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Molecular and Cellular Biochemistry 243: 23–28, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Preventive effects of Cassia auriculata L. flowerson brain lipid peroxidation in rats treated withstreptozotocin

Muniappan Latha and Leelavinothan PariDepartment 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 thebrain of streptozotocin diabetic rats. Significant increase in the activities of superoxide dismutase, catalase, glutathione per-oxidase, glutathione-S-transferase and reduced glutathione were observed in brain on treatment with Cassia auriculata flowerextract (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 membranedamage. Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating theantiperoxidative efficacy of medicinal plant, these findings are suggestions of possible antiperoxidative role played by Cassiaauriculata flower extract. (Mol Cell Biochem 243: 23–28, 2003)

Key words: Cassia auriculata, catalase, glutathione peroxidase, glutathione-S-transferase, lipid peroxidation

sequence of hyperglycemia [7]. Diabetes has also been foundto affect neurotransmitter metabolism in the brain [8]. Stegerand Kienast have found decreased non-adrenergic neuro-transmitters in streptozotocin induced diabetes [9].

For various reasons in recent years the popularity of com-plementary medicine has increased. Dietary measures and tra-ditional plant therapies as prescribed by ayurvedic and otherindigenous systems of medicine are used commonly in In-dia [10]. In recent times many traditionally used medicinallyimportant plants were tested for their antidiabetic potentialby various investigations in experimental animals [11].

Cassia auriculata L. commonly known as ‘Tanner’s Cassia’(Ceasalpinaceae) is a medicinal plant, which grows abundantlyall over India. It is widely used in Ayurvedic medicine as tonic,astringent and as remedy for diabetes, conjunctivitis and oph-thalmia [12]. The flowers and seeds of Cassia auriculata hasbeen reported to show a very significant antidiabetic effect [13].It is one of the main constituents of ‘Kalpa herbal tea’ and hasproven antidiabetic action. The five parts of the plant (leaves,

Introduction

Oxidative stress plays an important role in chronic compli-cations of diabetes and is postulated to be associated withincreased lipid peroxidation [1]. Under physiological condi-tions, a wide range of antioxidant defenses protects the bodyagainst the adverse effects of free radical production in vivo[2]. Diabetic patients have an increased incidence of vascu-lar disease and it has been suggested that free radical activ-ity increased in diabetes [3]. Several reports demonstratealtered brain energy metabolism during diabetes [4]. The im-pact of diabetes mellitus on the central nervous system hasbecome a field of interest recently. A number of reports areavailable on the structural and biochemical abnormalities ofthe brain in diabetes [5], which affects the central nervoussystem in several ways. Epidemiologic studies demonstratethat diabetes mellitus causes a 2–6-fold increase in the riskof thrombotic stroke [6]. Transportation of glucose into thebrain of diabetic animals has been found to decrease as a con-

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

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roots, flowers, bark and unripe fruits) are taken in equal quan-tity, dried and powdered to give ‘Avarai panchaga choornam’which gives a good effect in the treatment of diabetes. It estab-lishes good control of sugar levels in the treatment of diabetes[14, 15]. Nageswara rao et al. have reported that the Cassiaauriculata contains several active constituents such as flavo-noids, β-sitosterol-β-D-glucoside, polysaccharides, anthra-cene, dimeric procyanidins and myristyl alcohol [16]. We havealready reported the antiperoxidative effect of aqueous extractof Cassia auriculata flowers in diabetic rats [17]. A literaturesurvey showed that the antihyperglycemic activity of Cassiaauriculata has been demonstrated by Shrotri et al. in alloxaninduced diabetic rabbits [15]. Therefore, the primary objectiveof this study was to assess the antiperoxidative efficacy ofCassia auriculata against a diabetogenic agent. The effects pro-duced were compared with glibenclamide, a reference drug.

