signal at 72.50ppm is attributed to the oxy-methylene carbon. Another up field signal at

22.02ppm assigned to C-19 and C-20 confirmed two methyl groups carbons in almost same chemically environment. All the above evidences confirmed the structure of compound (104).

(table No 3.7).

3.5.3.5. Other chemical tests for identification Obtusifolate C (104)

It gave red solutions on reduction in alkaline solution (in aqueous sodium hydroxide, which distinguished anthraquinone from benzoquinones and naphthoquinones [165].

Table No 3.7 1HNMR and 13CNMR data of Obtusifolate C (δ=chemical shift in ppm) Obtusifolate C (104) Position δ (H) δ(C) 1 ------144.00 2 ------136.60 3 7.57d 127.80 4 7.55d 127.50 5 ---- 132.30 6 7.72d 129.40 7 ----- 117.00 8 7.74d 124.50 9 ------181.00 10 ------182.50 1a ------134.04 8a ------133.15 4a ------132.00 5a ------132.50 11 ------168.12 12 4.23q 72.50 13 1.28t 22.70 14 3.91s 48.40 15 ---- 206.01 16 2.13s 31.30 17 ------209.00 18 ----- 48.40 19 1.35s 22.02 20 1.35s 22.02 21 1.42q 34.50 22 0.93t 9.00 23 2.39s 20.01

3.5.4. Obtusifolate D (105)

O 21 O 8 1 19 13 8a 9 1a O 15 11 7 16 18 12 17 O 3 5a 10 4a 5 4 Obtusifolate D (105) 14 O O

F6 fraction was subjected to column chromatography over silica gel, eluting with n-hexane, n- hexane/DCM, DCM/AcOEt, AcOEt and MeOH in increasing order of polarity. Eluting with the solvent system AcOEt /DCM 1:1 Obtusifolate D (105) was obtained as an orange gummy solid.

3.5.4.1. UV and IR spectra of Obtusifolate D

The UV spectra confirm anthraquinones skeleton as in compound (104). The absorptions at

(1701–1728) and (1590) in cm-1 of the IR spectrum showed the presence of ester group and conjugated carbonyl groups of anthraquinone nucleus but a peak at 1715 cm-1 was found to be disappear which showed the absence of non conjugated carbonyl group in compound (105). An additional strong absorption at 1270--1230 cm-1 showed the ether functionality.

3.5.4.2. Mass spectrometry of Obtusifolate D (105)

The molecular formula was determined by HREI-MS as C25H26O6 (m/z =422.1719; calcd.

422.1729), which shows 10 degrees of unsaturation. Eight of them are eventually accounted for anthraquinone ring, one for ketonic and last one for ester carbonyls group.

3.5.4.3. 1H-NMR and 13C-NMR spectra of compound D (105)

The1H-NMR and 13C-NMR spectra of compound (105) (Table 3.8) was very similar to that of

(104), except for the presence of additional peaks due to ether group (H-14, 3.79s, 56.60=C-14) plus absence of carbonyl group peaks (H-14=3.91s, H-16=2.13s, C-14=48.40, C-15=206.01,C-

16=31.30) in ppm of compound (104). 1H-NMR and 13C-NMR spectra and molecular formula determined by HREI-MS showed that CH3O- group is present in compound (105) in place –CH2-

CO-CH3 group of compound (104).

3.5.4.4. Other chemical tests for identification of Obtusifolate D (105)

It also gave red solutions on reduction in alkaline solution (in aqueous sodium hydroxide, which distinguished anthraquinone from benzoquinones and naphthoquinones [165].

Table No 3.8 1HNMR and 13CNMR data of Obtusifolate D (δ=chemical shift in ppm) Obtusifolate D (105) Position δ (H) δ(C) 1 ------144.00 2 ------136.60 3 7.57d 127.80 4 7.55d 127.50 5 ---- 150.13 6 7.72d 129.40 7 ----- 117.00 8 7.74d 124.50 9 ------181.00 10 ------182.50 1a ------134.04 8a ------133.15 4a ------132.00 5a ------132.50 11 ------168.12 12 4.23q 72.50 13 1.28t 22.70 14 3.79s 56.60 15 ------209.00 16 ----- 48.40 17 1.35s 22.02 18 1.35s 22.02 18 1.42q 34.50 20 0.93t 9.00 21 2.39s 20.01

3.6. Free radical scavenging activity of the isolated compounds

The results of table No 3.9 show prominent radical scavenging activity for 102 & 103. There is a very little difference in RSA b/w the two compounds. Both of the compounds have greater radical scavenging property than ascorbic and gallic acid.

