EFFECT OF METHANOL EXTRACT OF ALOE VERA ON ACETIC ACID-INDUCED COLON ULCER

Transcription

1 EFFECT OF METHANOL EXTRACT OF ALOE VERA ON ACETIC ACID-INDUCED COLON ULCER

2 4. EFFECT OF METHANOL EXTRACT OF ALOE VERA ON ACETIC ACID -INDUCED COLON ULCER 4.1 INTRODUCTION Aloe rdensis Miller (Aloe ver) hs een used medicinlly for severl thousnds of yers with long nd illustrious history. The gel of Aloe ver contins out 99 to 99.5% wter with ph in the rnge of 4.4 to 4.7. The mjor components in the gel re glucomnnn, cemnnn, minerls, flvonoids, tnnic cid, lprogen, c- glucosyl chromone, etc. The solid mteril contins out 45 different ingredients including vitmins, minerls, enzymes, sugrs, nthrquinone or phenolic compounds lignin, sponins, sterols, mino cids nd slicylic cid (Burn, 2003). The plnt is reported possess numerous phrmcologicl properties like ortifcient effect, djuvnt ctivity, nlgesic ctivity, nticlstogenic, etc. (Akev nd Ayse, 1999; Yun et l., 2009). There re mny reports on the effect of consumption of extrcts of Aloe ver gel on gstric ulcer, gstric microcircultory chnges, nti-inflmmtory, heptoprotective, clinicl tretment of sepsis, etc. (Gdegesin et l., 2010; Emlknm et l., 2006). Aloe ver gel extrct ws reported to possess for ntioxidnt potentil in vivo (Anilkumr et l., 2010) nd exhiit rdicl scvenging ctivity (72.2%) which is higher thn tht of BHT (70.5%) nd α-tocopherol (65.65%). The thick fleshy leves of Aloe ver plnts contin not only cell wll crohydrtes such s cellulose nd hemicellulose ut lso storge crohydrtes such s cetylted mnnns (Ni et l., 2004). The im of our study ws to evlute the effect of methnol extrct of Aloe ver on the chnge in the oxidtive system nd suppressing cetic cid-induced colon ulcer in rts. 4.2 MATERIALS AND METHODS 4.2 MATERIALS This is erlier provided in the Chpter No.3

3 4.2 METHODS Tretment of experimentl rts The tretment of nimls is s given in the Chpter No Chemicl, iochemicl nd enzymtic ssys The spectrophotometric ssys of the enzymes were crried out using spectrophotometer (Schimtzu model UV 1601) doule em. For the flurometric ssys of enzymes, spectroflourometer (Elico model) ws used. All centrifugl seprtions were crried out in Sorvll RC-5B utomtic superspeed refrigerted centrifuge with SM-24 rotor. All the microweighings were done with Schimtzu AEL-160 electronic lnce nd the other weighings with the help of Kern or Anmed electronic lnce. Liquid nitrogen continer ws used for pre-freezing the tissue smples nd storge until nlysis ws crried out. Kelvintor deep-freezer (- 15 C) ws used for storing regents ment for enzymtic rections nd pre-cooling centrifuge rotors efore running t low tempertures Experimentl protocol Helthy mle Wistr strin Alino rts inred t Defence Food Reserch Lortory niml house weighing pproximtely g were llocted rndomly to 8 groups of 6 nimls ech, Group 1 (control group), group 2 (6% cetic cid group), group 3 (50 mg/kg.wt methnol extrct of Aloe ver group), group 4 (50 mg/kg.wt methnol extrct of Aloe ver group + 6% cetic cid group, group 5 (100 mg/kg.wt methnol extrct of Aloe ver group), group 6 (100 mg/kg.wt methnol extrct of Aloe ver group+ 6% cetic cid group), group 7 (200 mg/kg.wt methnol extrct of Aloe ver group) nd group 8 (200 mg/kg.wt methnol extrct of Aloe ver + 6% cetic cid group) received orlly fed methnol extrct of Aloe ver for 21 dys. Groups 2, 4, 6 nd 8 were dministered intrrectlly with 6% cetic cid nd groups 1, 3, 5 nd 7 were dministered intrrectlly with sline 24 hrs prior to scrifice. All the nimls were mintined on sterile, stndrd control diet nd wter, d liitum. Food intke nd weight gin were monitored weekly. On 21 st dy, ll the rts were scrificed under mild nesthesi nd orgns were quickly excised nd stored 93