Materials and methods

Chemicals

1-chloro-2, 4-dinitrobenzene (CDNB), 5,5′-dithiobis-2-nitro-benzoicacid (DTNB), reduced glutathione (GSH), bovineserum albumin (BSA), thiobarbituric acid (TBA), nicotina-mide adenine dinucleotide phosphate (NADP), xylenol orange,butylated hydroxy toluene (BHT), were obtained from SigmaChemicals (St. Louis, MO, USA). The rest of the chemicalsutilized were obtained from local firms (India) and were ofhighest purity grade.

Animals

Male albino Wistar rats, body weight 180–200 g, bred inCentral Animal House, Rajah Muthiah Medical College, Anna-malai University, were used in this study. The animals werefed on a pellet diet (Hindustan Lever, India) and water adlibitum. The animals used in the present study were main-tained in accordance with the guidelines of the National In-stitute of Nutrition, Indian Council of Medical Research,Hyderabad, India and approved by the ethical committee,Annamalai University.

Plant material

Cassia auriculata flowers were freshly collected from Nyeveli,Cuddalore District, Tamil Nadu, India. The plant was identi-fied and authenticated at the Herbarium of Botany Directoratein Annamalai University. A voucher specimen (No. 231) wasdeposited in the Botany Department of Annamalai University.

Preparation of the plant extract

500 g of Cassia auriculata flowers were extracted with 1500ml of water by the method of continuous hot extraction at60°C 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) in0.1 M citrate buffer, pH 4.5 was injected intraperitoneally ina volume of 1 ml/kg [19]. 48 h after streptozotocin adminis-tration, rats with moderate diabetes having glycosuria andhyperglycemia (i.e. with blood glucose of 200–300 mg/dl)were taken for the experiment.

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 groupsof 8 rats each.

Group 1: Normal untreated rats.Group 2: Diabetic control rats given 1 ml of aqueous solu-

tion daily using an intragastric tube for 30 days.Group 3: Diabetic rats given Cassia auriculata flower ex-

tract (CFEt) (0.15 g/kg body weight) in 1 ml of aqueoussolution 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 tubefor 30 days.

Group 5: Diabetic rats given CFEt (0.45 g/kg body weight)in 1 ml of aqueous solution daily using an intragastric tubefor 30 days.

Group 6: Diabetic rats given glibenclamide (600 µg/kg bodyweight) [20] in 1 ml of aqueous solution daily using anintragastric tube for 30 days.

Biochemical studies in brain

Animals were sacrificed at the end of 30 days by cervicaldislocation. Blood was collected in tubes containing potas-sium oxalate and sodium fluoride solution for the estimationof blood glucose and plasma was separated for assay of insu-lin. The entire brain was perfused immediately with ice-cold0.9% sodium chloride. TBARS, hydroperoxides, superoxidedismutase, catalase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione were estimated in brain.Brain was selected as it continuously generates large amountsof free radicals from mitochondrial oxidative activity and

25

catecholamine catabolism. At the same time brain containshigh levels of polyunsaturated fatty acids, which are the pre-ferred targets of free radical damage in cell [21].

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 andthe precipitate was removed after centrifugation. 1 ml of su-pernatant was mixed with 4 ml of O-toluidine reagent and keptin boiling water bath for 15 min and cooled. The absorbancewas 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 bythiobarbituric acid reactive substances (TBARS) and hydro-peroxides by the method of Nichans and Samuelson [23] andJiang et al. [24], respectively. In brief, 0.1 ml of tissue homoge-nate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1ratio) 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 1000rpm. The absorbance of clear supernatant was measured againstreference blank at 535 nm and expressed as mM/100 g tissue.

Hydroperoxides was expressed as mM/100g tissue. 0.1 mlof tissue homogenate was treated with 0.9 ml of Fox reagent(88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenolorange and 9.8 mg ammonium ion sulphate were added to90 ml of methanol and 10 ml 250 mM sulphuric acid) andincubated at 37°C for 30 min. The colour developed was readat 560 nm colorimetrically.

