Antihyperglycemic and Antioxidant potentials Psidium guajava leaf extract and its isolated fraction in alloxan-induced diabetic rats

M. Shakeera Banu¹, K. Sujatha², W. Sherly Beena³ and N. Divya⁴

¹P.G. Department of Biotechnology, Sree Narayana Guru College, Coimbatore, Tamilnadu, India.

²P.G. and Research Department of Zoology, Governement Arts College, Tamilnadu, India.

³P.G. Department of Biochemistry, M.I.E.T. Arts and Science College,Tiruchirappalli, Tamilnadu, India.

⁴P.G. Department of Biochemistry, Pavendar Bharathidasan College of Arts and Science, Tiruchirappalli, Tamilnadu, India.

*Corresponding Author E-mail: sakeeramsb@gmail.com

ABSTRACT:

In diabetes, hyperglycaemia generates reactive oxygen species, which in turn cause lipid peroxidation and membrane damage and these free radicals play an important role in the production of secondary complications in diabetes. Wistar albino rats aged 6-8 week were selected for the experiments and these were divided into five groups. Diabetes was induced by alloxan. Blood glucose and hepatic antioxidant levels were measured. Psidium guajava and its isolated fraction restored the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). It further decreased the hepatic lipid peroxidation as evidenced by decreased thiobarbituric acid reactive substances (TBARS) level with a concomitant increase in the reduced glutathione (GSH). Psidium guajava has a significant antihyperglycemic effect, and that this effect is associated with its antioxidative activity.

KEYWORDS: Diabetes, antioxidant, free radicals, Psidium guajava

INTRODUCTION:

The occurrence of diabetes mellitus, especially type 2 diabetes mellitus, is increasing at an epidemic rate worldwide ¹. Oxidative stress in diabetes leads to tissue damage, with lipid peroxidation, inactivation of proteins, and protein glycation as intermediate mechanisms ² for complications including retinopathy, nephropathy, and coronary heart disease ^{3, 4, 5, 6}.

Antioxidants have been shown to prevent the destruction of β-cells ^{7, 8}by inhibiting the peroxidation chain reaction and thus they may provide protection against the development of diabetes ^{9, 10, 11}. Implication of oxidative stress in the pathogenesis of diabetes is suggested not only by oxygen free radical generation but also due to non-enzymatic protein glycosylation, auto-oxidation of glucose, impaired antioxidant enzymes, and formation of peroxides ^{12,
13, 14}.

Since time immemorial the use of plants and plant based food materials in the management of diabetes has been prevalent in the Indian society.

Several medicinal plants have been reported to possess potential hypoglycemic activity in Indian system of medicine. The common guava tree (Psidium guajava Linn.) is a member of the Myrtaceae family, which is native to tropical and subtropical countries. Interestingly, guava leaves have also attracted attention as a folk remedy for diabetes not only in Japan and East Asia ^{15, 16, 17}. The present study aimed to investigate the possible antihyperlbycemic and antioxidative potentials of Psidium guajava in alloxan-induced diabetic rats.

MATERIALS AND METHODS:

Collection and preparation of plant material

Fresh leaves of Psidium guajava were collected in Coimbatore, during the months of April-May. The plant was authenticated by Botanist at the Government Arts College, Coimbatore. Plant material was dried under shade at room temperature, pulverized by a mechanical grinder and sieved through 40 meshes, then stored in airtight closed bottles until required.

Extraction and isolation

The coarse powder (100 g) was extracted successively with ethanol (250 ml) by hot continuous percolation method in a Soxhlet apparatus for 24 hrs.

The extracts was concentrated and re-concentrated in petroleum ether (40°-60°C) (fraction-I), diethyl ether (fraction-II) and ethyl acetate (fraction-III) in succession in the ratio of 1:2:1. Each of the steps was repeated three times to ensure complete extraction in each case. Fraction I was rejected since it was rich in fatty substances whereas fraction II was analysed for the free flavonoids in each of the samples. Fraction III of each of the test samples was hydrolysed by refluxing with 7% H₂SO₄ (10 ml/gm residue) for 5 hrs. The mixture was filtered and the filtrate extracted with ethyl acetate in a separating funnel. The ethyl acetate layer was washed with distilled water till neutrality and dried in vacuo. The residues were taken up in small volumes of ethanol separately and then the fraction was subjected to TLC to confirm the isolated fraction (quercetin).

Introduction of experimental diabetes

Hyperglycemia was induced by intraperitoneal injection of freshly prepared aqueous solution of alloxan monohydrate (SD fine Chemicals Pvt. Ltd., Biosar) 150 mg/kg, to overnight fasted rats except control group. Diabetes was confirmed after 48 hour, animals with plasma glucose level above 150 mg/dl (diabetic) were selected for the study. The diabetic animals were allowed free access to tap water and pellet diet and were maintained at room temperature in plastic cages.

