Diabetes mellitus is a complex syndrome involving severeinsulin dysfunction in conjugation with gross abnormalitiesin glucose homeostasis and lipid metabolism, which has affecting several millions of population all over the world.The individual with diabetes has a 25-fold increase in therisk of blindness, a 20-fold increase in the risk of renal fail-ure, a 20-fold increase in the risk of amputation as a result ofgangrene and a 2 to 6 fold increased risk of coronary heartdisease and ischaemic brain damage.1)

There is considerable evidence on the role of free radicalsin the etiology of diabetes and altered antioxidant defenses indiabetes.2) Oxidative stress has been reported to play an im-portant role in diabetes mellitus right from its genesis to thedevelopment of microvascular complications. Generation offree radicals by hyperglycemia is related to glucose auto-oxi-dation. Glucose auto-oxidation has been linked to non-enzy-matic glycosylation and glycosylated proteins have beenshown to be a source of free radicals.3)

Prior to 1950’s control of diabetes was based entirely oninsulin therapy. Unfortunately, some patients developed com-plications and thus need for some other therapy was realized.Presently control of diabetes mellitus relies on many chemi-cals and plant extracts.4) More than 400 traditional planttreatments for diabetes mellitus have been recorded, but onlya small number of these have received scientific and medicalevaluation to assess their efficacy. There was a gap in properunderstanding of medicinal plants for mankind in past because traditional medicines generally lacked scientific explanations.

Many plant extracts and plant products have been shownto reduce oxidative stress significantly,5) which may be an im-portant property of plant medicines associated with the treat-ment of several ill fated diseases including diabetes.6) It hasbeen found that compounds in their natural formulations aremore active than their isolated form.7) Among these Mo-mordica charantia is a good example of ignorant folk medi-cine out stripping scientific understandings in the therapeuticapplications is selected for this study. Consumption of bittergourd (Momordica charantia) by diabetic patients is a com-

mon practice in India, with the belief that it has a useful hy-poglycemic potential.8)

Momordica charantia (MC) LINN, commonly referred to asbittergourd or karela, belongs to the Curcurbitaceae family. Itis a climbing plant, cultivated throughout Southern Asia. Itsfruits are very cheap and available throughout the year. Im-mature fruits are used to prepare different dishes for humanconsumption, while highly matured fruits are considered asnot worthy for consumption. There are two varieties of thisvegetable based on size and shape. The large variety is long,oblong and pale green in color. The other one is small, littleoval and dark green in color. The yield of small variety perplant is much less when compared to large ones; as a resultthe cost of the small variety is almost thrice when comparedto the larger ones. Both the types are bitter in taste. The pulpis blood red or scarlet after dehiscence. The seeds are dap-pled, flat, thick notched margin, red aril in morphology and itis white color in raw fruits and become red when they areripe. Different parts of these plants have been used in the Indian system of medicine for a number of ailments besidesdiabetes.9,10) Our previous experimental results were highlyencouraging as they revealed that blood glucose level wassignificantly lowered after oral administration of aqueous ex-tract of Momordica charantia seeds in glucose load conditionand in streptozotocin induced diabetes.11) In view of theabove considerations, the present study was designed to ex-amine the effect of an aqueous extract from the seeds of Mo-mordica charantia on oxidative stress in plasma and pancreasof diabetic rats induced by streptozotocin, and their efficacywas compared with glibenclamide, a standard hypoglycemicdrug.

MATERIALS AND METHODS

Chemicals Streptozotocin was procured from Sigmachemical Co., St. Louis Mo, U.S.A. RIA kit for plasma in-sulin assay was purchased from Linco research Inc., U.S.A.All other chemicals were of analytical grade.