Determination of catalase and superoxide dismutase

Catalase (CAT) was assayed colorimetrically at 620 nm andexpressed as µmoles of H

2O

2 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 mlof tissue homogenate and 0.4 ml of 2M H

2O

2. The reaction

was stopped by the addition of 2.0 ml of dichromate-aceticacid reagent (5% potassium dichromate and glacial aceticacid were mixed in 1:3 ratio).

Superoxide dismutase (SOD) was assayed utilizing thetechnique of Kakkar et al. [26]. A single unit of enzyme wasexpressed as 50% inhibition of NBT (Nitroblue tetrazolium)reduction/min/mg protein.

Determination of glutathione peroxidase and reducedglutathione

Glutathione peroxidase (GPx) activity was measured by themethod described by Rotruck et al. [27]. Briefly, reactionmixture 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 mlglutathione, 0.1 ml of 0.2 mM hydrogen peroxide. The con-tents were incubated at 37°C for 10 min. The reaction wasarrested by 0.4 ml of 10% TCA, and centrifuged. Superna-tant was assayed for glutathione content by using Ellmansreagent (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 methodof Ellman [28]. 1.0 ml of supernatant was treated with 0.5ml of Ellmans reagent and 3.0 ml of phosphate buffer (0.2M, pH 8.0). The absorbance was read at 412 nm. Glutath-ione peroxidase activity was expressed as µg of GSH con-sumed/min/mg protein and reduced glutathione as mg/100g of tissue.

Determination of glutathione-S-transferase

The glutathione-S-transferase (GST) activity was deter-mined spectrophotometrically by the method of Habig et al.[29]. The reaction mixture (3 ml) contained 1.0 ml of 0.3 Mphosphate buffer (pH 6.5), 0.1 ml of 30 mM CDNB and 1.7ml of double distilled water. After pre-incubating the reac-tion mixture at 37°C for 5 min, the reaction was started bythe addition of 0.1 ml of tissue homogenate and 0.1 ml ofglutathione as substrate. The absorbance was followed for5 min at 340 nm. Reaction mixture without the enzyme wasused as blank. The activity of glutathione-S-transferase isexpressed as mmoles of GSH-CDNB conjugate formed/min/mg protein using an extinction coefficient of 9.6 mM–1 cm–

1.

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 wasperformed using ANOVA followed by Duncan’s MultipleRange Test (DMRT). A value of p < 0.05 was considered toindicate a significant difference between groups [31].

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Results

The results of the present investigation are depicted in Tables1–4. The treatment with aqueous extract of Cassia auriculataflowers reversed the weight loss in diabetic rats.

Table 1 demonstrates the levels of blood glucose and plasmainsulin in normal and experimental animals. There was a sig-nificant increase in blood glucose and a significant decreasein the level of plasma insulin in diabetic rats. The effects ofadministration of CFEt at 0.45 g/kg body weight of CFEt andglibenclamide tend to bring the blood glucose and plasmainsulin towards normal value. The effect of CFEt at a doseof 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 forfurther biochemical studies.

TBARS and hydroperoxides (Table 2) from brain homoge-nate were significantly decreased with CFEt and glibenclamidetreatment whereas, diabetic control rats showed significantlyincreased levels of lipid peroxidation products.

For studying the effect of CFEt on free radical production,the activities of superoxide dismutase, catalase, glutathioneperoxidase, glutathione-S-transferase and reduced glutathionewere measured (Tables 3 and 4). They presented significantincreases in CFEt treatment when compared with diabeticcontrol rats.

Table 4. Change in levels of glutathione peroxidase, glutathione-S-transferase and reduced glutathione in brain of normal and experimental animals

Groups Glutathione peroxidase Glutathione-S-transferase Reduced glutathione(UnitsA/mg protein) (UnitsB/mg protein) (mg/100 mg tissue)

Normal 3.13 ± 0.25a 5.55 ± 0.38a 34.39 ± 2.24a

Diabetic control 1.06 ± 0.08b 0.89 ± 0.03b 19.50 ± 1.74b

Diabetic + Cassia auriculata (0.45 g/kg) 2.85 ± 0.17c 2.65 ± 0.24c 29.79 ± 2.83c,e

Diabetic + glibenclamide (600 µg/kg) 1.70 ± 0.14d 1.64 ± 0.15d 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). Duncanprocedure, range for the level 2.95, 3.09, 3.20. Aµg of GSH consumed/min. Bµmoles of CDNB–GSH conjugate formed/min.

