Antiglycation and Hypolipidemic Effects of Polyphenols from Zingiber officinale Roscoe (Zingiberaceae) in Streptozotocin-Induced Diabetic Rats
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1 Tropicl Journl of Phrmceuticl Reserch Jnury 2015; 14 (1): ISSN: (print); (electronic) Phrmcotherpy Group, Fculty of Phrmcy, University of Benin, Benin City, Nigeri. All rights reserved. Originl Reserch Article Avilble online t Antiglyction nd Hypolipidemic Effects of Polyphenols from Zingiber officinle Roscoe (Zingibercee) in Streptozotocin-Induced Dibetic Rts Mutiu I Kzeem 1,2, Musbu A Aknji 2, Mus T Ykubu 2 nd Anofi OT Ashf 1 * 1 Phytomedicine nd Phytophrmcology Reserch Group, Deprtment of Plnt Sciences, University of the Free Stte, Qwqw Cmpus, Phuthditjhb 9866, South Afric, 2 Deprtment of Biochemistry, University of Ilorin, PMB 1515, Ilorin, Nigeri *For correspondence: Emil: shfot@qw.ufs.c.z; Tel: ; Fx: Received: 26 August 2014 Revised ccepted: 6 December 2014 Abstrct Purpose: To evlute the ntiglyction nd hypolipidemic potentil of polyphenols from Zingiber officinle in streptozotocin-induced dibetic rts. Methods: Dibetes ws induced in mle Wistr rts by single intrperitonel injection of 50 mg/kg body weight (bw) of streptozotocin. This ws followed by orl dministrtion of 500 mg/kg ech of free nd bound polyphenol extrcts of Z. officinle to the rts dily for 42 dys. Distilled wter nd glibenclmide (5 mg/kg) were used s norml nd positive controls, respectively. Results: Significnt increses (p < 0.05) in blood glucose level ( mg/dl), serum dvnced glyction end-products (AGEs) (6.80 µg/ml), lipid profile nd therogenic indices, with decrese in high density lipoprotein cholesterol (HDL-C) (15.55 mg/dl) were observed in dibetic rts compred to control. Free polyphenol extrcts of Z. officinle significntly reduced (p < 0.05) blood glucose ( mg/dl), serum AGEs (1.98 µg/ml), lipid profile nd therogenic indices while it significntly incresed HDL-C (23.28 mg/dl). However, bound polyphenol extrct did not cuse ny significnt chnge in the lipid profile of the dibetic rts except for LDL-C. Conclusion: This study indictes tht free nd bound polyphenols from Z. officinle cn meliorte dibetes s well s its complictions, nd its effect is comprble to tht of the stndrd drug, glibenclmide. Keywords: Zingiber officinle, Dibetes, Lipid profile, Atherogenic index, Polyphenol, Glyction, Streptozotocin Tropicl Journl of Phrmceuticl Reserch is indexed by Science Cittion Index (SciSerch), Scopus, Interntionl Phrmceuticl Abstrct, Chemicl Abstrcts, Embse, Index Copernicus, EBSCO, Africn Index Medicus, JournlSeek, Journl Cittion Reports/Science Edition, Directory of Open Access Journls (DOAJ), Africn Journl Online, Bioline Interntionl, Open-J-Gte nd Phrmcy Abstrcts INTRODUCTION Dibetes mellitus is group of metbolic diseses chrcterized by hyperglycemi nd dyslipidemi which results from defects in both insulin secretion nd/or insulin ction [1]. The disese is ssocited with reduced qulity of life nd incresed risk fctors for mortlity nd morbidity. Long-term hyperglycemi is n importnt fctor in the development nd progression of micro- nd mcro-vsculr complictions, which include neuropthy, nephropthy, crdiovsculr nd cerebrovsculr diseses [2]. Zingiber officinle Roscoe (fmily: Zingibercee) is commonly known s ginger (English), gingembre (French), Afu (Hus) nd At-ile (Yorub). It is used widely for indigestion, stomch-che, mlri nd fevers [3]. Its Trop J Phrm Res, Jnury 2015; 14(1): 55
