Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 ISSN 0976 – 044X Research Article Protective Effects of Cistanche tinctoria Aqueous Extract on Blood Glucose and Antioxidant Defense System of Pancreatic β-cells in Experimental Diabetes in Rats 1 1 2 2 Amina Bouzitouna * , Khierddine Ouali , Samah Djeddi Laborat ory of Biochemist ry and Environmental Toxicology, Facult y of Sciences, Universit y of Badji M okht ar, Annaba, Algeria. 2 Depart ment of Biology, Facult y of Science Universit y Badji M okht ar, BP 12 Sidi Amar Annaba, Algeria. * Corresponding author’s E-mail: bouzitouna_bio@yahoo.fr Accepted on: 29-04-2015; Finalized on: 31-05-2015. ABSTRACT The aim of the present st udy is t o evaluat e t he possible prot ect ive effects of ‘ Cistanche tinctoria ’ ext ract on t he ant ioxidant defense syst ems of pancreas in st rept ozotocin (STZ) induced diabet es in rat s. The levels of blood glucose and TBARS in pancreas were estimat ed in cont rol and experiment al groups of rat s. The aqueous ext ract (ACE) was administered daily in doses of 200mg/ kg body weight t o st rept ozot ocin induced diabet ic rat s for a period of 21 days. Evaluat e t he changes in t he cellular ant ioxidant defense syst em such as t he level of reduced glut at hione and activit ies of superoxide dismut ase, cat alase, glut at hione peroxidase and glutat hione-s-t ransferase were assayed in pancreatic t issue homogenat e. The aqueous ext ract exert ed a significant (P<0.000) ant idiabet ic effect in st reptozotocin diabetic rat s. Daily t reat ment wit h 200mg/ kg body weight of ACE for 21 days not only brought a significant decrease on blood glucose level in STZ-induced diabet ic rat s, but also increased t he ant ioxidant enzymes’ act ivit ies. From t his study it can be concluded t hat the aqueous ext ract of C.tinctoria causes antidiabet ic and ant ioxidant activit y in St rept ozot ocin induced in diabet ic rat s. Keywords: Ci stanche tinctoria , Ant idiabet ic effect , diabetes, oxidative st ress, st rept ozocin. 7 INTRODUCTION F ree radicals are cont inually produced in t he body as a result of norm al m et abolic processes and int eract ion w ith environment al st im uli 1 com plicat ions. These unst able m olecules are capable of causing cellular dam age, w hich leads t o cell death and t issue injury. The ROS can bind w ith m ost norm al cellular com ponent s; t hey react w it h unsat urat ed bonds of m em brane lipids, denat ure prot eins, and at t ack nucleic 2 acids. The concentrat ions of ROS are m odulat ed by ant ioxidant enzym es and non-enzymat ic scavengers, such as superoxide dism ut ase (SOD), cat alase (CAT) and 3 glut at hione peroxidase (GSH-Px). A dist urbance of t he balance bet ween form at ion of active oxygen met abolit es and t he rate at w hich t hey are scavenged by enzym ic and nonenzym ic antioxidant s is referred t o as oxidat ive 4 st ress. It has been est ablished t hat oxidat ive st ress lies at t he root of a num ber of pathological processes and diseases such as cancers, at herosclerosis, rheum atic art hrit is, haem at ological and neurodegenerat ive disorders are not exem pt , wit h more m aking t he list among w hich is diabet es mellit us. Diabetes m ellit us is a het erogeneous m et abolic disorder characterized by high levels of blood glucose wit h dist urbances of carbohydrat e; lipid and prot ein m et abolism resulting from defect s in insulin secret ion, 5 insulin action or bot h. This serious, m et abolic disorder affect s approxim at ely 4% of t he populat ion w orldwide and is expected t o increase 6 by 5.4% in 2025. Ihara , exam ined oxidative st ress m arkers in experim ent al diabet ic rat s and found increased reactive oxygen species (ROS) in pancreat ic islet s. The pancreatic bet a-cells, have required intricat e mechanism s t o defend against ROS t oxicit y. How ever, t he reduced ant ioxidant capacit y potentially makes pancreatic β-cells