Phytochemical and Biological Study of some Species (Rumex vesicarius) Family

Thesis submitted for fulfillment of the requirement of the master degree of the pharmaceutical science in pharmacognosy by Marwa Yehia El Harriry National Organization of Drug Control and Research (NODCAR)

Under the Supervision of

Dr. Seham El Hawary Dr. kamilia Fouly Taha Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Applied research center for Cairo university medicinal NODCAR Dr. Nadia Sokkar Associate Professor of Pharmacognosy Faculty of Pharmacy Cairo university

Pharmacognosy department Faculty of Pharmacy Cairo University 2012

Acknowledgement

I’m grateful to God by the grace of whom this work was accomplished.

I would like to express my deep appreciation and sincere gratitude to Prof. Dr. Seham Salah El-Din El-Hawary, Professor of Pharmacognosy, Faculty of pharmacy, Cairo University for her super way of supervision, valuable scientific guidance and her useful efforts throughout this work.

I would like to express my deep appreciation and sincere gratitude to Prof. Dr. Kamilia Foluy Taha, Professor of Pharmacognosy, Applied Research Center for Medicinal Plants, National Organization for Drug control and Research, for her supervision and useful efforts throughout this work.

I would like to express my sincere everlasting gratitude to Dr. Nadia H. Sokkar, Associate Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University for her kind and patient supervision, stainless support and unlimited help throughout the course of this work.

My deepest thanks to Dr.ZeinabYousef, Assistant Professor of Biochemistry Department, National Organization for Drug control and Research for her kind help in carrying out the pharmacological and toxicological testing of the extracts.

Finally, my deepest thanks to my parents, my family, my colleagues and friends who play a very special part in my life.

Marwa Yehia El Harriry

Contents

Subject Page Introduction 1 Review of literature 3 18 Material, apparatus and techniques 21 Part One: DNA profiling of Rumex vesicarius. 32

Part Two: Phytochemical investigation of Rumex vesicarius Chapter I: Prelimenary phytochemical screening and proximate 37 analysis Chapter II: investigation of volatile constitueunts of rumex 41 vesicarius. Chapter III: Investigation of lipoidal conistituents of Rumex 46 vesicarius. Chapter IV: Quantitative estimation of total phenolic and 55 flavonoid content in leaves of Rumex vesicarius Chapter V: Qualitative and quatitative investigation of flavonoids in Rumex vesicarius 60 Chapter VI: Quantitative estimation of anthraquinones in Rumex vesicarius 97

Chapter VII: Study of the vitamin content of Rumex vesicarius. 102 Part Three: Biological study on different organs of Rumex 106 vesicarius A- Serum biochemical analysis 109 I- Assessment of liver function 109

II- Assessment of antioxidant activities and lipid peroxidation 113

III- Assessment of liver fibrosis 120 IV- Assessment of the permeability of hepatic lysosomal 121 membrane B- Histopathological studies in liver 129 Summary 133 Recommendations 139 References 140

List of Figures

Figure Page Some selected photographs of Rumex vesicarius L Figure (1) 20 wildly grown in Egypt Figure (2) RAPD analysis of the well known Rumex cultivated in 35 Egypt using five different primers Figure (3) TIC for GC/MS chart of volatile oil prepared from 42 Rumex vesicarius L Figure (4) Total Ion Chromatogram (TIC) of GC/MS chart of the 48 unsaponifiable matter of leaves of Rumex vesicarius L Figure (5) Total Ion Chromatogram (TIC) of GC/MS chart of the 52 fatty acid methyl esters of Rumex vesicarius L. Figure (6) Standard calibration curve of Gallic acid 57 Figure (7) Standard calibration curve of Quercetin 58 Figure (8) Structure of compound F1 65 Figure (9) Full mass spectrum of compound F1 66 Figure (10) Mass fragmentation of compound F1 67 Figure (11) HNMR spectrum of compound F1 68 Figure (12) Structure of compound F2 70 Figure (13) Full mass spectrum of compound F2 71 Figure (14) Mass fragmentation of compound F2 72 Figure (15) HNMR spectrum of compound F2 73 Chromatogram of ethyl acetate fraction using S5, (A) LC/PDA chromatogram and (B) LC/MS, 1;vitexin, Figure (16) 2;orientin, 3;hexosyl-quercetin, 4;rutin, 5; 7-O-rhamno- 76 hexosyl-diosmetin, 6; 7-O-rhamno-acetyl hexosyl- diosmetin LC/MS/MS chromatogram of n-butanol fraction using S6, (A) LC/PDA chromatogram and (B) LC/MS,7;catechin, 8;epicatechin, 9;feruloyl hexoside, Figure (17) 77 10;naringenin 6-C- glucoside, 11;epigallocatechin gallate, 12; catechin 6-C- glucoside and epicatechin gallate (A)Hexose cross-ring cleavages, (B) Structures of Figure (18) vitexin, orientin, 6-C- hexosyl quercetin, catechin-6-C- 79 glucoside and naringenin-6-C-glucoside Mass fragments of vitexin, orientin, 6- c- hexosyl Figure (19) quercetin, naringenin 6-c- glucoside and catechin 6-c- 82

