The Effect of Ethanolic Extract of Larpotea ovalifolia Plants Growing in Calabar on Antioxidants Status of Streptozocin Induced Diabetic Rats

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1 Glol Journl of Phrmcology 4 (1): 01-05, 2010 ISSN IDOSI Pulictions, 2010 The Effect of Ethnolic Extrct of Lrpote ovlifoli Plnts Growing in Clr on Antioxidnts Sttus of Streptozocin Induced Dietic Rts Theres S. Iffen nd Chinyere A.O. Usoro Deprtment of Chemicl Pthology, College of Medicl Sciences, University of Clr, P.M.B. 1115, Clr, Nigeri Astrct: In this study, the effect of the dministrtion of extrct of Lporte ovlifoli, on the ntioxidnt sttus of dietic rts ws determined. Thirty (30) mle lino rts with men weight of ±1.93g were divided into three groups thus: Group I (norml control), Group II (dietic control) nd Group III, (Lporte ovlifoli tretment). Tretment with Lporte ovlifoli significntly reduced (P<0.001) the level of plsm glucose nd significntly incresed (P<0.05) levels of the ntioxidnt enzymes when compred with the untreted group. The results indicte tht Lporte ovlifoli, in ddition to eing ntihyperglycemic, is effective in reducing oxidtive stress in dietes. Key words: Lrpote ovlifoli Antioxidtive potentil Ethnolic extrct Dietes INTRODUCTION leding to lipid peroxidtion, protein oxidtion, enzyme inctivtion, DNA se modifictions nd DNA strnd Dietes is heterogeneous group of syndromes reks [6]. Becuse much of this dmge occurs within chrcterized y n elevtion of fsting lood glucose cells, the ntioxidnt ctivity in tissue is considered to e cused y reltive or solute deficiency in insulin [1]. more relevnt thn tht in plsm. The liver is the min In line with the ove, [2] defines dietes s group of orgn involved in the metolism of iologicl toxins nd metolic diseses chrcterized y hyperglycemi medicinl gent nd such metolism is lwys ssocited resulting from defects in insulin secretion, insulin ction with the disturnce of heptocyte iochemistry nd or oth. genertion of rective oxygen species [7]. Therpy using Dietes is ssocited with numer of ntioxidnts s contined in medicinl plnts cn prevent, complictions including ketocidosis, recurrent dely or meliorte these complictions. infections, weight loss, crdiovsculr disese, dietic The mechnisms of ction of ntidietic plnts hve neuropthy, dietic nephropthy nd dietic een identified y severl reserchers to include one or retinopthy. The oxidtive stress which is ssocited with comintion of the following: inhiition of crohydrte dietes mellitus might ply n importnt role in the hydrolyzing enzymes in the GIT [8], insulin-like ctivity, initition nd progression of dietic complictions [3]. insulinse inhiition, incresed glycogen storge, Free rdicls trigger ctrct, one of the degenertive improved peripherl glucose utiliztion nd ntioxidnt mnifesttions of dietes [3]. Ptients with dietes hve ctivity [9]. decresed ntioxidnt defenses with lower levels of There is n incresing interest mong dietic ntioxidnts such s vitmin C nd E or reduced ctivities ptients nd helth professionls in using medicinl of ntioxidnt enzymes such s ctlse, superoxide hers. These nturl products re ville in undnce dismutse nd glutthione peroxidse [4]. One promising nd cn provide sfe, chep, stle, stndrdized nd spect of understnding the role of oxidtive stress in efficcious medicinl preprtions [10]. The plnt dietes-medited disorders is the ility of ntioxidnt Lporte ovlifoli elongs to the nettle fmily, supplementtion to ttenute dietes dverse effects [5]. Urticcee). The people of Iiio trie in Nigeri use the Severl studies hve reported tht rective oxygen leves nd tender shoots of the plnt s pot her or species cuse dmge to iologicl mcromolecules vegetles in soups [11]. Some households use the plnt Corresponding Author: S. Theres Iffen, Deprtment of Chemicl Pthology, College of Medicl Sciences, University of Clr, G.P.O. Box 3385 Clr (540001) Nigeri, Tel:

