THE PHENOLIC COMPOSITION OF THE HEPATOPROTECTIVE AND ANTIOXIDANT FRACTIONS OF Albizia lebbeck L.

Transcription

1 Quim. Nov, Vol. 39, No. 8, , THE PHENOLIC COMPOSITION OF THE HEPATOPROTECTIVE AND ANTIOXIDANT FRACTIONS OF Albizi lebbeck L. Ndi M. Sokkr,b, *, Sehm M. El-Hwry b, Amny M. Slem c nd Zeinb Tlt d Nturl Products nd Alterntive Medicine Dept. Fculty of Phrmcy, King Abdulziz University, Jeddh, 80200, Kingdom of Sudi Arbi b Phrmcognosy Dept., Fculty of Phrmcy, Ciro University, Ciro, 11562, Egypt c Ntionl Reserch Institute Dokki, Giz, Egypt d Ntionl Orgniztion for Drug Control nd Reserch, Giz, Egypt Artigo Recebido em 01/02/2016; ceito em 07/04/2016; publicdo n web em 17/06/2016 An investigtion of the heptoprotective nd ntioxidnt ctivities of the chloroform, ethyl cette nd n-butnol frctions of the leves of Albizi lebbeck L. ws performed. The first two frctions expressed the best results regrding the suppression of the incresed levels of plsm mino-trnsferses nd lkline phosphtse in liver-dmged mice (fter intoxiction with CCl 4 ) compred with silymrin nd the significntly incresed GSH content of lloxn- induced dibetic rts compred with vitmin E (tests nd reference drugs were orlly dministered). The bioctive frctions of Albizi lebbeck L. were subjected to chromtogrphic nlysis to investigte their phenolic contents using HPLC-PDA-ESI-MS/MS technique in the negtive ion mode. The results constitute the first report of the presence of seven compounds in the genus Albizi, three of which were identified s 3-O-cffeoylquinic cid, cfeic cid, myricetin; four other flvonoids (in mg 100 g -1 dry powder ± SD), myricetin 3-O-rhmnoside (0.129 ± ), quercetin 3-O-dideoxypentoside (0.011 ± 0.001), kempferol 3-O-glucoside (0.015 ± 0.002), quercetin 3-O-dihexoside (0.138 ± 0.002); quercetin 3-O-rutinoside t level of ± 0.004; nd the glycones quercetin, luteolin nd kempferol. Method vlidtion ws performed, providing n nlyticl technique tht cn be used to detect trce mounts of the identified compounds in Albizi extrcts with rpid smple preprtion. Keywords: Albizi lebbeck; heptoprotection; ntioxidnt; phenolics; LC-PDA-ESI-MS/MS INTRODUCTION The Albizi lebbeck L. tree is ntive to tropicl Afric, Asi, nd northern Austrli nd is widely plnted nd nturlized throughout the tropics. It occurs s populr ornmentl tree in Egypt nd is described s potent herbl drug. 1 The tree is trditionlly used in the tretment of ophthlmi, bronchil sthm nd other llergic disorders, including chronic cough nd bronchitis. 2 Biologiclly, Albizi lebbeck L. hs nticonvulsnt, 3,4 nthelmintic, 5,6 ntioxidnt, 7 ntifertility, 8,9 nlgesic, 10 spermicidl, 2 nootropic nd nxiolytic, 11 ntimicrobil 12 nd in vitro ntiprotozol 13 effects. Previous phytochemicl studies hve reported the presence of flvonoids, sponins, β-lctm derivtives, triterpenes, sterols nd hydrocrbons, 16 mcrocyclic lkloids 22,23 nd tnnins 24 in different tissues of the tree. The flower of A. lebbeck L. hs been used in chronic cough, helped in removing blck spots, used s n nti-llergenic, for urine retention nd lso s sex tonic. Anlysis of the florl odor hs reveled its chemicl composition. In continution to the work tht focused on the medicinl vlue of the different orgns of the A. lebbeck L. tree cultivted in Egypt, 28 n investigtion of its frctions ws performed, showing suppressive effects ginst crbon tetrchloride (CCl 4 )-induced liver injury in mice nd n ntioxidnt effect on lloxn-induced dibetic rts. In this study, we investigted the heptoprotection nd ntioxidnt ctivity of the chloroform, ethyl cette nd n-butnol frctions prepred from n ethnol extrct of the leves of A. lebbeck L. nd crried out liquid chromtogrphy tndem mss spectrometry (LC-MS/MS) nlysis to explore their phenolic contents. The occurrence of phenolic cid derivtives, myricetin, quercetin nd kempferol glycosides *e-mil: ndisokkr@yhoo.com other thn those previously published in the bioctive frctions re reported for the first time, nd no reserch on the heptoprotection nd ntioxidnt properties of ctive frctionsof A. lebbeck L. hs previously been crried out. EXPERIMENTAL Plnt mteril Leves of A. lebbeck L. were obtined from