sensitive to ROSm ediat ed signal transduction and cellular response. Thus, m aint enance of β-cell oxidant st at us and their prot ection against oxidative dam age might delay t he onset of diabet es as well as t he evolution of it s 8 com plicat ions. Cist anche tinct oria is a parasit ic plant (Orobanchaceae) t hat is at t ached underground t o t he root s of t he m ain host plant s (Tamarix gallica , Calligonum comosum and Pulicaria sp) and grow s by absorbing nut rient s from t he host plant . The parasit e is widely dist ribut ed in Nort h Africa, Arabia, and Asian countries. As a rare traditional m edicinal plant , t he dried w hole plant is used for t he t reatm ent of abdominal pains, diarrhoea, M uscle contractions, bruises, gynaecological diseases; st im ulant of lactat ion and Diabetes. No inform ation w as found on t he pharm acological of t his plant , w hile a search on it s t oxicit y appears negat ive. The present st udy is conduct ed t o syst em at ically evaluate t he antihyperglucem ic effect of C.t inct oria aqueous ext ract in STZ-induced diabetic rat s. In addit ion, t his w ork det erm ines w het her t he pancreas w as subject ed t o oxidat ive dam age during experim ent al diabet es as w ell as t o exam ine t he associat ed changes in ant ioxidant st at us. International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed. 243 © Copyright pro Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 ISSN 0976 – 044X On t he last day of experim ent at ion, t he anim als w ere deprived of food overnight and sacrificed by cervical decapit ation. M ATERIALS AND M ETHODS Collection of Plant M aterial Ariel part of C. Tinctoria (Desf.) Beck. (Orobanchaceae) w ere used in t his st udy. Low er part s of t he st em w ere collect ed in M ars, 2012, in t he region of Ouregla, Algeria. 9-10 The plant w as ident ified by t he bot anist s in t he Departm ent of Biology (Annaba, Algeria). Chemical Reagents All chemicals w ere purchased from Sigm a (USA), Aldrich (M ilw aukee, USA), Fluka (Buchs, Sw it zerland), Tokyo Chem ical Indust ry (TCI) and M erck (Germ any). Preparation of Extract C.t inct oria aqueous extract (ACE) w as prepared by boiling 50 g of t he pow der of t he aerial part of t he plant in a flask cont aining 1 L of w ater for 5 m in. The ext ract w as agit at ed and covered until it reached room temperature. The residue w as removed by filt ration and t he ext ract w as t hen suit ably concentrat ed in a rotary evaporat or (final concentrat ion: 50 mg/ mL). A sam ple w as separat ed in order t o det ermine t he solid concent ration, and t hen t he ext ract w as divided int o aliquot s st ored at −20° C un l furt her use. (Yield of aqueous ext ract w as about 13.5%). Animals and Experimental Design Tissue preparation The Pancreat ic tissue w as excised, rinsed in ice-cold physiological saline and hom ogenized in 0.1mM phosphat e buffer (pH 7.4). The hom ogenat e w as t hen cent rifuged at 9000 ×g for 30 min at 4°C, and aliquot s of supernat ant w ere kept at -20°C unt il used for assa ys. Assay of Non-enzymatic Antioxidants M easurement of Reduced Glutathione (GSH) Pancreas GSH content w as det ermined by elim an m ethod 12 13 of Ellm an m odified by Jollow , based on t he developm ent of yellow colour w hen DTNB (5, 5’ dit hiobis(2-nitrobenzoic acid) is added t o com pounds cont aining sulfhydryl groups. In brief, 0.8 m l of hom ogenat e supernat ant w as added t o 0.2 m l of 10% t richloroacet ic acid, and t hen tubes w ere cent rifuged at 3000 ×g for 5 min. Supernat ant (0.5 m l) w as mixed w it h 0.025ml of 0.01 M DTNB and 1 ml phosphat e buffer (0.1 M , pH = 7.4). The absorbance at 412 nm w as recorded. Finally, t ot al GSH content w as expressed as U GSH/ g prot ein. M easurement of Ascorbic Acid Animal Wist ar rat s (body w eight 220 ± 20 g) used for experim ent s w ere obt ained from Past eur Inst it ute (Algiers, Algeria). The rat s w ere