U.S.Mahadeva Rao et al./ Journal of Pharmacy Research 2011,4(10),3630-3634 Research Article ISSN: 0974-6943 Available online through www.jpronline.info Comparative Biochemical Evaluation of Hypoglycemic and Free Radical Scavenging activities of Noni and Spirulina in Streptozotocin-Induced Diabetic Rat Tissues R.Babujanarthanam 1, R.Balabhaskar 2, & U.S.Mahadeva Rao*3, Department of Biochemistry, K.M.G. College of Arts and Science, Gudiyattam, India. 2. PG Department of Biochemistry, SRM Arts and Science College, Kattankulathur,India. 3. Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia. 1. Received on: 19-06-2011; Revised on: 08-07-2011; Accepted on:01-10-2011 ABSTRACT The present study was aimed to investigate the hypoglycemic property and free radical scavenging activity (FRSA) of aqueous extracts of Noni fruit and Spirulina evaluated in Streptozotocin (STZ) induced diabetic rat tissues. Dosage : The Noni fruit and Spirulina aqueous extracts at a concentration of 300mg and 15mg /kg of body weight/rat/day respectively was orally administrated to STZ –induced diabetic rats for a period of 30 days. Methods : The blood glucose, plasma insulin, glycosylated hemoglobin, serum creatinine, and blood urea estimations were carried out. Histopathological studies were done in pancreatic tissues. Lipid peroxidation as well as anti-oxidant activity (enzymatic and non-enzymatic) assays were performed. Results and Discussion: The elevated levels of blood glucose, glycosylated hemoglobin, serum creatinine, and blood urea and diminished concentration of insulin in diabetic rats reverted back to near normal after treatment with aqueous extracts. Histopathological studies of the pancreas showed the protective role of Noni and Spirulina aqueous extracts. Elevation of Lipid peroxidation products and depletion of anti-oxidant activity in diabetic rats reverted back to near normal after treatment with aqueous extracts confirm the potent FRSA. Conclusion: From the comparative observation, it was hypothesized that aqueous extract of Noni fruit shows more effective antidiabetic property as well as diminished lipid peroxidation and elevated anti-oxidant activity than Spirulina extract. Both the extracts were compared with reference drug Metformin. Keywords : Noni, Spirulina, Metformin, Streptozotocin, Diabetes mellitus. INTRODUCTION Diabetes mellitus is one of the most common endocrine metabolic disorders. It is the most prevalent chronic diseases in the world affecting markedly 25% of the population (1). All forms of diabetes are due to a decrease in the circulating concentration of insulin and a decrease in the response of peripheral tissues to insulin, that is, insulin resistance. (2). According to the world health organization projections, the prevalence of diabetes is likely to increase by 35% by the year 2025. Several drugs such as biguanides and sulphonylureas are presently available to reduce hyperglycemia in diabetes mellitus. These drugs have side effects and thus searching for a new class of compounds is essential to overcome these problems (3). Plants have always been usable sources of drugs and many currently available drugs are directly or indirectly derived from plants (4). Noni (Morinda citrifolia) is the most popularly used medicinal plant for the treatment of diabetes mellitus because of its hypoglycemic and free radical scavenging property (5). It belongs to Rubiaceae family and commonly known as Indian mulberry. Noni is a small evergreen tree that grows widely across Polynesia. All parts of the plant have been used in traditional medicine. The fruit juice is high in demand in alternative medicine for different kinds of illness such as arthritis, cancer, AIDS, gastric ulcers, mental depression and poor digestion. However scientific evidences for the pharmacological properties of Noni are limited. Their natural habitats are volcanic terrains, and lava coast (6).Spirulina is a microscopic blue – green algae in the shape of a spiral coil, belongs to Phormidiaceae family living both in sea and fresh water. It is produced primarily from two species of cyanobacteria, Arthrospira platensis and Arthrospira maxima. It occurs naturally in tropical and subtropical lakes with high pH. It helps in strengthening the immune system, making the body produce red and white cells, which kills viruses and germs, increasing free radical scavenging protection. Spirulina contains high amount of protein, between 55% and 77% by dry weight, depending upon