Molecular and Cellular Biochemistry 243: 2328, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Preventive effects of Cassia auriculata L. flowers on brain lipid peroxidation in rats treated with streptozotocin
Muniappan Latha and Leelavinothan Pari
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
Received 12 April 2002; accepted 19 July 2002

Abstract
The effect of aqueous extract of the flowers of Cassia auriculata were examined on antioxidants and lipid peroxidation in the brain of streptozotocin diabetic rats. Significant increase in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione were observed in brain on treatment with Cassia auriculata flower extract (CFEt) and glibenclamide. Both the treated groups showed significant decrease in thiobarbituric reactive substances (TBARS) and hydroperoxide formation in brain, suggesting its role in protection against lipid peroxidation induced membrane damage. Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antiperoxidative efficacy of medicinal plant, these findings are suggestions of possible antiperoxidative role played by Cassia auriculata flower extract. (Mol Cell Biochem 243: 2328, 2003) Key words: Cassia auriculata, catalase, glutathione peroxidase, glutathione-S-transferase, lipid peroxidation

Introduction
Oxidative stress plays an important role in chronic complications of diabetes and is postulated to be associated with increased lipid peroxidation [1]. Under physiological conditions, a wide range of antioxidant defenses protects the body against the adverse effects of free radical production in vivo [2]. Diabetic patients have an increased incidence of vascular disease and it has been suggested that free radical activity increased in diabetes [3]. Several reports demonstrate altered brain energy metabolism during diabetes [4]. The impact of diabetes mellitus on the central nervous system has become a field of interest recently. A number of reports are available on the structural and biochemical abnormalities of the brain in diabetes [5], which affects the central nervous system in several ways. Epidemiologic studies demonstrate that diabetes mellitus causes a 26-fold increase in the risk of thrombotic stroke [6]. Transportation of glucose into the brain of diabetic animals has been found to decrease as a con-

sequence of hyperglycemia [7]. Diabetes has also been found to affect neurotransmitter metabolism in the brain [8]. Steger and Kienast have found decreased non-adrenergic neurotransmitters in streptozotocin induced diabetes [9]. For various reasons in recent years the popularity of complementary medicine has increased. Dietary measures and traditional plant therapies as prescribed by ayurvedic and other indigenous systems of medicine are used commonly in India [10]. In recent times many traditionally used medicinally important plants were tested for their antidiabetic potential by various investigations in experimental animals [11]. Cassia auriculata L. commonly known as Tanners Cassia (Ceasalpinaceae) is a medicinal plant, which grows abundantly all over India. It is widely used in Ayurvedic medicine as tonic, astringent and as remedy for diabetes, conjunctivitis and ophthalmia [12]. The flowers and seeds of Cassia auriculata has been reported to show a very significant antidiabetic effect [13]. It is one of the main constituents of Kalpa herbal tea and has proven antidiabetic action. The five parts of the plant (leaves,

Address for offprints: L. Pari, Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India (Email: paribala@sancharnet.in; paribalaji@rediffmail.com)

24 roots, flowers, bark and unripe fruits) are taken in equal quantity, dried and powdered to give Avarai panchaga choornam which gives a good effect in the treatment of diabetes. It establishes good control of sugar levels in the treatment of diabetes [14, 15]. Nageswara rao et al. have reported that the Cassia auriculata contains several active constituents such as flavonoids, -sitosterol--D-glucoside, polysaccharides, anthracene, dimeric procyanidins and myristyl alcohol [16]. We have already reported the antiperoxidative effect of aqueous extract of Cassia auriculata flowers in diabetic rats [17]. A literature survey showed that the antihyperglycemic activity of Cassia auriculata has been demonstrated by Shrotri et al. in alloxan induced diabetic rabbits [15]. Therefore, the primary objective of this study was to assess the antiperoxidative efficacy of Cassia auriculata against a diabetogenic agent. The effects produced were compared with glibenclamide, a reference drug. Preparation of the plant extract 500 g of Cassia auriculata flowers were extracted with 1500 ml of water by the method of continuous hot extraction at 60C for 6 h and the extract was then evaporated to dryness. The residue was dissolved in water and used in the study [18].

