Comparative Biochemistry and Physiology Part C 135(2003) 357–364

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Antidiabetic effects of quercetin in streptozocin-induced diabetic rats

Mahmood Vessal *, Mina Hemmati , Mohammad Vaseia, a b

Department of Biochemistry, Shiraz University of Medical Sciences, P.O. Box 1167, Shiraz 71345, Irana

Department of Pathology, Shiraz University of Medical Sciences, Shiraz 71345, Iranb

Received 7 February 2003; received in revised form 12 June 2003; accepted 18 June 2003

Abstract

Effects of the intraperitoneal injection of quercetin in streptozocin-induced diabetic and normal rats were investigatedand compared. Although quercetin had no effect on plasma glucose level of normal animals, it significantly and dose-dependently decreased the plasma glucose level of streptozocin-induced diabetic rats. Glucose tolerance tests of thediabetic animals approached those of normal rats, their plasma cholesterol and triglycerides were reduced significantly,while their hepatic glucokinase activity was significantly increased upon quercetin treatment. In normal rats, quercetindid not affect the glucose tolerance test, but resulted in an increase of plasma cholesterol and triglycerides and a decreasein hepatic glucokinase activity. No significant pathologic changes were noted in hepatocytes or kidney tubules andglomeruli, while the number of pancreatic islets significantly increased in both treated normal and diabetic groups. It isconcluded that quercetin, a flavonoid with antioxidant properties brings about the regeneration of the pancreatic isletsand probably increases insulin release in streptozocin-induced diabetic rats; thus exerting its beneficial antidiabeticeffects. However, it may be of little value in normoglycemic animals.� 2003 Elsevier Inc. All rights reserved.

Keywords: Cholesterol; Diabetes; Glucose tolerance; Hepatic glucokinase; Pancreatic islets; Quercetin; Streptozocin; Triglycerides

1. Introduction

The most common substances inducing diabetesin the rat are alloxan and streptozocin(streptozo-tocin, STZ). STZ is taken up by pancreaticb-cellsvia glucose transporter GLUT2. The main causeof STZ-inducedb-cell death is alkylation of DNAby the nitrosourea moiety of this compound. How-ever, production of NO and reactive oxygeno

species may also be involved in DNA fragmenta-tion and other deleterious effects of STZ. Thetoxic action of alloxan on pancreaticb-cellsinvolves several processes such as oxidation ofessential –SH groups, inhibition ofb-cell gluco-

*Corresponding author. Tel.:q98-711-230-3029; fax:q98-711-230-3029.

E-mail address: mahmoodv@yahoo.com(M. Vessal).

kinase, generation of free radicals and disturbancesin intracellular calcium homeostasis. Calcium doesnot play a significant role in necrosis ofb-cellsby STZ since calcium channel antagonists do notprotect b-cells against this drug(Szkudelski,2001). Interestingly, STZ is not equally effectivein all vertebrates and mechanisms of action maydiffer between groups(Wright et al., 1999).

b-Cells are affected by many immunologicaland chemical agents leading to local inflammationsproducing IL-6 and glucocorticoids. IL-6ygluco-corticoid stimulation produces an active transcrip-tional complex for Reg, ab-cell regeneratingfactor gene, in which poly(ADP-ribose) synthe-taseypolymerase(PARP) is involved. In the pres-ence of PARP inhibitors such as nicotinamidewhen PARP is not itself poly(ADP-ribosyl)-ated,

358 M. Vessal et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 357–364

the transcriptional complex is stabilized and Reggene transcription and subsequent Reg proteinformation occurs inb-cells. This protein acts as agrowth factor onb-cells via Reg receptor. DNAreplication inb-cells takes place andb-cell regen-eration is accomplished. DNA damaging sub-stances such as superoxide(O 2) and nitric oxideo