Table 1. Blood glucose, plasma insulin and changes in body weight of normal and experimental animals

Groups Body weight Fasting blood glucose (mg/dl) Plasma insulin (µU/ml)

Initial Final

Normal 196 ± 10.40 208 ± 9.80 97.50 ± 8.04a 16.03 ± 1.04a

Diabetic control 201 ± 15.70 151 ± 13.66* 232.00 ± 15.40b 4.35 ± 0.95b

Diabetic + Cassia auriculata (0.15 g/kg) 193 ± 17.70 198 ± 15.33** 216.66 ± 20.80b 4.90 ± 0.41b

Diabetic + Cassia auriculata (0.30 g/kg) 198 ± 18.30 208 ± 10.32** 158.60 ± 14.20c 7.05 ± 0.64c

Diabetic + Cassia auriculata (0.45 g/kg) 202 ± 19.68 214 ± 12.72** 113.30 ± 10.30a,d 14.16 ± 0.67d

Diabetic + glibenclamide (600 µg/kg) 195 ± 11.80 206 ± 13.43** 124.60 ± 10.32d 12.70 ± 0.65e

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 groupswere compared with diabetic control, **p < 0.001.

Table 2. Change in the levels of brain TBARS and hydroperoxides in nor-mal and experimental animals

Groups TBARS Hydroperoxides(mM/100 g tissue) (mM/100 g tissue)

Normal 1.00 ± 0.09a 110.70 ± 5.20a

Diabetic control 1.75 ± 0.06b 127.90 ± 1.70b

Diabetic + Cassia auriculata(0.45 g/kg) 1.20 ± 0.08c 115.12 ± 4.26c

Diabetic + glibenclamide(600 µg/kg) 1.39 ± 0.08d 119.20 ± 4.05d

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

Table 3. Change in activities of catalase (CAT) and superoxide dismutase(SOD) in brain of normal and experimental animals

Groups Catalase Superoxide dismutase(UnitsA/mg protein) (UnitsB/mg protein)

Normal 3.08 ± 0.30a 8.75 ± 0.58a

Diabetic control 0.96 ± 0.07b 5.57 ± 0.40b

Diabetic + Cassia auricu-lata (0.45 g/kg) 2.63 ± 0.25c 7.46 ± 0.56c

Diabetic + glibenclamide(600 µg/kg) 1.96 ± 0.18d 6.50 ± 0.40d

Values are given as mean ± S.D. for 6 rats in each group. Values not shar-ing a common superscript letter differ significantly at p < 0.05 (DMRT).Duncan procedure, range for the level 2.95, 3.09, 3.20. Aµmole of H2O2

consumed/min. BOne unit of activity was taken as the enzyme reaction,which gave 50% inhibition of NBT reduction in 1 min.

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Discussion

Health and disease are parameters of the effectiveness withwhich human groups adapt to their environments. The herbalsoccupied a distinct place in the life right from the primitivetill today [32]. NIDDM is characterized by a deficiency ininsulin secretion associated with an insulin resistance of pe-ripheral tissue [33]. The involvement of free radicals in dia-betes and the role of these toxic species in lipid peroxidationand the antioxidant defense system have been studied. Forthe study of antidiabetic agents, streptozotocin induced hyper-glycemia in rodents is considered to be good preliminaryscreening type II diabetic model [34] and is widely used.Sptreptozotocin, N-{methylnitrocarbamoyl}-D-glucosamineis a potent methylating agent for DNA and acts as nitric ox-ide donor in pancreatic cells. β-cells are particularly sensi-tive to damage by nitric oxide and free radical because of theirlow levels of free radical scavenging enzymes [35, 36].

