Indian Journal of Experimental Biology Vol. 40, September 2002, pp. 1 005- 1 009

Antidiabetic and antioxidant effects of S-methyl cysteine sulfoxide isolated from onions (Allium cepa Linn) as compared to standard drugs in alloxan diabetic rats

Kumud Kumari & K T Augusti*

Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 69558 1 , India

Received 26 February 2002; revised 25 June 2002

Antidiabetic and antoxidant effects of S-methyl cysteine sulfoxide (SMCS) isolated from A. cepa and two standard drugs, glibenclamide and insulin were studied and compared in alloxan diabetic rats after using each of them for treatment

:;. for two months. These drugs amel iorated the diabetic condition significantly, viz. maintenance of body weight and control of blood sugar in rats. Further they lowered the levels of malondialdehyde, hydroperoxide and conjugated dienes in tissues exhibiting antioxidant effect on lipid peroxidation in experimental diabetes. Thi s is achieved by their stimulating effects on glucose utilization and the antioxidant enzymes, viz. superoxide di smutase and catalase. The probable mechanism of action of SMCS and glibenclamide may be partly dependent on the stimulation of insulin secretions and partly due to their individ­ual actions. In the amelioration of diabetes the standard drugs showed a better action, but as an antioxidant SMCS proved to be a belter one.

Oxidation and production of free radicals are an inte­gral part of metabolism. At high concentrations, free radicals can initiate l ipid peroxidation and also dam­age other i mportant molecules including proteins, carbohydrates and DNA. Free radicals and l ipid per­oxides are generated in vivo under various pathologi­cal conditions including hyperglycemia l . With respect to the role of l ipid peroxidation in diabetes, it has been postulated that the etiology of the complications of diabetes involves oxidative stress, as a result of hyperglycemia . It has been demonstrated that glucose can undergo oxidation in blood when catalyzed by trace metals, generating free radicals, hydrogen per­oxide and reactive ketoaldehydes2. Although the rate of glucose autoxidation is slow, it is relevant to the tissue damage in diabetes.

Living organisms have developed complex anti­oxidant systems to control production and reduce damage from free radicals. Endogenous components include GSH and Se-glutathione peroxidase, Fe­catalase, NADPH, ubiquinol- l O, Mn, Cu, Zn-SOD and uric acid. Decreased levels of antioxidants are reported in diab�tes3,4.

Some natural products l ike cysteine sulfoxide de­rivatives, thiosulfinates, flavanoids and beta-carotene are also reputed for their free radical scavenging ac-

* Address for correspondence Dr. K T Augusti, Professor of B iochemistry, School of Medical Education, Kottayam 686 008, India. E-mai l : ktaugusti@yahoo.co.uk.

tions5,6. As S-methyl cysteine sulphoxide (SMCS) isolated from onion showed s ignificant antidiabetic and hypolipidemic activities7•8, a study of i ts effects as compared to that of standard drugs on l ipid peroxida­tion i n diabetic rats was envisaged. A brief description of the experimental procedures and the results are presented here.

Materials and Methods SMCS was prepared from fresh onions using ion­

exchange resins9. Fresh onion was sliced into bol ing water to inactivate the enzyme al l i inase, which con­verts the sulfoxide amino acids (al l i ins) to their al­l ic in-type thiosulfinate compounds. It was then ground with 80% methanol, filtered and centrifuged. SMCS was i solated from the clear supernatant by ion­exchange chromatography using three successive col­umns of ion-exchange resins, viz. Amberlite IR 120 supplied by Romali , Bombay, India; Amberlite IRC 50 and Amberlite IRA 93 suppl ied by S igma Chemi­cal Company, USA. Al l the onion amino acids were first adsorbed on IR 120 column of a strong cat ion exchanger, and e luted with O. I N ammonium hydrox­ide. The fractions were analysed by paper chromato­graphy using butanol/acetic acid/water ( l 2 :3 :5v/v) system. Fractions containing the amino acids were pooled and concentrated on a rotary evaporator at 40°-43°C whereby ammonia was also removed.