2 combintion with lime juice nd rock slt increses ppetite nd stimultes the secretion of gstric juice [4]. The rhizomes of Z. officinle re importnt ingredients of mny trditionl medicines worldwide. The fresh (or dried) rhizomes of Z. officinle re lso used s tonics to tret indigestion, dyspepsi, fltulence nd nuse It is lso used to tret vriety of humn ilments such s rheumtism, sthm, stroke, constiption nd dibetes [5]. Polyphenolic compounds re ubiquitous in ll plnt nd re, therefore, n integrl prt of the humn diet [6]. Until recently, most of the nutritionl interest in polyphenolic compounds ws in the deleterious effects cused by the bility of certin polyphenols to bind nd precipitte mcromolecules, such s dietry protein, crbohydrte, nd digestive enzymes, thereby reducing food digestibility. Recent interest, however, in phenolics hs incresed gretly becuse of the ntioxidnt nd free rdicl-scvenging bilities ssocited with some phenolics nd their potentil effects on humn helth [7]. Though, severl studies hd been conducted on the possible ntidibetic potentil of Z. officinle [8,9], none hs been done on the ntiglyction potentil of its polyphenols in dibetic rts. It is bsed on this premise tht the ntiglyction nd hypolipidemic potentil of polyphenols from Z. officinle ws performed in streptozotocininduced dibetic rts. EXPERIMENTAL Plnt mteril Zingiber officinle ws purchsed from the Centrl Spices Mrket in Mile 12 re, Ketu, Lgos, Nigeri in My Identifiction nd uthentiction of the smple ws done by Dr. A. B. Kdiri of the Deprtment of Botny, University of Lgos, Akok, Lgos nd voucher specimen (LUH 4730) ws deposited in the university s herbrium. Chemicls Streptozotocin ws product of Alexis Biochemicl, Sn Diego CA 92101, USA. The ssy kits for glucose nd lipid profile were products of Rndox Lbortories, Antrim BT41, United Kingdom while dvnced glyction endproducts (AGEs) ELISA kit ws obtined from Cusbio Biotech. Co. Ltd, Hubei Province , Chin Animls Albino rts were obtined from the Animl House of the Deprtment of Biochemistry, Lgos Stte University, Ojo, Lgos. All the nimls were mintined under lbortory conditions of temperture (22 ± 2 o C), humidity (45 ± 5 %) nd 12 h dy: 12 h night cycle under ir-conditioner. They were lso llowed ccess to food (stndrd pellet diet) nd wter d libitum. All procedures were performed in complince with the Guide for the Cre nd Use of Lbortory Animls [10], s pproved by the Reserch ethicl committee of Lgos Stte University (ref no. RC/BCH/0720). Preprtion of free polyphenol A known mss (3 kg) of the Zingiber officinle ws crushed in 80 % cetone (1 : 2 w/v) using Wring blender (Wring Commericl, Torrington, CT) for 5 min [11]. The smple ws homogenized using Polytron homogenizer (Glen Mills Inc., Clifton, NJ) for 3 min. The homogentes were filtered under vcuum using Buchner funnel nd Whtmn no. 2 filter pper. The filtrte ws concentrted using rotry evportor under vcuum nd lter freeze-dried in lyophilizer (Ilshin Lb. Co. Ltd, Seoul, Kore). The extrct ( g) ws stored frozen t - 20 o C until the commencement of the experiment. Preprtion of bound polyphenol Residue from the free polyphenol extrction ws drined nd hydrolyzed with 2 L of 4 M NOH for 1 h with constnt shking [11]. The mixture ws cidified with concentrted hydrochloric cid to ph 2, extrcted six times with ethylcette, pooled nd concentrted using rotry evportor nd subsequently freeze-dried. The extrct (97.35 g) ws stored frozen t - 20 o C until the commencement of the experiment. Induction of experimentl dibetes Dibetes mellitus (Type 2) ws induced by single intrperitonel injection of freshly prepred STZ (50 mg/kg bw) in 0.1 M citrte buffer (ph 4.5). Dibetes ws confirmed in these STZ-treted rts over period of 7 dys. In ll experiments, rts were fsted for 18 h prior to STZ injection. After 7 dys, the blood ws collected from the til nd the blood glucose level of ech rt determined. Rts with fsting blood glucose rnge of mg/dl were considered dibetic nd included in the study. Trop J Phrm Res, Jnury 2015; 14(1): 56
