Biosci. Biotechnol. Biochem., 68 (8), 1640–1648, 2004

Effects of Chard ( vulgaris L. var cicla) on the Liver of the Diabetic Rats: A Morphological and Biochemical Study

Ozlem OZSOY-SACAN,1 Omu¨rKARABULUT-BULAN,2 Sehnaz BOLKENT,2 y Refiye YANARDAG,1; and Yasemin OZGEY1

1Department of Chemistry, Faculty of Engineering, Istanbul University, 34850-Avcilar, Istanbul, Turkey 2Department of Biology, Faculty of Science, Istanbul University, 34459-Vezneciler, Istanbul, Turkey

Received January 8, 2004; Accepted May 13, 2004

Chard ( L. var cicla) is one of the systems.3) The increased production of ROS has been medicinal herbs used by diabetics in Turkey. It has been attributed to glycation and/or glucose auto- reported to reduce blood glucose. We have investigated oxidation owing to a hyperglycemic environment. An the effect of chard extracts on the liver by biochemical impaired radical scavenger function has been linked to and morphological investigation. The plant extract was decreased activity of enzymatic and non-enzymatic administered by the gavage technique to rats at a dose of scavengers of free radicals.4) 2 g/kg every d for 28 d, 14 d after experimental animals The aim of antidiabetic therapy is to normalize the were made diabetic. In the diabetic group, some blood glucose level and prevent diabetes-induced com- degenerative changes were observed by light and plications. The antihyperglycemic effect of antidiabetic electron microscope examination, but degenerative drugs and their effect on diabetic complications are still changes decreased or were not observed in the diabetic being evaluated. Recently, the role of plant extract in the group given chard. In the diabetic group, blood glucose treatment of diabetes mellitus has been undergoing levels, serum alanine, aspartate transaminase, alkaline extensive investigation.5,6) phosphatase activities, total lipids, sialic and uric acid Chard is one of the plants used as an alternative levels, liver lipid peroxidation (LPO), and nonenzymatic hypoglycemic medication by diabetic people in Tur- glycosylation (NEG) levels increased, while blood glu- key.7) The hypoglycemic effect of chard was reported by tathione, body weight, and liver glutathione (GSH) Yanardagˇ et al.8–10) Phytochemical screening of chard levels decreased. The diabetic group given chard, serum revealed the presence of some fatty acids,11) phospho- alanine, aspartate transaminase, alkaline phosphatase lipids, glycolipids,12) polysaccarides,13) ascorbic acid,14) activities, total lipid level, sialic and uric acid levels, folic acid,15) and pectin.16) In addition, saponins17) and blood glucose levels, and liver LPO and NEG levels flavonoids18) were reported to have hypoglycemic decreased, but the other values increased. As a result of effects, which are present in various Beta vulgaris all the morphological and biochemical findings ob- species. No studies of the effects of chard extract on tained, it was concluded that the extract of this plant has liver are to be found in the literature. In the present a protective effect on the liver in diabetes mellitus. study, the effects of chard extracts on the liver of STZ- induced diabetic rats were examined by biochemical and Key words: Beta vulgaris L. var cicla; streptozotocin; morphological investigation. liver; chard; hepatic regeneration Materials and Methods Diabetes is recognised as one of the leading causes of morbidity and mortality in the world. About 2.5–3% of Plant material. Chard were purchased from the world’s population suffers from this disease, a open markets. They were carefully washed with tap portion which in some countries reaches 7% or more.1) water, then sliced into small pieces and dried under Hyperglycemia is an important factor in the develop- shade at room temperature. They were stored in well ment and progression of the complications of diabetes closed cellophane bags. mellitus.2) Diabetes mellitus has been shown to be a state of increased free radical formation. Oxidant stress Preparation of aqueous plant extract. Dried chard may increase in diabetes owing to a higher production of leaves (50 g) were extracted by adding 500 ml distilled reactive oxygen species (ROS) such as superoxide water and boiling for 30 min. The extract was then radical (O2 ), hydroxide radical (HO ), hydrogen per- filtered and the filtrates were evaporated using a rotary oxide (H2O2), and/or deficiency in antioxidant defense evaporator under reduced pressure until drying. Then the