Induction of experimental diabetes A freshly prepared solution of streptozotocin (45 mg/kg) in 0.1 M citrate buffer, pH 4.5 was injected intraperitoneally in a volume of 1 ml/kg [19]. 48 h after streptozotocin administration, rats with moderate diabetes having glycosuria and hyperglycemia (i.e. with blood glucose of 200300 mg/dl) were taken for the experiment.

Materials and methods

Chemicals 1-chloro-2, 4-dinitrobenzene (CDNB), 5,5-dithiobis-2-nitrobenzoicacid (DTNB), reduced glutathione (GSH), bovine serum albumin (BSA), thiobarbituric acid (TBA), nicotinamide adenine dinucleotide phosphate (NADP), xylenol orange, butylated hydroxy toluene (BHT), were obtained from Sigma Chemicals (St. Louis, MO, USA). The rest of the chemicals utilized were obtained from local firms (India) and were of highest purity grade.

Experimental protocol In the experiment, a total of 48 rats (40 diabetic surviving rats, 8 normal rats) were used. The rats were divided into 6 groups of 8 rats each. Group 1: Normal untreated rats. Group 2: Diabetic control rats given 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 3: Diabetic rats given Cassia auriculata flower extract (CFEt) (0.15 g/kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 4: Diabetic rats given CFEt (0.30 g/kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 5: Diabetic rats given CFEt (0.45 g/kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 6: Diabetic rats given glibenclamide (600 g/kg body weight) [20] in 1 ml of aqueous solution daily using an intragastric tube for 30 days.

Animals Male albino Wistar rats, body weight 180200 g, bred in Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used in this study. The animals were fed on a pellet diet (Hindustan Lever, India) and water ad libitum. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee, Annamalai University.

Biochemical studies in brain Animals were sacrificed at the end of 30 days by cervical dislocation. Blood was collected in tubes containing potassium oxalate and sodium fluoride solution for the estimation of blood glucose and plasma was separated for assay of insulin. The entire brain was perfused immediately with ice-cold 0.9% sodium chloride. TBARS, hydroperoxides, superoxide dismutase, catalase, glutathione peroxidase, glutathione-Stransferase and reduced glutathione were estimated in brain. Brain was selected as it continuously generates large amounts of free radicals from mitochondrial oxidative activity and

Plant material Cassia auriculata flowers were freshly collected from Nyeveli, Cuddalore District, Tamil Nadu, India. The plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No. 231) was deposited in the Botany Department of Annamalai University.

25 catecholamine catabolism. At the same time brain contains high levels of polyunsaturated fatty acids, which are the preferred targets of free radical damage in cell [21]. Determination of glutathione peroxidase and reduced glutathione Glutathione peroxidase (GPx) activity was measured by the method described by Rotruck et al. [27]. Briefly, reaction mixture contained 0.2 ml of 0.4 M phosphate buffer pH 7.0, 0.1 ml of 10 mM sodium azide, 0.2 ml of tissue homogenate (homogenised in 0.4 M phosphate buffer pH 7.0), 0.2 ml glutathione, 0.1 ml of 0.2 mM hydrogen peroxide. The contents were incubated at 37C for 10 min. The reaction was arrested by 0.4 ml of 10% TCA, and centrifuged. Supernatant was assayed for glutathione content by using Ellmans reagent (19.8 mg of 5,5-dithiobisnitro benzoic acid (DTNB) in 100 ml of 0.1% sodium nitrate). Reduced glutathione (GSH) was determined by the method of Ellman [28]. 1.0 ml of supernatant was treated with 0.5 ml of Ellmans reagent and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. Glutathione peroxidase activity was expressed as g of GSH consumed/min/mg protein and reduced glutathione as mg/100 g of tissue.