The concentrate was passed through IRC 50 col­umn of weakly acidic cation exchanger. The neutral effluent obtained from the column was collected, con-

1 006 INDIAN J EXP S IOL, SEPTH1SER 2002

centrated and loaded on to the last column of IRA 93, a weakly basic anion exchanger. The amino acids were eluted with O. I M formic acid and collected in fractions. Fractions containing SMCS were detected by chromatography on s i l ica gel G using bu­tanoVacctic acid/water system as cited above. SMCS was precipitated by 50% acetone. Recrystall i sation was can'ied out to improve the purity of the sample. The pure sample had melting point of 1 64°C. It gave Rr value of 0. 1 7 in TLC on sil ica gel G using the same solvent system, agreeing with that of an authentic sample obtained from the B iochemical Institute, Hel­sienki .

In a pilot study we found that SMCS at a dose of 250 mg/kg body weight i mproved the glucose toler­ance and reduced fasting blood glucose in moderately diabetic rats by 1 3%. A smaller dose of 200mg/kg was selected for the long-term (60 days) treatment. Male albino rats (Sprague-Dawley strain) weighing 1 20-1 80g were made diabetic by a subcutaneous injection of alloxan monohydrate ( 1 80 mg/kg body weight). After a fortnight rats with moderate diabetes exhibit­ing glycosuria as detected by Benedict 's test and hy­perglycemia with a fasting blood glucose (FBG) of 1 90-280 mg/l OO ml were selected and divided into four groups of six . Their initial body weights, glyco­suria and FBG were determined before the start of the experiment. A control group of six normal rats were also taken. The rats were maintained on laboratory rat feed (Gold Mohur) supplied by Lipton Indian Ltd. , Bangalore, India and treated as given below:

Group I Normal rats fed with normal sal ine 10 mVkg /day

Group I I Diabetic rats fed with normal saline as above

Group III

Group IV

Group V

Diabetic rats treated with SMCS 200 mg/kg /day as a solution i n normal saline Diabetic rats treated with glibencla­mide 500 Ilg/kg /day as a suspension in normal sal ine. Diabetic rats injected (sc) with plain insul in 5 IU/kg /day in normal saline

Gl ibenclamide was supplied by Boehringer Knoll Ltd. , Bombay, India and insulin by Boots Company Ltd. , India. SMCS and glibenclamide were adminis­tered orally with a gastric tube. Water was given to the rats ad libitum. The experimenl was carried out �'Jr 60 days. Their f00c\ intak� was measured daily. Their fasting bloed gl 'JCo�e and urine sugar were (lS-

sessed after one month , ,ind also after two months. Then their final weights were taken and they were decapitated. Their l iver and heart were col lected for the estimation of malondialdehyde (MDA), hydroper­oxides (HP), conjugated d ien,�s (CD) I O, super oxide dismutase (SOD) I I and catalase l 2 by standard meth­ods. Data were analysed statisticall y by S tudent' s t­test and Analysis of Variance (ANOVA) qJong with Ducan ' s multiple range test.

Results The daily food consumption by different groups of

rats was more or less the same (8- 1 0g/rat/day). The changes in their body weights, however differed sig­nificantly. The diabetic rats showed a significant re­duction in their body weights and a significant in­crease in blood sugar level (P < 0.00 1 ) after two months. After the first month of treatment, the body weights of the treated groups were more or less main­tained. However after two months, the treated groups showed a significant (P < O.OO l ) decrease in blood sugar and urine sugar and a just significant increase (P < 0.05) in body weight as against the untreated con­trol (data not presented).

Urine sugar showed a gradational decrease (+++ to +) during two months treatment. In the diabetic con­trol group, the body weight steadily decreased and the FBG increased during the period. Loss of weight was prevented and the FBG was significantly reduced by treatment. The urine sugar for al l the treated groups was controlled to negligible levels (traces +) at the end of two months treatment. The effects of treatment was in the order insulin > glibenclamide > SMCS.