glucoside. Figure (20) Structures of rutin, 7-O-rhamno-hexosyl-diosmetin and 83 7-O-rhamno-actyl hexosyl-diosmetin Mass fragments of 7-O-rhamno-hexosyl-diosmetin and Figure (21) 84 7-O-rhamno-acetyl hexosyl-diosmetin. Structures of catechin, epicatechin , epicatechin gallate, Figure (22) 86 epigallocatechin gallate and feruloyl hexoside. Mass fragments of catechin, epicatechin , epicatechin Figure (23) 88 gallate and epigallocatechin gallate. Standard curves of authentics, (1) naringin, (2) diosmin, Figure (24) 95 (3) catechin, (4) orientin and (5) vitexin Figure (25) Standard calibration curve of rhein 98 TLC chromatogram of, A: roots, B: leaves and C: fruits Figure (26) of Rumex vesicarius L, 1, Aloe-emodin; 2 , rhein; 3, 100 emodin; 4, chrysophanol and 5, physcione. Effect of root, leaves and fruits ethanolic extracts of Rumex vesicarius L on serum enzymes (AST, ALT, Figure (27) 112 ALP, T.B, T.P) compared to silymarin in liver damaged rats Effect of root, leaves and fruits ethanolic extracts of Figure (28) Rumex vesicarius L on (MDA, SOD, CAT, GST and 119 GSH) compared to silymarin in liver damaged rats Effect of root, leaves and fruits ethanolic extracts of Figure (29) Rumex vesicarius on Liver Index, MDA, 124 Hydroxyproline(HYP) and lysosomal enzymes (ACP, β-GAL and β-NAG) Compared to Silymarin A photomicrograph of liver sections in the normal Figure (30) control rats (A) showing cords of hepatocytes radiating 130

from the central vein CCl4-intoxicated rats showing fatty degeneration, Figure (31) necrotic areas and portal loss of architecture with mild 130 congestion of blood vessels (Haematoxylin-Eosin stain, x150) (A) rats treated with (L.E+CCl4) showing improvement in the liver, (B): rats treated with (F.E+CCl4) showing mild pathological changes as fatty degenerative Figure (32) changes, (C): rats treated with (R.E+CCl4) showing mild 131 fatty degenerative changes and (D): rats treated with (Sily+CCl4) showing more or less normal architecture of liver lobules

List of Tables

Table Page Distribution of active constituents in different Table (1) 7 organs of genus Rumex The RAPD analysis of the species of Rumex Table(2) dentatus L. and Rumex vesicarius L. with five 36