3 Experimentl design A totl of 40 rts (8 norml; 32 STZ-dibetic rts) were used for this experiment. The rts were rndomised into five groups of eight nimls ech. Group 1 consisted of rts dministered with vehicle lone (distilled wter) while group 2 comprises STZ-induced dibetic rts only; Groups 3 nd 4 consisted of STZ-induced dibetic rts dministered 500 mg/kg bw free nd bound polyphenol extrcts of Z. officinle respectively. Group 5 comprises STZ-induced dibetic rts dministered glibenclmide (5 mg/kg bw). The extrct ws suspended in the vehicle (distilled wter) nd ws dministered everydy orlly using n orogstric tube. Following 42 dys of dministrtion [12], nimls were decpitted, their blood ws collected nd serum seprted immeditely. This ws done by centrifuging the blood smples t 1282 g for 5 min with Uniscope Lbortory Centrifuge (model SM800B, Surgifriend Medicls, Essex, Englnd). Biochemicl mesurements Glucose ws determined by the glucose oxidse method. Glycted hemoglobin (HbA1c) ws determined using Micromt II HbA1c nlyzer (Bio-Rd, Deeside, UK) using Glycosl test crtridge. Serum dvnced glyction end products ws determined using rt dvnced glyction end products (AGEs) ELISA kit (Cusbio Biotech Co., Chin). Totl cholesterol (TC), HDL cholesterol nd tricylglycerol (TG) were mesured using colorimetric methods while LDL-cholesterol ws clculted by Friedwld s formul [13]. Sttisticl nlysis Sttisticl nlysis ws performed using GrphPd Prism 5 sttisticl pckge (GrphPd Softwre, Sn Diego MA, USA). The dt were nlysed by one wy nlysis of vrince (ANOVA) followed by Bonferroni test. All the results were expressed s men ± SEM (n = 8) nd were considered sttisticlly significnt when p < RESULTS Effect on fsting blood glucose Tble 1 shows the result of fsting blood glucose (FBG) level of the nimls during the experiment. The dibetic control nimls hd incresing fsting blood glucose throughout the period of the experiment rising from mg/dl on the 1st dy to mg/dl t the end of the experiment. The dministrtion of free nd bound polyphenol extrcts of Z. officinle to dibetic rts significntly decresed (p < 0.05) FBG compred to the dibetic control rts by the 42nd dy. However, the dministrtion of glibenclmide to the dibetic nimls produced the best result by significntly reducing (p < 0.05) FBG drsticlly by the end of the experiment. Effect on HbA1c The effect of the dministrtion of polyphenol extrcts of Z. officinle on the HbA1c of the streptozotocin induced dibetic rts is shown in Figure 1. The HbA1c level of dibetic control rts (10.33 %) ws significntly higher (p < 0.05) thn the norml control (4.52 %). The dministrtion of free nd bound polyphenols from Zingiber officinle significntly reduced (p < 0.05) HbA1c of the dibetic rts. However, it ws only glibenclmide treted dibetic nimls tht exhibited vlues tht were similr to the control group. Tble 1: Effect of dministrtion of polyphenols from Z. officinle on the fsting blood glucose (mg/dl) of norml nd streptozotocin-dibetic rts Group Glucose (mg/dl) Initil Finl % chnge Norml Control ± ± ± 0.16 Dibetic Control ± 8.46 b ± b ± 3.02 b Dibetic + Free polyphenol ± 6.72 b ± 9.34 c ± 4.63 c Dibetic + Bound polyphenol ± 9.06 b ± 7.15 d ± 1.75 d Dibetic + Glibenclmide ± 8.94 b ± 6.04 c ± 3.78 c Vlues re men ± SEM (n = 8); vlues down the verticl columns crrying different superscripts re significntly different from ech other (p < 0.05) Trop J Phrm Res, Jnury 2015; 14(1): 57