(NO ) are produced in inflammatory processes byo

cytotoxic agents such as STZ. When DNA isdamaged, PARP senses the nicks and autopoly(ADP-ribosyl)-ates itself for DNA repair. Auto-poly (ADP-ribosylation) of PARP inhibits theformation of Reg gene transcriptional complex andtranscription of this gene stops(Akiyama et al.,2001).Chemicals with antioxidant properties and free

radical scavengers in particular prevent autopoly(ADP-ribosyl)-ation of PARP and by stabilizingReg gene transcriptional complex, result in theregeneration ofb-cells and protect pancreatic isletsagainst cytotoxic effects of STZ or alloxan(Szku-delski, 2001).Recently, there has been renewed interest in the

use of plant compounds as antidiabetic compounds(Pari and Saravanan, 2002). Flavonoids are natu-rally occurring phenolic compounds that are wide-ly distributed in plants. Due to the presence ofaromatic hydroxyl groups, flavonoids have strongantioxidant properties. They are scavengers ofreactive oxygen and nitrogen species and, there-fore, inhibit peroxidation reactions. They also pro-tect macrophages from oxidative stress by keepingglutathione in its reduced form(du Thie andCrozier, 2000; Fuhrman and Aviram, 2001).Flavonoids have the capacity to inhibit enzymes

such as cyclooxygenases and protein kinasesinvolved in cell proliferation and apoptosis(For-mica and Regelson, 1995). It was reported that aflavonoid, (y)-epicatechin, protects normal ratislets from alloxan, normalizes blood glucose lev-els and promotesb-cell regeneration in islets ofalloxan—treated rats(Chakravarthy et al., 1981,1982a,b). Tritiated thymidine incorporation intoislet cell DNA was also enhanced by this flavonoidin an in vitro study (Hii and Howell, 1984).However, the beneficial effects of(y)-epicatechinin STZ—diabetic animals could not be demonstrat-ed (Bone et al., 1985). A different flavonoid,quercetin, used in doses of 10–50 mgykg bodymass was capable of normalizing blood glucoselevel, augmenting liver glycogen content and sig-nificantly reducing serum cholesterol and LDL

concentration in alloxan–diabetic rats(Nuralievand Avezov, 1992).Hii and Howell (1985) showed that exposure of

isolated rat islets to certain flavonoids such as(y)-epicatechin or quercetin enhances insulinrelease by 44–70%. They argue that such flavo-noids may act on islet function, at least in part,via alteration in Ca fluxes and in cyclic nucle-2q

otide metabolism.Due to differences in the mechanism of action

of streptozocin and alloxan and the presence ofcontroversial reports on the antidiabetic effects ofdifferent flavonoids in alloxan-treated and STZ-induced diabetes, we decided to re-evaluate theeffects of two different doses of quercetin in STZ-induced diabetic rats and compare the results withthe effects of this flavonoid on normoglycemicanimals.This study compares the action of quercetin on

blood glucose concentration, glucose tolerance test,pancreatic islet regeneration, activity of an insulin-induced enzyme(hepatic glucokinase), and thelevels of plasma cholesterol and triglycerides(TG)in normoglycemic and diabetic rats.

2. Materials and methods

2.1. Reagents

Glucose and fatty acid free bovine serum albu-min were purchased from Roche chemical com-pany (Germany). Leuconostoc mesenteroidesglucose 6-phosphate dehydrogenase(G6PD),Na .ATP, quercetin, HEPES buffer and dithiothrei-2

tol were obtained from Sigma Chemical Company(St. Louis, MO, USA). Na .NAD was from Fluka2

chemical company(Switzerland), and streptozocinvials containing 1 g streptozocin and 220 mg citricacid was obtained from Upjohn Co.(Kalamazoo,MI, USA). Streptozocin was reconstituted with 9.5ml of 0.9% NaCl solution to pH 3.5–4.5 accordingto manufacturer’s instructions. Furthermore, dilu-tion of the drug was performed in 0.9% NaClsolution immediately before use. The enzymatickits for the determination of glucose, cholesteroland triacylglycerol(TG) were purchased from ParsAzemoon Co.(Tehran, Iran).

2.2. Animal experiments

Adult male Sprague–Dawley derived rats bredand raised at the University animal quarters with

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a mass ranging from 200 to 220 g were used. Fiveanimals each were housed in a cage and fed a ratchow diet(Pars Dam Co, Tehran, Iran) and givenwater ad libitum. Diabetes was induced uponinjection of streptozocin(40 mgykg body mass)through the caudal vein two weeks prior to theinitiation of treatments. Blood was obtained fromthe tail vein using heparinized microhematocrittubes. Plasma glucose of all groups was measuredweekly. After 2 weeks, animals with plasma glu-cose level exceeding 16.6 mM were considered asdiabetic. Normoglycemic and streptozocin diabeticrats were employed for the tests described below.