trees grown in the Agriculturl Reserch Center nd EL-Ormn grden during The txonomicl identity ws kindly verified by Dr. M. Abd El Hfez, Agriculturl Reserch Center, Giz. A voucher specimen (A-123) hs been deposited in the Herbrium, Phrmcognosy Deprtment, Fculty of Phrmcy, Ciro University. Stndrds nd regents 3-O-Cffeoylquinic cid, cffeic cid, quercetin, luteolin, kempferol, myricetin, quercetin 3-O-rutinoside, myricetin 3-O-rhmnoside, nd kempferol 3-O-glucoside were kindly supplied by the Lbortory of Phytochemistry, Nturl Products Deprtment, NODCAR, Giz-Egypt; silymrin, from Grnd Phrm Co.; vitmin E (dl-α-tocopherol cette), from Phrco Phytophrmceuticl Co., Egypt; loxn, from Sigm Co., USA; glutthione kit, from Wok Co., Germny; nd biodignostic kits for estimtion of serum liver enzymes (AST, ALT, nd ALP), from Biomerieux, Frnce. Solvents Acetonitrile, methnol nd formic cid were HPLC grde

2 974 Sokkr et l. Quim. Nov (Merck), nd deionized H 2 O ws treted with pure qu RC655. The chemicl regents nd solvents were ll of nlyticl grde (BDH). Extrction of A. lebbeck leves Air-dried nd deftted (using petroleum ether) powdered leves (1 kg) of Albizi lebbeck L. were exhustively extrcted with 70% ethnol. The combined extrct ws evported under reduced pressure (93 g, dry residues). The residue ws suspended in wter nd portioned successively with chloroform, ethyl cette nd n-butnol sturted with wter to fford g (CFL - chloroform frction of the leves), 7.6 g (EAFL - ethyl cette frction of the leves) nd 5.5 g (BFL - n-butnol frction of the leves) dry residues, respectively. LC-MS/MS-ESI nlysis Mss detection ws performed on Thermo LCQ Advntge Mx ion trp mss spectrometer (Thermo Finnign, Sn Jose, CA, USA). The nlysis ws crried out pplying the following settings: the heted cpillry nd voltge were mintined t 400 C nd 4 kv, respectively; the nebulizer gs ws ir; the curtin gs ws N 2 ; the collision gs ws He; ioniztion ws performed in the negtive mode; nd the collision energy ws 35%. The full-scn mss infusion ws performed using syringe pump (Hmilton syringe, 500 μl) connected to the ESI unit t flow rte of 10 μl ml 1. The totl ionmpping experiment ws used s LC-MS/MS technique, in which the production scns for ech prent ion were used to determine which prent ions lost frgment to yield prticulr product ion. The HPLC nlysis ws performed using PDA detector with n Intersil ODS-2 C18 column (2.1 mm 50 mm, prticle size 3 μm, Alltech). Mobile phse: (A) 0.1% formic cid-wter; nd (B) cetonitrile-methnol (60:40, v/v). The grdient progrm ws 30% B (0-2 min), 50% B (2-6 min), 70% B (6-9 min), nd 70% A (9 12 min) t flow rte of 0.2 ml min 1 nd injection volume of 20 μl. The Xclibur softwre (version 1.4) linked to the instrument ws used for the clcultion of the corresponding concentrtions. Smple preprtion for qulittive nlysis of phenolics by HPLC CFL (250 mg) ws chromtogrphed by vcuum liquid chromtogrphy (VLC) using silic gel nd eluted with grdients of chloroform to methnol incresing the polrity with 5% stepwise ddition of methnol to give five min frctions (200 ml ech): (40 mg), b (25 mg), c (32 mg), d (34 mg) nd e (28 mg) by TLC profile, performed with cyclohexne:dichloromethne:formic cid:ethyl formte (35:30:5:30, v/v) (S1) nd ethyl cette:formic cid:cetic cid:wter (100:11:11:26, v/v) (S2) s mobile phses. An liquot of ech frction (5 mg) ws seprtely dissolved in 5 ml of methnol nd filtered through 0.45-µm membrne filter before injection. Smple preprtion for the quntittive nlysis of flvonoids by LC-ESI-MS Air-dried powdered leves (10 g) were deftted with petroleum ether in Soxhlet extrctor for 12 h; the plnt residue ws extrct with 70% ethnol by soniction t room temperture (1 h). The extrct ws filtered, concentrted under reduced pressure, suspended in 25 ml of wter nd frctionted between chloroform nd ethyl cette (4 x 100 ml, ech). The concentrted EAFL residue ws quntittively dissolved in 5 ml of methnol nd filtered through membrne filter (0.45 pore size) before injection. Quntittive determintion of flvonoids Compounds used s externl stndrds were prepred t different dilutions. Rutin nd kempferol-3-o-glucoside were diluted to 5 