primers Table (3) Results of preliminary phytochemical screening of 37 different organs of Rumex vesicarius L. Table (4) Pharmacopeial constants of the leaves of Rumex 40 vesicarius L. Table (5) GC/MS of the essential oil of fruits of Rumex 43 vesicarius L Table (6) The different chemical classes of the identified 46 compounds Table (7) GC/MS of the unsaponifiable matter of leaves of 49 Rumex vesicarius L. Table (8) GC/MS of the fatty acid methyl esters of leaves of 53 Rumex vesicarius L. Table (9) The absorbance measured for different 56 concentrations of standard gallic acid solution Table (10) The absorbance measured for different 58 concentrations of standard quercetin solution Table (11) TLC screening of the n-butanol extract of the 62 leaves of Rumex vesicarius L Table (12) data of compound F1 64 Table (13) data of compound F2 69 The retention times, UV absorbance and masses of Table (14) 89 the identified compounds Table (15) Quantitative estimation of phenolics in Rumex 93 vesicarius L Table (16) The peak areas for different concentrations of the 98 standard rhein Table (17) Identified anthraquinones in roots, leaves and fruits 99 of Rumex vesicarius L by HPTLC Table (18) Anthraquinone content in roots, leaves and fruits of 101 Rumex vesicarius L Vitamin content in different organs of Rumex Table (19) 105 vesicarius L. Effect of root, leaves and fruits ethanolic extracts Table (20) of Rumex vesicarius L on serum enzymes (AST, 111 ALT, ALP, T.B, T.P) compared to silymarin in liver damaged rats. Effect of root, leaves and fruits ethanolic extracts Table (21) of Rumex vesicarius L on (MDA, SOD, CAT, 118 GST and GSH) compared to silymarin in liver damaged rats Effect of root, leaves and fruits ethanolic extracts Table (22) of Rumex vesicarius L on Liver Index, Liver MDA, 123 Hydroxyproline and Lysosomal Enzymes (ACP, β-GAL and β-NAG) Compared to Silymarin

List of abbreviations: a P: Probability < 0.05 b P: Probability < 0.01 c P: Probability < 0.001

ALP: Alkaline phosphatase

ALT: Alanin amino transeferase

AST: Aspartate amino transeferase

ACP: Acid phosphatase

β -GAL: β- galactosidase

β-NAG: N- acetyl-β –glucosaminsidase

CAT: Catalase

CDNB: 1- Chloro dinitrobenzene

CTAB: N-cetyl-N,N,N-tri methyl ammonium bromide

CYP2E: Cytochrome P 450 system

DNA: Deoxyribonucleic acid

DMSO: Dimethyl sulfoxide

EEF: Ethanolic extract of fruits

EEL: Ethanolic extract of leaves

EER: Ethanolic extract of roots

ESI: Electrospray ionization

IL: Interleukin

GAE: Gallic acid equivelant

GC/MS: Gas chromatography / mass spectroscopy GC/FID: Gas chromatography/flame ionization detector