4 in -20 C freezer until nlyses. The experiments were designed nd conducted fter clernce from the Institutionl Animl Ethicl Committee, following the ethicl guidelines for rts Chemicl nlysis Hydroperoxides Aout 0.05 g of the lipid isolted from liver ws thoroughly mixed with 5.0 ml of chloroform: methyl lcohol solvent mixture (2:1). This ws followed y centrifugtion t 800 x g for 5 min seprting the upper phse distinctly. Upper phse is removed using syringe. Lower lyer is recovered into test tue nd tken to dryness under strem of nitrogen. While still under the strem of nitrogen 1ml of cetic cid: chloroform solvent mixture (3:2) is dded followed y 0.05 ml of potssium iodide (dissolve 6.0 g of KI in 5.0 ml of wter on ice, which hs previously een uled with nitrogen gs for 5 min). The test tues re then stoppered nd kept in drk for 5 minutes exctly. This is followed y the ddition of 3 ml of cdmium cette (0.5%). The solution mixture is then centrifuged t 800 x g for 15 min. The opticl density of the upper phse is red t 353 nm ginst lnk contining the complete ssy mixture minus the lipid. Stndrdistion of the rection ws done y using cumene hydroperoxide s the peroxide stndrd. The molr extinction coefficient of cumene hydroperoxide is 1.73x10 4 /M (Buege nd Aust, 1978) Conjugted dienes Aout 0.05 g of the lipid isolted from liver ws mixed thoroughly with 5.0 ml of chloroform: methnol solvent mixture (2:1) followed y centrifugtion t 800 x g for 10 min. This ided in seprtion of phses. Most of the upper phse ws removed either y suction or y the id of syringe. The lower chloroform lyer ws plced in test tue nd ws tken to dryness in strem of nitrogen gs. The lipid residue ws dissolved in 1.5 ml of cyclohexne nd the sornce t 233 nm ws determined ginst cyclohexne lnk. The mount of conjugted dienes produced ws clculted using molr extinction coefficient of 2.52x10 4 /M (Buege nd Aust, 1978). 94

5 4.2.4c Thiorituric cid rective sustnce (TBARS) mesured s mlondildehyde (MDA) Aout 0.5 ml of tissue homogente in phosphte uffer, ph 7.0 ws dded to 0.5 ml of 10% TCA. 2.0ml 0f TBA mixture (0.35% TBA, 0.2% sodium luryl sulphte, 50 µm FeCl 3 nd 0.5 mm BHT in 0.1 M glycine HCl uffer, ph 3.6) ws dded to the homogente contining TCA. The ove rection mixture ws heted in oiling wter th for 10 min. After cooling the mixture ws centrifuged t 1000 x g for 10 min. The OD of the superntnt ws mesured t 532 nm. The concentrtion of MDA ws estimted using the molr extinction coefficient, 1.56x10 5 / M/cm (Girotti nd Deziell, 1983) d Myeloperoxidse (EC ) Myeloperoxidse (MPO) is n enzyme found undntly in neutrophils nd t very low concentrtion in mcrophges nd monocytes. This enzyme oxidizes the electron donor (hlides etc) y H 2 O 2. The concentrtion of neutrophils could e indirectly estimted y mesuring the enzyme ctivity from its suspension. Briefly, 100 mg of colonic segments were minced finely in 10 ml of 50 mm potssium phosphte uffer (ph 6.0) contining 14 mm hex-decyltrimethyl-mmonium romide (HETAB) using scissors. Tissues were homogenized for 1 min, nd then the lystes were sonicted for 30seconds followed y centrifugtion for 10 min t 4 C (15000 x g). Myeloperoxidse ctivity ws estimted in the superntnts in presence of O-dinisidine-HCl nd % H 2 O 2 y monitoring opticl density t 415 nm using Shimdzu spectrophotometer. The results were expressed s units/g wet tissue.1 unit of enzyme ws defined s tht degrding 1 µm hydrogen peroxide/min t 25 C Krwisz et l. (1984) e Ctlse (EC ) Ctlse ws ssyed in tissue homogentes y following the spectrophotometric degrdtion of H 2 O 2 t 20 o C, ccording to the method of Cohen et l. (1970). Aout 1.0 g of liver or colon tissue ws homogenised in the cold in 4 volumes of 0.05 M potssium phosphte uffer in 0.9% sline solution (ph 7.4), 95