mg ml -1 using 1/10 dilutions ech. Myricetrin ws diluted to 0.087, 0.87, 4.35, 8.7, 43.5, nd 87 μg ech. The stock solutions were stored t -20 ºC. The stndrd solutions were filtered through 0.45-μm filters before injection into the HPLC nd diluted s necessry with methnol. Ech concentrtion of the stndrds ws nlyzed in triplicte. Quntifiction of the flvonoid glycosides ws chieved by the externl stndrd method. The nlyticl curves were prepred with six concentrtions of ech stndrd, nd ech smple ws nlyzed in triplicte. Quntittive determintions of rutin, quercetin 3-O-dideoxypentoside nd quercetin 3-O-dihexoside were expressed s rutin; myricetrin s myricetrin; nd kempferol 3-O-glucoside s kempferol 3-O-glucoside. The limits of detection were clculted s the concentrtions corresponding to three times the signl-noise rtio. The precision test ws crried out by injecting ech smple solution six times. The mesurements of intr- nd inter-dy vribility were utilized to determine the repetbility of the developed method. The intr-dy vribility ws determined by nlyzing ech smple three times within the sme dy, nd the inter-dy reproducibility ws performed on three different dys. The RSD (reltive stndrd devition) ws considered s the mesure of precision. Smple preprtion for the heptoprotective effect Aliquots of the residues of CFL, EAFL, nd BFL of leves (4 g, ech) of A. lebbeck L. were seprtely dissolved in distilled wter (20%, w/v) contining few drops of TWEEN 80 for ntiheptotoxicity testing. Experimentl nimls Albino mice (25-30 g) nd dult mle lbino rts of the Sprgue Dwley Strin ( g) were used. The nimls were kept on stndrd lbortory diet nd under the sme hygienic conditions. Wter ws supplied d lib. Toxicity The medin lethl dose (LD 50 ) vlues of the frctions CFL, EAFL, nd BFL prepred from leves of A. lebbeck L. were determined ccording to OECD. 29 Heptoprotective effect The experimentl procedures were performed ccording to the recommendtions of the Ethicl Committee of the Ntionl Reserch Centre nd followed the guidelines of the proper cre nd hndling of nimls. The tested frctions (CFL, EAFL, BFL) of A. lebbeck were evluted nd compred with stndrd smple of silymrin ( powerful heptoprotective drug tht ws used s positive control), 30,31 ccording to previously published method. 32 Fifty dult mle lbino rts were rndomly divided into 5 groups (10 nimls ech) s follows: the first group received 1 ml of sline nd ws kept s the reference control group; the second group received silymrin (25 mg kg -1 b. wt.); nd groups 3-5 received the three test solutions of A. lebbeck L. (100 mg kg -1 b. wt.), respectively. The extrcts nd silymrin were orlly dministered for two weeks, followed by induction of heptotoxicity through n intrperitonel injection of

3 Vol. 39, No. 8 The phenolic composition of the heptoprotective nd ntioxidnt frctions of Albizi lebbeck L. 975 Tble 1. Effect of CFL, EAFL, BFL prepred from A. lebbeck L. nd silymrin on the serum AST (u/l) ALT (u/l) ALP (KAU) Zero 15wks 72h 15wks Zero 15d 72h 15d Zero 15d 72h 15d Control 29.5± ± ± ±5.2 b 31.4± ± ± ±4.7 b 6.9± ± ± ±2.3 b CFL 29.4± ± ± ±2.4 b (25.7%) EAFL 28.7± ± ± ±1.5 b (46%) BFL 27.6± ± ± ±3.1 b (23.2%) Sily. 30.8± ± ± ±0.8 b (48 %) 29.7± ± ± ±1.2 b (23.2%) 32.4± ± ± ±0.6 b (37.7%) 28.9± ± ± ±2.3 b (20.9%) 27.5± ± ± ±0.3 b (49.1%) 6.9± ± ± ±0.7 b (21.5%) 7.1± ± ± ±0.1 b (31.8%) 7.2± ± ± ±0.6 b (15.9%) 7.1± ± ± ±0.1 b (57.9%) Sttisticlly significnt from time zero t p < 0.01; b sttisticlly significnt from 72 h fter CCl 4 t p < A dily dose of 100 mg kg -1 b. wt. of different extrcts nd 50 mg kg -1 b. wt. of silymrin.; d, dy; h, hour; Sily., silymrin s positive control; wks, weeks. 