The results indicate significant radical scavenging activity for 104 & 105. Both the compounds have round about same RSA. Both of the isolated compounds have less radical scavenging property than ascorbic and gallic acid. %DPPH inhibition has been shown graphically for all the four isolated compounds (fig 3.1).

Table 3.9 Percentage of RSA of compounds 102, 103, 104 and 105 from Rumex Obtusifolius

S.No Compounds/standards % DPPH inhibition±SEM 1 A 97.55±1.51 2 B 96.00±0.83 3 Gallic acid 93.00±0.29 4 Ascorbic acid 92.33±0.55 5 C 80.12±0.91 6 D 79.00±1.17

Fig 3.4 % DPPH inhibibition zone of compound the four isolated compounds from Rumex obtusifolius

% DPPH inhibition±SEM 120

100

0 A B Gallic acid Ascorbic C D acid

Chapter No 4

Experimental

4.1. Plant collection, Identification and Grinding

The whole plant except fruit and seed of Rumex obtusifolius (Polygonaceae) was collected from

Bannu, KPK, Pakistan at December 2015 and identified by Prof. Abdur Rehman, Botany

Department, GPG College Bannu. The plant was washed, shade dried and was then grinded into powders using electrical grinder.

4.2. Extraction and Fractionations

The powders of the grinded plant were extracted with ethanol for 14 days at room temperature.

Ethanolic extract was evaporated by rotary to obtain gummy crude. The crude was successively fractionated with increasing order of polarity i.e. n-hexane, dichloromethane, ethylacetate and ethanol by separating funnel as shown in scheme 1.

4.3. Preliminary qualitative phytochemical analysis [166, 167].

4.3.1. Alkaloids

Each extract was evaporated in a boiling water bath and the residue was mixed with 2N HCl.

Then the mixture was filtered. The filtrate was divided into three equal parts. One part was mixed with a few drops of Mayer's reagent; one portion was treated with equal amount of

Wagner's reagent and the other portion was reacted with equal amount of Dragondroff’s reagent.

The creamish, brown and orange precipitate indicated the presence of respective alkaloids [168].

4.3.2. Flavonoids

The existence of flavonoids was analyzed by Shinoda experiment. Each extract was reacted with a few drops of concentrated Hydrochloric acid and Mg- ribbon. The appearance of tomato red or pink colour within a few minutes confirmed the presence of flavonoids [169].

4.3.3. Tannins

Each extract dry powder form was treated with alcoholic FeCl3 reagent. Appearance of blue color indicated the presence of tannins [170].

4.3.4. Cardiac glycosides

Keller-kiliani experiment was carried out to judge the existence of cardiac glycosides. The crude

3 dry powder of each extract was mixed with 1cm of FeCl3 reagent (mixture of 1 part of 5 percent

FeCl3 solution and 99 part of glacial acetic acid). To this solution a few drops of concentrated

H2SO4 was added. Greenish blue color within a few minutes indicated that cardiac glycosides was present [171].

4.3.5. Anthraquinones

About 500mg of each extract was mixed with 10% HCl and boiled on water bath. It then filtered and filtrate was allowed to cool. Equal volume of chloroform was added the filtrate and then few drops of 10% NH3 was added to mixture and heated. The appearance of Rose-pink color indicates the presence of anthraquinones.

4.3.6. Steroids

Liebermann-Burchard test was conducted to evaluate the presence of steroids. A chloroform solution of the crude dry powder of each extract was treated with acetic anhydride and a few drops of concentrated H2SO4 were added. The appearance of blue green ring indicated the presence of steroids.

4.3.7. Saponins

The existence of saponins was tested by Frothing procedure. The crude dry powder of each extract was vigorously shaken with distilled water and was allowed to stand for ten minutes. No froth indicates absence of saponins while stable froth more than 1.5 cm indicated that saponins are present [172].