6 contining 0.1% (v/v) ethnol, in motor driven potter-elvehjem tissue homogenizer equipped with teflon pestle. The homogente ws centrifuged t 4 o C for 10 min t 700 x g to sediment nuclei nd cell deris. The sediment ws resuspended in the uffer, homogenised nd centrifuged. The superntnts were pooled to give 1:10 finl dilution. The superntnt ws kept in crushed ice for 30 min. To n liquot of the superntnt, 10% triton X-100 (w/v) ws dded to finl concentrtion of 1% nd mixed with gentle shking. Cold isotonic uffer ws then dded to produce desired dilutions. One ml of the ssy mixture contined 10 mm potssium phosphte uffer, 6 mm H 2 O 2 nd 0.1ml of the diluted enzyme. The rection ws strted y dding the H 2 O 2 to the ssy mixture nd monitored the rte of decrese in sornce t 240 nm every 15 sec ginst regent lnk devoid of the sustrte. One unit of ctivity ws defined s the mount of enzyme ctlticlly degrding H 2 O 2 producing decrese in sornce of 0.1 per min. The enzyme concentrtion ws so djusted to provide n initil sorption etween 0.5 to 0.6 units when the stedy stte ws reched. Men of three determintions ws tken for clculting enzyme ctivity. Any non-ctltic degrdtion of H 2 O 2 ws controlled y repeting the experiment in the presence of 1 mm sodium zide, potent inhiitor of ctlse f Glutthione peroxidse (EC ) Glutthione peroxidse (GSH-Px) ctivity ws ssyed oth in liver nd colon homogentes y the spectroflurometric method of Weiss et l. (1980). Aout 0.5 g of fresh tissue ws homogenised in ice cold 0.5 M phosphte uffer ph 7.0 in motor driven Potter-Elvehjem tissue homogeniser equipped with teflon pestle. The homogente ws centrifuged t 4 o C for 10 mins t 700 x g to sediment nuclei nd cell deris. The sediment ws resuspended in the uffer, rehomogenised nd recentrifuged. The superntnts were pooled to finl 1:5 dilution with the homogenising uffer nd kept in crushed ice for 10 mins. 0.1 ml liquot of 300 mm solution of reduced glutthione (GSH) in phospte uffer ph 7.0 (prepred fresh nd flushed with N 2 ), 25 mm of nicotinmide denine dinucleotide phospte reduced (NADPH N 4 ), 200 mm of sodium zide (to inhiit ny ctlse ctivity), 400 mm of ethylene dimine tetrcetic cid (EDTA N 2 ),