Plant Material Fresh fruits of Momordica charantia

∗ To whom correspondence should be addressed. e-mail: subbus2020@yahoo.co.in © 2005 Pharmaceutical Society of Japan

Beneficial Effects of Momordica charantia Seeds in the Treatment of STZ-Induced Diabetes in Experimental Rats

Dhanasekar SATHISHSEKAR and Sorimuthu SUBRAMANIAN*

Department of Biochemistry and Molecular Biology, University of Madras; Chennai-25, India.Received December 27, 2004; accepted February 28, 2005

The aim of the present study was to evaluate the effect of the aqueous extract of seeds of two varieties,namely a country and hybrid variety of Momordica charantia (MCSEt1 and MCSEt2) on oxidative stress inplasma and pancreas of streptozotocin (STZ) induced diabetic rats. Oral administration of each of the seed extracts at a dosage of 150 mg/kg body weight for 30 d resulted in a significant reduction in plasma glucose, thio-barbituric acid-reactive substances, lipid-hydroperoxides, alpha-tocopherol and significant improvement inascorbic acid, reduced glutathione and insulin. The treatment also resulted in a significant reduction in thiobar-bituric acid reactive substances, lipid-hydroperoxides, superoxide dismutase, catalase, glutathione peroxidaseand significant improvement in reduced glutathione in pancreas of drug treated diabetic rats when compared tothe untreated diabetic rats. On the basis of results obtained, it may be concluded that the treatment of Momordica charantia seed varieties may effectively normalize the impaired oxidative stress in streptozotocin induced-diabetes than the glibenclamide treated groups.

Key words Momordica charantia; oxidative stress; streptozotocin; diabetes

978 Biol. Pharm. Bull. 28(6) 978—983 (2005) Vol. 28, No. 6

were procured from a vegetative farm of Chengalpattu, India.Authentication of the plant was carried out by Prof. V. Kavi-yarasan, Centre for Advanced Studies in Botany, Universityof Madras and the voucher specimens of the plants have beenretained in the department herbarium.

Preparation of Seed Extracts The fruits were slicedinto two halves and the seeds were selectively collected man-ually, washed with fresh water and dried in shade at roomtemperature. The dried seeds were grounded into fine powderby an electrical mill and mesh (mesh number 50). The pow-dered seeds were kept in airtight containers in a deep freezemaintained at 4 °C until the time of further use. The seed ex-tract was prepared by dissolving the seed powder in distilledwater using a magnetic stirrer. It was then filtered and evapo-rated to dryness under reduced pressure. An aqueous suspen-sion, which is the form customarily, used in folk medicine,was prepared to facilitate easy handling. The drug solutionswere prepared freshly each time and administrated intragas-trically. The dosage schedule for the drug was once a day.

Animals Male albino rats of Wistar strain weighingaround 160—180 g were purchased from Tamilnadu Veteri-nary and Animal Sciences University (TANUVAS), Chennaifor the present study. They were acclimatized to animalhouse conditions, fed with commercial pelleted rat chow(Hindustan Lever Ltd., Bangalore) and had free access towater. The experiments were designed and conducted in ac-cordance with the ethical norms approved by Ministry of So-cial Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines (Approvalnumber: 360/01/A/CBCSEA).

Induction of Diabetes STZ-induced hyperglycemia hasbeen described as a useful experimental model to evaluatethe activity of hypoglycemic agents.12,13) After overnight fast-ing (deprived of food for 16 h had been allowed free access to water), diabetes was induced in rats by intraperi-toneal injection of STZ dissolved in 0.1 M cold sodium citratebuffer (pH 4.5) at a dose of 55 mg/kg body weight.14) The an-imals were allowed to drink 5% glucose solution overnight toovercome the drug-induced hypoglycemia. Control rats wereinjected with citrate buffer alone. After a week time for thedevelopment of diabetes, the rats with moderate diabeteshaving glycosuria and hyperglycemia (blood glucose range ofabove 250 mg/dl) were considered as diabetic rats and usedfor further experiments.15) The treatment was started on the8th day after STZ-injection and this was considered as 1stday of treatment. The treatment is continued for 30 d.

Experimental Set Up The animals were divided intofive groups with six animals in each group as follows.

Group I : Normal control ratsGroup II : Diabetic control ratsGroup III : Diabetic rats treated with MCSEt1

(150 mg/kg b.w/d) in aqueous solutionorally for 30 d.

Group IV : Diabetic rats treated with MCSEt2(150 mg/kg b.w/d) in aqueous solutionorally for 30 d.

