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Journal of Aquatic Biology and Fisheries Vol. 2/2014/ pp. 133 to 140 ANTIDIABETIC AND ANTIOXIDANT ACTIVITY OF PADINA
TETRASTROMATICA IN HIGH CALORIE FED/STREPTOZOTOCIN
TREATED RATS
Divya S. Mohan, Mini Saraswathy, Muraleedhara Kurup and Gopala Kurup*
Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram-695581, Kerala, India. Received on: 10 October 2013, accepted on: 12 December 2013 Abstract: Type 2 diabetes mel itus is a complex, heterogeneous, and polygenic disease and the major sources of morbidity
and mortality in diabetes mellitus can be attributed to the direct and indirect effects of chronic hyperglycemia on human vasculature. Chronic hyperglycemia condition produces multiple biochemical sequels and diabetes induced oxidative stress could play an important role in the onset and progression of the disease. The present study was designed to simulate the natural history and metabolic characteristics of human T2DM in male Wistar rats and its treatment with 80 % aqueous methanolic extract of Padina tetrastromatica (PME). The rats, except the controls were fed with high calorie/energy diet for two months and then intraperitoneally injected with streptozotocin (STZ) at a dose of 15 mg/kg body weight. Effect of oral administration of graded doses of PME viz, 150, 300, 450 and 600 mg/kg body weight for 45 days on body weight, fasting blood glucose, glycated haemoglobin, serum toxicity markers, hepatic and renal antioxidant enzymes and concentration of lipid peroxidation products in serum and tissues were evaluated using standard protocols. Treatment with the extract significantly decreased the blood glucose and glycated haemoglobin in a dose dependent manner. However a more significant effect was produced by treatment with 450 mg/kg, suggesting that it is the optimum dose for acquiring good glycemic control. Activities of various toxicity markers demonstrated the non toxic nature of the extract at these doses. PME restored the diabetes induced alterations in the activities of antioxidant enzymes as well as the levels of lipid peroxidation products. The present findings clearly demonstrated the invivo antidiabetic and antioxidant activities of PME. Since no work is available in this regard, further clinical studies are warranted to recommend P. tetrastromatica as a source of drug for the management of type II diabetes mellitus.
Key words: Type 2 diabetes mellitus, Insulin resistance, Oxidative stress, Antioxidant defence system, Lipid
peroxidation, Edible brown seaweed INTRODUCTION
Type 2 Diabetes mellitus accounts for 90 to 95% rised by resistance to the action of insulin (in
of all the diagnosed cases, affecting 10 to 20% of liver, muscle and adipose tissue) that results in adults in many developed countries (Engelgau an impairment of glucose uptake, leading to et al., 2004; Bell and Polonsky, 2001). T2DM insulin hyperstimulation and subsequently to a results from a complex interaction of multiple dysfunction of pancreatic -cells (decreasing factors, namely genetic and environmental secretion of insulin) (Yki, 1995; Cheng and components, such as high-energy diets and Fantus, 2005).
sedentary lifestyle (Conget, 2002). Diabetic abnormalities include post prandial glucose When high plasma glucose concentration production by the liver (in an unregulated persists for a long time, the physiological fashion), diminished glucose uptake by the pathways of glucose (glycolysis and oxidative skeletal muscle and liver, increased fatty acid phosphorylation) saturate and glucose is generation (lipolysis) by the adipose tissue and shunted to other pathways such as polyol increased levels of digestive enzymes and pathway, diacylglycerol pathway and absorptive cells in the intestine (Bell and intracellular advanced glycation end-products Polonsky, 2001). The risk to develop macro and (AGE) pathway (Nishikawa et al., 2007; microvascular complications in T2DM is very Robertson and Harmon, 2006). All of these high, since in addition to hyperglycaemia, there pathways lead to the increase in the production is an abnormal lipid profile, (Smith, 2007; Boden of reactive species (reactive oxygen species – and Pearson, 2000). Type 2 diabetes is characte- ROS and reactive nitrogen species – RNS) which in turn increase oxidative stress and oxidative invivo antidiabetic and antioxidant activities of damage in proteins, lipids and DNA of the cells, PME in experimental diabetes mellitus.
particularly damaging pancreatic -cells and endothelial cells (Robertson and Harmon, 2006; MATERIALS AND METHODS
Jay et al., 2006). The control of postprandial Seaweed material: Padina tetrastromatica was
hyperglycemia and inhibition of oxidative stress collected freshly from the coastal rocks of have often been suggested as important Vizhinjam, Thiruvananthapuram, India (Lat. 8° measures for the treatment of diabetes.