GLC: Gas liquid chromatography

GSH: Reduced glutathione

GST: Glutathione S- transferase HNMR: Proton nuclear magnetic resonance

HD: Hydrodistillation

HPLC: High performance liquid chromatography

HPTLC: High performance thin layer chromatography

HSC: Hepatic stellate cells

LC-MS/MS-ESI: Liquid chromatography equipped with mass electrospray ionization

LC/PDA: Liquid chromatography equipped with photodiode array

MDA: Malondialdehyde

MSD: Mass spectrometric detector

MRM: Multiple reaction monitoring

MUFA: Mono unsaturated fatty acid

NP/PEG: Natural product /Polyethylene glycol

PCR: Polymerase chain reaction

PDMAB: p- Dimethyl amino benzaldhyde

PFTBA: Perflurotributylamine

PUFA: Poly unsaturated fatty acid

PVP: Poly vinyl providone

RAPD: Random amplified polymorphic DNA

RNA: Ribonucleic acid Rt: Retention time RI: Relative index

ROS: Reactive oxygen species

SaFA: Saturated fatty acid

S.E: Standard error

Sily: Silymarin

SOD: Superoxide dismutase

SRM: Selective reaction monitoring

SIM: Selective ion monitoring

Tris-HCl: Hydroxy methyl amino methane with HCl

TE: Tris- HCl with EDTA

TCA: Tri chloro acetic acid

TBA: Thiobarbituric acid

T.B: Total bilirubin

TGF-β1: Transforming growth factor

THF: Tetrahydrofurane

TIC: Total ion chromatogram

TPC: Total poly phenol content

TMS: Trimethyl sulfone

TNF-α: Tumor necrosis factor

T.P: Total protein

UV: Ultra violet

Introduction

Rumex vesicarius L, (Al Hommade), family Polygonaceae, is a wild edible Egyptian herb (Boulos, 1999). It is distributed all over the different geographical regions of Egypt. It is an annual glabrous herb with an erect to ascending stem branching from base, fleshy leaves, greenish to purplish bisexual flowers, purplish-red-veined fruits and flowers in March and April (Al-Yemeny, 1999). In folk medicine, it is used for treatment of hepatic diseases, constipation, bad digestion, pains, heart troubles, spleen disorders, flatulence, asthma, bronchitis, dyspepsia, vomiting, piles, scabies, leucoderma, toothache, appetizer, diuretic, laxative, stomachic, tonic and analgesic (Batanouny, et al, 1999 & Ibn Sina, 1411 H). Previous phytochemical investigation revealed the presence of anthraquinones mainly: chrysophanol, emodin, physcione, rhein and their glycosides (Al- Gindy, et al, 1998), vitamin C (Alfawaz, 2006) and minerals (Al-Rumaih, 2003). However, the biochemical basis and mechanism of hepatoprotective action of Rumex vesicarius L. extract were not scientifically studied. Despite of its importance, only few studies have been conducted on the plant.

1 The present investigation was aimed to fulfill the following: 1- Obtain a bioactive profile of constituents' viz., flavonoids, catechins, vitamins, lipid, and volatile constituents in different organs of Rumex vesicarius L. 2- Quantitative estimation of the major identified flavonoids using sensitive, accurate and specific methods: coupling HPLC with an ion-trap mass spectrometer equipped with electrospray ionization mass spectrometry (ESI- MS), HPLC and GC/MS. 3- Prove the hepatoprotective, anti-fibrogenic and antioxidant activities of different organs of the plant through biochemical and histopathological studies.

2 Review of Literature

Rumex L., is a genus of about 200 species of annual, biennial and perennial herbs in the buckwheat family Polygonaceae (IUCN, 2005). Members of this family are very common perennial herbs growing mainly in the northern hemisphere, but various species have been introduced almost everywhere. The leaves of most species contain oxalic acid and tannin, and many have astringent and slightly purgative qualities. Some species with particularly high levels of oxalic acid are called including: sheep's , Rumex acetosella, common sorrel, Rumex acetosa and French sorrel, Rumex scutatus, and some of these are grown as pot herbs or garden herbs for their acidic taste. Most of Rumex species contain flavonoids, anthraquinones and minerals. (www.botanical.com & Łuczaj, 2008). I. Chemistry a) Chemistry of Genus Rumex: Anthraquinones: In 1972, Buchalter, isolated and identified emodin (1,3,8-tri-hydroxy-6- methyl anthraquinone) from Rumex hymenosepalus. In 1972, Fairbairn, et al, studied the distribution of anthraquinones (aglycones, O-glycosides and C-glycosides) in all parts of the plant of 19 representative species of Rumex. All species contained emodin, chrysophanol and physcion and in addition some contained aloe-emodin and others rhein or rhein-like substances. In 1977, Harborne, et al, isolated a sulphated anthraquinone emodin 8-O-

β-D-glucopyranosyl-6-O-sulfate derivatives in Rumex pulcher

3 In 1981, Kasai, et al, isolated and identified from the methanolic extract of Rumex obtusifolius 6-O-malonyl-β-methyl--glucopyranoside and ascorbalamic acid. In 1993, Saleh, et al, investigated the anthraquinones, emodin and chrysophanol in eight Rumex species, R. aegyptiacus,R.cispus, R.pulcher, R. dentatus, R. vesicarius, R.pictus, R.simpiflorus and R. cyprius. In 2009, Fan, et al, obtained from the ethyl acetate fraction of Rumex crispus, chrysophanol, physcione, emodin, chrysophanol-8-O-beta-D- glucopyranoside, physcion-8-O-beta-D-glucopyranoside and emodin-8O- beta-D-glucopyranoside. In 2009, Sandra, et al, obtained demethylmacrosporine I, an anthraquinone derivative from a chemical reaction-guided isolation on Rumex obtusifolius. In 2009, Zhang, et al, two compounds, hastatusides A and B were isolated from the roots of Rumex hastatus. In 2010, Liang, et al, isolated from the roots of Rumex nepalensis two naphthalene acylglucosides, rumexneposides A and B. In 2011, Guo, et al, purified three anthraquinones emodin, chrysophanol, and physcion from the dichloromethane extract of the Chinese medicinal herb Rumex japonicus by high-speed counter-current chromatography (HSCCC). Flavonoids: In 1993, Saleh, et al, investigated the flavonoids, kampferol-3-glucoside, quercetin-3- glucoside, vitexin and orientin in eight Rumex species, R. aegyptiacus, R.cispus, R.pulcher, R. dentatus, R. vesicarius, R.pictus, R.simpiflorus and R. cyprius.