Figure 1 shows the levels of MDA, HP and CD in the l iver and heart of normal rats, diabetic control and treated rats. The l iver of the diabetic control rats showed elevated levels of MDA, HP and CD (P < O.OO I ) as compared to normal levels . These pa­rameters were significantly lowered in the treated groups (P < 0.05 - 0.00 1 ) . Both insulin and SMCS showed to be better than gJibenclamidc in this respect. The decreases of MDA in the treated groups were 1 1 .6%, 8% and 2 1 % for SMCS, gl ibenclamide and insulin respectively. The corresponding reductions of HP in these groups were 34%, 23% and 3 1 % respec­tively. The order of reductions of CD was the same as that [or HP, i .e with SMCS giving the highest reduc­tion of 1 2%, fol lowed by insul iil with 1 0% and

glibencJamide with 8% reduction ·:. In the case of heart, only CD shuv, ,;:d :1 significant

(P .:: 0 .U0 1 ) increase 0[ 6'2% in ti : ·; ::, i ;.' belic c. f )(· t r, ' !

t

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KUMARl & AUGUSTl : ANTIDIABETIC & ANTiOXIDANT EFFECTS OF S-METHYL CYSTEINE SULFOXIDE 1 007

rats. Treatment with the drugs significantly lowered this parameter by 1 5 - 1 8% (P < 0.05-0.0 1 ) .

On the contrary, IVIDA alld H P levels were just sig­nificantly (P < 0.05) lower in the diabetic rats by 7% and 13% respectively. Treatment with the three drugs showed only a non-sign ificant change in the former. However all treatment with SMCS, glibenclamide and insul in the I-IP levels showed a just significant in­crease (P < 0.05).

Figure 2 shows the acti vities of SOD and catalase in the liver and heart of normal rats, diabetic control and treated diabetic rats. The enzymes showed sig­nificant decreases in their activities both in l iver and heart of the diabetic control rats (P < O.OO l ) . On treat­ment with each of the drugs the activ ities of these en­zymes i ncreased to signi fi cant levels (P < 0.05-0.00 1 ) except that for SOD in the heart of glibencl am ide treated group.

Discussion The resul ts show that although all the three drugs

have antidiabetic action, only SMCS has a definite antioxidant action. SMCS seemed to be only a mild antidiabetic drug but a strong antioxidant agent, as i t most effectively reduced the peroxidation products,

Q) � .. .. i= 1> � C) 8 i E

120

D Normal Control- 1 0m! normal saline.

1 00 [ll]]] Diabetic Control - l Oml normal saline

• Diabetic +SMCS -200mg.

80 � Diabetic +Glibenclamide -SOOllg

60 • Diabetic +Insulin -SIU.

40

20

MDA (liver) MDA (heart) HP (liver)

viz. MDA, HP and CD in both l i ver and heart of the treated diabetic rats.

The rise of the l ipid peroxidation level observed in the diabetic rats agrees with the results of several other studies conducted on diabetic rats and human sUbjects l , \ 3 . Under normal conditions some amount of peroxidation also occurs. Scavenging enzymes l ike SOD, catalase and glutathione peroxidase metabolize the superoxide ions thus formed to harmless interme­diates. A change in this condi tion in diabetes leads to overproduction of superoxide ion and hydrogen per­oxide which i n turn form the harmful hydroxyl radi­ca\ . As found in this study a decrease has been ob­served in the activ ities of SOD and catalase in some tissues of diabetics by Sekar et al. 1 4 and also in a pre­vious study from this department which may explain the reasons for the over production of l ipid peroxide in diabetes. As observed in the benefi cial effects of S­allylcysteine sulfoxide (SACS) isolated from Allium sativum Linn 1 5 in diabetic condition, here also we find that SMCS isolated from Allium cepa Linn is superior to glibenclamide and insul in for the antioxidant ef­fects. The lower effects of SMCS as compared to SACS may be because of the unsaturated bonds pre­sent i n the latter.