4 12 b Dibetic rts treted with bound polyphenol extrct, GBN: Dibetic rts treted with glibenclmide HbA1c (%) CON DBT c Free c Bound GBN Figure 1: Effect of orl dministrtion of polyphenols from Z. officinle on the HbA1c level in norml nd streptozotocin-induced dibetic rts. Vlues re men ± SEM (n = 8); brs crrying different superscripts re significntly different (p < 0.05) from ech other. CON: Norml Control, DBT: Dibetic Control, Free: Dibetic rts treted with free polyphenol extrct, Bound: Dibetic rts treted with bound polyphenol extrct, GBN: Dibetic rts treted with glibenclmide Effect on AGEs Advnced glyction endproducts (AGEs) were significntly higher (p < 0.05) in the dibetic untreted rts when compred with norml control rts (Figure 2). Administrtion of free nd bound polyphenol extrcts of Z. officinle to dibetic rts reduced the AGEs significntly (p < 0.05) when compred to the dibetic control rts nd their vlues re comprble to the norml control s well s glibenclmide treted dibetic nimls. AGE concentrtion (µg/ml) CON b DBT Free Figure 2: Effect of orl dministrtion of polyphenols from Z. officinle on the formtion of dvnced glyction endproducts (AGEs) in norml nd streptozotocin-induced dibetic rts. Vlues re men ± SEM (n = 8); brs crrying different superscripts re significntly different (p < 0.05) from ech other. CON: Norml Control, DBT: Dibetic Control, Free: Dibetic rts treted with free polyphenol extrct, Bound: Bound GBN Effect on lipid profile Tble 2 depicts the effect of polyphenolic extrcts of Z. officinle on the serum totl cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) nd tricylglycerol (TG) concentrtion of STZ-induced dibetic rts. The serum TG, TC nd LDL-C levels were significntly higher (p < 0.05) in the dibetic control rts compred to the norml control rts while HDL-C ws significntly reduced (p < 0.05). Tretment with free polyphenols from Z. officinle significntly reduced (p < 0.05) the level of TC, LDL-C nd TG but incresed HDL-C nd this result is comprble to the vlues obtined for glibenclmide-treted dibetic rts. Bound polyphenol extrct only significntly reduced (p < 0.05) LDL-C compred to the dibetic control group. Effect on therogenic indices Dibetic control rts displyed the highest level in ll therogenic indices clculted nd re significntly different (p < 0.05) from the norml control group except for log (TG/HDL-C) (Tble 3). The dministrtion of free nd bound polyphenols from Z. officinle to dibetic rts significntly decresed (p < 0.05) these indices nd their vlues re comprble to stndrd drug glibenclmide. DISCUSSION Streptozotocin-induced dibetes hs been described s useful experimentl model to study ntidibetic ctivity of severl gents [14]. The mechnism by which streptozotocin brings bout dibetes includes selective destruction of pncretic β-cells which mke cells less ctive, leding to poor sensitivity of insulin for glucose uptke by tissues [15]. The incresed levels of serum glucose in STZ-induced dibetic rts were lowered by the dministrtion of both free nd bound polyphenols from Z. officinle, though glibenclmide produced better effect. The reduced glucose levels suggests tht Z. officinle might be exerting insulin-like effect on peripherl tissues by promoting glucose uptke, inhibiting heptic gluconeogenesis or by bsorption of glucose into the muscle nd dipose tissues through the stimultion of regenertion process of the remining β-cells [16]. Trop J Phrm Res, Jnury 2015; 14(1): 58