acclim at ized for t hree week before st art ing t he experiment . Before and during t he experim ent t he rat s w ere housed under controlled environm ent al conditions of t em perat ure (22 ± 2°C) in a 12 h ligh t and dark cycle, and w ere m aint ained on (unless ot herwise st at ed) st andard food pellet s and t ap w ater ad libitum . Induction of Diabetes in Animals Diabetes w as induced by a single int raperit oneally (i.p.) injection of st rept ozot ocin (STZ) in fast ed rat s at dose of 11 60 m g/ kg body weight . STZ w as freshly dissolved in 0.1M cold sodium cit rate buffer, pH 4.5. Three days aft er STZ injection, t he glucose level of blood from the t ail vein w as det ermined, and hyperglycem ic rat s (blood glucose level > 200 mg/ dl) w ere used as t he diabet ic rat s for furt her experim ent s. Treatm ent w it h plant extract w as st art ed on t he t hird day aft er STZ inject ion and cont inued for 21 days. Experimental Design The STZ-induced diabet ic rat s (ment ioned above) w ere randomly divided int o t w o groups (8 rat s per group), and norm al rat s w ere used as t he cont rol group. Group I (n = 8): norm al control (NC), norm al rat s were received 1ml dist illed w ater Group II (n = 8): diabetic cont rol (DC), t he diabet ic rat s w ere received 1ml dist illed w at er; Group III (n = 8): diabetic rat s w ere t reat ed wit h 200m g/ kg/ d of ACE for 21days. Weekly body weight s w ere also recorded. Quant ificat ion of ascorbic acid w as performed according 14 t o Jagot a and Dani . In brief t w o hundred microlit res of t issue hom ogenat e or st andard preparat ion of ascorbic acid was precipitated on ice with 800 µl trichloroacetic acid for 5 m in. The sam ples w ere then centrifuged at 3000 rpm / min for five m inut es. Thereaft er, five hundred microlit ers of t he supernat ant w as subsequent ly diluted w it h double dist illed w at er t o 2 m L and m ixed wit h t w o hundred microliters of FolinCiocalt eus reagent , diluted in double distilled w at er (1:10), t he ascorbat e reduced t he Folin-Ciocalteau solut ion yielding a blue colour. Aft er 10 m in t he absorbance of t he samples w as m easured at 760 nm in a spect rophot om eter. Estimation of Lipid Peroxidation (malondialdehyde) Lipid peroxidation in the pancreat ic t issue w as estim ated colorim et rically by t hiobarbituric acid reactive subst ances 15 TBARS m et hod of Ohkaw a . A principle component of TBARS being m alondialdehyde (M DA), a product of lipid peroxidation. In brief, 2.5 ml of 20% t richloroacet ic acid and 1.0 m l of 0.67% TBA are added t o 0.5 m l of t issue hom ogenat e (KCl 1.15%), t hen t he mixt ure is heat ed in a boiling w ater bat h for 30 min. The result ing chrom ogen is ext ract ed w ith 4.0 m l of n-but yl alcohol and t he absorbance of t he organic phase is det ermined at t he w avelengt h of 530 nm. The M DA cont ent s w ere calculat ed using 1, 1, 3, 3-t etraet hoxypropane as st andard and t he result s are expressed as nmol of m alondialdehyde/ g of tissue. International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed. 244 © Copyright pro Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 ISSN 0976 – 044X Assay of Pancreas Enzymatic Antioxidants Statistical analysis Assay of Catalase (CAT) Activity The dat a w ere analyzed by one w ay analysis of variance (ANOVA) follow ed by Tukey’s t est . All t he result s w ere expressed as m ean± S.E.M . for eight rat s in each group. A difference in t he m ean values of p<0.05 w as considered t o be st at ist ically significant . Cat alase (E.C.1.11.1.6) activity w as m easured according t o 16 Aebi m et hod. The 0.1m l of t he t issue hom ogenat e w as pipet t e int o cuvet te cont aining 1.9 m L of 50mM phosphat e buffer, pH7.0. Reaction w as st arted by t he addit ion of 1.0mL of freshly prepared 30%(v/ v) hydrogen peroxide (H2O2). The rat e of decom posit ion of H2O2w as m easured spectrophot om etrically