4.4. Sub-fractionation of Dichloromethane soluble fraction

Dichloromethane (DCM) fraction was Sub-fractionated into several fractions (F1, F2, F3, F4, F5 and F6) using column chromatography as presented in table No 4.1. All the fractions were evaporated by rotary evaporator. These fractions were screened for biological activities.

Table No 4.1 Sub-fractionation of Dichloromethane soluble fraction

Sub- Eluents fractions

% DCM %n-hexane

F1 0 100

F2 25 75

F3 50 50

F4 75 25

F5 100 0

F6 100% ethyl acetate was eluted at the end.

4.5. Biological Screening of Dichloromethane Sub-fractions of R. obtusifolius

4.5.1. Antibacterial screening

Test bacteria: Antibacterial screening of different fractions was performed for eight pathogenic

bacteria i.e. Bacillussubtiliss (NCTC 10400), Escherichia coli (ATCC 8739), ampicillin-resistant

Escherichia coli (NCTC 10418), Bacillus cereus (ATCC 11778), Staphylococcus aureuss (NCTC

1803), Pseudomonas aeruginosas (ATCC 27853), Streptococcus pneumonia (ATCC 49619),

Salmonella typhii (NCTC 10203), obtained from Biotechnology Department of UST Bannu,

Pakistan.

Procedure: The antibacterial assay was performed by a common disc diffusion process [173,

174]. Briefly, the concentration was 500 × 10-6 g/disc in case of each extract. The sample discs and positive controls (cefotaxime, 10 × 10-6 g/disc) along with negative control discs were placed on Petri dishes having suitable agar medium seeded with the test microorganisms using sterile

transfer loop and kept at 4 °C to assist maximum diffusion s. All the plates were incubated at 37

°C for bacterial growth. The diameter of clear area in the Petri dish which was devoid of bacterial growth was calculated which showed antibacterial activity of the test agents in terms of mm.

4.5.2. Antifungal screening

Test fungi: , Aspergillus flavass, Fusarium solanii, Aspergillus nigers, Mueor species, Fusarium moniliformes, Alternaria alterata, Aspergillus fumigates.

Procedure: An agar tube dilution method was conducted for antifungal screening of the factions was investigated according to the procedure reported by Choudhary et al [175]. Briefly, to

3 prepare media for fungi 32.5 g savored dextrose agar was mixed with 500 cm distill H2O. Then it was heated to be dissolved and 5 cm3 was dispensed into screw cap tubes. The tubes were

marked and autoclaved at 121°C for 20 minutes. All the tubes were then allowed to cool. 100 ×

10-6 L of extract (0.02 g/cm3 in DMSO) and 83 × 10-6 L of terbinafine (0.012 g/cm3 in DMSO) were mixed just before solidification, as positive control in tubes to get concentration of 0.4 and

0.2 × 10-3 g/cm3, respectively. Add 0.1 mg per tube DMSO was used as negative control. Each tube was inoculated with a 4 mm diameter piece of inoculum from a culture seven days. All these tubes were incubated at 27 °C for 7 days. Fungal growth was calculated by measuring linear growth in terms of mm. % inhibition of fungal growth was finalized by the following equation.

A X = [100 − ] × 100 B

Where; A= linear growth in test (mm), B= linear growth in control (mm), X= Percentage inhibition of fungal growth.

4.5.3. Cytotoxic screening

Cytotoxic activity for DCM sub- fractions were analyzed by performing brine shrimps hatched in saline process, as reported by Meyer-Alber et al. [176], with slight modification. Briefly, 20 mg each sample was dissolved in 2cm3 of respective solvent. From this stock solution 5 × 10-6 L, 50

×10-6 L and 500 × 10-6 L was poured separately into 5mL; 20mL 50 mL vials respectively. They were placed open in continuous air flow for dryness. 3 cm3 artificial sea water was added in each vial and then 10 matured brine shrimp larvae were also added. At last sea water was poured up to the mark and 10, 100, 1000 ppm working solution were prepared by simple dilution method.

They were kept under illumination and after 24h of incubation survivors were counted by magnifying glass. LD50 value was calculated by probit analysis in finny computer software.

4.6. Secondary metabolites from R. Obtusifolius

As fractions (F1, F2, F3, F4, F5 and F6) have been screened for biological activities, in which F4,

F5 and F6 were proved to be the most active antibacterial, cytotoxic and antifungal agents [177], so they were further subjected to column chromatography to isolate various secondary metabolites.