HP (heart) CD (liver) CD (heart)

Fig. I -Effect of long-term treatment with S MCS, gl ibenclamide, and insul in on l ipid peroxidation. Values presented are mean of 6 rats. ANOYA calculated. Duncan' s procedure: Range for the 0.05 level . Significance of the results are given in the text.

1008 INDIAN J EXP mOL, SEPTEMBER 2002

70

� Normal control - I Oml saline.

60 � Diabetic Control - I Oml normal saline.

[Z3 Diabetic +SMCS-200mg.

50 � Diabetic +Glibenclamide 500llg.

_ Diabetic +lnsulin 5 IU.

20

1 0

oLailiR SOD (liver) SOD (heart) Catalase (liver) Catalase (heart)

Fig. 2 - Effect of long-term treatment with SMCS, glibcnclamide and insul in on the activ i ties of the scavcnging cnzymcs, superoxidc disl11utase and catalase in thc tissues. Values prcscnted are mean of six rats. Acti vity of catalase is exprcssed as valucs x 1 000 units/mg protein. Activity or SOD: Unit = Enzyme concentration required to i nhibit the optical density at 560 nm of chromogen produced by 50% in one min. Activity of catala:;e: Unit=Velocity constan[/sccond. ANOVA calculated. Duncan procedure: Range for the 0.05 Icvel. Sig­nificance of the results are given in thc text.

A lowered level of MDA and HP with the heart tis­sue of diabetic rats despite a decrease in the activ ities of SOD and catalase may be explai ned thus: under conditions of stress, the heart tissue util izes glucose in preference to fatty acids for i ts energy production t 6 .

The decreased uti l i zation of fatty acids may be one of the factors for the decreased formation of lipid perox­ides as well as the observed decreases in the activities of SOD and catalase in heart. Secondly an enhanced action of glutathione S-transferase as reported by some workers in heart ' 7 may also contribute to a de­crease in the level of l ipid peroxides in that tissue. This enzyme detoxifies fatty acid hydroperoxides to nontoxic alcohols.

The antioxidant activity of the sulfoxide R-S(O)­cysteine is cri tical ly dependent on the nature of the R side chain. SMCS and the sulfoxide isolated from gar­lic, viz. SACS are good scavengers of free radicals and this may be due to their stimulatory action on dif-

ferent antioxidant enzymes l 8 . Thus treatment of dia­betic rats with SMCS might have ameliorated the de­teriorated condition of diabetes by increasing the ac­tivities of SOD and catalase independently or through a stimulated secretion of insul in 7 that eventually lead to a control of l ipid peroxidation. Glibenclamide treat­ment might have gained the control of diabetes via its insulin secretary action and that too to a considerable extent. According to the papers presented at an international symposium on Al l iums 2000, the senior author and many others t9 proved that both garl ic and onion are full of active pri nc iples that protect us from diabetes, cancer and coronary heart disease.

Acknowledgement

The authors acknowledge the authorities of the University of Kerala for providing ai l lhe faci l i t ies for this research and particularly for the grant of a re­search fel lowship to the first author (K K).

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KUMARI & AUGUSTI : ANTIDIABETIC & ANTIOXIDANT EFFECTS OF S-METHYL CYSTEINE SULFOXIDE 1009

References I Sato Y, Hotta N, Sakamoto N, Matusoka S, Ohishi N &

Yagi, K. Lipid peroxide level in plasma of diabetic patient . Biochem Med, 2 1 ( 1 979) 1 04

2 Hunt J V, Dean R T & Wolf S P, Hydroxyl radical produc­tion and autooxidative glycosylation. Glucose autooxidation as the cause of protein damage in the experimental glycation model of diabetes and aging, Biochem J, 256 ( 1 988 )205.