2.3. Effect of quercetin on plasma glucose level

The required amount of quercetin was dissolvedin 0.5 ml of 60% ethanol prior to injection to rats.Five groups of rats including two normoglycemicand three diabetic groups(five rats per group)were used: (1) normoglycemic control groupreceiving the vehicle as one intraperitoneal(i.p.)injection of 0.5 ml 60% ethanol per day;(2)normoglycemic rats receiving one i.p. injection of10 mg quercetin per kg body mass per day;(3)diabetic control rats receiving one i.p. injection of0.5 ml 60% ethanol per day;(4) diabetic experi-mental animals receiving one i.p. injection of 10mg quercetin per kg body mass per day; and(5)diabetic experimental animals receiving one i.p.injection of 15 mg quercetin per kg body mass perday. Such treatments were continued for 10 daysat 13:00 h every day. Blood was collected fromthe tails using heparinized microhematocrit tubesand used for plasma glucose determination priorto quercetin treatment(day 0) and every other daythereafter.

2.4. Glucose tolerance test

Glucose tolerance test was performed accordingto the procedures of Young et al.(1995) on thefive different groups mentioned above. Followingtreatment for 10 days, plasma glucose was deter-mined, the animals were treated with anotherrespective dose of quercetin and deprived of foodfor 24 h. After this period, blood was collectedthrough heparinized tubes for plasma glucose(0time glucose level), cholesterol and triglyceridedeterminations. The fasting plasma glucose levelsof the diabetic control rats, the diabetic experimen-tal group receiving 10 mg quercetin per kg and

the diabetic animals receiving 15 mg quercetin perkg were 26.8"1.2, 5.9"0.17 and 5.8"0.44 mM,respectively. The fasting plasma glucose levels ofthe normoglycemic control and the normoglycemicexperimental groups were in the range of 4.4–5.0mM. Fasted animals were fed 1 ml of a glucosesolution containing 3.5-mmol glucose(3 gykgbody mass) through a gavage. Tail blood werecollected at 45, 90 and 135 min after glucosefeeding and plasma glucose levels were determinedand used for establishing glucose tolerance curves.

2.5. Hepatic glucokinase and hexokinase assay

Five groups of five rats each consisting of twonormoglycemic and three diabetic groups wereinjected i.p. with various levels of quercetin asdescribed above except that injections were doneinto normoglycemic animals, diabetic animalsreceiving 10 mg quercetin per kg body mass, anddiabetic animals receiving 15 mg quercetin per kgbody mass were continued for 8, 10 and 7 days,respectively. Plasma glucose levels were monitoredduring the treatments. At the times speficied above,when the plasma glucose level of the quercetintreated diabetic animals in the fed state reachedapproximately 7.0 mM, the animals were killed bydecapitation and blood collected for serum alanineaminotransferase (ALT) assay. Livers wereremoved; a portion was placed in buffered 10%formalin and the rest was frozen aty70 8C andlater employed for hexokinase and glucokinaseassays. The pancreas and kidneys from these ratswere placed in buffered 10% formalin and usedfor histological studies.Frozen liver tissue(1 g) from each rat, was cut

into small pieces and homogenized at 48C in 9ml of a cold buffer solution(pH 7.4) containingNa-HEPES, 50 mM; KC1, 100 mM; EDTA, 1mM; MgCl , 5 mM and dithiothreitol, 2.5 mM2

using a glass–Teflon Potter homogeniser with 20up and down motions at half maximum speed. Thesuspension formed was centrifuged at 12 000=gfor 1 h at 48C. The clear supernatant solution wasimmediately employed for the measurement ofhexokinase and glucokinase activities by the cou-pled enzyme assay procedures of Davidson andArion (1986). The incubation mixtures for theassay of these enzymes in both control and testtubes contained the following ingredients in a finalvolume of 1.0 ml: incubation buffer, pH 7.4containing HEPES, 50mmol; KC1, 100 mmol;