5 ml kg -1 of 25% CCl 4 in liquid prffin; then, the tretments were continued for nother two weeks. After n overnight fst, blood ws obtined from the rt orbitl venous plexus through the eye cnthus of the nesthetized niml. The blood smples were collected t time zero nd fter two nd four weeks. Serum ws isolted by centrifugtion nd divided for the determintion of the biochemicl mrkers sprtte mino-trnsferse (AST), lnine mino-trnsferse (ALT) nd lkline phosphtse (ALP). Antioxidnt ctivity Rts were divided into 6 groups (6 nimls, ech). The first group ws kept s negtive control nd received 1 ml of sline orlly. For the other groups, dibetes mellitus ws induced by n intrperitonel injection of single dose of lloxn (150 mg kg -1 b. wt.) in ech niml followed by n overnight fst. 33 The second group of dibetic rts remined untreted, the third group received vitmin E s reference drug (7.5 mg kg -1 b. wt.) nd the other three groups received CFL, EAFL, nd BFL (100 mg kg -1 b. wt., orlly) of leves of A. lebbeck L. Blood glutthione ws determined fter one week. 34 RESULTS AND DISCUSSION Biochemicl nlysis The toxicity study reveled tht orl dministrtion of CFL, EAFL, or BFL of A. lebbeck in doses up to 1.3 g kg -1 b. wt. did not cuse ny signs of toxicity. Previous work hs discussed the heptoprotective ctivity of 70% ethnolic extrct of leves of A. lebbeck in experimentl liver dmge induced by thiocetmide (100 mg kg -1 ) in lbino rts 35,36 nd it ws demonstrted tht the extrct hd positive effect in lowering serum enzymes. In the present study, n experiment ws performed to define the bioctive frctions prepred from 70% extrct of leves fter induction of liver dmge using CCl 4, highly heptotoxic industril chlorinted solvent. The effects of CCl 4 intoxiction (Tble 1) were high recorded levels of liver enzymes, (AST, ALT nd ALP) 15 dys fter intoxiction. The heptotoxicity of CCl 4 hs been reported to be due to its biotrnsformtion by the cytochrome P450 system (CYP2E1) in the endoplsmic reticulum of the liver to generte highly rective trichloromethyl (CCl 3 ) rdicl tht rects rpidly with oxygen to form trichloromethyl peroxy (CCl 3 O 2 ) rdicl. 37 These rective rdicls possibly ttck the polyunsturted ftty cids of the endoplsmic reticulum, thereby stimulting lipid peroxidtion nd disrupting C 2+ homeostsis, 38,39 resulting in cellulr lekge, loss of functionl integrity of the cell membrne nd relese of the heptic enzymes. 40 Menwhile, the orl dministrtion of silymrin (25 mg kg -1 b. wt.) decresed the level of the liver enzymes AST, ALT nd ALP by 48, 49.1 nd 57.9%, respectively, compred to the control group. However, simultneous tretment of liverdmged rts with A. lebbeck frctions or silymrin were significntly ble to preserve biochemicl chnges during CCl 4 intoxiction nd confirmed their potentil heptoprotection ctivity to ccelerte the regenertion of prenchyml cells, which decresed in the order of silymrin > EAFL > CFL > BFL. The reduction in the serum enzymes ctivities reveled the stbiliztion of the plsm membrne nd the severity of the heptopthy. Tble 2. Effect of CFL, EAFL, nd BFL of A. lebbeck L. leves on the blood glutthione level of lloxn-induced dibetic rts (n=6). Group (mg %) Blood glutthione % Potency Control (1 ml sline) 36.4±1.1 _ Dibetic control 21.9± Dibetic + vitmin E 35.9± Dibetic + CFL 31.2± Dibetic + EAFL 35.6± Dibetic + BFL 29.7± Sttisticlly significnt difference from the control t p < A dily dose of 100 mg kg -1 b. wt. of different extrcts. The results (Tble 2) demonstrted tht intoxiction with lloxn cused disturbnce in the ntioxidnt defence systems nd oxidtive stress, s evident from mrked decrese in the GSH content of 39.84%. In ddition, the depleted level of GSH my lso be due to decresed synthesis or incresed utiliztion to counterct the excess free rdicls produced. 41 In our investigtion, the orl dministrtion (100 mg kg -1 b. wt.) of CFL, EAFL, nd BFL of the leves of A. lebbeck or vitmin E significntly incresed the GSH content (% chnge from control 14.28, 2.20, 18.41, respectively), consequently preventing oxidtive stress nd estblishing potentil therpeutic role of frctions prepred from the leves of A. lebbeck in free-rdicl- -medited diseses in the order of vitmin E > EAFL > CFL > BFL. The ntioxidnt properties of the phenolics in the EAFL nd CFL re relted to their redox properties nd their chemicl structures, which llow them to ct s hydrogen donors nd singlet oxygen quenchers. Some of them lso disply metl cheltion effect, which hinders the oxidtionpromoting effect of trnsition metls. For this reson, our chromtogrphic investigtion ws directed towrds the highly bioctive EAFL nd CFL.