y To whom correspondence should be addressed. Tel: +90-212-473-70-37; Fax: +90-212-591-19-97; E-mail: [email protected] Effects of Chard on Diabetic Rats 1641 extract was dissolved in distilled water before admin- homogenizer to make up 10% (w/v) homogenizate. The istration to normal and diabetic rats. homogenates were centrifuged and the clear super- natants were used for glutathione (GSH), lipid perox- Preparation of diabetic rats. Diabetes was induced by idation (LPO), nonenzymatic glycosylation (NEG), and intraperitoneal injection of streptozotocin (STZ) in a protein analysis. Liver glutathione (GSH) levels were single dose of 65 mg/kg body weight.19) STZ was determined according to the method of Beutler using dissolved in a freshly prepared 0.01 M citrate buffer Ellman’s reagent.27) LPO levels were determined with (pH ¼ 4:5). malondialdehyde (MDA) by the method of Ledwozyw et al.28) NEG levels were determined by thiobarbituric Animals. The experiments were reviewed and ap- acid methods.29) Protein content in the supernatants was proved by the Institute’s Animal Care and Use Com- determined by the Lowry method.30) mittee of the University of Istanbul. Six–6.5 months old female Swiss albino rats weighing 150–200 g were used. Statistical analysis. The results were evaluated using The animals were fed laboratory pellet chow and given an unpaired t-test and ANOVA variance analysis using water ad libitum. All rats were clinically healthy. The the NCSS statistical computer package.31) animals were randomly divided into four groups. Group I: Intact animals (normal,control). Group II: Animals Results given chard extract (control). Group III: Diabetic animals. Group IV: Diabetic animals given chard Light microscope results extract. Beginning on d 14 of the study, chard extract In the hepatocytes of the diabetic group in comparison was given to the group II (normal + chard) and IV to the control group (Fig. 1), picnotic nuclei, vacuoliza- (diabetic + chard) animals at a dose of 2 g/kg daily for tion, many big cytoplasmic granules, rupturing in the 28 days by gavage through an intragastric tube. Twenty- central vein, moderate hyperemia and dilation in some four female rats were used for light and electron sinusoids were detected (Figs. 2A, B, C), while in the microscopic studies and 96 for biochemical analyses. hepatocytes of diabetic group given the plant extract, it was observed that vacuolization, picnotic nuclei, and Histological assays. Small pieces of liver tissue taken hyperemia decreased and that little sinusoidal dilation from fasted animals for one night under ether anesthesia was present in some places (Figs. 3A, B). in the 42nd d, were fixed by Bouin’s fixative. Paraffin sections of 6 m thickness were taken from liver tissue Electron microscope results and stained by Haematoxylin-eosin and Masson’s triple In the diabetic group in comparison to the control dyes and examined with a light microscope. group, an expansion of the granular endoplasmic reticulum (GER) cisterns, picnotic nuclei, an increase Cytologic assays. Small liver tissues at +4 C were in smooth endoplasmic reticulum, lipid accumulations, a prefixed for two hours in So¨rensen’s phosphate buffered decrease in glycogen amount in some cells, mitochon- (pH ¼ 7:2) 2% glutaraldehyde. Then the tissues were dria surrounded by dilated granular endoplasmic retic- postfixed in phosphate buffered osmium tetraoxide for ulum, dark cells including picnotic nuclei, many one hour (pH ¼ 7:2), dehydrated in a graded series of ethanol solutions, and embedded in epon. Ultrathin sections were cut using a Reichert Om U3 ultramicro- tome with glass knives, stained with uranly acetate and lead citrate, and examined using a Carl Zeiss EM 9 S-2.

Biochemical assays. Blood samples were collected through the tail vein of the experimental animals at 0, 14 and 42 d. In all samples, the 18 h fasting blood glucose and glutathione levels were determined by o-toluidine20) and the Beutler, Duron, Kelly method21) respectively. Serum AST and ALT activities were determined by Reitman22) methods. Serum ALP activity and serum total lipid levels were determined by two point23) and sulfophosphovanilin24) respectively. At 42 d, serum sialic acid and uric acid levels were determined by the Lorentz25) and Caraway26) method respectively. At the end of the experimental period, liver tissues were taken from animals sacrificed under ether anes- thesia after an overnight fast. They were homogenized in Fig. 1. Liver of the Control Rat. cold 0.9% serum physiologic by means of a glass Hematoxylin-eosin. 400. 1642 O. OZSOY-SACAN et al.

Fig. 2. Liver of the Diabetic Rat. Vacuolization ( ), picnotic nuclei (!), many large cytoplasmic granules ( ), rupturing in the central vein (CV), moderate hyperemia ( ), dilations in sinusoids (). A, B: Haematoxylin-eosin. C: Masson’s tri-dye. 400.