2 Glol J. Phrmcol., 4 (1): 01-05, 2010 s vegetles when prepring food for ies. Severl Chemicls nd Regents: All chemicls used for this studies hve shown tht nettles hve een used to tret reserch were of nlyticl grde. Ethnol ws from Jmes mny infections. When dministered 30 minutes efore Burrough Limited, Englnd. Streptozotocin used for glucose loding queous extrct of stinging nettle induction of dietes ws otined from Sigm, St Louis, (Urtic dioc), plnt of the sme fmily s Lporte Mo, U.S.A. Glucometer strips were from Lifescn Inc., ovlifoli, showed strong glucose lowering effect [12]. USA. Assy kits for the determintion of ctlse (CAT), This effect ccording to [12] my e cused in prt y the superoxide dismutse (SOD), glutthione peroxidse (GPx) reduction of intestinl glucose sorption. In the dietic nd mlondildehyde (MDA) were otined from OXIS rts, 2 weeks of dily, intrgstric tretment with Interntionl, Inc., USA. An ssy kit for glucose Lporte ovlifoli not only produced significnt determintion from Rndox lortories Ltd, United reduction in the fsting serum glucose concentrtions ut Kingdom ws used. lso lowered the serum concentrtions of totl cholesterol, triglycerides nd low-density lipoprotein Collection nd Preprtion of Plnt Mterils: cholesterol, [13]. The plnt used for this study ws identified nd It is importnt therefore to crry out study on the uthenticted s Lporte ovlifoli with voucher ntihyperglycemic nd ntioxidtive potentil of this numer 359, in the herrium of the Deprtment of common plnt. The result otined from this study will go Botny, University of Clr, Clr. Fresh long wy to helping the poor nd uneducted folks in leves of Lporte ovlifoli were collected from our loclity who tend to prefer herl therpy to the otnicl grden of the Deprtment of Botny, conventionl medicine to mnge dietes t lower cost. University of Clr. The leves were rinsed with severl chnges of clen tp wter nd llowed to drin MATERIALS AND METHODS completely. The plnt mterils of Lporte ovlifoli (700g) Mterils nd Equipment: The mterils nd equipment were chopped into its with knife then homogenized used in the course of this study include: wooden cges, in 1400 liters of 80% (v/v) ethnol using n electric wter ottles, feeding tue, syringes (1ml nd 5ml), test lender. The homogenized mixtures were kept for 48hours tues, ekers, glss pipettes, utomtic micropipettes, in the refrigertor t 4 C, fter which the mixtures were glucometer, lncet, cnnul, electric lender, centrifuge, filtered with cheese cloth then with Whtmn No. 1filter wter th, refrigertor, deep freezer, wter th, timer, pper. The filtrtes were concentrted t C using rotry evportor (RE-52A, Shngi Y Rong rotry evportor. The concentrtes were plced in wter Biochemistry Instrument Compny, Englnd), visile th t 40 C for complete dryness. Lporte ovlifoli spectrophotometer (SP-300 Optim, Jpn). yielded 23.3g of extrct. Tle 1: Weights of rts efore nd fter tretment Groups Before Tretment (g) After Tretment (g) Weight Chnge (g) Percentge Weight Chnge N NC ± ± ± ± DC ± ± ± ± LO ± ± ± ± NC: Norml control; DC: Dietic control; IN: Insulin treted group; LO: Lrpote ovlifoli treted group. Mens on the sme column with different superscripts re significntly different (P<0.05) Tle 2: Glucometer redings of fsting lood glucose efore nd fter tretment Groups Before Tretment (mmol/l) After Tretment (mmol/l) N NC 3.08±0.16 NS 2.88± DC 11.12±1.12 NS 12.12± LO 11.56± ±0.43* 10 NC: Norml control; DC: Dietic control; IN: Insulin treted group; LO: Lrpote ovlifoli treted group; Results re presented s men ± stndrd error; * = Significntly different when compred with lood glucose efore tretment (P<0.001); NS= Not Significnt Tle 3: Effect of tretment on the levels of ntioxidnt enzymes, mlondildehyde nd fsting plsm glucose Groups CAT (U/ml) SOD (U/ml) GPx (mu/ml) MDA (µmol/l) Glucose (mmol/l) N NC 12.56± ± ±22.14 c 7.11±2.56 c 2.09± DC 11.14± ± ± ± ± LO 12.57± ± ± ± ± NC: Norml control; DC: Dietic control; LO: Lrpote ovlifoli treted group; CAT: Ctlse; SOD: Superoxide Dismutse; GPx: Glutthione Peroxide; MDA: Mlondildehyde; GL: Fsting Plsm Glucose; Mens on the sme column with different superscripts re significntly different (P<0.05) 2

3 Animls: Thirty mle lino wistr rts of verge weight ±1.93g were used for this study. The nimls were otined from the niml house of the deprtment of phrmcology, University of Clr. The nimls were llowed to cclimtize for 7 dys in the niml house of the deprtment of Biochemistry where they were housed during the study. The nimls were housed in well ventilted cges nd kept under environmentl temperture of (25 ±5 C), reltive humidity of (50±5%) nd 12 hour light/ drk cycle. Induction of Dietes: The nimls were sujected to 12 hour fst fter which 65mg/kg ody weight streptozotocin (STZ) reconstituted in norml sline ws injected intrperitonelly to induce dietes. Dietes ws not induced in nimls tht were used s norml control. Confirmtion of dietes ws done fter seven dys of induction. Blood ws