5 Tble 2: Effect of orl dministrtion of polyphenols from Z. officinle on the lipid profile (mg/dl) of norml nd streptozotocin- dibetic rts Group Lipid (mg/dl) TC HDL-C LDL-C TG Norml Control ± ± ± ± 4.63 Dibetic Control ± b ± 2.55 b ± 7.31 b b Dibetic + Free ± ± ± 4.99 c ± 8.00 Dibetic+Bound ± 7.06 b ± 4.41 b ± 3.54 c ± 5.67 b Dibetic + GBN ± ± ± ± 5.95 Vlues re men ± SEM (n = 8); vlues down the verticl columns crrying different superscripts re significntly different (p < 0.05) from ech other; TC: totl cholesterol, HDL-C: high density lipoprotein cholesterol, LDL-C: low density lipoprotein cholesterol, TG: tricylglycerol Tble 3: Effect of dministrtion of polyphenols from Z. officinle on the therogenic indices of norml nd streptozotocin-induced dibetic rts Group Atherogenicity TC/HDL-C LDL-C/HDL-C TG/LDL-C Log(TG/HDLC) Norml Control 2.17 ± ± ± ± 0.08 Dibetic Control ± 2.00 b 7.48 ± 1.78 b ±2.09 b 0.99 ± 0.08 Dibetic + Free 3.19 ± ± ± ± 0.07 Dibetic+Bound 4.85 ± 0.05 c 1.83 ± ± 0.64 c 0.63 ± 0.06 Dibetic + GBN 2.78 ± ± ± ± 0.02 Vlues re men ± SEM (n = 8); vlues down the verticl columns crrying different superscripts re significntly different (p < 0.05) from ech other; TC: totl cholesterol, HDL-C: high density lipoprotein cholesterol, LDL-C: low density lipoprotein cholesterol, TG: tricylglycerol Glycted hemoglobin, often mesured s HbA1c hs been widely used s n index of glycemic control in most prts of the world [17]. HbA1c ws found to increse in ptients with dibetes mellitus due to glycosyltion of hemoglobin nd the mount of increse ws directly proportionl to the fsting blood glucose levels. In uncontrolled or poorly controlled dibetes, there is n incresed glycosyltion of number of proteins including hemoglobin [18]. This is consistent with the result of this study in which the HbA1c ws very high in dibetic control rts. However, dministrtion of free polyphenol extrcts of Z. officinle suppressed this index. This decrese in HbA1c levels in dibetic rts treted with free polyphenol extrcts of Z. officinle could be due to the decresed glucose levels (good glycemic control). Hyperglycemi is known to hve rnge of biologicl effects which include the production nd ccumultion of dvnced glyction endproducts (AGEs). Advnced glyction endproducts (AGEs) hve been implicted in dibetic complictions becuse they lter enzymic ctivity, decrese lignd binding, modify protein hlf-life nd lter immunogenicity [19]. The four-fold increse in the formtion of AGEs in the streptozotocin-induced dibetic control rts ws normlised in dibetic rts treted with both free nd bound polyphenol extrcts of Z. officinle nd re comprble to the norml control rts nd glibenclmide treted dibetic rts. The reduction in the formtion of AGEs in dibetic rts treted with polyphenols from Z. officinle my be due to the ntioxidnt potentil of these extrct nd/or their hypoglycemic potentil. Since hyperglycemi is the min precursor to ll dibetic-relted pthologies, blility of the extrcts to lower the glucose level my lso reduce the formtion of AGE nd subsequently dibetic complictions. Dibetes is ssocited with ltertions in the plsm lipid nd lipoprotein profile, with n incresed risk of coronry hert disese [20]. In this study, elevted levels of serum lipids such s cholesterol nd tricylglycerols were noticed in dibetic rts. Under norml circumstnces, insulin ctivtes lipoprotein lipse nd hydrolyzes tricylglycerols. It lso increses uptke of ftty cids into dipose tissue s well s inhibits lipolysis. In cses of insulin deficiency, lipolysis is not inhibited but rther proceeds t higher rte which finlly leds to hyperlipidemi. In this study, dibetic control rts hd elevtion in the Trop J Phrm Res, Jnury 2015; 14(1): 59