Estimation of blood glucose and plasma insulin Blood glucose was determined by the O-toluidine method [22]. 0.1 ml of blood was precipitated with 1.9 ml of 10% TCA and the precipitate was removed after centrifugation. 1 ml of supernatant was mixed with 4 ml of O-toluidine reagent and kept in boiling water bath for 15 min and cooled. The absorbance was read at 620 nm. Glucose was expressed as mg/dl of blood. Plasma insulin was assayed by ELISA, using Boeheringer Mannheim kit with a Boeheringer analyser ES300.

Estimation of lipid peroxidation Lipid peroxidation in brain was estimated colorimetrically by thiobarbituric acid reactive substances (TBARS) and hydroperoxides by the method of Nichans and Samuelson [23] and Jiang et al. [24], respectively. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25N HCl and 15% TCA) and placed in water bath for 15 min, cooled and centrifuged at room temperature for 10 min at 1000 rpm. The absorbance of clear supernatant was measured against reference blank at 535 nm and expressed as mM/100 g tissue. Hydroperoxides was expressed as mM/100g tissue. 0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37C for 30 min. The colour developed was read at 560 nm colorimetrically.

Determination of glutathione-S-transferase The glutathione-S-transferase (GST) activity was determined spectrophotometrically by the method of Habig et al. [29]. The reaction mixture (3 ml) contained 1.0 ml of 0.3 M phosphate buffer (pH 6.5), 0.1 ml of 30 mM CDNB and 1.7 ml of double distilled water. After pre-incubating the reaction mixture at 37C for 5 min, the reaction was started by the addition of 0.1 ml of tissue homogenate and 0.1 ml of glutathione as substrate. The absorbance was followed for 5 min at 340 nm. Reaction mixture without the enzyme was used as blank. The activity of glutathione-S-transferase is expressed as mmoles of GSH-CDNB conjugate formed/min/ mg protein using an extinction coefficient of 9.6 mM1 cm 1 .

Determination of catalase and superoxide dismutase Catalase (CAT) was assayed colorimetrically at 620 nm and expressed as moles of H2O2 consumed/min/mg protein as described by Sinha [25]. The reaction mixture (1.5 ml, vol.) contained 1.0 ml of 0.01M pH 7.0 phosphate buffer, 0.1 ml of tissue homogenate and 0.4 ml of 2M H2O2. The reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). Superoxide dismutase (SOD) was assayed utilizing the technique of Kakkar et al. [26]. A single unit of enzyme was expressed as 50% inhibition of NBT (Nitroblue tetrazolium) reduction/min/mg protein. Estimation of protein Protein was determined by the method of Lowry et al. [30] using bovine serum albumin (BSA) as standard, at 660 nm.

Statistical analysis of the data Results are presented as mean S.D. Statistical analysis was performed using ANOVA followed by Duncans Multiple Range Test (DMRT). A value of p < 0.05 was considered to indicate a significant difference between groups [31].

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Table 1. Blood glucose, plasma insulin and changes in body weight of normal and experimental animals Groups Body weight Initial Normal Diabetic control Diabetic + Cassia auriculata (0.15 g/kg) Diabetic + Cassia auriculata (0.30 g/kg) Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + glibenclamide (600 g/kg) 196 10.40 201 15.70 193 17.70 198 18.30 202 19.68 195 11.80 Final 208 9.80 151 13.66* 198 15.33** 208 10.32** 214 12.72** 206 13.43** 97.50 8.04 a 232.00 15.40b 216.66 20.80b 158.60 14.20 c 113.30 10.30a,d 124.60 10.32d 16.03 1.04 a 4.35 0.95b 4.90 0.41b 7.05 0.64c 14.16 0.67d 12.70 0.65e Fasting blood glucose (mg/dl) Plasma insulin (U/ml)

Values are given as mean S.D. for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT). Duncan procedure, range for the level 2.89, 3.03, 3.13, 3.20, 3.25. Diabetic control was compared with normal, *p < 0.001. Experimental groups were compared with diabetic control, **p < 0.001.