4 976 Sokkr et l. Quim. Nov Phenolics nlysis Generlly, the mobile phses tht were minly used in the HPLC nlysis of phenolics (flvonoids nd phenolic cids) re queous cetonitrile, queous methnol, or their mixtures in combintion with different concentrtions of n cid s proton source s needed for ioniztion, viz. formic cid (0.1 or 0.5%), cetic cid (0.25, 0.5, 1%), trifluorocetic cid (0.05%), phosphoric cid (2, 0.1%), mmonium cette (10 mmol L -1 ) or formte (10 mmol L -1 ). In the present study, the method development ws intended to provide relible rpid technique for the seprtion of 9 flvonoids using n Intersil ODS- 2 C18 column nd mobile phse consisting of mixtures of wter cidulted with formic cid nd cetonitrile-methnol (60:40, v/v). The nlysis of the phenolics in CFL nd EAFL ws performed on n HPLC-PDA (Figures 1 nd 2), nd the pek identities were further confirmed by LC-ESI-MSn (n = 2) system in the negtive mode. This nlysis ws used for the seprtion, detection nd chrcteriztion of the structure of the phenolic cids nd flvonoids in the bioctive frctions in which no extensive smple preprtion is required, nd this nlysis ws shown to hve higher sensitivity for the subject compounds thn MS nlysis in the positive mode. In Tble 3, the MS behvior of frctions (-e) prepred by VLC of the CFL reveled the presence of phenolic cids 1 nd 2, which were positively identified by comprison with vilble stndrds. The spectrum of compound 1 showed deprotonted moleculr ion t m/z 353 of monocffeoylquinic cid, which ws first detected through loss scn of 162 µ ( cffeic cid unit) nd gve bse pek t m/z 191 (for quinic cid). This frgmenttion is typicl of 3-O-cffeoylquinic cid (chlorogenic cid). 42 The mss spectrum of compound 2 showed typicl loss of CO 2 for cffeic cid, giving [M H 44] s chrcteristic ion. 43 MSn frgmenttion of the ion [M H] of the stndrds produced mjor frgment specific for flvone nd flvonols t m/z 151 with different intensities, originting from n RDA rection. 44 There ws mjor frgment for the glycones detected in frctions (-e) of the CFL. Compound 3 displyed n [M H] ion t m/z 301, while compounds 4 nd 5 hd deprotonted ion [M H] t m/z 285. Their frgmenttion ptterns mtch with those of stndrds for quercetin, luteolin nd kempferol. 44 The detection of free glycones is commonly n indiction of the presence of their glycosylted forms, but no glycosylted luteolin ws detected here. Compound 6 hs n [M H] ion t m/z 317, nd its frgmenttion behvior greed with tht of myricitin stndrd. 45 The LC-MS/MS chromtogrm of the EAFL (Tble 4) of A. lebbeck L. identified five flvonoid glycosides tht re O-glycosylted, nd their frgmenttion ws chrcterized by clevge of the glycosidic bonds nd elimintion of the sugr moieties with chrge retention on the glycone (Y 0- ). The dt for the retention times (R t ), UV, deprotonted molecules [M H] nd mss frgmenttion ptterns (MSn) of the flvonoids detected in the EAFL re listed in Figure 1. HPLC/PDA chromtogrm of VLC frctions (-e) prepred from CFL of A. lebbeck Tble 3. Retention times, UV spectr nd product ions of phenolic cids nd flvonoid glycones in frctions (-e) prepred from CFL of A. lebbeck L. Pek No. Compounds R t (min) UV mx (nm) MW MS/MS [M H] Frction m/z E d c b 1 3-O-Cffeoylquinic cid , 298 (sh), , 179, Cffeic cid (100%) Quercetin , (34%) Luteolin (65%) Kempferol , (55%) Myricetin , (58%)