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MgC1 , 7.5 mmol and dithiothreitol, 2.5mmol;2

fatty acid free bovine serum albumin, 10 mg;NAD , 0.5 mmol; G6PD, 4 units; liver superna-q

tant, 100ml for hexokinase assay or 10ml fortotal hexokinase and glucokinase assays; andD-glucose, 0.5mmol for hexokinase and 10mmolfor total enzyme activities. Tubes were preincubat-ed at 258C for 5 min. To the control tubes, 0.2ml of H O were added and to start the reactions2

in the test tubes, 0.2 ml of a solution containing0.5 mmol of ATP was added. The increase inabsorbance at 340 nm was followed for 15 minwhere the reaction was found to be linear withtime. Total enzyme activities(glucokinaseqhexo-kinase) and hexokinase activities were calculatedin terms of mUyml of the liver supernatant. Onemilliunit of the enzyme corresponds to the amountof the enzyme producing 1 nmol of NADH permin under assay conditions at 258C. Hexokinaseactivities were subtracted from the total hexoki-naseqglucokinase activities to obtain glucokinaseactivities. Protein concentration in the liver super-natants was measured using the biuret reagent(Gornall et al., 1949) using bovine serum albuminas a standard. Enzyme specific activities wereexpressed in mU per mg protein.

2.6. Plasma triglycerides, cholesterol and serumalanine transaminase

Plasma cholesterol and triglycerides(TG) weremeasured on tail blood obtained from fasted ani-mals using enzymatic kits(Pars Azemoon Co.,Tehran, Iran) prepared according to the proceduresof Allain et al. (1974) and Fossati and Prencipe(1982), respectively. Serum alanine aminotransfer-ase was measured on the blood obtained from fedanimals killed for hepatic glucokinase assay usinga kit (Zist Chemie, Tehran, Iran) prepared accord-ing to the procedure of Reitman and Frankel(1975) and the results were expressed in units perliter. One unit of the enzyme corresponds to thatamount of the enzyme producing 1mmol ofpyruvate per min under assay conditions.

2.7. Histological studies

Pancreas, liver and kidneys were removed andimmediately immersed in 10% formalin. Afterparaffin embedding, 5mm thick sections wereprepared and stained by hematoxylin and eosin.Pancreatic tissue was serially sectioned, the 5th,

10th and 15th sections of each pancreas werestained and the total number of islet cells in eachsection was counted under light microscope. Usinggraph paper, the total area of each stained sectionwas determined and the average number of pan-creatic islets per cm was calculated.2

2.8. Statistical analyses

Using SPSS version 10 software, the data werestatistically analyzed by one-way ANOVA andDuncan test. The level of statistical significancewas set atP-0.05.

3. Results

Quercetin had no effect on plasma glucoseconcentration of normoglycemic animals, but sig-nificantly reduced blood glucose level of diabeticrats in 8–10 days at the two doses used. The bloodglucose level of the diabetic animals treated withquercetin reached normal values at the end of thisperiod(Fig. 1).Quercetin exerted no effect on the glucose

tolerance curve of the normoglycemic animals, butat the two doses used, normalized the glucosetolerance curves of the streptozocin diabetic rats(Fig. 2). We noticed no significant differencebetween the effects of 10 or 15 mg quercetinykgbody mass. As seen in Fig. 2, ten days of i.p.injection of quercetin was able to normalize glu-cose tolerance curves of diabetic animals.Table 1 shows the effect of quercetin on nor-

moglycemic and STZ-diabetic rats. Quercetin treat-ment had no effect on hepatic hexokinase activityof normoglycemic animals, but significantlyincreased hexokinase activity of diabetic animalsand brought the activity to the level of normogly-cemic animals. Although quercetin treatmentdecreased the specific activity of hepatic glucoki-nase in normoglycemic animals, it significantlyand dose-dependently increased the activity of thisenzyme in diabetic animals and at 15 mg querce-tinykg b.m., glucokinase activity of the diabeticanimals reached that of normoglycemic rats. Sim-ilarly, plasma cholesterol and triglyceride levels ofnormoglycemic animals were significantlyincreased by quercetin treatment, while those ofdiabetic animals were significantly reduced by thisdrug. Serum ALT levels were not changed uponthe treatment of either normoglycemic or diabeticanimals with this antioxidant.