6 level of LDL-C (Tble 2). Insulin deficiency or insulin resistnce my lso be responsible for this becuse insulin hs n inhibitory ction on β- hydroxyl- β methyl glutryl CoA reductse (HMG-CoA reductse), key rte-limiting enzyme responsible for the metbolism of cholesterol-rich LDL prticles. Acute insulin deficiency initilly cuses n increse in free ftty cid mobiliztion from dipose tissue [21]. This results in incresed production of cholesterol-rich LDL prticles [22]. However, dministrtion of both free nd bound polyphenol extrcts of Z. officinle countercted this effect in dibetic rts nd is comprble to glibenclmide (Tble 2). HDL-C is n nti-therogenic lipoprotein which trnsports cholesterol from peripherl tissues into the liver nd thereby cts s protective fctor ginst coronry hert disese [21]. The concentrtion of HDL-C, which incresed fter Z. officinle free polyphenol extrcts dministrtion, might be due to the increse in the ctivity of lecithin cholesterol cyl trnsferse (LCAT), which my contribute to the regultion of blood lipids [23]. In this study, dministrtion of free polyphenol extrcts of Z. officinle lowers serum lipids, decreses LDL-C nd lso increses the serum HDL-C level in dibetic rts. The ntilipidemic effect of Z. officinle my be due to decresed cholesterogenesis nd ftty cid synthesis. Significnt lowering of totl cholesterol, tricylglycerols, LDL-cholesterol nd rise in HDL-cholesterol is very desirble biochemicl stte for prevention of therosclerosis nd ischemic conditions [23].. This ws chieved by dministrtion of free polyphenol extrct of Z. officinle to the dibetic rts. In individuls with dibetes, crdiovsculr risk is incresed by cluster of risk fctors such s bdominl obesity, impired fsting glucose, incresed blood pressure, low HDL-C, increse in both TGs nd LDL-C prticles [1]. Although, insulin resistnce is crucil to the pthogenesis of this disese, the ssocited therogenic lipoprotein phenotype considerbly enhnces the risk. Hence there ws the need to evlute therogenic indices from the lipid profile obtined during the experiment. Severl lipoproteinrelted indices hve been postulted to evlute the risk of crdiovsculr diseses in dibetes. These include TC/HDL-C, LDL-C/HDL-C, TG/HDL-C nd log (TG/HDL-C) molr rtios nd re djudged to be good predictive vlue for future crdiovsculr events [24]. In this study, dibetic control nimls hd the highest vlues in ll the therogenic indices evluted (Tble 3). However, tretment with both free nd bound polyphenol extrcts of Z. officinle reduces the therogenic indices of these rts nd the vlues were comprble to tht of glibenclmide. The high vlues of therogenic indices experienced by dibetic rts my be due to the decresed HDL-C vlues nd hypertricylglycerolemi which re ll due to derngement in lipid metbolism [25]. The difference in the ctivities of both free nd bound polyphenol extrcts of Z. officinle my not be unconnected to the difference in their structures nd compositions. Free polyphenols occur s phenolic cids nd flvonoids. They re freely vilble nd more redily bsorbed, nd thus, exert beneficil bioctivities in erly digestion [11]. Bound polyphenolic compounds, on the other hnd, re present s component of plnt cell wlls. They re present s monomeric, dimeric, or oligomeric compounds, which re esterified to the cell wll. Bound phytochemicls, minly s β-glycosides, could not be digested by humn enzymes, nd could survive stomch nd smll intestine digestion to rech the colon nd be digested by bcteri flor relesing phytochemicls with helth benefits [11]. CONCLUSION The results of the present study clerly indicte tht free polyphenols from Z. officinle rhizome possess the ntiglyction nd hypolipidemic ctive principle(s) which probbly cts by improving the ltertions in the crbohydrte nd lipid metbolism of the nimls used. ACKNOWLEDGEMENT The uthors