from changes in absorbance at 240nm for 2m in. The enzym e act ivit y w as calculat ed by using an ext inct ion coefficient of 0.043 m M 1 -1 -1 cm . Act ivit y of enzym e w as expressed as unit s mg prot ein. Assay of Glutathion Peroxidase (GSH-Px) Activity Glut athion peroxidase (E.C.1.11.1.9) act ivit y w as m easured by t he met hod described by Floche and 17 Gunzler. The reaction m ixt ure cont ained 0.3m l of 0.1M phosphat e buffer, pH 7.4, 0.1m L of 10m M sodium azide, 0.3m l of enzym e, 0.2ml 2m M glut at hione and 0.1 ml of 1m M H2O2. The content s w ere incubat ed at 37°C for 10m in, follow ed by t he t erm ination of t he react ion by t he addit ion of 0.5 ml TCA 5%, cent rifuged at 5000 rpm for 5 m in. The supernat ant w as collected. 0.2 m l phosphate buffer (0.1 M , pH 7.4) and 0.7 ml DTNB (10 m M ) w ere added t o 0.1 ml supernat ant . Aft er mixing, t he absorbance of t he product w as read at 420 nm and -1 expressed as nm ol mg prot ein. Assay of Superoxide Dismutase (SOD) Activity Pancreat ic SOD (E.C.1.15.11) activit y w as m easured by inhibit ion of t he form azan form ation according t o t he m et hod (xant hine/ xant hine oxidase t est ) of Beaucham p 18 and Fridovich. The react ion mixt ure cont ained t he following solut ions: 2.25 ml of 0.05 M Tris/ HCl buffer, pH 8.3, including 0.15 m M Na2EDTA, 0.2 m l nitroblue t et razolium chloride (3 m g per 10 m l buffer), 0.1 ml xant hine oxidase solut ion, and 0.1 m l xant hine solution (23 m g xant hine, 0.3 m l 1 N NaOH). The reaction w as st art ed by adding an aliquot e of t he xant hine oxidase solut ion. Aft er incubation for 1-2 min at 25° C t he act ivit y w as follow ed for 5 m in at 560 nm. One unit of SOD is defined as t he am ount of enzyme w hich inhibit s t he form azan form ation t o 50 %. Assay of Glutathione-S-transferase (GST) Activity Glut athione-S-t ransferase (GST) (EC2.5.1.18) cat alyzes t he conjugation react ion w it h glut athione in t he first st ep of m ercapt uric acid synt hesis. The act ivit y of GST w as 19 m easured according t o t he m et hod of Habig . The react ion mixt ure cont ained 0.05 m l of 1-chloro-2,4dinitro benzene (20 mM ), 0.84m l phosphat e buffer (0.1M , pH 6.5), 0.01 pancreas supernat ant and 0.1 m l of GSH (0.3 m g GSH/ ml in 0.1 M phosphat e buffer, pH 7.4) change in color w as m onit ored by recording absorbance (340 nm ) at 30 s int ervals for 5 min. The enzym e act ivit y w as expressed in µmole conjugate/min/mg protein. RESULTS Effect of ACE on changes of body weight, pancreas weight, and blood glucose levels in diabetic rats. Table 1 present s t he effect of aqueous extract of C.t inct oria on changes on body weight , pancreas w eight and blood glucose levels in diabetic rat s. There w as no significant int ra-group variation in t he basal body w eight of t he rat s. How ever, t he body w eight of the anim als in t he NC group increased significantly from 211±4.058 g t o 231±5.19 g, w hile t he w eight of t he diabet ic rat s decreased rem arkably from 215±4.34 g t o 185±10.04 g. Oral adm inist rat ion of ACE at t hat dose of 200mg/ kg bw significantly increased t he body w eight , com pared t o unt reat ed diabetic rat s, t hough t here is no st at ist ical significance. There were no significant changes in t he panaceas w eight of t he test group com pared t o t he diabet ic cont rol. As illust rated in Table 1, t he changes in fasting blood glucose levels of different experim ent al groups during t he experiment al period. There w as a significant elevat ion in blood glucose level in STZ-diabetic rat s com pared t o norm al rat s. The adm inist rat ion of ACE ext ract produced m arked antihyperglycem ic effect in diabet ic rat s. The fast ing blood glucose decreased by 59.97% aft er t reatm ent. The difference betw een t he experiment al and diabet ic control rat s in low ering t he fasting blood glucose levels w as st atist ically significant. Table 