Group V : Diabetic rats administered with gliben-clamide (600 mg/kg b.w/rat/d) in aque-ous solution orally for 30 d.15)

After 30 d of treatment, the fasted rats were sacrificed bycervical decapitation. Blood was collected into heparinized

tubes. Plasma was separated and used for the estimation ofglucose,16) Vitamin C,17) and Vitamin E.18) Insulin was esti-mated by using radio immuno assay. The pancreatic tissueswere excised, rinsed in ice-cold saline and homogenized in0.1 M Tris–HCl buffer (pH 7.4).

The Tissue Homogenate and Plasma Were Used for theFollowing Estimations Lipid peroxidation was estimatedusing thiobarbituric acid reactive substances (TBARS) by themethod of Ohkawa et al.19) Lipid-hydroperoxides was as-sayed by the method of Jiang et al.20) Reduced glutathionewas estimated by the method of Ellman.21)

Assay of Antioxidant Enzymes in Tissue HomogenateSuperoxide dismutase (SOD) was estimated using themethod of Misra and Fridovich.22) Catalase (CAT) was as-sayed by the method of Takahara et al.23) Glutathione peroxi-dase (GPx) was assayed by the method of Rotruck et al.24)

Total protein present in the tissue homogenate was estimatedby the method of Lowry et al.25)

Histopathological Studies A portion of the pancreatictissue was fixed in 10% buffered neutral formal saline forhistological studies. After fixation, tissues were embedded inparaffin, solid sections were cut at 5 mm and stained withaldehyde. The sections were examined under light micro-scope and photomicrographs were taken.26)

Statistical Analysis All the grouped data were statisti-cally evaluated with SPSS/7.5 software. Hypothesis testingmethods included one way analysis of variance (ANOVA)followed by least significant difference (LSD) test. p valuesof less than 0.05 were considered to indicate statistical sig-nificance. All the results were expressed as mean�S.D. forsix animals in each group.

RESULTS

Table 1 shows the levels of blood glucose and plasma in-sulin in normal and experimental groups of rats. The in-creased levels of blood glucose and decreased level of insulinin diabetic rats were restored to near normal levels in MC-seed extract and glibenclamide treated diabetic rats.

Table 2 and Fig. 1 shows the levels of TBARS, lipid-hy-droperoxides and reduced glutathione in plasma and pan-creas of normal and experimental groups of rats. There was asignificant increase in the levels of TBARS, lipid-hydroper-oxides and a significant decrease in the level of reduced glu-

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Table 1. Effect of Momordica charantia on Blood Glucose and Plasma In-sulin of Diabetic Rats

Blood glucose (mg/dl)Plasma insulin

GroupsBefore treatment After treatment

(mU/ml)

Normal control 81.76�5.81 85.25�4.96 16.18�1.48Diabetic control 263.91�18.42*,a) 330.18�20.63*,a) 5.23�0.52*,a)

Diabetic�MCSEt1 270.12�15.56NSb) 93.52�4.34*,b) 15.05�1.45*,b)

Diabetic�MCSEt2 270.41�17.11NSc) 108.16�6.00*,c) 14.87�1.32*,c)

Diabetic�267.57�16.79NS

d) 121.32�6.09*,d) 12.73�0.74*,d)

glibenclamide

Values are given as mean�S.D., for six rats per group. Values are statistically signifi-cant at ∗ p�0.05; NS, non significant. Statistical significance was compared with in thegroups as follows: a) diabetic control rats were compared with normal control rats, b) MCSEt1 treated diabetic rats were compared with diabetic control rats, c) MCSEt2treated diabetic rats were compared with diabetic control rats, d) glibenclamide treateddiabetic rats were compared with diabetic control rats.

tathione in both plasma and pancreas of diabetic rats whencompared to normal rats. Treatment with Momordica cha-rantia seed extracts and glibenclamide significantly reversedthese levels to near normal levels.

Figure 2 demonstrates the levels of vitamin C and vitaminE in plasma of normal and experimental groups of rats. Thedecreased level of vitamin C and increased level of vitamin Ewere observed during diabetes, when compared to normalcontrol group. Treatment with Momordica charantia seed ex-tracts and glibenclamide significantly reversed these levels tonear normalcy.