the source. It is a complete protein, containing all essential amino acids, though with reduced amounts of methionine, cysteine, and lysine when compared to the proteins of meat, eggs, and milk. The free radicals generated in human body can lead to ailments like cancer, arthritis, cataracts and premature ageing. The antioxidants like Vitamins A, E, and minerals like Zinc and Selenium effectively neutralise these free radicals (7). Metformin hydrochloride is a biguanide antidiabetic agent used as a reference drug. Recently Zhou et al demonstrated the activation of 5’- AMP activated protein kinase (AMPK) by Metformin in both hepatocytes and skeletal muscles. It decreases hyperglycemia by activating AMPK pathway (8). The present comparative pharmacological efficacy study was aimed to investigate the hypoglycemic property and free radical scavenging activity (FRSA) of aqueous extracts of Noni fruit and Spirulina with reference to Metformin drug, evaluated in Streptozotocin (STZ) induced diabetic rat tissues. MATERIALS AND METHODS Experimental animals Male albino Wister rats (180 ± 20g) were used in this study. The animals were fed on a standard pellet diet (Pranav Agro Industries, Pune, India) and water. The pellet diet consisted of 22.02% crude protein, 4.25% crude oil, 3.02% crude fiber, 7.5% ash,1.38% sand silica), 8% calcium, 0.6% phosphorus, 2.46% glucose, 1.8% vitamins and 56.17% carbohydrates. It provides a metabolisable energy of 3600kcal/kg. They were maintained in a controlled environment (12/ 12h light/dark cycle) and temperature (30 ±?20C). The experiment was carried out according to the guidelines of the Committee for the purpose of Control and Supervision of Experiment of Animals (CPCSEA), New Delhi, India. Toxicity and dosage fixation studies Acute toxicity studies with Noni fruit and Spirulina aqueous extracts were performed in experimental rats. Graded doses of Noni fruit and Spirulina (100, 300, 500, and 1000 mg Noni/kg body weight) (5, 10, 15, 20, 50, and 100 mg Spirulina / kg b.w.) were administered orally, and the animals were subsequently observed for 2 weeks. Changes in body weight, food consumption, haematological, and biochemical findings, including the activities of enzymes, were noted (Data not shown). Dosage fixation studies were carried out by virtue of unequally long administration of graded doses of Noni fruit and Spirulina aqueous extract, given to rats introduced into STZ-induced hyperglycaemia; it was found that these two extracts showed their maximal hypoglycaemic and FRSA effect at the concentration of 300 mg and 15 mg respectively /kg body weight administered orally for 30 days. Hence, the dosage was fixed at 300 mg/kg body weight /rat/day for Noni and 15 mg/kg body weight /rat/day for Spirulina pursued for 30 days. *Corresponding author. Associate Professor, Faculty of Medicine and Health Science, Universititi Sultan Zainal Abidin, 20400 Kuala Terengganu, Terengganu, Malaysia. Phone: 00 60 09 6275680(Direct) Hand phone: 00 60 11 16547654 Fax: 00 60 09 622 0021 E-mail :
[email protected] Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3630-3634 Biochemical assay Whole blood was used for glucose (9) and urea (10) estimation.35±0.68c* 6. Levels of glycosylated hemoglobin were estimated according to the methods of Nayak & Pattabiraman (11). the animals were grouped as follows: Group I: Normal control. (13).05) increase in the activities of these antioxidant enzymes in the tissues (Table 3).Mahadeva Rao et al.3630-3634 Induction of experimental diabetes Rats were fasted overnight and experimental diabetes was induced by single intra peritoneal injection of STZ with a dose of 50mg/kg b.37±0. blood glucose concentration >250mg/ dl) that exhibited glycosuria were selected for the experiment. Group V: Diabetic + Metformin (Reference drug) aqueous extract (25mg/kg b.1M cold citrate buffer. TBARS and Hydroperoxides increased significantly (p<0.46±0.74±1. /day) treated for 30days.98±0. and then homogenized in Tris-HCl buffer (pH 7.21c* 7.65±0. All of the results are expressed as means ± standard deviation (SD) referring to six rats in each group.75±0.46±0.57a* 57±2. Experimental design For the present study. /day) treated for 30days. Blood Urea and Serum Creatinine in the Control and Experimental Groups Groups Blood Glucose(m g/dl) 95.67±0.31b* 5. glycosylated haemoglobin. After 72 h of STZ injection.1M cold citrate buffer pH 4.55d* GST 7.4. Spirulina and Metformin on lipid peroxidation profile in