In our present study we have observed that an aqueous ex-tract of CFEt can reverse these effect. The possible mechanismby which CFEt brings about its antihyperglycemic action maybe by stimulation of surviving β-cells to release more insulin.This was clearly evidenced by the increased level of insulin indiabetic rats treated with CFEt. In this context a number ofother plants have also been observed to have antihypergly-cemic and insulin-release stimulatory effect [20, 37].

Earlier studies in this lab have demonstrated a defectivemetabolism of lipid peroxides in other tissues of diabeticanimal [37]. TBARS and hydroperoxides (lipid peroxidativemarkers) showed high lipid peroxidation. This may be be-cause, the brain contains relatively high concentration ofeasily peroxidizable fatty acids [38]. In addition, it is knownthat certain regions of the brain are highly enriched in iron,a metal that, in its free form, is catalytically involved in pro-duction of damaging oxygen free radical species [39]. Vul-nerability of brain to oxidative stress induced by oxygen freeradicals seems to be due to the fact that, on one hand, the brainutilizes about one fifth of the total oxygen demand of the bodyand on the other, that it is not particularly enriched, whencompared with other organs, in any of the antioxidant en-zymes. Relatively low levels of these enzymes may be re-sponsible in part for the vulnerability of this tissue [39].

Associated with the changes in lipid peroxidation diabeticbrain showed decreased activity of the key antioxidant en-zymes, superoxide dismutase, catalase, glutathione peroxi-dase, glutathione-S-transferase and reduced glutathione,which play an important role in scavenging the toxic inter-mediates of incomplete oxidation. A decrease in the activityof these enzymes can lead to an excess availability of super-oxide anion (O2.–) and hydrogen peroxide in the biologicalsystems, which in turn generate hydroxyl radicals, resultingin initiation and propagation of lipid peroxidation [40]. Treat-

ment with CFEt increased the activity of enzymes and mayhelp to control free radicals, as Cassia auriculata has beenreported to be rich in flavonoids [16], well-known antioxi-dant, which scavenge the free radicals generated during dia-betes. The increase in superoxide dismutase activity mayprotect catalase and glutathione peroxidase against inactiva-tion by O2.– anions as these anions have been shown to inac-tivate catalase [41] and glutathione peroxidase [42].

The pathophysiological consequences owing to depletionof GSH have been well studied. The depletion of GSH pro-motes generation of reactive oxygen species and oxidativestress with cascade of effects thereby affecting functional aswell as structural integrity of cell and organelle membranes[43]. Depletion of GSH results in decreased activity of en-zymes namely, GPx and GST. The GST catalyses the conju-gation of GSH with a large number of electrophiles as a stepto detoxify these species. It has been proposed that GPx isresponsible for the detoxification of H

2O

2 in low concentra-

tion whereas catalase comes in to play when GPx pathway isreaching saturation with the substrate [44]. Administration ofCFEt increased the GSH content. The elevated level of GSHprotects cellular proteins against oxidation through glutathioneredox cycle and also directly detoxifies reactive oxygen spe-cies generated from exposure to streptozotocin [45]. The sig-nificant increase in GSH content and GSH dependent enzymesGPx and GST in diabetic rats treated with CFEt indicates anadaptive mechanism in response to oxidative stress.

Although a causal relationship is not yet established thereis an inverse correlation between the lower content of GSHand the higher level of lipid peroxides [45]. Accordingly, inthis study we have found that CFEt treatment increased theGSH levels and other enzymatic antioxidants significantlyand diminished lipid peroxidation in brain.

It may be concluded that in diabetes, brain tissue wasmore vulnerable to oxidative stress and showed increasedlipid peroxidaton. The antioxidant responsiveness mediatedby Cassia auriculata may be anticipated to have biologi-cal significance in eliminating reactive free radicals thatmay otherwise affect normal cell functioning. Detection ofantihyperglycemic activity in CFEt along with protectiveeffect against streptozotocin challenge and preventive actionon lipid peroxidation provides a scientific rationale of use ofCassia auriculata as an antidiabetic plant.

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