4.6.1. Instrumentation

Melting points were measured by using Gallenkamp and Kofler hot-stage apparatus. Glass capillaries were used and melting points are uncorrected. All the solvents were distilled before use .1H-NMR and 13C-NMR spectra were recorded with a Brucker spectrometer operating at 500

MHz and 100 MHz respectively, using CDCl3 (δH 7.26, δC 77.00 ppm) as a solvent. Chemical shift values are reported relative to TMS. The Mass spectra (MS) were determined using

Micromаss ZMD and Varian MAT 312 double focusing mass spectrometer coupled to DEC–

PDP 11 / 34 computer system. Shimadzu 460 and Shimadzu UV-2401PC spectrometers were used for IR and UV spectra determination respectively.

4.6.2. Chromatography

Silica gel (230–400 mesh) for column chromatography and aluminum sheets coated with silica gel 60F245 (20cm × 20 cm) 0.2mm thick; were used for TLC (E.Merck).Visualization of the spots on TLC plates was carried out by UV at 254 and 366 nm.

4.6.3. Isolation, Purification and Characterization of compounds

F5 fraction was subjected to column chromatography over silica gel, eluting with increasing polarity n-hexane, n-hexane/DCM, DCM /AcOEt, and MeOH. Eluting with the solvent system n- hexane/DCM (1:9) to afford compound 102 while compound 103 was obtained in semi-pure form from the column by using DCM/AcOEt (9:1) as eluent. It was purified by pencil column chromatography.

Fraction (F6) was also subjected to column chromatography over silica gel, eluting with n- hexane, n-hexane/DCM, DCM/AcOEt, AcOEt and MeOH in increasing order of polarity.

Eluting with the solvent system AcOEt /DCM 1:1 to afford comound105 and 104 was obtained in semi-pure form from the column by using DCM/AcOEt 1:2 as eluent as given in scheme 2.

R. obtusifolius 15 Kg

Ethanolic Extract (Crude) 780g

Extracted with n-hexane Water

n- Hexane soluble fraction (A, 70g) Aqueous layer

Extracted with DCM

DCM soluble Aqueous layer fraction (B, 170g)

extracted with Ethyl acetate

Ethyl acetate soluble Aqueous layer fraction (C, 60g)

Extracted with Ethanol

Ethanol soluble fraction (D, 51g) Aqueous layer

Scheme 1 Extraction and Fraction of R. obtusifolius

DCM soluble fraction (B, 170g)

Column Chromatography

F1 F6 F5 F4 F3 F2 !00% EtOAc 100%DCM DCM:n-hex DCM:n-hex 100% n-haxane DCM:n-hex 50%:50% 75%:25% 25%:75%

Re-Chromatographed

AcOEt :DCM DCM:AcOEt DCM:AcOEt n-hexane:DCM 1:1 1:2 9:1 1:9

Compound D Compound C Compound B Compound A (Obtusifolate D) (Obtusifolate C) (Obtusifolate B) (Obtusifolate A) 105 104 103 102

Scheme 2 Chromatographic resolution on Si-gel column eluted with n-hexane, DCM and EtOAs as solvent system.

4.6.4. Obtusifolate A (102)

19 O OH O OH

21 18 17 1 9a 9 8a 8 O 13 6' 3 6 4a 10 10a 16 1' 5 12

4' 2' O 11 14 1'' 4'' H 5'' 2'' O 3'' Obtusifolate A (102) O 7'' 6'' 8''

Physical State: yellow amorphous solid.

UV Spectra: λmax 211, 239, 252, 275, and 366 nm

-1 IR (dry KBr) νmax cm : (3441, 3425), (1701–1728), (1590) and (1619)

Mass Spectra: HREI-MS as C39H42O8 (m/z =638.2880; calcd: 638.2870)

1 H-NMR (CDCl3, 500 MHz) δ (ppm): δ =12.31(1H, s, H-1), 7.02 (1H, s, H-4), 7.12 (1H, s, H-

7), 12.13 (1H, s, H-8), 2.38 (3H, s, H-11), 1.28 (6H, s, H-13 & H-14), 1.20 (2H ,q, H-15), 0.85

(3H, t, J=6.7Hz, H-16), 4.20 (1H, m, H-18), 1.35 (3H, d, J=6.6Hz, H-19), 1.75(2H, m, H-20),

0.92 (3H, t, J=6.7Hz, H-21), 7.66 (1H, d, J=3Hz, H- 3'), 7.72 (1H, dd, J=3 & 8.8, H-5'), 7.56

(1H, d, J=8.8, H-6'), 3.62 (2H, s, H-1''), 2.13 (3H, s, H-3'' ), 6.69(1H, s, H-5''), 1.65 (3H, s, H-

7''), 1.70 (3H, s, H-8'').