5 Vol. 39, No. 8 The phenolic composition of the heptoprotective nd ntioxidnt frctions of Albizi lebbeck L. 977 Tble 4. Retention times, UV spectr nd product ions of flvonoid glycosides in EAFL of A. lebbeck L. Pek No. Compounds R t (min) UV mx (nm) MW [M H] - LC-MS/MS m/z 7 Quercetin 3-O-rutinoside , 267 (sh), , 447, 301([A-H] - 80%) 8 Myricetin 3-O-rhmnoside (60%) 9 Quercetin 3-O-dideoxypentoside , 267 (sh), (42%), 301([A-H] - 100%) 10 Kempferol 3-O-glucoside , (43%) 11 Quercetin 3-O-dihexoside , 267(sh), , 301(100%) Tble 4. The ions corresponding to the deprotonted glycone (Y 0- ) products were compred with deprotonted molecules [M H] of the glycones detected in the CFL frctions; similr dt re present in the literture nd hve been compiled for known stndrds tht confirmed the identifiction of glycosides of the glycones 3, 5, 6 s quercetin 3-O-rutinoside (rutin) (7), 16 myricetin 3-O-rhmnoside (8) 46 nd kempferol 3-O-β-glucoside (10). 47 Becuse reference smples of compounds 9 nd 11 (Tble 4) were not vilble, LC- MS proved to be extremely helpful for their ssignment nd further chrcteriztion of individul substnces with the id of literture dt. Compound 9 hd deprotonted ion [M- H] - t m/z 593 nd (MS2, [M-H] - ) nd other ions t m/z 447 (23%, [M-146] - ) nd m/z 301 [100%, Y 0- ] due to the successive loss of two deoxypentoses from the glycone quercetin (compred with compound 3). The two sugrs re ttched to the sme glycone crbon. This ws minly demonstrted by compring their pek intensities to tht of rutin, indicting their link t the 3-position. 47 These ions rising from the clevge of the glycosidic bonds re wek, nd the presence of qusi-moleculr ion with low bundnce (< 25) indicted the ttchment of the two sugrs t the sme position s noticed from rutin. The flvonoid is identified s quercetin 3-O-dideoxypentoside. Compound 11 showed MS t m/z 625 [M-H] - nd pek t m/z 301 (100%, Y 0- ) due to the loss of two hexose moieties (324 mu) linked to the glycone nd frgment t m/z 463 [M-H-162] -. By compring these frgments with those of compound 7 nd other similr compounds in the literture, 46 in which the two hexoses re mostly ttched to C-3, we propose the structure to be quercetin 3-O-dihexoside. The ttchment of the sugrs t this position is recurrent chrcteristic of quercetin glycosides in Albizi species. 14,47 Nturl products derived from nturl resources worldwide hve potentil for protection nd re successfully used to tret liver diseses Approximtely hlf of the phrmceuticls in use tody re derived from nturl products. 51 Their positive effects re minly due to the presence of different phenolics viz., flvonoids, coumrins, nd phenolic cids. 52 The heptoprotective effect of silymrin is minly due to its bioctive flvonoid content. 31,53 Similrly, the promising heptoprotective effect of Albizi lebbeck is minly ttributed to the flvonoids content in the frction prepred from 70% ethnol extrct. Evidence for this resoning is tht the ethyl cette frction, which contins minly flvonoid glycosides tht re polyphenols with heptoprotective nd ntioxidnt properties, hs highest ctivity. Previous publictions discussed the identifiction of quercetin nd kempferol 3-O-α rhmnopyrnosyl (1 6)-β-glucopyrnosyl (1 6)-β-glctopyrnosides in leves, 14 in ddition to rutin nd kempferol-3-o-rutinoside in flowers 16 of A. lebbeck.top of Form To our knowledge the two phenolic cids 1 nd 2, the glycone myricetin (6) nd the glycosides myricetin 3-O-rhmnoside (8), quercetin 3-O-dideoxypentoside (9), kempferol 3-O-glucoside (10), nd quercetin 3-O-dihexoside (11) hve not previously been reported in the genus Albizi. As EAFL exhibited the highest potency in the heptotoxicity ssys, it is resonble