collected from the til y prick with lncet for fsting lood glucose estimtion. Estimtion of fsting lood glucose ws done using one touch glucometer nd nimls with fsting lood glucose >126 mg/dl (7.0 mmol/l) were regrded s dietic. Experimentl Design : After induction nd confirmtion of dietes, rts were rndomly divided into 3 groups contining 10 rts ech. Ech group received different tretments twice dily for 28 dys s follows: Group 1: Norml control (Received 0.2ml of distilled wter). Group II: Dietic control (Received 0.2ml of distilled wter). Group III: Lporte ovlifoli treted group (Received 200mg/kg ody weight of the extrct). Smple Collection nd Processing: After 28 dys of tretment, the nimls were sujected to 12 hour fst. The rts were euthnized y nesthesi under chloroform vpor nd dissected. Blood ws collected y crdic puncture, trnsferred into fluoride oxlte ottle nd mixed thoroughly. After two hours this ws centrifuged t 4000g for 10 minutes nd the plsm ws otined nd stored frozen until used. The liver ws removed surgiclly, immeditely lotted using filter pper to remove trces of lood. The liver ws first rinsed with ice-cold heprinized sline (0.9% NCl contining 0.16mg/ml heprin) to remove lood cells nd therefter lotted with lotting pper. One grm of the tissue ws weighed nd thoroughly homogenized in 10ml of ice-cold phosphte uffered sline (20mM; ph 7.4). The homogente ws then centrifuged t 3,000g for 10 minutes nd the superntnt decnted into clen tues nd stored frozen until used for the ssy of ctlse, SOD, GPx nd MDA. Sttistics: The results were expressed s men ± stndrd devition (SD). Student t-test ws used to compre group mens efore nd fter tretment. The men vlues for ll prmeters etween groups were compred using nlysis of vrince (ANOVA) followed y Fisher s Lest Significnt Difference (LSD). The difference ws considered to e significnt when p vlue ws less thn RESULTS Tle 1 shows the men weight efore tretment nd fter tretment for norml control (NC), dietic control (DC), Lporte ovlifoli treted group (LO). The men weights efore tretment were: ± 6.36g, ± 3.98g, ± 3.35g for NC, DC nd LO respectively. While the men weights fter tretment were: 182 ± 5.56g, ± 6.08g, ± 5.40g for NC, DC nd LO respectively. The differences in weights efore tretment nd fter tretment were used to determine the percentge weight increse. Vlues otined for weight chnge nd percentge weight chnge for NC, DC, LO, GL nd IN were: (14.35 ± 3.46g, 8.94 ± 2.24%); ( ± 4.86g, ± 2.80%); (13.25 ± 4.05g, 7.48 ± 2.17%). The result shows tht untreted dietic rts recorded reduction in weight wheres norml control nd rts receiving Lporte ovlifoli tretment recorded increse in weight. The percentge weight gin of untreted dietic rts ws significntly lower (P<0.05) when compred with norml control nd treted group. No significnt difference in percentge weight gin (P<0.05) ws oserved etween norml control nd Lporte ovlifoli treted group fter tretment. Tle 2 shows the result otined for glucometer redings of the lood glucose of rts efore tretment nd fter tretment The men levels of lood glucose efore tretment were: 3.08 ± 0.16mmol/L, 11.12±1.12mmol/L, ± 0.90mmol/L for NC, DC nd LO respectively. After tretment, the lood glucose levels recorded were: 2.88 ± 0.20mmol/L, 12.12±0.94mmol/L, 3.61 ± 0.43mmol/L for NC, DC nd LO respectively. Tretment with, Lporte ovlifoli significntly reduced (P<0.001) nd returned the high level of lood glucose found efore tretment to norml. On the other hnd, untreted dietic rts recorded high level of lood glucose even t the end of the experiment. The result of the effect of tretment with Lporte ovlifoli on the levels of ctlse, superoxide dismutse (SOD), glutthione peroxidse (GPx), mlondildehyde (MDA) nd plsm glucose is shown on Tle 3. The men levels of ctlse in the heptocytes were: 3