13 C-NMR (CDCl3, 100 MHz) δ (ppm): 165.42 (C-1), 117.00 (C-2), 138.90 (C-3), 124.50 (C-4),

144.00 (C-5), 150.13 (C-6), 144.40 (C-7), 159.00 (C-8), 190.50 (C-9), 181.00 (C-10), 134.04 (C-

4a), 114.20 (C-8a), 113.10 (C-9a), 133.15 (C-10a), 20.01 (C-11), 34.50 (C-12), 29.60 (C-13 &

C-14), 37.60 (C-15), 9.00 (C-16), 168.12 (C-17), 74.00 (C-18), 22.02 (C-19), 29.80 (C-20), 8.01

(C-21), 138.70 (C- 1'), 132.30 (C-2'), 129.40 (C-3'), 136.60 (C-4'), 127.80 (C-5'), 127.50 (C-6')

48.4 (C-1''), 206.01 (C-2''), 31.10 (C-3''), 192.10 (C-4''), 120(C-5''), 133.10 (C-6''), 18.5 (C-7''),

25.5 (C-8'').

4.6.5. Obtusifolate B (103)

O OH O OH 18 1 9a 9 8a 8 O 17 13 6' 3 6 10 10a 4a 16 1' 5 12 4' O 11 14 3' 7' 1'' Obtusifolate B (103) O 2'' 4'' H 3'' 5''

Physical State: yellow amorphous solid.

UV Spectra: λmax 211, 252, 275, and 366 nm

-1 -1 IR (dry KBr) νmax cm : (3441, 3425), (1701–1728), (1590) and (1619) cm

Mass Spectra: HREI-MS as C34H34O7 (m/z =554.2305; calcd.554.2296),

1 H-NMR (CDCl3, 500 MHz) δ (ppm): δ =12.31 (1H, s, H-1), 7.02 (1H, s, H-4), 7.12 (1H, s, H-

7), 12.13 (1H, s, H-8), 2.38 (3H, s, H-11), 1.28 (6H, s, H-13 & H-14), (2H, 1.20, q, H-15), 0.85

(3H, t, J=6.7Hz, H-16), 4.23 (3H, s, H-17), 7.66 (1H, d, J=3Hz, H-3'), 7.72 (1H, dd, J=3 &

8.8Hz, H-5'), 7.56 (1H, d, J=8.8, H-6'), 2.47 (3H,s,H-7'), 6.69 (1H, s, H-2''), 1.65 (3H, s, H-4''),

1.70 (3H, s, H-5'').

13 C-NMR (CDCl3, 100 MHz) δ (ppm): 165.00 (C-1), 116.01(C-2), 138.60 (C-3), 124.55 (C-4),

144.00 (C-5), 150.14 (C-6), 144.40 (C-7), 159.00 (C-8), 190.50 (C-9), 181.00 (C-10), 134.04 (C-

4a), 114.20 (C-8a), 113.10 (C-9a), 133.15 (C-10a), 20.00 (C-11), 34.50 (C-12), 29.60 (C-13 &

C-14), 37.60 (C-15), 9.00 (C-16), 166.10 (C-17), 51.60 (C-18), 139.50 (C- 1'), 136.70 (C-2'),

130.00 (C-3'), 136.60 (C-4'), 127.80 (C-5'), 127.50 (C-6'), 22.5 (C-7'), 192.10 (C-1''), 120.00 (C-

2''), 133.10(C-3''), 18.5 (C-4''), 25.5 (C-5'').

4.6.6 Obtusifolate C (104)

O 23 O 8 1 21 13 17 7 8a 9 1a O 11 18 20 12 19 O 3 5a 10 4a 5 4

O 14 O 15 Obtusifolate C(104)

Physical State: orange amorphous solid.