Experimental design

Animals were classified into five groups of six rats each. Group I: served as control and received normal saline (2 ml/kg body weight). Group II: treated with alloxan monohydrate 150 mg/kg served as diabetic control. Group III: treated with ethanolic leaf extract of Psidium guajava alone (500 mg/kg body weight). Group IV: treated alloxan monohydrate (150 mg/kg) and ethanolic leaf extract of Psidium guajava (500 mg/kg body weight). Group V treated alloxan monohydrate (150 mg/kg) and isolated fraction of Psidium guajava 10 mg/kg body weight). At the end of the treatment period (21^st day), the animals in all the groups were sacrificed, dissected, and bled using the orbital technique ¹⁸ and cardiac puncture. Blood samples for the different tests were collected.

Sample preparation for biochemical estimations

The blood sample was allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm at 30°C for 15 min. Haemolysis-free serum samples were stored at -70°C before determination and utilized for the estimation of various biochemical parameters. Livers were excised, washed thoroughly in ice-cold saline to remove the blood. They were then gently blotted between the folds of a filter paper and weighed in an analytical balance. Ten percent of homogenate was prepared in 0.05 M phosphate buffer (pH 7) using a polytron homogenizer at 20°C. The homogenate was centrifuged at 3000 g for 20 min to remove the cell debris, unbroken cells, nuclei, erythrocytes and mitochondria. The supernatant was used for further hepatic biochemical assays.

The level of glucose in serum was analyzed by the method of Sasaki and Matsui ¹⁹. Hepatic superoxide dismutase (SOD) by the method of Kakkar et al. ²⁰, catalase (CAT) by the method of Sinha ²¹, glutathione peroxidase (GPx) by the method of Rotruck et al. ²² and reduced glutathione (GSH) by the method of Ellman ²³. Lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) by the method of Niehaus and Samuelsson ²⁴.

DISCUSSION:

Oral administration of the Psidium guajava leaf extract showed a significant decrease in blood glucose level. It also showed an improved antioxidant potential as evidenced by decreased lipid peroxidation and a significant increase in the activity of various antioxidant enzymes such as CAT, SOD, GPx and GSH.

Mechanistically, Psidium guajava protected pancreatic tissues, including islet β-cells, against lipid peroxidation and DNA strand breaks induced by STZ, and thus reduced the loss of insulin-positive β-cells and insulin secretion. Moreover, Psidium guajava also markedly inhibited pancreatic nuclear factor-kappa B protein expression induced by STZ and restored the activities of antioxidant enzymes, including SOD, CAT, and GPx ²⁵.

The present results showed that administration of fraction of Psidium guajava leaves significantly decreased fasting blood glucose levels and improved the antioxidant status. Lipid peroxidation formation was suppressed by isolated fraction of Psidium guajava. The findings of the present study indicate that isolated fraction exerts protective effects on alloxan-induced diabetes and its complications. Administration of 15 mg/kg/day of isolated quercetin-3-O-glucoside for 10 consecutive days to the hyperglycemic animals showed the decrease serum glucose and increase insulin level and simultaneously inhibited the activity of hepatic glucose-6-phosphatase. It further decreased the hepatic and renal LPO with a concomitant increase in the activities of antioxidative enzymes, such as CAT and SOD and GSH, indicating its safe and antiperoxidative effects ²⁶.

Quercetin treatment of diabetic rats reversed only the diabetic effects on brain oxidized glutathione concentration and on hepatic GPx activity. By contrast, a 20% increase in hepatic lipid peroxidation, a 40% decline in hepatic glutathione concentration, an increase in renal (23%) and cardiac (40%) GPx activities, and a 65% increase in cardiac CAT activity reflect intensified diabetic effects after treatment with quercetin ²⁷.

Treatment with quercetin significantly attenuated renal dysfunction and oxidative stress in diabetic rats ²⁸. Oral administration of quercetin to diabetic rats resulted in a decrease in the levels of blood glucose, plasma TBARS and hydroperoxides. Quercetin also resulted in the activities of SOD, CAT coming to near normal, along with the levels of vitamin C and vitamin E ²⁹.

Coskun et al. ³⁰ evaluated the possible protective effects of quercetin against beta-cell damage in experimental STZ-induced diabetes in rats. Quercetin treatment has shown protective effect possibly through decreasing lipid peroxidation, NO production and increasing antioxidant enzyme activity (SOD, CAT, and GPx). Increased staining of insulin and preservation of islet cells were apparent in the quercetin-treated diabetic rats.

CONCLUSION:

Thus the data of the results of studies conducted in the present work clearly depicted that the Psidium guajava leaf extract and its isolated fraction possess significant antihyperglycemic and antioxidant against alloxan-induced diabetes. This may be due to the phytochemical content of the plants and as such make them potential candidates as natural chemoprophylactic agents.

ACKNOWLEDGEMENT:

I gratefully acknowledge Dr. G. Sridharan, Assistant Professor, Department of Biochemistry, Srimad Andavan Arts and Science College, Tiruchirappalli, for providing me the facility to do the experiments and his useful guidance.

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Received on 23.03.2012 Modified on 30.03.2012

Research J. Pharm. and Tech. 5(4): April 2012; Page 541-546