to stndrdize the extrcts from Albizi bsed on the contents of these mjor glycosides, which re probbly relted to the biologicl ctivity in question. Tble 5. Quntittive determintion of the identified flvonoids in the EAFL of A. lebbeck Flvonoid glycosides mg 100 g -1 dry powder ± SD Quercetin 3-O-rutinoside 0.135±0.004 Myricetin 3-O-rhmnoside 0.129±0.005 Quercetin 3-O-dideoxypentoside 0.011±0.001 Kmpferol 3-O-glucoside 0.015±0.002 Quercetin 3-O-dihexoside 0.138±0.002 Sum of the determined flvonoid glycosides Ech result is the men of three determintions ± stndrd devition. Vlidtion Figure 2. HPLC/PDA chromtogrm of the EAFL frction of A. lebbeck The stndrd compounds quercetin 3-O-rutinoside (rutin), myricetin 3-O-rhmnoside (myricetrin) nd kmpferol 3-O-glucoside showed good linerity, with r 2 = , , , respectively, over reltively wide concentrtion rnge. The inter-dy precision showed tht the RSD (reltive stndrd devition) vlues were 2.21, 1.98, nd 2.12% nd the intr-dy vritions were 2.22, 1.688, nd 1.36%, respectively. Their verge regression equtions were y = x (rutin); y = x (myricitrin); nd y = x (kmpferol 3-O-glucoside). The limits of detection were 2.14 (rutin), 1.22 (myricetrin), nd 6.44 (kmpferol 3-O-glycoside) ng ml -1. The percentge ccurcy rnged between nd 99.98%. The glycosides were quntittively estimted (in units of mg 100 g -1 dry powder ± SD) s being from the EAFL

6 978 Sokkr et l. Quim. Nov of A. lebbeck by LC/MS, with the results in Tble 5, quercetin 3-O-dihexoside ws present in the highest concentrtion (c mg 100 g -1 ) followed by quercetin 3-O-rutinoside (c mg 100 g -1 ). CONCLUSION Air-dried powdered leves were extrcted with 70% ethnol. The concentrted residue ws frctionted with different orgnic solvents (chloroform, ethyl cette nd n-butnol) to yield the CFL, EAFL nd BFL residul frctions, respectively. The frctions were exmined for their heptoprotection on induced liverdmged mice nd compred to silymrin s well s for their ntioxidnt ctivity on the dibetic rts compred with vitmin E. The results showed the following decresing order of ctivity: silymrin > EAFL > CFL > BFL nd vitmin E > EAFL > CFL > BFL, respectively. The bioctive frctions were subjected to LC-ESI-MS/MS nlysis in the negtive ion mode to explore their phenolics content. The results reveled the presence of 11 compounds: two phenolic cids, four flvonoid glycones nd five flvonoid glycosides. Seven of these compounds were identified here for the first time in the genus Albizi. Quntifiction of the identified flvonoid glycosides reveled tht quercetin 3-O-rutinoside ws predominnt. ACKNOWLEDGMENTS This work ws funded by the Denship of Scientific Reserch (DSR), King Abdulziz University, Jeddh, under grnt no. ( d1434). The uthors cknowledge the technicl nd finncil support from DSR. REFERENCES 1. Fisl, M.; Singh, P. P.; Irchhiy, R.; Int. Res. J. Phrm. 2012, 3, Orw, C.; Mutu, A.; Kindt, R.; Jmndss, R.; Simons, A.; Agroforestree Dtbse: tree reference nd selection guide, World Agroforestry Centre, Keny, Ksture, V. S.; Chopde, C. T.; Deshmukh, V. K.; J. Ethnophrmcol. 2000, 71, Ksture, V. S.; Ksture, S. B.; Pl, S. C.; Indin J. Exp. Biol. 1996, 34, Gll, M.; Bshir, A. K.; Slih, A. M.; Adm, S. E.; J. Ethnophrmcol. 1991, 31, El Grhy, M. F.; Mhmoud, L. H.; J. Egypt. Soc. Prsitol. 2002, 32, Resmi, C. R.; Venukumr, M. R.; Lth, M. S.; Indin J. Physiol. Phrmcol. 2006, 50, Gupt, R. S.; Kchhw, J. B.; Chudhry, R.; Asin J. Androl. 2004, 6, Gupt, R. S.; Chudhry, R.; Ydv, R. K.; Verm, S. K.; Dobhl, M. P.; J. Ethnophrmcol. 