Figure 3 shows the activities of superoxide dismutase(SOD), catalase (CAT) and glutathione peroxidase (GPx) inpancreas of normal and experimental groups of rats. A sig-nificant increase in the activities of SOD, CAT and GPx inpancreas was observed in diabetic rats when compared withnormal rats. Treatment with Momoridca charantia seed ex-

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Table 2. Effect of Momordica charantia on the Levels of ThiobarbituricAcid-Reactive Substances (TBARS), Lipid-Hydroperoxides and ReducedGlutathione in Plasma of Diabetic Rats

TBARS Lipid- Reduced

Groups(nmol/ml)

hydroperoxides glutathione(10 nmol/dl) (mg/dl)

Normal control 3.32�0.18 9.20�0.20 24.12�0.66Diabetic control 7.12�0.11*,a) 14.00�0.17*,a) 16.30�0.61*,a)

Diabetic�MCSEt1 3.65�0.14*,b) 10.08�0.26*,b) 23.49�0.51*,b)

Diabetic�MCSEt2 3.90�0.20*,c) 10.80�0.25*,c) 22.48�0.71*,c)

Diabetic�4.24�0.22*,d) 12.02�0.54*,d) 31.50�0.14*,d)

glibenclamide

Values are given as mean�S.D., for six rats per group. Values are statistically signifi-cant at ∗ p�0.05; NS, non significant. Statistical significance was compared with in thegroups as follows: a) diabetic control rats were compared with normal control rats, b) MCSEt1 treated diabetic rats were compared with diabetic control rats, c) MCSEt2treated diabetic rats were compared with diabetic control rats, d) glibenclamide treateddiabetic rats were compared with diabetic control rats.

Fig. 1. Effect of Momordica charantia on the Levels of Thiobarbituric Acid-Reactive Substances, Lipid-Hydroperoxides and Reduced Glutathione in Pan-creas of Diabetic Rats

Values are given as mean�S.D., for six rats per group. Values are statistically significant at ∗ p�0.05; NS, non significant. Statistical significance was compared with in thegroups as follows: a) diabetic control rats were compared with normal control rats, b) MCSEt1 treated diabetic rats were compared with diabetic control rats, c) MCSEt2 treated dia-betic rats were compared with diabetic control rats, d) glibenclamide treated diabetic rats were compared with diabetic control rats.

Fig. 2. Effect of Momordica charantia on the Levels of Vitamin C and Vitamin E in Plasma of Diabetic Rats

Values are given as mean�S.D., for six rats per group. Values are statistically significant at ∗ p�0.05; NS, non significant. Statistical significance was compared with in thegroups as follows: a) diabetic control rats were compared with normal control rats, b) MCSEt1 treated diabetic rats were compared with diabetic control rats, c) MCSEt2 treated dia-betic rats were compared with diabetic control rats, d) glibenclamide treated diabetic rats were compared with diabetic control rats.

tracts and glibenclamide decreased the activities of SOD,CAT and GPx in diabetic groups of rats.

Figures 4a—e show the section of pancreas of control andexperimental groups of rats. Fig. 4a presents the section ofpancreas from normal control group showing normal islets.Diabetic pancreas showing atrophy of b-cells and vasculardegenerative changes in the islets (Fig. 4b). MCSEt1 (Fig.4c), MCSEt2 (Fig. 4d) and glibenclamide (Fig. 4e) treateddiabetic pancreas showing increase in the islets as comparedto diabetic pancreas.

DISCUSSION

The traditional medicine all over the world is now-a-daysrevalued by an extensive activity of research on differentplant species and their therapeutic principles. Experimentalevidence suggests that free radicals (FR) and reactive oxygenspecies (ROS) are involved in a high number of diseases.Free radicals may play an important role in causation andcomplications of diabetes mellitus.27)

STZ is a valuable agent for experimental induction of typeI diabetes. In type I diabetes, the insulin is markedly depletedbut not absent28) It has been reported that STZ-diabetic ani-mals may exhibit most of the diabetic complications medi-ated through oxidative stress, and the involvement of freeradicals in pancreatic cell destruction.6) Keeping these inview, in the present study, the oxidative stress has been as-sessed in rats made diabetic by streptozotocin. Preliminarystudies conducted by us revealed the non-toxic nature of MCseeds on normal rats. In the present study oral administrationof MC seeds extract decreased the blood glucose level tonear normal in diabetic rats. The possible mechanism bywhich extracts brings about decrease in blood glucose maybe by stimulation of surviving b-cells of islets of langerhansto release more insulin. This was clearly evidenced by the increased level of plasma insulin in diabetic rats treated withMC seeds extract. The activation of b cells with bitter gourdseed treatment was reported in mildly STZ diabetic animalsin which some b cells were found active and granulation returns to normal giving insulinogenic effect.29) In this con-text a number of other plants have also been reported to have

antihyperglycemic and insulin stimulatory effects.30)