1: Effect of oral administ ration of ACE on body w eight , Pancreas w eight and blood glucose in st rept ozot ocine-induced diabet ic rat s on 21st day. Parameters studied NC DC DC + ACE (200mg/ kg bw ) Init ial body w eight (g/ day) 211.61 ± 4.05 215.15 ± 4.76 226.94 ± 4.34 Final body w eight (g/ day) 231.71 ± 5.19* 185.8 ± 10.4 231.53 ± 6.67* Pancreas w eight (g/ 100g bw ) 0.40 ± 0.01 0.36 ± 0,06 0.48 ± 0,07 Blood glucose levels (mg/ dl) 101.32 ± 4.04 442.0 ± 1,8 a 176.9 ± 33,6 b Each value is mean ± SEM of eight rat s in each group. * P < 0.01. Init ial vs. Final body w eight a P < 0.01.by comparison w it h normal rat s. b P < 0.01.by comparison w it h st rept ozot ocin diabet ic rat s. Evaluation of Redox Status in Pancreatic Tissue To m easure oxidative st ress m arkers in pancreatic t issue, w e evaluat ed oxidative dam age to lipids, specifically, TBARS. A significant increase in the concentrat ion of International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed. 245 © Copyright pro Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 TBARS w as observed in t he pancreas of diabetic anim als com pared t o controls (Figure 1). Diabet ic anim als t reated w it h 200 mg/ kg of ACE show ed a reduct ion in t he concentrat ion of TBARS com pared t o unt reat ed diabet ic anim als. ISSN 0976 – 044X decreased activities of t hese enzym es w ere observed in STZ-induced diabetic rat s. The oral adm inist rat ion of ACE t o diabet ic rat s show ed a significant increase in t he act ivit ies of SOD, CAT, GsT and GSH-Px and rest ored t hese act ivit ies t o near control levels. Table3: Ant ioxidant enzymes act ivit ies (SOD, CAT, GSH-Px and GsT) in t he pancreas tissue of adult rat s (controls and experiment al groups). Figure 1: Effect of ACE on the level of TBARS in pancreas of experim ent al groups of rat s. Dat a are presented as t he m ean ± SEM (n = 8). N C, cont rol (untreated); DC, diabet ic; DC+ACE, diabetic treat ed w it h 200m g/ kg body m ass. St at ist ically significant differences (p ≤0.01) are bet w een the following groups: (a) NC and DC; (b) DC and DC+EAC. Effect of EAC on Antioxydants the Levels of Non NC DC SOD 3.51 ± 0.15 1.95 ± 0.26 CAT 20,24 ± 1.02 8,15 ± 0.45 GSH-Px 8.08 ± 0.88 3.60 ± 0.43 GST 6.31 ± 0.22 3.39 ± 0.24 DC+ ACE (200mg/ kg bw ) a a a a 3.345 ± 0.49 16,31 ±0.89 6.01 ± 1.64 6.14 ± 0.56 b b b b Act ivity is expressed as: 50 % of inhibit ion of form azan form at ion / m in/ m g of prot ein for SOD; µM of hydrogen peroxide decom posed/ m in/ mg of prot ein for cat alase; nM of glut at hione oxidized/ m in/ m g of protein for GSHPx, U/ m in/ mg of protein for GST. Values are given as m ean ± SEM for groups of eight rat s in each. Values are st at ist ically significant at P<0.01. St at ist ical significance w as com pared wit hin the groups as follow s: a) com parison w ith norm al rat s. b) com parison wit h st rept ozot ocin diabet ic rat s. enzymatic DISCUSSION The change in t he levels of nonenzym atic ant ioxidant s such as vit amin C and GSH in pancreat ic tissue of experiment al and control groups of rat s are represented in Table 2. Diabet ic rat s show ed a significant (P<0.01) decrease in t he levels of vit amin C and GSH com pared t o cont rol rat s. Treatm ent wit h C.t inct oria aqueous ext ract reversed t he level of GSH t o near cont rol levels w hen com pared t o diabetic rat s. How ever a m arked a sm all unsignificat ion increase in t he concent ration of vit am in C is observed. Table 2: Effect of ACE on non enzymat ic antioxidant s (GSH, Vit C) in experim ent al groups of rat s. Parameters studied NC DC GSH (U / g protein) 8.20 ± 0.86 3.72 ± 0.42 Ascorbic acid (mg/ g w et tissue) 51.59 ± 2.95 28.06 ± 4.10 DC+ ACE (200mg/ kg bw ) a 7.31 ± 1.42 a b 37.11 ± 2.70 a Each value is M ean ± SEM of eight rat s in each group. a P < 0.01.by comparison w it h normal rat s. b Parameters studied P < 0.05.by comparison w it h st rept ozot