2005, 96, Sh, A.; Ahmed, M.; Pk. J. Phrm. Sci. 2009, 22, Une, H. D.; Srveiy, V. P.; Pl, S. C.; Ksture, V. S.; Ksture, S. B.; Phrmcol. Biochem. Behv. 2001, 69, Rhul, C.; Pnkj, P.; Srwn, S. K.; Mhesh, J. K.; J. Chem. Phrm. Res. 2010, 2, Al-Musyeib, N. M.; Mothn R. A.; Al-Mssrni, S.; Mtheeussen, A.; Cos, P.; Mes, L.; Molecules 2012, 17, El-Mousllmy, A. M. D.; Phytochemistry, 1998, 48, Rshid, R. Chwdhury R. B.; Jbbr, A.; Hsn, C. M.; Rshid, M. A.; Sudi phrm. J. 2003, 11, El Hlim, A.; Mohmed, F. A.; El Gml, A. A.; Mster Thesis, College of Phrmcy, King Sudi University, Sudi Arbi, Nzneen, B.; Wesely, E. G.; Johnson, M.; Int. J. Adv. Phrm. Res. 2012, 3, Sulimn, C. T.; Blchndrn, I.; Indin J. Phrm. Sci. 2012, 74, Pl, B. C.; Achri, B.; Yoshikw, K.; Arihr, S.; Phytochem. 1995, 38, Ued, M. L.; Tokung, T. O.; Okzki, M. St, P; N. U.; Ued, K. O.; Ymmur, S. P.; Nt. Prod. Res. 2003, 17, Scott, J. P.; Tinto, W. F.; Reynolds, W. F.; Nt. Prod. Commun. 2008, 3, Misr, L. N.; Dixit, A. K.; Wgner, H.; Phytochemistry 1995, 39, Dixit, A. K.; Misr, L. N.; J. Nt. Prod. 1997, 60, M, Y. T.; Hsiob, S. C.; Chen, H. F.; Hsu, F. L.; Phytochem. 1997, 46, Jin, A. S.; Mishr, K. L.; Tetrhedron Lett. 1963, 1, Holmn, R. T.; In North Americn Terrestril Orchids Symposium II, Proceedings nd Lecture, Michign Orchid Society, Livoni, 1981, pp Aiyelgbe, O. O.; Oyewole, A. E.; Oldosu, I. A.; J. Essent. Oil-Ber. Plnts 2010, 13, El- Hwry, S.; El- Fouly, K.; Sokkr, N. M.; Tlt, Z; Asin J. Biochem. 2011, 6, OECD; Guidelines for the testing of chemicls, Orgnistion for economic co-opertion nd development: Pris, Pndey, G.; Shni Y. P.; Int. J. Res. Ayurved Phrm. 2011, 2, Abenvoli, L.; Cpsso, R.; Milic, N.; Cpsso, F.; Phytother. Res. 2010, 24, Klssen, C. D.; Pl, G. L.; Biochem. Phrmcol. 1969, 18, Elisson, S. G.; Smet, J. M.; Life Sci. 1969, 8, Beutler, E.; Duron, O.; Kelly, B.M.; J. Lb. Clin. Med. 1963, 61, Shirode, D. S.; Hirode Jin, B. B.; Mhendr Kumr, C. B.; Setty, S. R.; J. Chem. Phrm. Sci. 2012, 5, Kokil, K.; Priydhrshini, S. D.; Sujthi, V.; Int. J. Phrm. Sci. 2013, 5, Smudrm, P.; Vsuki, R.; Rjeshwri, H.; Geeth, A.; Moorthi, P.; J. Medicinl Plnts Res. 2009, 3, Heibtollh, S.; Rez, N. M.; Izdpnh, G.; Sohill, S.; Afr. J. Biomed. Res. 2008, 2, Gutiérrez, R.; Solís, R.; Rec. Nt. Prod. 2009, 3, Tiwri, B. K.; Khos, R. L.; The Internet Journl of Tropicl Medicine 2010, 6, Dng, S. S.; Zhng, X.; Ji, X. L.; Cheng, Y. A.; Song, P.; Liu, E. Q.; He, Q.; Li, Z. F.; Chin. Med. J. 2008, 121, Rbned, F. S.; Jáuregui, O.; Csls, I.; Lcuev, C. A.; Pulido, M. I.; Lmuel-Rventós, R. M.; Mss Spectrom. 2003, 38, Querlt, A. V.; Uregui, O. J.; Remo, A. M.; Lcuev, C. A.; Rvento, R. M. L.; Rpid Commun. Mss Spectrom. 2010, 24, Justesen, U.; J. Chromtogr. A 2000, 902, Hifnwy, M.; Sokkr, N.; Ezzt, S.; Rsln, M.; Sleim, A.; Asin J. Plnt Sci. 2012, 11, Ferreres, F.; Llorch, R.; Gil-Izquierdo, A.; J. Mss Spectrom. 2004, 39, Melek, F. R.; Ghly, N. S; El-Kdy, M.; Nbil, M.; Egypt. J. Pure Appl. Sci. 2011, Seeff, L. B.; Lindsy, K. L.; Bcon, B. R.; Kresin, T. F.; Hoofngle, J. H.; Heptology 2001, 34, Sntillán, E. M.; Bujidr, E. M.; González, I. Á.; Mrtínez, M. T. S.; Slins, J. G.; Butist, M.; González, Á. M.; Rubio, M. G. L. G.; Fisl, L. A.; González, J. A. M.; World J. Gstroenterol. 2014, 20, Zchrih, S. M.; Aleykutty, N.; Vishwnd, V.; Hlim, O. A.; Res. J. Phrm. Technol. 2012, 5, Syed, S. H.; Nmdeo, A. G.; Int J. Phytophrm. 2014, 4, Zhng, A.; Sun, H.; Wng, X.; Eur. J. Med. Chem. 2013, 63, Bll, K. R.; Kowdley, K.; J. Clin. Gstroenterol. 2005, 39, 520.