7 µg of glutthione reductse (GSSGR) nd 400 mm of nicotinmide were dded in the test tues contining 0.5 ml of the pooled superntnt. The rection mixture ws preincuted t room temperture for 10 mins to ctivte the sulfhydryl enzyme, GSSGR. The rection ws initited y dding H 2 O 2 to finl concentrtion of 0.05 mm (0.58 ml of H 2 O 2 freshly diluted in 100 ml of the phosphte uffer). The smples were incuted t room temperture for 30 mins. 0.2 ml of 1 N HCl ws dded nd vortexed thoroughly to stop the rection nd destroy the unoxidized NADPH. The precipitte ws removed y centrifugtion t 800 x g for 10 min. 0.5 ml of the superntnt ws diluted to 2.0 ml with uffer nd rected with 1.0 ml of 6 N NOH contining 0.03% H 2 O 2 s the ctlyst. The tues contining the rection mixture were heted t 60 C for 10 min in shking wter th. The smples were cooled nd red in the spectrofluorometer t excittion wvelength of 360 nm nd emission wvelength of 460 nm. Two regent lnks were set, one contining the rection mixture devoid of tissue homogente while the other devoid of H 2 O 2 (sustrte). 2 mm of NADP contining 3.0 mm glutthione oxidized (GSSG) ws used for the stndrd curve g Superoxide dismutse (EC ) Superoxide dismutse (SOD) is mesured y the inhiition of cytochrome C reduction medited vi superoxide nions generted y xnthine-xnthine oxidse nd monitored t 550 nm. One unit of of SOD is defined s the mount required to inhiit the reduction of cytochrome C y 50% (Flohe nd Otting, 1984). Pipetted 2.9 ml of solution A (consisting of 0.76 mg xnthine in 10 ml of N N 2 HPO 4 nd 24.8 mg cytochrome C mixed with 100 ml of 50 mm phosphte uffer, ph 7.8 contining 0.1 mm EDTA) into 3 ml cuvette. Added 50 µl of tissue superntnt, prepred s in the cse of other enzyme ssys, in 50 mm phosphte uffer, ph 7.8. For stndrd curve using pure SOD, 100, 200, 300, 400 nd 500 ng were dded. The rection ws strted with 50 µl of solution B (consisting of xnthine oxidse in 0.1 m M EDTA, 0.2 U/ml). After mixing, the chnge in OD ws recorded t 550 nm for 1 min. A plot of 1/ E min -1 derived from the liner plot of the rection versus concentrtion of SOD stndrds. The ctivity ws tken from the clirtion curve lredy mde with stndrd SOD. 97

8 4.2.4h Glucose 6 phosphte dehydrogense (EC ) The superntnt of the tissue homogente prepred in 100 mm Tris uffer ph 8.2, ws ssyed for glucose-6-phosphte dehydrogense (G-6-PDH) y monitoring spectrophotometriclly the mount of NADPH formed under specified conditions of ph nd temperture in given time. The ssy mixture consisted of 2 mm glucose-6- phosphte, 0.3 mm NADP nd 100 mm Tris uffer ph 8.2. After pre-incution t 25 C for 5 min, the rection ws strted y dding the enzyme nd monitoring the increse in opticl density t 340 nm. The procedure ws essentilly ccording to Blinsky nd Bernstein (1963). One enzyme unit ws defined s 1 µmol NADPH formed per minute under conditions of the ssy i Glutthione S-trnsferse (EC ) Glutthione S-trnsferse (GST) ws ssyed in liver nd colon homogentes y the spectrophotometric method of Hig et l. (1974). Freshly excised tissues (1 g) were homogenised in ice cold phospte uffer, 0.1 M (ph 6.5) keeping the tue cool in eker contining crushed ice. The homogente ws centrifuged t 800 x g for 10 min nd the residue ws rehomogenised nd recentrifuged. The pooled superntnt ws mde upto 14.0 ml. This superntnt ws diluted 10 times using phosphte uffer tht served s the enzyme source for the rection. The rection ws strted y dding 0.1 ml of GSH (5 mm) nd 0.1 ml of 1 mm 1-chloro-2, 4- dinitroenzene (CDNB, s the sustrte) to 0.1 ml of the enzyme. The rection mixture ws mde upto 1.0 ml with the phosphte uffer. The rte of increse in sornce t 340 nm ws monitored every 15 sec ginst regent lnk devoid of CDNB. One unit of the ctivity ws defined s the mount of enzyme in mg protein ctlysing the formtion of 1µm of product formed per minute under condition of ssy. The enzyme concentrtions were so djusted s to provide n initil sorption etween 0.5 to 0.6 sorption units when the stedy stte ws reched. Men of determintion ws tken for clculting enzyme ctivity. GSH solution ws flushed with pure nitrogen gs efore ddition. The rection mixture ws thoroughly shken efore the ddition of CDNB so tht H 2 O 2 formed ws fully decomposed nd did not interfere with CDNB. 98