ocin diabet ic rat s. Effects of Cistanche tinctoria extract on STZ-induced changes in the antioxidant enzyme activities Table 3 show s t he changes in t he activities of enzym at ic ant ioxidant s such as superoxide dism ut ase (SOD), Cat alase (CAT), glut at hione perioxidase (GSH-Px), and glut at hione-s-transferase (GST) in pancreatic t issues of cont rol and experim ent al groups of rat s. Significantly Diabetes is a m etabolic defect charact erized wit h developed hyperglycemia after t he insufficiency of insulin release from t he pancreas, increased oxidat ive st ress, non-enzym atic glycolizat ion, lipid peroxidation and changed ant ioxidat ive defence syst em aft er being 20 exposed t o free radicals. The use of phyt ochem icals com pounds on t issues w hich regulat es glucose m et abolism , is an int erest ing area t o explore. In t he current st udy, STZ-induced diabetes w as characterized by a severe loss in body w eight , w hich has 21 also been report ed by ot her investigat ors. The decrease in body w eight s com pared t o norm al rat s could be due t o poor glycem ic cont rol. The excessive cat abolism of prot ein t o provide am ino acids for gluconeogenesis during insulin deficiency result s in m uscle w ast ing and 22 w eight loss in diabetic untreat ed rat s. Oral adm inist ration of ACE partially im proved t he body weight in diabet ic rat s. An increase in t he body w eight of diabet ic rat s m ight be due t o an im provement in insulin secretion 23 and glycem ic cont rol. Likewise, C.t inct ria aqueous ext ract decreased polyphagia and polydipsia in treat eddiabet ic rat s compared wit h diabetic group. Hyperglycemia, as t he m ost predom inant characteristics of diabet es, is very dangerous for diabet ic pat ient s and 24 anim als. In t his st udy, it is observed a significant increase in t he concent ration of blood glucose in st rept ozot ocin-induced diabetic rat s. Oral adm inist ration of ACE might reverse t he blood glucose in diabetic rat s. Ant i-hyperglycemic effect of m edicinal plant ext ract s is International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed. 246 © Copyright pro Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 generally depen dent upon the degree of β-cell 25 dest ruction. We suggest ed also that ACE ext ract m ight reverse t he cat abolic feat ures of insulin deficiency by (i) st im ulat ing peripheral glucose ut ilizat ion, (ii) increasing glucose rem oval from blood or (iii) reducing glucose absorpt ion from t he gast roint estinal t ract . Oxidat ive st ress in diabet es coexist ed w it h a reduct ion in t he ant ioxidant capacit y, w hich could increase t he delet erious effect s of free radicals. The accum ulation of free radical observed in diabetic rat s is at t ribut ed t o chronic hyperglycemia t hat alt ers ant ioxidant defense 7-3 syst em as dem onst rated by previous st udies. Lipid peroxidation of unsat urated fat t y acids is frequently used as an indicat or of increased oxidat ive st ress and 21 subsequent oxidative damage. Lipid peroxidation im pairs cell membrane funct ion by decreasing m em brane fluidit y and causes free radical induced m em brane lipid peroxidation including increased mem brane rigidit y, decreased cellular deform abilit y and alt ers activit y of m em brane-bound enzym es and recept ors, leading t o 26 disease. The high level of t he lipid peroxidation m arker TBARS in diabetic rat s is a reflection of insufficient ant ioxidant defenses in com bating ROS-mediated dam age. The result s show t hat t he pancreas of diabet ic anim als has increased oxidat ive dam age, exem plified by t he increased concentrat ion of TBARS. Several st udies also show ed an increase in t he concentrat ion of TBARS in 27-21 t he pancreatic t issue of diabet ic rat s. In t he present st udy, t he increased form ation of lipid peroxidation in pancreas t issue of diabet ic rat s support ed t hese