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Fig. 1. Plasma concentration–time profile of glucose in normoglycemic and streptozocin—diabetic rats after i.p. administration ofquercetin. Each point represents the mean"S.E.M. of five rats.d, normoglycemic control;s, normoglycemicq10 mg quercetin perkg; m, diabetic control;n, diabeticq10 mg quercetin per kg;j, diabeticq15 mg quercetin per kg.

A significant increase in the number of pancre-atic islets was seen in both normoglycemic anddiabetic rats treated with quercetin.Fig. 3a and b demonstrate typical pancreatic

sections of the diabetic and quercetin treated dia-betic groups, respectively. There was considerablyhigher number of micro-islet formation in treatedcases and no apparent mitotic figures were notedin these sections. Occasionally some islets wereseen adjacent to the excretory ducts. The numberof large and small size islets was also higher inthe quercetin treated animals. Except for the pres-ence of scattered and focal eosinophils in pancre-atic septa, no specific pathologic change was notedin the quercetin treated pancreas(Fig. 3b). Nosignificant pathological damage to hepatocytes,kidney tubules and glomeruli was observed. Minorsubtle changes such as mild degree of hydropicchange and higher frequency of one or two

macronucleoli were noted in liver tissues treatedwith quercetin.

4. Discussion

The plasma glucose lowering effects of querce-tin in streptozocin-induced diabetic rats and itslack of effects on plasma glucose level of normo-glycemic animals(Fig. 1) is in agreement withthe results of Chakravarthy et al.(1981, 1982a,b)on similar effects of(y)-epicatechin, in alloxandiabetic rats and also in accord with the data ofNuraliev and Avezov(1992) on hypoglycemiceffects of quercetin in alloxan diabetic animals.However, the latter authors used 10 and 50 mg ofquercetin per kg in their treatment of diabeticanimals, instead of the 10 and 15 mg used in thisexperiment. Normalization of glucose tolerancecurves in diabetic animals treated with either 10

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Fig. 2. Glucose tolerance curves of normoglycemic and streptozocin—diabetic rats after i.p. administration of quercetin. Each pointrepresents the mean"S.E.M. of five rats.d, normoglycemic control;s, normoglycemicq10 mg quercetin per kg;m, diabetic control;n, diabeticq10 mg quercetin per kg;j, diabeticq15 mg quercetin.

Table 1Effect of quercetin on normoglycemic and STZ-diabetic rats

Experimental Hexokinase Glucokinase Plasma Plasma Serum Number ofgroups1 Specific activity Specific activity cholesterol TG ALT pancreatic islets

(mUymg protein) (mUymg protein) (mmolyl) (mmolyl) (Uyl) (cm )y2

Normoglycemic 0.89"0.052a 1.22"0.11a 1.5"0.11a 0.70"0.02a 13.4"0.24 43.4"3.9a

controlNormoglycemicq10 mgquercetinykg b.m. 0.74"0.07a 0.83"0.05b 2.1"0.10b 0.79"0.01b 14.2"0.12 61.8"4.1b

Diabetic control 0.31"0.03b 0.23"0.03c 5.1"0.08c 1.60"0.08c 14.7"0.30 14.8"1.6c

Diabeticq10 mgquercetinykg b.m. 0.75"0.08a 0.50"0.04c 2.5"0.06b 0.70"0.01a 14.9"0.40 22.6"1.2d

Diabeticq15 mgquercetinykg b.m. 0.80"0.02a 1.37"0.20a 2.1"0.08b 0.60"0.05a 13.8"0.50 33.2"3.1a

For details of experimental conditions and definition of enzyme units see the text. Quercetin treatment for normoglycemic animals,1

diabetic rats receiving 10 mgykg b.m. per day and diabetics receiving 15 mgykg b.m. per day were continued for 8, 10 and 7 consecutivedays, respectively.