9 4.2.5 Histopthologicl studies All the scrificed rts were necropsied. Specimen viz. colon were collected from ll the rts nd fixed in 10% neutrl uffer formlin. Prffin sections (6-8 microns) were prepred nd stined with Hrris Hemtoxylin nd eosin (Crleton nd Drury, 1967) for microscopic exmintion Sttisticl nlysis Results were expressed s men ± stndrd devition (S.D). Sttisticl significnce determined y one-wy nlysis of vrince (ANOVA) (Grph Pd Prism version 5 softwre). Significnce or non-significnce of difference etween men vlues ws determined (p<0.01) level of significnce. 4.3 RESULTS AND DISCUSSION Figs 25 nd 26 presents the food intke nd weight gin pttern of rts fed with methnol extrct of Aloe ver respectively. The results show tht the feeding of the methnol extrct of Aloe ver or the dministrtion of cetic cid did not ffect the dily food intke nd weight gin of rts. Tle 8 presents the orgn weights viz. liver, kidney, hert, rin nd colon. The results show tht the feeding of Aloe ver frction or the dministrtion of cetic cid did not ffect it. Figs 27, 28, 29, 30, 31 nd 32 gives the colonic enzyme nd ntioxidnt /detoxifying enzyme viz. MPO, SOD, ctlse, GSH-Px, G-6-PD, GST. MPO ctivity in colon did not show ny chnge y the feeding of methnol extrct of Aloe ver per se. There ws lso significnt decrese in colonic GSH-Px with ssocited decrese in G-6-PD nd GST ctivities. The comined dministrtion of cetic cid nd methnol extrct of Aloe ver (200 mg/kg.wt.) showed the reversl of cetic cid-induced myeloperoxidse, GSH-Px, G-6-PD nd GST ctivities. A decline in colonic ctlse nd SOD ctivities ws lso noted in the group of rts dministered with cetic cid nd methnol extrct of Aloe ver (200 mg/kg.wt). Figs 33, 34, 35, 36 nd 37 gives the effect of feeding of methnol extrct of Aloe ver on cetic cid-induced chnges in heptic ctlse, GSH Px, SOD, G-6-PD, GST. Administrtion of cetic cid reduced the heptic ctlse, GSH-Px, SOD nd G- 6-PD ctivities nd there ws rise in the heptic GST ctivity significntly. 99

10 Fig 25. Effect of methnol extrct of Aloe ver on cetic cid induced chnges on food intke of rts (n=6) g/100g Group 1 Group 2 Group 3 Vlues re expressed s Men ± SD Group 4 Group 5 Group 6 Group 7 Group 8 7th dy 14 th dy 21st dy Fig 26. Effect of methnol extrct of Aloe ver on cetic cid -induced chnges on weight gin of rts (n=6) g/100g th dy 7th dy 14th dy 21st dy 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Vlues re expressed s Men ± SD 100