Normal and Experimental groups of rats Groups Group I Group II Group III Group IV Group V TBARS (mM/100 g of tissue) 0.55c* 128.25 11.w.55±1. Noni and Spirulina treated rats exhibited a significant (p<0.21±0. STZ-administrated rats were provided with 10% glucose solution after 6h for the next 24h to prevent hypoglycemia.3a* 122.74± 0.5.69 5.59 98.w.36c* 79±5.. pH 4.28b* 9.12±0.23±1.03 1.63±0.34b* 34.09a* 8. Enzymatic antioxidants such as superoxide dismutase (SOD) (18).21±1. units per mg of protein for GST II.68±0. For Histopathological studies.31a* 14.24±7. the activities of SOD. the sections were observed under light microscope.71±0. Table 1 : Levels of Blood Glucose.05) in the activities of non enzymatic antioxidants (Table 4).37 289.26±0.48a* 75±5.18±0.43b* 85±4.08c* 4.12b* 3. /day) treated for 30days.4) using a Teflon homogenizer.49b* 140±6.17±0. washed in ice-cold saline.05) decreased. the rats were fasted overnight and sacrificed by cervical decapitation.06b* 1. Liver homogenate was then centrifuged at 5. µ moles of glutathione oxidized per min per mg of protein for GPx.09c* 1. and glutathione (GSH) were determined using the methods of Omaye et al.06 c* 0.46d* GPx 12.58±0. while hydro-peroxides were estimated using the method established by Jiang et al.07a* 5.08±3.97±0.52±0. The levels of non enzymatic free radical scavengers such as ascorbic acid. Plasma was separated and used for insulin radioimmunoassay (RIA) kit for rats (Linco Research. After staining. (14).28d* CAT 73.05).73c* 51±1. Plasma Insulin.07d* Hydroperoxides (mM/100 g of tissue) 72. RESULTS Effect of Noni and Spirulina aqueous extracts on biochemical parameters : The blood glucose levels.05 b* 0. Glycosylated Hemoglobin.000 x g to remove cellular debris.w.17 2.78±0.. catalase (CAT) (19).03±0.58±0. Because STZ is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release. blood was withdrawn from animals fasted over night in tubes containing potassium oxalate and sodium fluoride as anticoagulant. Journal of Pharmacy Research Vol.17±0. blood urea and serum creatinine levels and brought the levels to near normal.18±6. Statistical analysis All-group data were statistically evaluated using SPSS 16.45b* 5.45 43. Spirulina and Metformin on enzymatic free radical scavenging profile in Normal and Experimental groups of rats Groups Group I Group II Group III Group IV Group V SOD 12.09a* 8.56±1. Desai (16). CAT. blood urea and serum creatinine levels were significantly increased. Vitamin E and GSH) was found to be significantly decreased (p<0.56±0. Control rats were similarly injected with 0.79±0. The p-value of <0.47±0. USA). and photographs were taken. Lipid peroxidation was determined using thiobarbituric acid reactive substances (TBARS) by the method of Ohkawa et al.72b* 11. and plasma insulin levels significantly decreased in diabetic control rats. The hypothesis-testing methods included One-Way Analysis of Variance followed by the Least Significant Difference (LSD) test. Group II: Normal + Streptozotocin (50mg/kg b.23d* After the treatment period of 30 days. Pancreases were removed immediately from the rats.w.45±1.e. paraffin sections of 5 µ m thickness were made and stained with hematoxylin – eosin (H&E) stain. rats with moderate diabetes (i. Noni and Spirulina treated diabetic rats showed significant increase (p<0.03 1. Group IV: Diabetic + Spirulina aqueous extract (15mg/kg b.46±0.05 was considered statistically significant..89c* 13. Neither death nor any other adverse effect was observed.81 33.02±0.29d* Plasma Insulin (µU mL-1 16.52±0. October 2011 3630-3634 .) induced (Diabetic) (single dose) Group III: Diabetic + Noni fruit aqueous extract (300mg/kg b.35c* 7. The plasma insulin levels were significantly increased in diabetic rats treated with Noni and Spirulina (Table 1). After 3 days for the development and aggravation of diabetes.42d* Serum Creatinine (mg/dl) 0.98a* 26. GPx and GST were significantly (P<0.5.U.08±0.43a* 6.05±0.w. Table 2:Effect of Noni.60±0. glutathione peroxidase (GPx) (20) and glutathione-S-transferase (GST) (21) were assayed in the liver supernatant. The separated serum was used for the purpose of determination of creatinine levels (12).93±0. Values are given as means ±SD for six rats in each groupValues are statistically significant at *p<0.Effect of Noni and Spirulina on non enzymatic antioxidants: The concentration of non enzymatic antioxidants (Vitamin C.71d* Blood Urea (mg/dl) 25. the supernatant was thence used for the determination