4 In 1995, Hasan, et al, characterized besides rutin, quercetin 3-rhamnoside and kaempferol 3-rhamnosyl(1 → 6) galactoside, a flavonol glycoside, quercetin 3-glucosyl(1→ 4)galactoside, and 1,6,8-trihydroxy-3-methyl anthraquinone (emodin) from leaves of Rumex chalepensis. In 2008, Jang, et al, isolated five flavonoids, kaempferol-3-O-beta-D- glucoside, quercetin, quercitrin, and isoquercitrin from an ethyl acetate soluble extract of the fruits of Rumex japonicas In 2009, Fan, et al, obtained from ethyl acetate fraction of Rumex crispus, kaempferol, quercetin, kaempferol-3-O-alpha-L-rhamnopyranoside and quercetin-3-O-alpha-L-rhamnopyranoside. In 2009, Zhang, et al, isolated rutin from the roots of Rumex hastatus. In 2011, Hawas, et al, isolated and identified six pure compounds of flavonol, kaempferol 3-O- β -galactoside, kaempferol 3-O-β-glucoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O- β -galactoside, isorhamnetin 3-O- β - glucoside, isorhamnetin 3-O-rutinoside, from Rumex dendatus L. Polyphenols: In 1993, Makkar, et al, determined various phenolic constituents in stem, bark and root of mature Rumex hastatus. The total polyphenols, tannins and condensed tannins in the bark were evaluated. In 2004, Stoggl, et al, carried out structure elucidation of catechin and epicatechin in sorrel leaf extracts using liquid chromatography coupled to photodiode array-, fluorescence-, and mass spectrometric detection. In 2009, Fan , et al, obtained ethyl acetate fractions of Rumex crispus, gallic acid and (+)-catechin. In 2009, Zhang, et al, isolated resveratrol and nepodin from the roots of Rumex hastatus.

5 Lipids and sterols: In 2009, Taveira, et al, studied the volatile compounds of Rumex induratus leaves. The different extracts were analyzed by gas chromatography/mass spectrometry (GC/MS) which allowed the identification of 81 compounds, distributed by several chemical classes: esters, terpenes, aldehydes, acids, norisoprenoids, ketones, naphthalene derivatives, steroids derivatives and alcohols. In 2009, Fan, et al, obtained from the petroleum ether fraction of Rumex crispus, beta-sitosterol, hexadecanoic acid and hexadecanoic-2,3-dihydroxy propyleste. b) Chemistry of Rumex vesicarius: In 1998, Al- Gindy, et al, made a macro- and micromorphological study of the root, stem, leaf and fruit of Rumex vesicarius L. (Polygonaceae), with the aim of identifying the entire and the powdered forms. TLC and HPLC analysis of the anthraquinone fraction revealed the presence of emodin and /or chrysophanic acid in all organs. A fast high-performance liquid chromatographic and two selective (spectrophotometric and colorimetric) methods have been proposed for the determination of emodine and chrysophanol in crude drugs. In 2006, Alfawaz determined protein, moisture, ash and lipids analysis, mineral and organic acids composition, ascorbic acid, and tocopherols of Rumex vesicarius L. leaves grown in the central and northern regions of Saudi Arabia. Table (1) shows the species, part used, and the chemical structure of the isolated compounds from genus Rumex.

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