11 Tle 7. Effect of Aloe ver methnol extrct on cetic cid -induced chnges on orgn weights of rts (n=6) Rt Groups Colon (g/100g ody wt.) Liver (g/100g ody wt.) Kidney (g/100g ody wt.) Hert (g/100g ody wt.) Control 0.33 ± ± ± ± % Acetic cid 0.44 ± ± ± ± mg AME 0.38± ± ± ± mg AME + Acetic cid 0.36 ± ± ± ± mg AME 0.34 ± ± ± ± mg AME + Acetic cid 0.31 ± ± ± ± mg AME 0.35 ± ± ± ± mg AME + Acetic cid 0.34 ± ± ± ± AME- Aloe ver -methnol extrct Vlues re expressed s Men ± SD Fig 27. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in myeloperoxidse ctivity in rts M/cm/mg protein c c c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 101

12 Fig 28. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic superoxide dismutse ctivity in rts units/min/mg protein Rt groups Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) Fig 29. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic ctlse ctivity in rts A of 0.1 /min/mg protein c c c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 102

13 Acetic cid induced colitis model is similr to humn ulcertive colitis in terms of histologicl fetures. The comined dministrtion of cetic cid nd methnol extrct of Aloe ver (200 mg/kg.wt.) showed the reversl of cetic cidinduced chnges in MPO ctivity. There were no chnges oserved on feeding of 50 nd 100 mg/kg ody wt. of Aloe ver extrct. Myeloperoxidse is n importnt enzyme of neutrophils, relted to oxidnt urst for cteril killing. Neutrophils re one of the professionl phgocytes in humns. They mnufcture O 2 - y the oneelectron reduction of oxygen t the expense of NADPH (Nito nd Yoshikw, 2002; - Hmpton et l., 1998; Winterourn, 2002). Most of the O 2 rects with itself to form H 2 O 2. From these gents lrge numer of highly rective microicidl oxidnts re formed, including hypochlorous cid, OH, peroxynitrite nd mny others (Bior, 2000). Uniquely, myeloperoxidse redily oxidizes chloride ions to the strong non rdicl oxidnt, hypochlorous cid, which hve severl cytotoxic effects on cteril cells (Winterourn, 2002; Bior, 2000; Murkmi et l., 1995; Alrich nd Hurst, 1982). Tles 8 nd 9 gives the effect of Aloe ver methnol extrct on cetic cidinduced chnges in colonic nd heptic ntioxidnt nd lipid peroxidtion respectively. Oxidtive stress nd its consequent lipid peroxidtion re le to ggrvte free rdicl chin rections, disrupt the integrity of intestinl mucos rrier nd ctivte inflmmtory meditors, resulting in incresed colonic MDA contents, s shown in oth humn nd experimentl niml studies (Girgin et l., 2000; Ek et l., 2008). Prefeeding of methnol extrct of Aloe ver t the level of 200 mg/kg ody wt. rought down the cetic cid-induced rise in heptic MDA, conjugted dienes nd hydroperoxides. As hydroperoxides re the primry products of lipid oxidtion nd ply centrl role in the further utoxidtion of lipids, the inhiition of formtion nd/or ction of these unstle species y ntioxidnts cn e used (Mn nd Tn, 1999; Jswir et l., 2000) s mens of ssessing ntioxidnt ctivity. Glutthione is one of the most undnt nturlly occurring tripeptids, non-enzymtic iologicl ntioxidnt present in liver (Gul et l., 2000). Perturtion of GSH sttus of iologicl system hs een reported to led to serious consequences (Udy et l., 1999). The comined dministrtion of cetic cid nd methnol extrct of Aloe ver (200 mg/kg.wt.) showed reduction in heptic GSH content. Ctlse is hemeprotein; it protects the cells from the ccumultion of H 2 O 2 y dismutting it to 103