of lipid peroxide and both enzymatic and non-enzymatic antioxidant levels.Effect of Noni and Spirulina on enzymatic anti oxidants: In diabetic rats.56c* 28. (15).50d* Glycosylated Hemoglobin (% Hb) 5. The liver tissue was excised. Blood was collected and plasma was obtained after centrifugation and used for biochemical estimations. Inc.24±4. µ moles of hydrogen peroxide decomposed per min per mg of protein for CAT.08a* 1. / Journal of Pharmacy Research 2011.05 Statistical significance was compared within the groups as follows :a Diabetic rats were compared with control rats b Noni treated diabetic rats were compared with diabetic rats c Spirulina treated diabetic rats were compared with diabetic rats d Metformin treated diabetic rats were compared with diabetic rats Effect of Noni and Spirulina on anti oxidant status I.. STZ was dissolved in a freshly prepared 0. and Sedlak and Lindsay (17).35±0.05 Statistical significance was compared within the groups as follows : a Diabetic rats were compared with control rats b Noni treated diabetic rats were compared with diabetic rats c Spirulina treated diabetic rats were compared with diabetic rats d Metformin treated diabetic rats were compared with diabetic rats Enzyme activities were expressed as 50% inhibition of epinephrine auto oxidation per min for SOD. Table 3: Effect of Noni. Diabetic rats when treated with Noni and Spirulina decreased the glucose.05) in diabetic rats. tocopherol.02 a* 0.S. glycosylated haemoglobin. rats from control and experimental groups were perfused with 10% neutral formalin solution.04 d* Group I Group II Group III Group IV Group V Values are given as means ±SD for six rats in each groupValues are statistically significant at *p<0.33 4.4(10)..00 software.27 3.05 Statistical significance was compared within the groups as follows :a Diabetic rats were compared with control rats b Noni treated diabetic rats were compared with diabetic rats c Spirulina treated diabetic rats were compared with diabetic rats d Metformin treated diabetic rats were compared with diabetic rats Effect of Noni and Spirulina on Lipid Peroxidative products: In diabetic rats.19±6. respectively.19±6. The treatment of diabetic rats with Noni and Spirulina returned the concentration of TBARS and hydroperoxides in these tissues to near normal (Table 2).06d* Values are given as means ±SD for six rats in each group Values are statistically significant at *p<0.85±6.Issue 10.21b* 40±1. Histopathology of Pancreas Figure 5: Reference drug treated DISCUSSION Diabetes mellitus is characterized by hyperglycemia. it has been shown that dietary supplementation with natural non enzymatic antioxidants such as ascorbic acid and tocopherol and reduced glutathione attenuated the oxidative stress and diabetic state induced by STZ (26). because of the relatively low expression of free radical scavenging enzymes such as CAT.45± 0. October 2011 3630-3634 . In recent years. Also.78± 0.4(10).29± 0.48± 0.21 0.23c* 36± 1. / Journal of Pharmacy Research 2011. a classical state of diabetes mellitus (27).18b* 1.Citrofolia fruit and Spirulina extracts prevent the formation of glycosylated hemoglobin.19± 4. hypertension.87± 0. The histopathological findings of Noni and Spirulina were compared with the reference drug (Figure 5).77± 1.3630-3634 Table 4: Effect of Noni.Citrofolia fruits extract and Spirulina may be due to its potent antihyperglycemic activity.75± 0. obesity. may lead to disruption of cellular function. Treatment with Noni fruit and Spirulina extracts lowered the level of blood glucose and improved the insulin level in the STZinduced diabetic rats.14d* GSH (mg/100g of tissue) 48.05 Statistical significance was compared within the groups as follows : a Diabetic rats were compared with control rats b Noni treated diabetic rats were compared with diabetic rats c Spirulina treated diabetic rats were compared with diabetic rats d Metformin treated diabetic rats were compared with diabetic rats Figure 4: Spirulina treated Effect of Noni and Spirulina on the histopathology of pancreas The histopathological examination of STZ induced diabetic pancreas showed the shrinkage of islet cells and the growth of adipose tissues in the pancreas (Figure 2). which usually produces many complications.cells may be vulnerable to ROS attack when the system is under oxidative stress situation (24). SOD. The supra-physiological concentration of glucose in diabetic state causes severe derangement in protein metabolism that