findings. Oral administrat ion of C.t inct oria aqueous extract t o SZT diabet ic rat s abrogat ed t he increased M DA levels suggest ing t hat ACE might have a high ant ioxidant capacity t o scavenge free radicals generat ed by react ive oxygen species and prevent radical dam age. Glut athione (GSH) is an im port ant intracellular pept ide w it h m ult iple functions ranging from ant ioxidant defense 28 t o m odulat ion of cell proliferation. It is well know n t hat GSH is involved in t he prot ect ion of norm al cell st ruct ure and function by m aint aining t he redox hom eost asis, quenching of free radicals and participat ing in det oxificat ion react ions. It is a direct scavenger of free radicals as w ell as a co-subst rat e for peroxide 29 det oxificat ion by glut at hione peroxidises. Decreased levels of reduced glut at hione are reported in t he 30 pancreas of the STZ-induced diabet ic rat s. This reduction could be explained, according t o previous 31 st udies, by a decrease of GSH synt hesis or an increase of 30 it s degradation induced by STZ oxidat ive st ress. In t he present st udy, t he elevat ion of GSH levels in pancreas w as observed in t he ACE treated diabet ic rat s. This indicat es t hat t he ACE can eit her increase t he biosynt hesis of GSH or reduce t he oxidat ive st ress leading t o less degradation of GSH, or could have bot h effect s. Vit am in C or ascorbic acid is an excellent hydrophilic ant ioxidant in plasm a and disappears fast er t han ot her 32 ant ioxidant s on exposure t o react ive oxygen species. ISSN 0976 – 044X Hypoinsulinem ia and/ or hyperglycem ia inhibit ascorbic acid and cellular t ransport . As t he chemical st ruct ure of ascorbic acid is sim ilar t o t hat of glucose, it shares t he m em brane t ransport syst em w ith glucose and hence 30 com pet es wit h it for it s transport. The decreased level of ascorbic acid in diabet ic rat s m ay be due t o eit her increased ut ilizat ion as an ant ioxidant defense against increased reactive oxygen species or t o a decrease in glut at hione level, since glut at hione is required for t he 25 recycling of ascorbic acid. The diabet ogenic act ion of st rept ozot ocin is associated w it h t he generat ion of reactive oxygen species, w hich causes oxidative dam age. This dam age m ight play an im port ant role in t he progression and development of 33-34 diabet es and it s com plicat ions. This can be prevented by t he scavenging activity of enzym atic ant ioxidant s such as superoxide dism ut ase, cat alase, and glut at hione peroxidase. M oreover, t he deleterious react ive oxygen electrophiles are neut ralized by t he act ion of nonenzym atic ant ioxidant , reduced glut at hione, w hich is form ed by t he act ivities of glut athione reduct ase and 35 glut at hione-S-transferase, respectively. Superoxide dism ut ase, an im port ant int racellular ant ioxidative enzym e, plays a pivot al role in oxygen defense met abolism by intercept ing and reducing 20-26 superoxide t o hydrogen peroxide. Several st udies have reported a reduced activit y of SOD in experim ent al 3-35-38 diabet es. This diminished act ivit y of SOD is t he result of t he over accum ulation of superoxide anion in t he 39 40 cellular organelles, inactivat ion by hydrogen peroxide 41 or by glycation of t he enzym e. Oral t reatm ent of ACE caused a significant increase in SOD activit ies of t he diabet ic rat s. This m eans t hat t he ACE extract can reduce reactive oxygen free radicals and im prove t he activit ies of t he t issue antioxidant enzym es. Cat alase, Anot her im port ant free radical scavenger enzym e t hat breaks dow n hydrogen peroxide, w hich w as produced by SOD, int o w at er and reactive oxygen 20 species. The decrease in Cat alase act ivit y in diabet es could result from uncontrolled product ion of hydrogen peroxide due t o t he aut o-oxidation of glucose, prot ein 35 glycation and lipid oxidation. The