The sulfonylureas such as glibenclamide have been usedfor many years to treat diabetes, to stimulate insulin secretionfrom pancreatic b cells principally by inhibiting ATP-sensi-tive KATP channels in the plasma membrane.31) Courtois etal., have reported that oral administration of glibenclamide tothe STZ-induced diabetic rats, decreased the blood glucoselevel and also could be considered as a standard antidiabeticdrug to compare the efficacy of hypoglycemic com-pounds.32,33)

Almost all the major classes of biomolecules are attackedby free radicals but lipids are probably the most susceptible.Cell membranes are rich sources of polyunsaturated fattyacids (PUFAs), which are readily attacked by oxidizing radi-cals. The oxidative destruction of PUFAs, known as lipidperoxidation, is particularly damaging because it proceeds asa self-perpetuating chain-reaction.34) Increased lipid peroxi-dation impairs membrane functions by decreasing membranefluidity and changing the activity of membrane bound en-zymes and receptors.35) The most popular and easiest methodused to assay lipid peroxidation and free radical activity inbiological sample is TBARS.36) A significant increase inTBARS and lipid-hydroperoxides in plasma and pancreas ofdiabetic rats suggest an increase in reactive oxygen species,that could be due to either their increased production or de-creased destruction.37,38)

In the present study, the plasma and pancreas TBARS andlipid-hydroperoxides levels were significantly lowered in theextract treated group compared to diabetic control group.The protective effect of extract was also confirmed by thehistopathological examination.

GSH has a multifaceted role in antioxidant defence. It is adirect scavenger of free radicals as well as a co-substrate forperoxide detoxification by glutathione peroxidases.39) Studieshave shown that the plasma and pancreas GSH concentrationof STZ-induced diabetic rats are significantly lowered whencompared to normal rats.40) Decreased glutathione in diabetescould be the result of decreased synthesis or increased degra-dation of GSH by increased oxidative stress.38) Administra-tion of Momordica charantia seed extracts and glibenclamideincreased the content of GSH in plasma and pancreas of dia-

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Fig. 3. Effect of Momordica charantia on the Activities of Superoxide Dismutase, Catalase and Glutathione Peroxidase in Pancreas of Diabetic Rats

Values are given as mean�S.D., for six rats per group. Values are statistically significant at ∗ p�0.05; NS, non significant. Statistical significance was compared with in thegroups as follows: a) diabetic control rats were compared with normal control rats, b) MCSEt1 treated diabetic rats were compared with diabetic control rats, c) MCSEt2 treated dia-betic rats were compared with diabetic control rats, d) glibenclamide treated diabetic rats were compared with diabetic control rats. Activity is expressed as : 50% of inhibition ofepinephrine auto oxidation per min for SOD; mmoles of hydrogen peroxide decomposed per min per mg of protein for catalase; mmoles of glutathione oxidized per min per mg ofprotein for GPx.

betic rats.Vitamin C is one of the four dietary antioxidants, the other

three being vitamin E, vitamin A precursor beta-carotene,and selenium. Frei reported that the ability of vitamin C topreserve the levels of other antioxidants in human plasma.41)

Also, vitamin C regenerates vitamin E from its oxidizedform.42) Hypoinsulinemia and/or hyperglycemia inhibitascorbic acid and cellular transport.43) As the chemical struc-ture of ascorbic acid is similar to that of glucose, it shares themembrane transport system with glucose and hence com-petes with it for its transport.44) We observed lowered levelsof plasma vitamin C in diabetic rats. Thus, the elevation inglucose concentration may depress natural antioxidant likevitamin C45) or due to decrease in GSH levels, since GSH isrequired for recycling of vitamin C.46) The elevated levels ofvitamin E in plasma may be due to increased intake of vita-min E per unit weight or increase in serum lipid levels orboth in the diabetic rats.47)