14 from H 2 O nd O 2 (Bhkt et l., 1999). SOD is the ntioxidnt enzyme tht ctlyses the dismuttion of the highly rective superoxide nion to O 2 nd to the less rective oxygen species H 2 O 2 (Hung et l., 1997; Okdo-Mtsumoto nd Fridovich, 2001). A decline in colonic ctlse nd SOD ctivities ws noted in the group of rts dministered with cetic cid. The decrese in the ctivity of SOD due to the dministrtion of cetic cid could e due to feedck inhiition or oxidtive inctivtion of enzyme proteins due to excess ROS genertion. Ctlse cts s preventive ntioxidnt nd plys n importnt role in protecting ginst the deleterious effects of lipid peroxidtion (Pigeolot et l., 1990).The inhiition of ctlse ctivity is suggestive of enhnced synthesis of superoxide nion during the dministrtion of cetic cid since superoxide nion is powerful inhiitor of ctlse. However the present study demonstrted the ility of methnol extrct of Aloe ver on enhncing the ctlse ctivity in colon nd liver of the rts. GSH-Px is seleno-enzyme; two third of which (in liver) is present in the cytosol nd one third in the mitochondri (Freemn nd Crpo, 1982). It ctlyses the rection of hydroperoxides with reduced glutthione to form glutthione disulphide nd the reduction product of the hydroperoxide (Meister nd Anderson, 1983). 104

15 Tle 8. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic ntioxidnt nd lipid peroxidtion Rt Groups GSH (µmoles/g) MDA (nmoles/g) Control 42.8± ±12.8 6% Acetic cid 27.9± ± mg Aloe ver frction 43.7± ± mg Aloe ver frction+ Acetic cid 37.07±6.0 c 168.3±04.2 c 100 mg Aloe ver frction 44.58± ± mg Aloe ver frction+ Acetic cid 38.33±3.2 c 159.2±08.3 c 200 mg Aloe ver frction 48.37± ± mg Aloe ver + Acetic cid 44.81±4.3 c 146.3±09.6 c Vlues re Men ± SD for 6 rts.vlues ering different superscripts in the sme column re significntly different (p<0.01) Tle 9. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic ntioxidnt nd lipid peroxidtion Rt Groups GSH (µmoles/g) MDA (nmoles/g) CDx10-1 nmoles/g HP moles/gx 10-5 Control 11.4± ± ± ± % Acetic cid 05.6± ± ± ± mg Aloe ver frction 50mg Aloe ver frction+ Acetic cid 100mg Aloe ver frction 100mg Aloe ver frction+ Acetic cid 200mg Aloe ver frction 11.3± ± ± ± ±0.59 c 488.3±15.0 c 0.019± ± ± ± ± ± ±0.53 c ±15.4 c 0.017± ± ± ± ± ± mg Aloe ver + Acetic cid 10.4±0.38 c 354.3±14.7 c 0.014± ±0.001 Vlues re Men ± SD for 6 rts.vlues ering different superscripts in the sme column re significntly different (p<0.01) 105

16 Fig 30. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic glutthione peroxidse ctivity in rts m moles NADP reduced/ min/mg protein Rt groups Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) Fig 31. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic glucose-6-phosphte dehydrogense ctivity in rts µ moles NADP formed / min/ mg protein c Rt groups c c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 106

17 GST plys n essentil role in liver y eliminting toxic compounds y conjugting them with glutthione. The comined dministrtion of cetic cid nd methnol extrct of Aloe ver (200 mg/kg ody wt.) showed the reversl of cetic cid-induced chnges in GSH-Px, GST nd G-6-PD ctivities. Enzymes such s SOD, GSH-Px nd ctlse s well s non enzymtic ntioxidnts such s GSH constitute the ntioxidnt system. The synergy of numer of compounds viz. rloin, glucomnnn, cemnnn, tnnic cid, c-chromosyl chromone, minerls, flvonoids, etc. present in the extrct of Aloe ver my contriute towrds the oserved reduction in inflmmtion. The plnt is reported to contin lprogen, n nti - llergic glycol protein nd c-glycosyl chromone, novel nti-inflmmtory compound (Akev nd Ayse, 1999). MPO is n enzyme present in neutrophils nd t much lower concentrtion in monocytes nd mcrophges. The level of MPO ctivity is directly proportionl to the neutrophil concentrtion in the inflmed tissue. Therefore, mesurements of MPO ctivity hve een considered quntittive nd sensitive ssy for cute intestinl inflmmtion. The present study implies tht the methnol extrct of Aloe ver hs good potentil to suppress the cetic cid-induced colon ulcer nd oxidtive stress in rts. 107