UV Spectra: λmax intense absorption at 240-260 nm, medium absorption at 320-330 nm

-1 IR (dry KBr) νmax cm : (1701–1728, 1715 and (1590)

Mass Spectra: HREI-MS as C27H28O6 (m/z= 448.1886; calcd.448.1876)

1 H-NMR (CDCl3, 500 MHz) δ (ppm): 7.57 (1H, d, J=8.8, H-3), 7.55 (1H, d, J=8.8, H-4), 7.72

(1H, d, J=3.3, H-6), 7.74 (1H, d, J=3.3, H-8), 4.23(2H, q, H-12), 1.28 (3H, t, J=6.5Hz, H-13),

3.91 (2H, s, H-14), 2.13 (3H, s, H-16), 1.35 (6H, s, H-19 & H-20), 1.42 (2H, d, H-21), 0.93 (3H, t, J=6.5Hz, H-22), 2.39 (3H, s, H-23)

13 C-NMR (CDCl3, 100 MHz) δ (ppm): 144.00 (C-1), 136.60 (C-2), 127.80 (C-3), 127.50 (C-4),

132.30 (C-5), 129.40 (C-6), 117.00 (C-7), 124.50 (C-8), 181.00 (C-9), 182.50 (C-10), 134.04 (C-

1a), 133.15 (C-8a), 132.00 (C-4a), 132.50 (C-5a), 168.12 (C-11), 72.50 (C-12), 22.70 (C-13),

48.40 (C-14), 206.01 (C-15), 31.30 (C-16), 209.00 (C-17), 48.40 (C-18), 22.02 (C-19 & C-20),

34.50 (C-21), 9.00 (C-22), 20.01 (C-23).

4.6.7. Obtusifolate D (105)

O 21 O 8 1 19 13 8a 9 1a O 15 11 7 16 18 12 17 O 3 5a 10 4a 5 4 Obtusifolate D (105) 14 O O

Physical State: Brown amorphous solid

UV Spectra: λmax intense absorption at 240-260 nm, medium absorption at 320-330 nm

-1 IR (dry KBr) νmax cm : (1701–1728), (1590) and (1270—1230)

Mass Spectra: HREI-MS as C25H26O6 (m/z =422.1729; calcd.422.1719).

1 H-NMR (CDCl3, 500 MHz) δ (ppm): 7.57 (1H,d, J=8.8, H-3), 7.55 (1H,d, J=8.8, H-4), 7.72

(1H, d, J=3.3, H-6), 7.74 (1H, d, J=3.3, H-8), 4.23 (2H, q, H-12), 1.28 (3H, t, J=6.5Hz, H-13),

3.79 (2H, s, H-14), 1.35 (6H, s, H-17 & H-18), 1.42 (2H, d, H-19), 0.93 (3H, t, J=6.5Hz, H-20),

2.39(3H, s, H-21).

13 C-NMR (CDCl3, 100 MHz) δ (ppm): 144.00 (C-1), 136.60 (C-2), 127.80 (C-3), 127.50 (C-4),

150.13 (C-5), 129.40 (C-6), 117.00 (C-7), 124.50 (C-8), 181.00 (C-9), 182.50 (C-10), 134.04 (C-

1a), 133.15 (C-8a), 132.00 (C-4a), 132.50 (C-5a), 168.12 (C-11), 72.50 (C-12), 22.70 (C-13),

56.60 (C-14), 109.00 (C-15), 48.40 (C-16), 22.02 (C-17 & C-18), 34.50 (C-19), 20.01 (C-21).

4.7. Free radical scavenging activity (RSA) of the isolated compounds

TLC-DPPH assay was used for qualitative analysis of radical scavenging screening as described in literature [178] and for quantitative estimation of radical scavenging activity (RSA) according to the standard assay [179]. In brief 2.5cm3 of 0.04% DPPH radical solution in methanol was added to each sample solution (100×10-6L) ranging from 192.50 to 6×10-6g/cm3. The mixtures were vortex-mixed and placed in dark room for half an hour. The absorbance was recorded at

517nm using spectrometer. MeOH was used as baseline control and ascorbic and gallic acid as positive controls. RSA was calculated as decrease in absorbance (samples V DPPH standard solution). %RSA was measured by the following formula. %RSA= [(A-B)/A)] ×100, where A is absorbance of control and B is absorbance of sample.

Chapter No 5

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