deficiency of t his enzym e in bet a cells causes increased oxidative stress and 42 dam aged bet a cells. In t he present st udy, t he reduction in CAT level in diabet ic condit ion w as observed. Our result 3-35-37-38 is in agreem ent w ith t he ot her result s, w ho reported a decrease in CAT level in diabet es m ellit us. In our st udy, it w as observed t hat t he oral adm inist ration of ACE caused a significant increase in t he act ivit y of CAT of t he diabet ic rat s and it m ay be due t o t he ant ioxidant act ivit y of extract. In experim ent al diabet es, it is observed reduced activities of t he GSH-m et abolizing enzymes, GSH-Px and GST. Glut athione peroxidise w as a selenium-cont aining t et ram eric glycoprotein involved in t he det oxificat ion of 3 hydrogen peroxide int o w at er and m olecular oxygen, in t he presence of reduced glut at hione (GSH) form ing International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright prot ect ed. Unaut horised republicat ion, reproduct ion, dist ribut ion, dissem inat ion and copying of t his docum ent in whole or in part is st rict ly prohibit ed. 247 © Copyright pro Int. J. Pharm. Sci. Rev. Res., 32(2), M ay – June 2015; Article No. 40, Pages: 243-249 oxidized glut at hione (GSSG), and t hus, it prot ect s cell 37 prot eins and cell mem branes against oxidat ive st ress. Glut athione peroxidases represent t he m ajor enzym at ic defense against oxidat ive st ress caused by hydroperoxides. They reduce hydrogen peroxide and organic hydroperoxides, such as fat t y acid 43 hydroperoxides, t o t he corresponding alcohols. GST show s broad subst rat e specificit y and det oxify a variet y of electrophiles by conjugat ion t o reduced GSH. GST also plays an im port ant alt hough indirect role in ant ioxidant defense, by eliminat ing t oxic subst ances and prevent ing 44 t heir subsequent delet erious effect. The decrease of GSH-Px and GST m ay also be due t o t he decreased availabilit y of it s subst rat e, GSH, w hich has been show n t o 37 be depleted during diabet es. In t he present st udy, t reatm ent of diabet ic rat s w it h aqueous ext ract of C.t inct oria induced an increase in GSH-Px and GST activit y in pancreatic t issues t o levels higher t han t hose in cont rol anim als by 66.9% and 81% respectively. M oreover, t he result s of t he present invest igat ion are consist ent wit h 3 35 t he result s of sefi , Sivakum ar and Subram anian, and El45 M issiry and El Gindy, reporting t he correlat ion bet w een t he increased lipid peroxides and decreased enzym at ic ant ioxidant s in experim ental diabet es. The enhanced act ivit y of antioxidant enzymes, such as superoxide dism utase, cat alase, glut athione peroxidase and glut at hione-s-transferase, can be very effect ive in scavenging t he various t ypes of oxygen free radicals and their products. Pancreatic β-cells m ay be prot ect ed from oxidat ive dam age (induced by STZ) according t o this reciprocal m echanism . Suggest ing t hat t he ACE has a direct or indirect preventive and prot ect ive effect in diabet es by decreasing oxidative st ress and by preserving t he integrit y of pancreatic β-cell. These m echanism s m ay help explain w hy ACE has a prot ect ive effect in STZinduced diabetic rat s. ISSN 0976 – 044X induced oxidative stress and β-cell damage in rat pancreas, Journal of Ethnopharmacology, 135, 2011, 243–250. 4. Papas A M , Determinant s of ant ioxidant st at us in humans, Lipids, 31, Suppl, 1996, 77-82. 5. Akpan H B, Adefule A K, Fakoya F A, Caxt on-M artins EA, Evaluat ion of LDH and G6-PDH activit ies in audit ory relay centers of st reptozotocin-induced diabetic wist ar rat s, Journal of Analyt ical Sciences, 1, 2007, 21–25. 6. Kim S H, Hyun S H, Choung S Y, Ant i-diabet ic effect of cinnamon ext ract on blood glucose in db/ db mice, Journal of Ethnopharmacology, 104, 2006, 119–123. 7. 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