The endogenous antioxidant enzymes (e.g.: SOD, CAT andGPx) are responsible for the detoxification of deleteriousoxygen radicals. SOD has been postulated as one of the mostimportant enzymes in the enzymatic antioxidant defense sys-tem which catalyses the disputation of superoxide radicals toproduce H2O2 and molecular oxygen,35) hence diminishingthe toxic effects caused by their radical. Catalase (CAT) is ahemoprotein which catalyses the reduction of hydrogen per-oxides and protects the tissues from highly reactive hydroxylradicals. The superoxide anion has been known to inactivateCAT, which is involved in the detoxification of hydrogen per-oxide.35,48) GPx plays a primary role in minimizing oxidativedamage. It has been proposed that GPx is responsible for thedetoxification of H2O2 in low concentration whereas catalasecomes into play when GPx pathway is reaching saturationwith the substrate.48)

The present study shows that increased activity of SOD,CAT and GPx in diabetic rats, when compared to normalrats, which may be due the increase in the activity of theseabove enzymes and may result from radical induced activa-tion.49) The obtained results are in agreement with earlierdata.50) Significant increase in the activity of antioxidant enzymes in diabetic pancreas indicates an adaptive mecha-nism in response to oxidative stress.

In conclusion, the results of this study represent that ad-ministration of Momordica charantia seeds showed hypo-glycemic effect, which controls blood glucose level andthereby prevents the formation of free radicals or it mayscavenge the reactive oxygen metabolites through various an-tioxidant compounds in them. Further studies are in progressto isolate the active components in Momordica charantiaseeds and their role in controlling diabetes.

REFERENCES

1) Wolff S. P., Br. Med. Bull., 49, 642—652 (1993)2) Garg M. C., Singh K. P., Bansal D. D., Indian J. Exp. Biol., 35, 264—

266 (1997).3) Ceriello A., Quatraro A., Giugliano D., Diabet Med., 9, 297—299

(1992).4) Jahodar L., Cesk farm., 42, 251—259 (1993).5) Anjali P., Manoj K. M., Ancient Sci. Life 15, 27—29 (1995).6) Garg M. C., Ojha S., Bansal D. D., Indian J. Exp. Biol., 34, 264—266

(1996).

982 Vol. 28, No. 6

4a

4b

4c

4d

4e

Fig. 4. Histopathological Observations Made on the Pancreatic Tissue ofcontrol and Experimental Groups of Rats and the Photomicrographs Pre-sented Are the Representatives of the Six Rats Used in Each Group (Alde-hyde Fuschin, 320�)

(a) Presents the section of pancreas from normal control rat showing normal isletswith clusters of purple stained b-cells. (b) Pancreas of STZ-induced diabetic rat show-ing atrophy of b-cells and vascular degenerative. A reduction in number and size ofislets were observed. (c) MCSEt1, (d) MCSEt2 and (e) glibenclamide treated diabeticpancreas showing near normal architecture.

7) Sabu M. C., Ramadasan Kuttan., J. Ethnopharmacol., 81, 155—160(2002).

8) Sastri B. N., “Wealth of India—Raw Materials,” Vol. 6, Council of Sci-entific and Industrial Research, India, 1962, p. 376.

9) Ganguly C., De S., Das S., Eur. J. Cancer Prev., 9, 283—288 (2000).10) Jayasooriya A. P., Sakono M., Yuxizaki C., Kawano M., Yammoto K.,

Fukuda N., J. Ethnopharmacol., 72, 331—336 (2000).11) Sathishsekar D., Sivagnanam K., Subramanian S., Die Pharmazie

(2005) (In press)12) Junod A., Lambert A. E., Staufacher W., Renold A. E., J. Clin. Invest.,

48, 2129—2139 (1969).13) Ledoux S. P., Woodley S. E., Patton N. J., Wilson L. G., Diabetes., 35,

866—872 (1986).14) Sekar N., Kanthasamy S., William S., Sulramanian S., Govindasamy

S., Pharmacol. Res., 22, 207—217 (1990).15) Ravi K., Sathishsekar D., Subramanian S., Biol. Trace Elem. Res., 99,