Results
The results of the present investigation are depicted in Tables 14. The treatment with aqueous extract of Cassia auriculata flowers reversed the weight loss in diabetic rats. Table 1 demonstrates the levels of blood glucose and plasma insulin in normal and experimental animals. There was a significant increase in blood glucose and a significant decrease in the level of plasma insulin in diabetic rats. The effects of administration of CFEt at 0.45 g/kg body weight of CFEt and glibenclamide tend to bring the blood glucose and plasma insulin towards normal value. The effect of CFEt at a dose of 0.45 g/kg body weight was significant than 0.15 and 0.30

g/kg body weight and therefore the higher dose was used for further biochemical studies. TBARS and hydroperoxides (Table 2) from brain homogenate were significantly decreased with CFEt and glibenclamide treatment whereas, diabetic control rats showed significantly increased levels of lipid peroxidation products. For studying the effect of CFEt on free radical production, the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione were measured (Tables 3 and 4). They presented significant increases in CFEt treatment when compared with diabetic control rats.
Table 3. Change in activities of catalase (CAT) and superoxide dismutase (SOD) in brain of normal and experimental animals

Table 2. Change in the levels of brain TBARS and hydroperoxides in normal and experimental animals Groups Normal Diabetic control Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + glibenclamide (600 g/kg) TBARS (mM/100 g tissue) 1.00 0.09a 1.75 0.06b 1.20 0.08c 1.39 0.08d Hydroperoxides (mM/100 g tissue) 110.70 5.20a 127.90 1.70b 115.12 4.26c 119.20 4.05d

Groups Normal Diabetic control Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + glibenclamide (600 g/kg)

Catalase Superoxide dismutase (UnitsA/mg protein) (UnitsB/mg protein) 3.08 0.30a 0.96 0.07b 2.63 0.25c 1.96 0.18d 8.75 0.58a 5.57 0.40b 7.46 0.56c 6.50 0.40d

Values are given as mean S.D. for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT). Duncan procedure, range for the level 2.95, 3.09, 3.20.

Values are given as mean S.D. for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT). Duncan procedure, range for the level 2.95, 3.09, 3.20. Amole of H2O2 consumed/min. BOne unit of activity was taken as the enzyme reaction, which gave 50% inhibition of NBT reduction in 1 min.

Table 4. Change in levels of glutathione peroxidase, glutathione-S-transferase and reduced glutathione in brain of normal and experimental animals Groups Normal Diabetic control Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + glibenclamide (600 g/kg) Glutathione peroxidase (UnitsA/mg protein) 3.13 0.25a 1.06 0.08b 2.85 0.17c 1.70 0.14d Glutathione-S-transferase (UnitsB/mg protein) 5.55 0.38a 0.89 0.03b 2.65 0.24c 1.64 0.15d Reduced glutathione (mg/100 mg tissue) 34.39 2.24a 19.50 1.74b 29.79 2.83c,e 26.90 2.20d,e

Values are given as mean S.D. for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p < 0.05 (DMRT). Duncan procedure, range for the level 2.95, 3.09, 3.20. Ag of GSH consumed/min. Bmoles of CDNBGSH conjugate formed/min.