Data are expressed as mean"S.E.M. of 5 rats in each group. In each column, figures bearing different superscripts are significantly2

different atP-0.05 (one way ANOVA and Duncan test).

or 15 mg quercetin per kg body mass and the lackof the effects of this flavonoid on glucose tolerancecurve of normoglycemic animals are apparent fromFig. 2. No information on the effects of thisflavonoid on glucose tolerance curves of diabetic

animals is available in the literature, however,improvement of the glucose tolerance curves ofstreptozocin—diabetic rats by a plant extract(Teu-crium polium), known to contain several flavo-noids(Oganesyan and Minatsakanyam, 1985; Rizk

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Fig. 3. (a). Pancreatic section from STZ-induced diabetic rat.(b). Pancreatic section from a diabetic rat treated with 15 mg quercetinykg body mass per day for 7 days. White arrows, medium sized islets; black arrows, microislets. Magnification=250. Note the presenceof 3 medium sized and two microislets in quercetin treated diabetic rats.

et al., 1986; Verykokidov-Vitsaropoulou andVajias, 1986), was previously shown in our lab-oratory(Vessal et al., 2003).Treatment of normoglycemic animals with 10

mg quercetin per kg lowered hepatic glucokinaseactivity by 32% and resulted in a significantincrease in plasma cholesterol and TG(Table 1).Quercetin treatment of diabetic animals increasedhexokinase activity up to 2.6-fold, glucokinaseactivity up to six-fold, the number of pancreaticislets up to 2.2-fold and decreased plasma choles-terol level up to 2.4-fold and TG up to 2.8-fold(Table 1). The effects of quercetin on decreasingthe level of cholesterol and LDL were also dem-onstrated by Nuraliev and Avezov(1992). Regen-eration of the islets of Langerhans by quercetin(Table 1) is in agreement with the data of Chak-ravarthy et al.(1981, 1982a,b) who specificallydemonstrated the regeneration and the functionalactivity of regeneratedb-cells by(y)-epicatechin.The ability of quercetin to significantly increaseglucokinase activity of the liver and to reduceplasma cholesterol and TG in diabetic animalscould be explained by the work of Hii and Howell(1985) on the insulin releasing capacity of quer-cetin in isolated rat islets of Langerhans. Increasednumber of pancreatic islets in quercetin treatedanimals may be due to increased DNA replicationby the islet cells. This has been demonstrated byHii and Howell (1984) in an in vitro study.

Quercetin resulted in an increase of islet cellsin normoglycemic animals, however, it loweredglucokinase activity and increased plasma choles-terol and TG levels in those animals. Therefore, itappears that although quercetin exerts some bene-ficial effects on STZ-diabetic rats, but it may causesome disturbances in insulin signaling of the nor-moglycemic animals.It appears that quercetin as an antioxidant and a

free radical scavenger prevents autopoly(ADP-ribosyl)-ation of PARP, thereby stabilizing Reggene transcriptional complex and resulting in theregeneration ofb-cells and protection of pancreaticislets against STZ or alloxan(Akiyama et al.,2001; Szkudelski, 2001).In conclusion, it seems that quercetin, a flavo-

noid which produces an increase in the number ofpancreatic islets, probably increases insulin releasein STZ-diabetic rats and induces the hepatic glu-cokinase enzyme. The plasma glucose loweringproperty and its beneficial effects on correction ofglucose tolerance test and on lowering plasmacholesterol and TG could also be probably attrib-uted to its ability to regenerate pancreaticb-cellsand to increase insulin release.In this study, measurement of hepatic glucoki-

nase activity was considered as an indirect indexof insulin release byb-cells. However, insulinstaining of the pancreaticb-cells and serum insulinestimation are required to confirm the mode of

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action of quercetin in regeneration of functionalb-cells and in insulin release by quercetin treatedSTZ-rats.

Acknowledgments

This investigation was supported by Grant No.79–1038 from the office of Vice Chancellor forResearch, Shiraz University of Medical Sciences.

References

Akiyama, T., Takasawa, S., Nata, K., Kobayashi, S., Abe, M.,Shervani, N.J., et al., 2001. Activation of Reg gene, a genefor insulin—producing(b-cell regeneration: poly(ADP–ribose) polymerase binds Reg promoter and regulates thetranscription by autopoly(ADP–ribosyl)ation. Proc. Natl.Acad. Sci: USA 98, 48–53.

Allain, C.C., Poon, L.S., Chan, C.S., Richmond, S., Fu, P.C.,1974. Enzymatic determination of total serum cholesterol.Clin. Chem. 20, 470–475.

Bone, A.J., Hii, C.S., Brown, D., Smith, W., Howell, S.L.,1985. Assessment of the antidiabetic activity of epicatechinin streptozotocin—diabetic and spontaneously diabetic BByE rats. Biosci. Rep. 5, 215–221.