Fig. 3. Liver of the Diabetic Rat Given Chard Extract. An ordinary histologic appearance. A: Haematoxylin-eosin. B: Masson’s tri-dye. 400. Effects of Chard on Diabetic Rats 1643

Fig. 4. Electronmicrographs of Hepatocytes of the Diabetic Rats. Fig. 5. Electronmicrographs of Hepatocytes of the Diabetic Rats Expansion in the granular endoplasmic reticulum (GER), picnotic Given Chard Extract. nuclei (N), increase in the smooth endoplasmic reticulum (SER), Usually an ordinary cytologic appearance except for an increase lipid accumulations (L), decrease in glycogen (!), mitochondria in glycogen (!) and peroxisomes (P). A, B. 14800. (M) surrounded by dilated GER in dark cell. A, B. 14800.

glucose levels were still high in the diabetic animals, but mitochondria, and expanded intercellular spaces were in the diabetic + chard group it decreased significantly detected (Figs. 4A, B), and in diabetic group given the (p ¼ 0:0001). The maximum reduction in blood glucose plant extract, an increase in the glycogen amount was noted 42 d after the administration of chard extract. peroxisomes, a decrease in the lipid amount, an increase At the end of the study, blood glucose levels were in granular endoplasmic reticulum containing many similar in all groups except for the diabetic group ribosomes, dark cells including dense cytoplasm, pic- (Table 2). The control rats did not show any significant notic nuclei, and many mitochondria (Figs. 5A, B). variation in the blood glucose throughout the exper- imental period (p ¼ 0:163). Biochemical results Before diabetes was induced there were no significant The mean body weights of the four groups are given differences in blood glutathione levels among the groups in Table 1. Body weight was significantly lower in rats (Table 3). After administration of chard to the diabetic with STZ diabetes than in the control group during the + chard animals, blood glutathione levels were found to experiment (Table 1). The body weight was significant- increase significantly compared with the diabetic ani- ly higher in the diabetic + chard group than in the mals (Table 3). At the end of the study, blood diabetic group at 42 d (Table 1). glutathione levels were similar in all groups except for The mean blood glucose levels of the groups are the diabetic group (Table 3). given in Table 2. Before induction of diabetes, the blood The mean serum AST and ALT levels of the fasting glucose levels of all groups were similar (pANOVA ¼ animals at 0, 14, and 42 d are shown in Tables 4 and 5 0:441). Fourteen days after administration of STZ, the respectively. When AST values were determined, there diabetic and diabetic + chard groups had significantly was no significant difference between the 4 groups on higher blood glucose levels than at 0 d. At 42 d blood d0(pANOVA ¼ 0:268). Fourteen days after administration 1644 O. OZSOY-SACAN et al. Table 1. Mean Levels of Body Weights for All Groups (g)

Group n Day 0 Day 14 Day 42 PValues Control 23 179:8 31:3 184:3 25:1 179:4 22:2 0.783 Control + Chard 23 182:9 30:5 179:0 22:6 175:6 20:9 0.627 Diabetic 25 164:8 14:9 144:9 18:7 139:5 20:7 0.0001 Diabetic + Chard 25 175:0 14:4 156:1 20:0 155:1 23:9 0.0001 PANOVA 0.058 0.0001 0.0001

Mean SD. n ¼ Number of animals.

Table 2. Mean Levels of Blood Glucose for All Groups (mg/dl)

Group n Day 0 Day 14 Day 42 PValues Control 23 66:7 12:859:7 15:459:1 15:9 0.163 Control + Chard 23 66:6 17:259:5 18:761:4 13:6 0.353 Diabetic 25 62:3 11:8 206:1 63:0 141:3 54:1 0.0001 Diabetic + Chard 25 69:2 16:6 180:4 59:579:7 22:9 0.0001 PANOVA 0.441 0.0001 0.0001

Mean SD. n ¼ Number of animals.

Table 3. Mean Levels of Blood Glutathione for All Groups (mg/dl)

Group n Day 0 Day 14 Day 42 PValues Control 23 36:2 10:638:9 16:338:5 20:2 0.839 Control + Chard 23 38:9 13:833:4 14:247:1 23:3 0.050 Diabetic 25 42:5 9:336:5 20:036:5 21:6 0.529 Diabetic + Chard 25 45:1 13:131:0 17:150:7 17:4 0.001 PANOVA 0.118 0.5 0.075

Mean SD. n ¼ Number of animals.