145—156 (2004).16) Sasaki T., Matsy S., Sonae A., Rinsho Kagaku, 1, 346—353 (1972).17) Omaye S. T., Turnbull J. D., Methods Enzymol., 62, 3—11 (1979).18) Desai I. D., Methods Enzymol., 105, 138—147 (1984).19) Ohkawa H., Ohishi N., Yagi K., Anal. Biochem., 95, 351—358 (1979).20) Jiang Z. Y., Hunt J. V., Woltt S. P., Anal. Biochem., 202, 384—389

(1992).21) Ellman G. L., Arch. Biochem. Biophys., 82, 70—77 (1959).22) Misra H. P., Fridovich I., J. Biol. Chem., 247, 3170—3175 (1972).23) Takahara S., Hamiltan B. H., Nell J. V., Kobra T. Y., Ogura Y.,

Nishimura E. T., J. Clin. Invest., 29, 610—619 (1960).24) Rotruck J. T., Pope A. L., Gasther H. E., Hafeman D. G., Hoekstra W.

G., Science, 179, 588—590 (1973).25) Lowry O. H., Rosenbrough N. J., Farr A. I., Randall R. J., J. Biol.

Chem., 193, 265—275 (1951).26) Gordon K., Bradbury P., “Theory and Practice of Histological Tech-

niques,” 3 ed., Churchill Livingston, New York, 1990, pp. 61—8027) Boynes J. W., Diabetes, 40, 405—411 (1991).28) Omura T., Sato R., J. Biol. Chem., 239, 2370—2378 (1964).29) Padmini K., Chakrabati C. H., Indian J. Exp. Biol., 2, 232—235

(1982).30) Pari L., Latha M., Singapore Med. J., 43, 617—621 (2002). 31) Ashcroft F. M., Ashcroft S. J. H., Biochim. Biophys. Acta, 1175, 45—

59 (1992).32) Courtois P., Jijakli H., Ladriere L., Oguzhan B., Sener A., Malaisse W.

J., Int. J. Mol. Med., 11, 105—109 (2003).33) Andrade-Cetto A., Wiedenfeld H., Revilla M., Isals S., J. Ethnophar-

macol., 72, 129—133 (2000).34) Cheeseman K. A., Spater T. F., Br. Med. Bull., 49, 481—493 (1993).35) Baynes J. W., “Mechanistic Approach to Diabetes,” Vol. 2, Hertford-

shire-Ellis Horwood limited, Chichester, England, 1995, pp. 203—231.

36) Holley A. E., Cheeseman K. A., Br. Med. Bull., 49, 494—505 (1993).37) Griesmacher A., Kinder H. M., Andert S., Amer. J. Med., 16, 337—

335 (1995).38) Matkovies B., Kotoroman M., Varga I. S., Quy Hai D. Q., Varga C.,

Acta Physiol. Hung., 85, 29—38 (1998).39) Winterbourn C. C., “Biothiols in Health and Disease,” Marcet Dekker

Inc., New York, 1995, pp. 117—134.40) Krishnakumar K., Augusti K. T., Vijayammal P. L., Indian J. Physiol.

Pharmacol., 43, 510—517 (1999).41) Frei B., Am. J. Clin. Nutr., 54, 113s (1991).42) Buettner G. R., Arch. Biochim. Biophys., 300, 535—543 (1993).43) Mann G. V., Newton P., Ann. N.Y. Acad. Sci., 258, 243—248 (1945).44) Fay M. J., Bush M. J., Verlangieri A. J., Life Sci., 46, 619—624 (1990).45) Inaye M., Mio T., Sumino K., Clin. Chim. Acta, 285, 35—44 (1999).46) Chatterjee I. B., Nandi A., Indian J. Biochem. Biophys., 28, 233—236

(1999).47) Aoki Y., Yanagisawa Y., Yazaki K., Oguchi H., Kiyosawa K., Furuta

S., Diabetologia, 35, 913—916 (1992).48) Arunabh B., Shibnath C., Indian J. Exper. Biol., 38, 877—880 (2000).49) Gonzales R., Auclair C., Voisin E., Gantero H., Dhermy D., Boivin P.,

Cancer Res., 4, 4137—4139 (1984).50) Kakar R., Kalra J., Mantha S. V., Prasad K., Mol. Cell. Biochem., 151,

113—119 (1995).

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