3 Karpen C W, Cataland S & O'Dorisio T M, Panganamala R . Interrelation of platelet v i tamin E and thromboxane synthesis in type I diabetes mell i tus, Diabetes, 33 ( 1984) 239.

4 Som S, Basu D, Mukherjee S , Deb S , Choudhary P R , Muk­herjee S N & Chatterjee S N , Ascorbic acid metabolism in diabetes mellitus. Metabolism, 30 ( 1 98 1 ) 572.

5 Belman S, Onion and garlic o i l inhibit tumor production, Carcinogellesis, 4 ( 1 983) 1 063.

6 Kawaki shi S & Morimitsu Y, New inhibitor of platelet ag­gregation in onion oi l , Lance£ , 2(8608) ( 1 988) 330.

7 Kumud Kumari & Augusti K T, Antidiabetic effects of S­methyl cysteine sulphoxide on alloxan diabetes. Planta Medica , 6 1 ( 1 995) 72.

8 Kumud Kumari, B iju C, Mathew & Augusti K T, Antidia­betic and hypolipidemic effects of S-methyl cysteine sulphoxide isolated from Allium Cepa Linn. Indian J Bio­

chem_Biophys, 32 ( 1 995) 49. 9 Carson J F & Wong F F, Isolation of S-methyl-L-Cystiene

sulfoxide and of S-N-propyl .-L-cysteine sulfoxide from on­ion as their N-2,4-dinitrophenyl derivatives. J Org Chem, 26 ( 196 1 ) 4997.

10 John A B & Steven D A, Microsomal l ipid peroxidation. In :Colowick S P, Kaplan N 0, editors. Methods in Enzymol­ogy Vol . 52.Part C (Academic Press Inc; Ncw York) 1 978, 302.

1 1 Kakkar P, Das B & Vishwanathan P N , A modified spectro­photometric assay of superoxide dismutase. Indian. J Bio­

chem Biophys, 2 1 (2) ( 1 984) 1 30. 1 2 Maehly A C, Chance B, The assay of catalase and peroxi­

dase. In Methods of B iochemical Analysis, edited by David Cl ick, Vol I (Interscience Publ i shers Inc. New York) 1 954, 357.

13 Dohi T, Kawamura K, Morita K, Okamoto H & Tsuj imoto A, Alterations of plasma selenium concentrations and the ac­tivities of tissue peroxide metabolism enzymes in strepto­zotocin induced diabetic rats. Hor Metab Res, 20( I I ) ( 1 988) 67 1 .

1 4 Sekar N & Govindasamy S , Insulin mimetic role o f venadate on plasma membrane insul in receptor. J Biosci. 1 5 ( 1 990) 67.

1 5 Augusti K T & Sheela C G, Antiperoxide effect of S-al ly l cysteine sulfoxide, an insul in secretagogue, in diabetic rats. Experientia, 52 ( 1 996) 1 1 5 .

1 6 B itmer J T, Idell-Wenger J A, Rovet! M J & Neely J R , Con­trol of fatty acid metabol ism in ischemic and hypoxy hearts. J Biol Chem, 253( 1 2) ( 1 978) 4305.

17 Lawrence A R & B urk F R, Glutathione peroxidase activity i n selenium deficient rat l iver. Biochem Biophys Res Com­

mun, 7( 1 4) ( 1 976) 952. 1 8 Sparins V L, Barany G & Wattenberg L W, Effects of or­

ganosulfur compounds from garlic and onion on benzo(a) pyrene-induced neoplasia and glutathione-S-transferase ac­t ivity in the mouse. Carcinogenesis, 9 ( 1 988) 1 3 1 .

1 9 Wargovich M J , B lock E & Augusti K T, Alliums and health

colioqnium, with relation to chemistry and biological activ­

ity. In : B i l l Randle editor abstrats of 3'd International Sympo­sium on Edible A l liaceae. The University of Georgia Atlanta USA. Oct 29-Nov. 3 (2000) 18 .