Table 4. Mean Levels of Serum AST for All Groups (U/l)

Group n Day 0 Day 14 Day 42 PValues Control 23 63:7 8:160:7 26:770:3 31:7 0.516 Control + Chard 23 60:4 7:569:6 28:5 105:4 15:5 0.0001 Diabetic 25 60:8 9:091:3 36:5 113:3 17:5 0.0001 Diabetic + Chard 25 66:7 14:388:3 6:772:0 37:6 0.229 PANOVA 0.268 0.011 0.0001

Mean SD. n ¼ Number of animals.

Table 5. Mean Levels of Serum ALT for All Groups (U/l)

Group n Day 0 Day 14 Day 42 PValues Control 23 20:3 8:222:1 9:828:6 7:3 0.202 Control + Chard 23 20:6 4:317:0 8:127:2 7:2 0.001 Diabetic 25 27:4 5:750:5 14:889:4 15:9 0.0001 Diabetic + Chard 25 25:3 7:959:2 14:827:9 5:8 0.0001 PANOVA 0.130 0.0001 0.0001

Mean SD. n ¼ Number of animals. of STZ, the diabetic and diabetic + chard groups had but in the diabetic + chard group it decreased. Treat- significantly higher AST activities than at d 0. But at ment with chard extract for 28 d decreased serum AST 42 d AST activities were still high in the diabetic rats, activities in the diabetic rats. Effects of Chard on Diabetic Rats 1645 Serum ALT activities and total lipids levels were high the livers of the normal and experimental groups. In in the diabetic rats compared with the controls. These diabetic rats a significant decrease in liver GSH levels changes were reversed in diabetic animals that were was observed (p ¼ 0:0001). Administration of chard given the chard extract (Tables 5 and 7). extract caused elevation in liver GSH levels in the The changes in serum ALP values at 42 d are given in diabetic and control groups (p ¼ 0:0001). The liver LPO Table 6. There was a significantly difference in the content in the diabetic group was significantly increased serum ALP values between groups (Table 6). A signifi- as compared to the control group (p ¼ 0:0001). This cant increase was observed in the values for the diabetic level decreased significantly with chard extract treat- groups (pANOVA ¼ 0:001). The ALP value in the ment (p ¼ 0:0001). In the diabetic rats a significant diabetic + chard group exhibited a significant decrease increase in liver NEG levels was observed. Adminis- compared to the diabetic group (p ¼ 0:011) (Table 6). tration of chard extract was found to reduce liver NEG The serum sialic acid and uric acid levels in the STZ levels in them (Table 9). treated and nondiabetic control groups is shown in Table 8. In the diabetic rats, a significant increase in Discussion serum uric and sialic acid levels was observed. Treat- ment with chard extract for 28 d decreased the serum In many countries, medicinal plants are used in uric and sialic acid levels in diabetic rats significantly, traditional practice to control diabetes.32) Plant drugs are whereas no significant changes in normal rats were frequently considered to be less toxic and free of side detected (Table 8). effects than synthetic ones.32) In our previous study, Table 9 shows the content of GSH, NEG, and LPO in chard showed a hypoglycemic effect on diabetic rats.7–10) The functions of the liver and the kidney may also be affected by changes in the level of insulin, which Table 6. Mean Levels of Serum ALP for All Groups (U/l) provides rapid uptake, storage as glycogen, and usage of glucose, especially in the liver. In diabetes, as the Group n Day 42 P Values activities of glycogen synthase and hexokinase are Control 23 55:1 28:9 diminished as a result of insulin deficiency, glucose Control + Chard 23 65:8 38:1 0.293 cannot be transformed into glycogen and glycogenesis is 33) Diabetic 25 112:5 60:7 reduced and thus the amount of glucose increases. In Diabetic + Chard 25 68:9 49:3 0.011 another study done by us in which the pancreases of PANOVA 0.001 diabetic rats were examined under light and electron

Mean SD. microscopes, it was found that chard extract increased n ¼ Number of animals. insulin synthesis and release and that blood glucose

Table 7. Mean Levels of Serum Lipid for All Groups (mg/dl)

Group n Day 0 Day 14 Day 42 PValues Control 23 250:6 84:3 243:5 31:7 226:9 21:3 0.314 Control + Chard 23 198:4 17:7 249:8 35:7 241:6 27:5 0.040 Diabetic 25 224:8 15:4 260:7 30:1 291:2 29:6 0.001 Diabetic + Chard 25 221:5 23:9 278:6 13:8 224:2 19:4 0.0001 PANOVA 0.504 0.077 0.0001

Mean SD. n ¼ Number of animals.