4 12.56 ± 0.19 U/ml, ± 0.65 U/ml, ± 0.44 U/ml for In the heptocytes, Lporte ovlifoli hs NC, DC nd LO respectively. The men levels of SOD were: ± U/ml, ± U/ml, 63.22± U/ml for NC, DC nd LO respectively. The men levels of GPx were: ± mu/ml, ± 4.87 mu/ml, ± mu/ml for NC, DC nd LO respectively. The men levels of MDA in the heptocytes were: 7.11 ± 2.56 µmol/l, ± 4.17 µmol/l, ± 4.06 µmol/l efficiently reduced enzyme protein oxidtion which my occur s result of ccumultion of hydrogen peroxide nd other free rdicls. Superoxide dismutse converts superoxide rdicl to hydrogen peroxide nd the hydrogen peroxide is destroyed y ctlse or glutthione peroxidse. Tretment with Lporte ovlifoli hs therefore prevented depression of the ctivity of these for NC, DC nd LO respectively. Vlues otined for the ntioxidnt enzymes which would hve occurred s n men plsm glucose were: 2.09 ± 0.15 mmol/l, 9.38 ± 1.21 dptive response to oxidtive stress. Dietic control mmol/l, 3.73 ± 0.43 mmol/l for NC, DC nd LO rts on the other hnd recorded the lowest levels of these enzymes. The free rdicls generted in this group, in respectively. There ws significnt difference (P<0.001) ddition to protein glyction s result of hyperglycemi mong the groups for ll prmeters. The dietic control could inctivte the enzymes. In line with the findings of group hd significntly higher (P<0.05) plsm glucose this study, [16] from their reserch hd oserved tht thn ll other groups. Plsm glucose level of norml superoxide dismutse nd ctlse re inctivted y control ws significntly lower (p<0.05) thn those of LO sugrs. Also, [3], oserved tht superoxide dismutse, treted group. ctlse nd glutthione peroxidse decrese in liver, Dietic control rts recorded significntly lower kidney nd hert of tissues of ptients with dietes (P<0.05) levels of ntioxidnt enzymes when compred mellitus while the level of rective oxygen species such s with norml control nd LO treted. No significnt superoxide nion rdicls increse. difference (P>0.05) ws found in ctlse ctivity etween Minerl elements form prt of some enzymes. norml control nd LO treted. There ws significntly higher SOD ctivity in norml control when compred For exmple, ctlse contins iron, superoxide dismutse contins copper nd zinc while glutthione peroxidse is with dietic control nd LO treted. The difference in the selenium contining enzyme. Lporte ovlifoli level of Gpx etween norml control nd LO treted ws not significnt (P>0.05). There ws significntly lower (p<0.05) MDA in norml control thn dietic control nd treted group. Tretment with LO resulted in significntly lower MDA concentrtion in the heptocytes when compred with the untreted elongs to the plnt fmily, urticcee found to e rich in minerls [17]. The results otined for dietic rts treted with extrct of Lporte ovlifoli my e n indiction of the vilility of these metls for the formtion of the enzymes. It is interesting to discover from study crried out y [18] tht the excretion of these importnt metls dietic rts. is higher in dietic ptients thn in norml controls. DISCUSSION Weight reduction hs een known to e one of the symptoms of dietes mellitus. In the dietic control rts, deficiency of insulin led to decresed mino cids uptke y tissues with consequent reduction in the level of protein synthesis. Also insulin deficiency results in lipolysis in dipose tissues nd protein rekdown in tissues [14]. The increse in weight oserved in the group treted with Lporte ovlifoli nd norml control group t the end of the experiment, is reflection of efficient insulin ction. Hyperglycemi in dietes mellitus is responsile for the development of oxidtive stress vi glucose uto-oxidtion nd protein glyction [15]. Bioctive molecules present in Lporte ovlifoli extrct my proly possess insulin- like effect or stimulte the cells of the pncres to produce insulin which in turn lowers the lood glucose level. A possile mechnism of ction of Lporte ovlifoli could e y incresing pncretic secretion of insulin [13]. This oservtion could explin why there re significntly reduced levels of these metl contining ntioxidnt enzymes in the untreted dietic rts. The study shows tht MDA, lipid peroxidtion product nd mrker of oxidtive stress ws significntly lower in the group treted with Lporte ovlifoli when compred with dietic controls. This indictes tht the dietic rts were exposed to n incresed oxidtive stress nd lipid peroxidtion. Similr result ws lso found in dietic ptients y [19]. Anormlly high levels of free rdicls, lipid peroxidtion nd simultneous decline in ntioxidnt defense mechnisms cn led to dmge of cellulr orgnelles nd enzymes [19]. Lower levels of MDA in norml controls when compred with Lporte ovlifoli treted group could e reflection of increse in the formtion of free rdicls s result of dietes. The result otined from this study shows tht Lporte ovlifoli increses the levels of ntioxidnt enzymes nd inhiits lipid peroxidtion. Lporte ovlifoli therefore hs gret potentil to reduce compliction tht occurs in dietes s result of oxidtive stress. 4

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