result in the development of Figure 1: Normal control Figure 2: Diabetic control for SOD.05d* Vitamin E (µg/mg of protein) 2. Figure 3: Noni treated Journal of Pharmacy Research Vol. Grover and Vats (28) reported that the antihyperglycemic potential of medicinal plant extract is normally reliant on the degree of ß cell demolition.45d* Values are given as means ±SD for six rats in each group Values are statistically significant at *p<0.03b* 0. This condition favors the reduction in the level of hemoglobin and concomitant elevation in the glycosylated hemoglobin. Oxidative stress is produced under diabetic conditions and it is likely involved in progression of pancreatic ß-cell dysfunction (23). pancreatic ß. Treatment with Noni and Spirulina aqueous extracts on STZ induced diabetic rats reduced the abnormal changes in the pancreas (Figures 3 & 4).4. The antihyperglycemic potential of M. Blood glucose is a key marker for the diagnosis and prognosis of diabetes mellitus. hypoinsulinemia.Mahadeva Rao et al. Spirulina and Metformin on non enzymatic free radical scavenging profile in Normal and Experimental groups of rats Groups Group I Group II Group III Group IV Group V Vitamin C (µg/mg of protein) 1. which in turn shows that the M. such as hyperlipidemia.U.16a* 1.07c* 0. elevated levels of free radicals.87± 0. The observed decrease in the levels of glycosylated hemoglobin in diabetic rats treated with M.Issue 10.04 0. due to insufficiency of the free radical scavenging defense system. and even cardiovascular disease (22).34c* 1. Chronic glycemia results in glycosylation in which the excess glucose nonenzymatically reacts with hemoglobin to form glycosylated hemoglobin (29).98± 0.24b* 31± 1. No histopathological alterations were observed in pancreas (Figure 1) of normal control rats.02± 0.citrifolia fruit and Spirulina may be due to the stimulatory effect on the remnant ß cells to secrete more insulin or from regenerated ß cells. atherosclerosis.28a* 38± 2. GPx and GST. oxidative damages to membranes and enhance their susceptibility to lipid peroxidation (25). which is directly proportional to supra-physiological glucose (30). The insulin deficiency causes drastic elevation in the levels of blood glucose as a result of excessive production of endogenous glucose by hepatic as well as extra hepatic tissues through gluconeogenic and glycogenolytic pathway and reduced utilization of glucose by various tissues. Similarly.02a* 1.S.45± 0.49 23. tocopherol. also known as type 1 diabetes mellitus (T1DM). However. These observations suggest the anti oxidant and antilipid peroxidative potential of these extracts. The enzymatic anti oxidant enzymes such as SOD. Zhou SS. Diabetes Care. Hydroperoxides are potentially toxic molecules and capable of demolishing enzymes and cell membranes (42). (61) also reported that aqueous and alcoholic extracts of Spirulina had a free radical scavenging activity at 1 mg/ml concentration. In: The useful plants of India. Hypoglycemic activity that is found when Spirulina was given with a simultaneous glucose load in diabetic rats indicates that the extracts may interfere with the intestinal glucose absorption in the gut (34). 6. Impact of changing demography and disease prevelance in the U. Chem.Antioxidants and diabetes mellitus.E. H.Some information on the ethnobotanical properties of Noni (Morinda citrifolia). Phycocyanin has the ability to scavenge free radicals. 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The diminished activities of enzymatic antioxidants in diabetic rats have been reported (45). / Journal of Pharmacy Research 2011. The observed elevation in the level of hydroperoxides in liver may be due to diminished activities of anti oxidant enzymes. So it was inferred that Metformin has a protective effect on hepatotoxicity produced by STZ. phycocyanin protein has been expressed and the antioxidant activity is also demonstrated with the recombinant phycocyanin protein (58. The process of extraction and identification of active principles responsible for the observed pharmacological actions of both the extracts through bioactivity guided fraction is under progress to understand the possible mechanism of action. Beffe C.Opin. alkaloids.. American Diabetes Association (2005). Boyle J. 5. In our study. World J Gastroenterol. GPx. 10. Vasudha shukla et al.. Spirulina contains several active ingredients. King H. 3. and peroxyl radicals. Clarkson P. 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