wish to cknowledge the Reserch Committee, University of the Free Stte, Qwqw Cmpus for funding of this project. REFERENCES 1. Interntionl Dibetes Federtion. Dibetes Atls, 4th edn, Brussels, Belgium, Altn VM. The phrmcology of dibetic complictions. Curr Medicinl Chem. 2003; 10: Wng WH, Wng ZM. Studies of commonly used trditionl medicine - ginger. Zhongguo Zhong Yo Z Zhi 2005; 30: Lngner E, Greifenberg S, Gruenwld J. Ginger: History nd use. Adv Therpy 1998; 15: Bdreldin HA, Blunden G, Tnir MO, Nemmr A. Some phytochemicl, phrmcologicl nd toxicologicl properties of ginger (Zingiber officinle Roscoe): A review of recent reserch. Food Chem Toxicol. 2008; 46: Trop J Phrm Res, Jnury 2015; 14(1): 60
7 6. Chung SY, Shengmin S, Joshu DL, Mo-Jung L. Biovilbility issues in studying the helth effects of plnt polyphenolic compounds. Moleculr Nutr Food Res. 2008; 8 (52): S139 S Weichselbum E nd Buttriss JL. Polyphenols in the diet, Nutr Bull. 2010; 35: Shlby MA, Hmowieh AR. Sfety nd efficcy of Zingiber officinle roots on fertility of mle dibetic rts. Food Chem Toxicol. 2010; 48: Shnmugm KR, Mllikrjun K, Nishnth K, Kuo CH, Reddy KS. Protective effect of dietry ginger on ntioxidnt enzymes nd oxidtive dmge in experimentl dibetic rt tissues. Food Chem. 2011; 124: Ntionl Acdemy of Sciences, Guide for the cre nd use of lbortory nimls, 8th Edition, Ntionl Acdemic Press, Wshington, USA 11. Chu Y, Sun J, Wu X, Liu RH. Antioxidnt nd ntiprolifertive ctivity of common vegetbles. J Agric Food Chem. 2002; 50: Murli B, Updhyy UM, Goyl RK. Effect of chronic tretment with Enicostemm littorle in non - insulindependent dibetic (NIDDM) rts. J Ethnophrmcol. 2002; 81: Friedwld WT, Levy RI, Fredrickson DS. Estimtion of the concentrtion of LDL-cholesterol in plsm without the use of the preprtive ultrcentrifuge. Clin Chem. 1972; 18: Sezik E, Asl M, Yesild E, Ito S. Hypoglycemic ctivity of Gentin olivieri nd isoltion of the ctive constituent through biossy-directed frctiontion techniques. Life Sci. 2005; 76: Lenzen S. Oxidtive stress: the vulnerble bet-cell. Biochem Soc Trns. 2008; 36: Rokey B, Nhr N, Ali L, Hssn Z, Alm MNE, Choudhury SN, Azd AK, Rosenstock J, Rskin P. Antioxidnt effect of Aloe ver gel extrct in streptozotocin-induced dibetes in rts. Crdiol. 2005; 6: World Helth Orgnistion. Definition nd dignosis of dibetes mellitus nd intermedite hyperglycemi: report of WHO/IDF consulttion. Genev, Switzerlnd, Pri L, Srvnn G. Antidibetic effect of Cogent db, herbl drug in lloxn-induced dibetes mellitus. Comp Biochem. Physiol. C: Phrmcol. Toxicol Endocrinol. 2002; 131: Vlssr H, Plce MR. Dibetes nd dvnced glyction endproducts. J Internl Med. 2002; 251: Mghrni M, Lemhdri A, Zeggwgh NA, El Amroui M, Hloui M, Joud H, Eddouks M. Effects of n queous extrct of Triticum repens on lipid metbolism in norml nd recent-onset dibetic rts. J Ethnophrmcol. 2004; 90: Shirwikr A, Rjendrn K, Kumr CD, Bodl R. Antidibetic ctivities of queous lef extrct of Annon squmos in streptozotocin-nicotinmide type 2 dibetic rts. J Ethnophrmcol. 2004; 91: Murli B, Updhyy UM, Goyl RK Effect of chronic tretment with Enicostemm litorle in noninsulin dependent dibetic rts. J Ethnophrmcol. 2002; 81: Ptil UK, Srf S, Dixit VK. Hypolipidemic ctivity of seeds of Cssi tor Linn. J Ethnophrmcol. 2004; 90: Rder DJ. High-density lipoprotein nd therosclerosis. Amer J Crdiol. 2002; 92: 62i 72i. 25. Lehto S, Ronnem T, Pyorl K, Lkso M. Crdiovsculr risk fctors clustering with endogenous hyperinsulinemi predict deth from coronry hert disese in ptients with type II dibetes. Dibetologi 2000; 43: Trop J Phrm Res, Jnury 2015; 14(1): 61
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