Table 8. Mean Levels of Serum Sialic Acid and Uric Acid for All Groups

Sialic Acid Uric Acid Groups n P P (mmol/l) Values (mg /dl) Values Control 23 2:6 0:40:7 0:2 Control + Chard 23 3:0 0:7 0.063 0:8 0:1 0.500

Diabetic 25 3:0 0:6a 3:5 0:4b Diabetic + Chard 25 2:4 0:5 0.001 2:8 0:3 0.01

PANOVA 0.001 0.0001

Mean SD. n ¼ Number of animals. ap ¼ 0:002 versus control groups. bp ¼ 0:0001 versus control groups. 1646 O. OZSOY-SACAN et al. Table 9. Mean Levels of Liver Tissue Nonenzymatic Glycosylation Glutathione and Lipid Peroxidation for All Groups

GSH NEG LPO Groups (nmol GSH/ PValues (nmol Fructose/ PValues (nmol MDA/ PValues mg protein) mg protein) mg protein) Control 3:4 0:318:2 3:00:7 0:1 Control + Chard 5:0 0:1 0.0001 16:9 5:6 0.546 1:3 0:1 0.0001

Diabetic 2:9 0:6c 32:2 6:6d 1:94 0:1e Diabetic + Chard 5:1 0:0 0.0001 30:4 8:5 0.548 1:79 0:1 0.001