Chakravarthy, B.K., Gupta, S., Gambhir, S.S., Gode, K.D.,1981. Pancreatic beta-cell regeneration in rats by(y)-epicatechin. Lancet ii 1, 759–760.

Chakravarthy, B.K., Gupta, S., Gode, K.D., 1982a. Antidiabeticeffect of (y) epicatechin. Lancet ii 1, 272–273.

Chakravarthy, B.K., Gupta, S., Gode, K.D., 1982b. Functionalbeta cell regeneration in the islets of pancreas in alloxaninduced diabetic rats by(y)-epicatechin. Life Sci. 31,2693–2697.

Davidson, A., Arion, W.J., 1986. Factors underlying significantunderestimations of glucokinase activity in crude liverextracts: physiological implications of higher cellular activ-ity. Arch. Biochem. Biophys. 253, 156–167.

du Thie, G., Crozier, A., 2000. Plant derived phenolic antiox-idants. Curr. Opin. Clin. Nutr. Metab. 3, 447–451.

Formica, J.V., Regelson, W., 1995. Review of the biology ofquercetin and related bioflavonoids. Food Chem. Toxicol.33, 1061–1080.

Fossati, P., Prencipe, L., 1982. Serum triglycerides determinedcolorimetrically with an enzyme that produces hydrogenperoxide. Clin. Chem. 28, 2077–2080.

Fuhrman, B., Aviram, M., 2001. Flavonoids protect LDL fromoxidation and attenuate atherosclerosis. Curr. Opin. Lipidol.12, 41–48.

Gornall, A.G., Bardawill, C.S., David, M.M., 1949. Determi-nation of serum proteins by means of the biuret reaction. J.Biol. Chem. 177, 751–766.

Hii, C.S., Howell, S.L., 1984. Effects of epicatechin on ratislets of Langerhans. Diabetes 33, 291–296.

Hii, C.S.T., Howell, S.L., 1985. Effects of flavonoids oninsulin secretion and Ca handling in rat islets of Lan-45 q2

gerhans. J. Endocrinol. 107, 1–8.Nuraliev, I.N., Avezov, G.A., 1992. The efficacy of quercetinin alloxan diabetes. Eks. Klin. Farmakol. 55, 42–44.

Oganesyan, G.B., Minatsakanyam, V.A., 1985. Flavonoids ofcat thyme(Teucrium polium; L). Arm. Khim Zh. 38, 57–61.

Pari, L., Saravanan, G., 2002. Antidiabetic effect of cogentdb, a herbal drug in alloxan-induced diabetes mellitus.Comp. Biochem. Physiol. C 131, 19–25.

Reitman, S., Frankel, S., 1975. A colorimetric method for thedetermination of serum glutamic—oxaloacetic and glutam-ic–pyruvic transminase. Am. J. Clin. Pathol. 28, 56–63.

Rizk, A.M., Hammouda, F.M., Rimpler, H., Kamel, A., 1986.Iridoids and flavonoids ofTeucrium polium herb. PlantaMed. 2, 87–88.

Szkudelski, T., 2001. The mechanism of alloxan and strepto-zotocin action inb-cells of the rat pancreas. Physiol. Res.50, 536–546.

Verykokidov-Vitsaropoulou, E., Vajias, C., 1986. Methylatedflavones fromTeucrium polium. Planta Med. 5, 401–402.

Vessal, M., Zal, F., Vasei, M., 2003. Effects ofTeucriumpolium on oral glucose tolerance test, regeneration of pan-creatic islets and activity of hepatic glucokinase in diabeticrats. Arch. Iran Med. 6, 36–39.

Wright Jr, J.R., Abraham, C., Dickson, B.C., Yang, H., Mor-rison, C.M., 1999. Streptozotocin dose–response curve intilapia, a glucose–responsive teleost fish. Gen. Comp. Endo-crinol. 114, 431–440.

Young, P.W., Cawthorne, M.A., Coyle, P.J., Holder, J.C., Smith,S.A., 1995. Report on the treatment of obese mice withBRL-49653, a new and potent insulin sensitizer, enhancesinsulin action in white adipocytes. Association withincreased insulin binding and cell surface GLUT4 as meas-ured by photo affinity labeling. Diabetes 44, 1087–1092.