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Discussion
Health and disease are parameters of the effectiveness with which human groups adapt to their environments. The herbals occupied a distinct place in the life right from the primitive till today [32]. NIDDM is characterized by a deficiency in insulin secretion associated with an insulin resistance of peripheral tissue [33]. The involvement of free radicals in diabetes and the role of these toxic species in lipid peroxidation and the antioxidant defense system have been studied. For the study of antidiabetic agents, streptozotocin induced hyperglycemia in rodents is considered to be good preliminary screening type II diabetic model [34] and is widely used. Sptreptozotocin, N-{methylnitrocarbamoyl}-D-glucosamine is a potent methylating agent for DNA and acts as nitric oxide donor in pancreatic cells. -cells are particularly sensitive to damage by nitric oxide and free radical because of their low levels of free radical scavenging enzymes [35, 36]. In our present study we have observed that an aqueous extract of CFEt can reverse these effect. The possible mechanism by which CFEt brings about its antihyperglycemic action may be by stimulation of surviving -cells to release more insulin. This was clearly evidenced by the increased level of insulin in diabetic rats treated with CFEt. In this context a number of other plants have also been observed to have antihyperglycemic and insulin-release stimulatory effect [20, 37]. Earlier studies in this lab have demonstrated a defective metabolism of lipid peroxides in other tissues of diabetic animal [37]. TBARS and hydroperoxides (lipid peroxidative markers) showed high lipid peroxidation. This may be because, the brain contains relatively high concentration of easily peroxidizable fatty acids [38]. In addition, it is known that certain regions of the brain are highly enriched in iron, a metal that, in its free form, is catalytically involved in production of damaging oxygen free radical species [39]. Vulnerability of brain to oxidative stress induced by oxygen free radicals seems to be due to the fact that, on one hand, the brain utilizes about one fifth of the total oxygen demand of the body and on the other, that it is not particularly enriched, when compared with other organs, in any of the antioxidant enzymes. Relatively low levels of these enzymes may be responsible in part for the vulnerability of this tissue [39]. Associated with the changes in lipid peroxidation diabetic brain showed decreased activity of the key antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione, which play an important role in scavenging the toxic intermediates of incomplete oxidation. A decrease in the activity of these enzymes can lead to an excess availability of superoxide anion (O2.) and hydrogen peroxide in the biological systems, which in turn generate hydroxyl radicals, resulting in initiation and propagation of lipid peroxidation [40]. Treat-

ment with CFEt increased the activity of enzymes and may help to control free radicals, as Cassia auriculata has been reported to be rich in flavonoids [16], well-known antioxidant, which scavenge the free radicals generated during diabetes. The increase in superoxide dismutase activity may protect catalase and glutathione peroxidase against inactivation by O2. anions as these anions have been shown to inactivate catalase [41] and glutathione peroxidase [42]. The pathophysiological consequences owing to depletion of GSH have been well studied. The depletion of GSH promotes generation of reactive oxygen species and oxidative stress with cascade of effects thereby affecting functional as well as structural integrity of cell and organelle membranes [43]. Depletion of GSH results in decreased activity of enzymes namely, GPx and GST. The GST catalyses the conjugation of GSH with a large number of electrophiles as a step to detoxify these species. It has been proposed that GPx is responsible for the detoxification of H2O2 in low concentration whereas catalase comes in to play when GPx pathway is reaching saturation with the substrate [44]. Administration of CFEt increased the GSH content. The elevated level of GSH protects cellular proteins against oxidation through glutathione redox cycle and also directly detoxifies reactive oxygen species generated from exposure to streptozotocin [45]. The significant increase in GSH content and GSH dependent enzymes GPx and GST in diabetic rats treated with CFEt indicates an adaptive mechanism in response to oxidative stress. Although a causal relationship is not yet established there is an inverse correlation between the lower content of GSH and the higher level of lipid peroxides [45]. Accordingly, in this study we have found that CFEt treatment increased the GSH levels and other enzymatic antioxidants significantly and diminished lipid peroxidation in brain. It may be concluded that in diabetes, brain tissue was more vulnerable to oxidative stress and showed increased lipid peroxidaton. The antioxidant responsiveness mediated by Cassia auriculata may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning. Detection of antihyperglycemic activity in CFEt along with protective effect against streptozotocin challenge and preventive action on lipid peroxidation provides a scientific rationale of use of Cassia auriculata as an antidiabetic plant.

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