PANOVA 0.0001 0.0001 0.0001

Mean SD. n ¼ Number of animals. cp ¼ 0:001 versus control groups. d,ep ¼ 0:0001 versus control groups. levels were reduced.34) In our present study, with controlled and long term cases.38) In our diabetic rats, a administration of chard extract, it was observed that significant increase in serum total sialic acid levels were blood glucose levels decreased in diabetic rats. The observed when compared with the control group. increase in the amount of liver glycogen can be Various factors might cause an elevation in the concen- explained by an increase of glucose intake which tration of serum sialic acid. Among these factors are an resulted in storage as glycogen. increase in the synthesis of sialic acid in insulin- Insulin is associated with protein as well as carbohy- independet tissues such as the liver and the brain and an drate metabolism. In the diabetic group given chard increase in the activity of sialytransferase, which trans- extract, an increase in regular parallel GER cisterns fers the sialic acid residues to the glycolipids and containing many ribosomes showed an increase in glycoproteins. In our study, administration of chard protein synthesis. In diabetes, protein synthesis ceases extract decreased the content of sialic acid in serum of and protein catabolism increases. Liver cell destruction diabetic rats. shows itself as an impairment in the permeability of Streptozotocin has been widely used to induce liver cell membranes. As a result, cytoplasmic enzymes diabetes in experimental animals. It causes selective such as AST and ALT pass into blood plasma and their destruction of pancreatic B cells, probably by a free activities in serum increase.35) The increase in serum radical-mediated mechanism.39) Free radicals formed activities of secretion enzymes (AST, ALT, ALP) which during the reduction of O2, such as peroxy-, hydroxy-, were formed in the liver and released into the bile is a and superoxide radicals, are known to cause cell damage sign of a loss of the secretion function of the liver.36) In directly or indirectly.40) Previous animal studies show our study, a decrease in the serum AST, ALT, and ALP that persistent hyperglycemia causes increased produc- values of diabetic rats administered chard extract shows tion of reactive oxygen species through glucose auto- the beneficial effect of the extract on liver tissue. Insulin oxidation and NEG, which in turn causes increased affects lipid as well as and protein oxidative stress.41) metabolism.33) The increase in lipid accumulation in GSH, the most important biomolecule against chemi- the diabetic group and the decrease in the chard extract- cally induced toxicity, can participate in the elimination treated group shows a parallelism with the effect of of reactive intermediates by reduction of hydroperoxides 42) insulin on lipid metabolism. While insulin deficiency in the presence of GPx. GSH also functions as a free causes the usage of lipid stores in adipose tissue, it also radical scavenger and in the repair of radical-caused causes lipid accumulation in the liver by increasing the biological damage.42) We observed a decrease in the amount of triglyceride stores there. The increase in level of GSH in the liver and blood in diabetes. The peroxisomes in the diabetic group treated with chard decrease in GSH levels represents increased utilization extract may be related to cholesterol and lipid metab- due to oxidative stress.42) Administration of chard olism. Peroxisomes play an important role in the extract increased the content of GSH in the liver and oxidation of fatty acids. blood of the diabetic rats. Sialic acids represent a group of 30 derivates of Nonenzymatic glycosylation (NEG) of liver neuraminic acid with different substituents at the amino causes alteration in their structure and function. Levels residue and alcoholic hydroxy groups. They are located of NEG were found to increase in the STZ-diabetic at the nonreducing ends of glycoproteins, glycolipids, groups in comparison with the untreated controls.43) and polysaccharides.37) Alteration in the contents of Various means of preventing this increase have been sialic acid in certain tissues and in the serum of diabetic investigated both in vitro and in vivo. It has been patients and animals has been reported.38) In diabetes reported that GSH, various antioxidant and mellitus, the serum concentration of sialic acid was compounds, trace elements such as vitamins C and E, found to increase significantly, especially in poorly flavanoids, saponins, vanadium, and selenium prevent Effects of Chard on Diabetic Rats 1647 the increase of tissue NEG levels.44) We found a Oxidative stress parameters in type I, type II and insulin- reduction of the NEG increase in the tissues of the treated type 2 diabetes mellitus; insulin treatment animals to which chard extract was administered, efficiency. Clin. Chim. Acta, 321, 89–96 (2002). producing a significant decrease in the diabetic group. 2) Arun, N., and Nalini, N., Efficacy of turmeric on blood Lipid peroxidation has been implicated in the patho- and polyol pathway in diabetic albino rats. Plant Foods Human Nutr., 57, 41–52 (2002). genesis of many degenerative disorders, including 45) 3) Kesavulu, M. M., Kameswara Rao, B., Giri, R., Vijaya, chemically induced diabetes. In diabetes, increases J., Subramanyam, G., and Apparao, C., Lipid peroxida- 46) in lipid peroxidation can cause tissue damage. Lipid tion and antioxidant enzyme status in Type 2 diabetics 47) peroxide levels increased in STZ diabetic rat tissue. In with coronary heart disease. Diabetes Res. Clin. Prac., our study, we have observed an increase in the levels of 53, 33–39 (2001). LPO in the liver in diabetes. Administration of chard 4) Hunt, J. V., Smith, C. C., and Wolff, S.P., Autooxidative extract significantly decreased the level of LPO in the glycosylation and possible involvement of peroxides and diabetic rats. This indicates that the extract may be free radicals in LDL modification by glucose. Diabetes, helpful in the prevention of damage caused by oxygen 39, 1420–1424 (1999). free radicals. 5) Grover, J. K., Yadav, S., and Vats, V., Medicinal plants Hypoglycemic effects have been reported for some of Indiana with anti-diabetic potential. J. Ethnopharma- col., 81, 81–100 (2002). plants that contain saponins, alkaloids, tannins, and 48,49) 6) Neef, H., Declercq, P., and Laekeman, G., Hypoglyce- quinovic acid. It is well established that some mic activity of selected European plants. Phytother. Res., saponins have hypoglycemic activity. This may be due 9, 45–48 (1995). to the inhibition of liver gluconeogenesis or glycoge- 7) Yanardagˇ, R., and C¸ olak, H., Effect of Chard (Beta nolysis.49) Flavonoids have also been found to possess vulgaris L. var cicla) on blood glucose levels in normal various other biological properties, for example, hep- and alloxan-induced diabetic rabbits. Pharm. Pharma- atoprotective, antithrombotic, and antiviral activity.50) col. Commun., 4, 309–311 (1998). Many actions have been correlated with their ability to 8) Yarat, A., O¨ zc¸elik, F., Yanardagˇ, R., Tunali, T., O¨ zsoy, scavenge oxygen-generated free radicals and to inhibit O¨ ., Emekli, N., and U¨ stu¨nel, A., The effect of chard lipid peroxidation in vitro.50) (Beta vulgaris L. var. cicla) on the protein and Phytochemical results showed that the chard extracts antioxidant systems in lenses of streptozotocin-induced diabetic rats. Pharm. Pharmacol. Commun., 4, 271–274 were rich in flavonoids18) and saponins,17) which are (1998). known to have strong antioxidant and free radical 9) Tunali, T., Yarat, A., Yanardagˇ, R., O¨ zc¸elik, F., O¨ zsoy, 50) scavenging properties. Our results indicate that chard O¨ ., Ergenekon, G., and Emekli, N., The effect of chard extract is effective in controlling hyperglycemia in STZ (Beta vulgaris L. var. cicla) on the skin of streptozotocin diabetic rats. In our other study, we observed the induced diabetic rats. 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