18 Fig 32. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in colonic glutthione S- trnsferse ctivity in rts m moles conjugte formed /min/mg protein c c c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) Fig 33. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic superoxide dismutse ctivity in rts 2.5 units/min/mg protein Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 0 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 108

19 Fig 34. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic ctlse ctivity in rts 2.5 A of 0.1 /min/mg protein Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) Fig 35. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic glutthione peroxidse ctivity in rts 7.00 m moles NADP reduced/min/mg protein Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 109

20 Fig 36. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic glucose-6-phosphte dehydrogense ctivity in rts c µ moles NADP formed/min/mg protein c Rt groups c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) Fig 37. Effect of Aloe ver methnol extrct on cetic cid -induced chnges in heptic glutthione S- trnsferse ctivity in rts m moles conjugte formed/min/mg protein c c c Control 6% AA 50mg AME 50mg AME+AA 100mg AME 100mg AME+AA 200mg AME 200mg AME+AA Rt groups Vlues re Men ± SD for 6 rts. Vlues ering different superscripts in the sme column re significntly different (p<0.01) 110

21 4.3 Histopthologicl results Severity of the inflmmtion in colon, liver, kidney, smll intestine nd hert ws ssessed nd grded semi quntittively (Al-Awdi et l., 2001) s follows; Grde 1- mild cute inflmmtion in wll, Grde 2- moderte cute inflmmtion, smll ulcers nd Grde 3- severe cute inflmmtion+ulcertion/necrosis of wll. Results of histopthologicl exmintions showed tht control group nimls did not show ny symptoms for inflmmtion wheres, cetic cid treted group showed moderte cute inflmmtion, smll ulcers nd some showed severe cute inflmmtion + ulcertion/necrosis of wll. The methnol extrct of Aloe ver treted group showed reduced intensity of lesions in colon lone without ny evidence of necrosis nd regenertion or inflmmtory rection (200 mg/kg ody wt.) (Fig 38). The interprettion of the histology results were done with the support of Pthologist. 111

22 Fig 38. Microgrphs showing the histopthologicl nlysis of the colonic tissue in rts : control group, no cute inflmmtion. : 6% cetic cid group, severe cute inflmmtion + ulcertion/necrosis of wll. c: Aloe ver methnol extrct (50mg/kg.wt) group, no cute inflmmtion. d: Aloe ver methnol extrct (50mg/kg.wt) + cetic cid group, Moderte cute inflmmtion ± smll ulcers. e: Aloe ver methnol extrct (100mg/kg.wt) group, no cute inflmmtion. f: Aloe ver methnol extrct (100mg/kg.wt) + cetic cid group, moderte cute inflmmtion ± smll ulcers. g: Aloe ver methnol extrct (200mg/kg.wt) group, no cute inflmmtion. h: Aloe ver methnol extrct (200mg/kg.wt) + cetic cid group, mild cute inflmmtion in wll or seros. () control group () 6% cetic cid group (c) Aloe ver methnol extrct group (d) Aloe ver methnol extrct +cetic (50mg/kg.wt) cid group (50mg/kg.wt) 112

23 (e) Aloe ver methnol extrct group (f) Aloe ver methnol extrct +cetic (100mg/kg.wt) cid group (100mg/kg.wt) (g) Aloe ver methnol extrct group (h) Aloe ver methnol extrct+cetic (200mg/kg.wt) cid group (200mg/kg.wt) 113