22' N; Long. 76°59' E) during the month of One therapeutic approach to decrease January-February. Immediately after collection, postprandial hyperglycemia is to retard the the algae were thoroughly washed in tap water absorption of glucose via inhibition of to remove the epiphytes, sand and other extraneous matter. The specimen was identified carbohydrate-hydrolyzing enzymes, such as by Dr. M.V.N Panikkar, (Department of Botany, -amylase and -glucosidase, in the digestive S.N.College, Kollam, Kerala, India). A voucher organs (Yki 1990; Holman et al., 1999; Tewari et specimen (KUBH 5804) was deposited in the al., 2003). The negative effects of oxidative stress Department of Botany, University of Kerala.
may be mitigated by antioxidants (Larson, 1995; Halliwell et al., 1995). Due to concerns on the Chemicals: All the chemicals used were
toxic and carcinogenic effects of synthetic analytical grade reagents purchased from Sisco chemicals, the search for alternatives from Research Laboratories Ltd, India.
natural sources has received much interest. Preparation of seaweed extracts: The
Traditionally, due to ease of access terrestrial extract was prepared according to the sources have provided humans with these procedure of Kim et al. (2008). Briefly, the much-needed cures. However, the oceans cover algal sample (1 kg) was soaked in MeOH: H O 70% of the earth's surface and are the habitat of (8:2, v/v, 1l) and extracted under conditions an extremely rich and varied fauna and flora. where the solvent was heated until reflux Marine algae are exposed to an extreme began, this being maintained for 3 h. Each environment in a combination of light and resulting residue was dissolved in MeOH: oxygen, but the absence of oxidative damage in H O (8:2, v/v, 500 ml) and extracted twice as their structural components (Matsukawa et al., above, with the extracts individually f iltered 1997) and their stability to oxidation during using Whatman No. 1 paper. Each combined storage (Ramarathnam et al., 1995) suggest they supernatant was then concentrated under have good antioxidant defence system.
reduced pressure at a temperature of 40°C, Padina tetrastromatica Hauck is a striped, defatted with hexane and again concentrated.
yellowish brown, fan shaped alga, which grows The extract thus obtained was kept at -20°C in the coastal rocks of tropical areas. It is used and used for present study.
as seasoning in dried flake form and as table Experimental animals: Male Wistar rats
salt replacement for high blood pressure patients from the same breed with a mean body weight (Novaczek and Athy, 2001). Studies from our of about 100 g were selected for the study. The laboratory reported that sulphated fucan from animals were bred and kept in the animal P.tetrastromatica possesses anti-oxidative and house, Department of Biochemistry, University anti-inflammatory activities against carrageenan of Kerala. They were housed in polypropylene induced paw edema in rats (Mohsin et al., 2011). cages under standard environmental However, to the best of our knowledge, no conditions (25±2°C; 12/12h light/ dark cycle).
reports are available regarding the antidiabetic Prior to the beginning of the experiment, all and antioxidant activities of 80 % aqueous rats were fed with basal diet at least for one methanolic extract of P.tetrastromatica (PME). week. Body weights of rats were recorded once Moreover we found that PME possessed in every two weeks. All studies were conducted disaccharidase inhibitory as well as antioxidant after obtaining prior approval f rom the activities invitro (unpublished data). So the institutional ethics committee [IEAC-KU-22/ present study was designed to evaluate the 2010-11 BC GMK (10)].
Experimental design: Rats were divided into Diagnostics Pvt. Ltd. India. Serum urea was
six groups with six animals each. Normal determined by enzymatic method using urease control was fed with the standard laboratory according to the procedure of Webster, (1977) diet throughout the experimental period. All using the kit from Accurex Biomedical Pvt. Ltd., other groups were fed with high calorie/ India. Serum creatinine was determined by energy diet (HCD) prepared with a slight modif ied Jeffe's method using the kit from modif ication of Wang et al. (2007), by adding Agappe diagonostics, India (Allen et al., 1982).
20% sucrose (w/w) and 10% groundnut oil (w/ The activities of superoxide dismutase (SOD) w) into the basal diet in order to develop and catalase (CAT) in the liver and kidney were insulin resistance. After two months of HCD assayed as described by Kakkar et al. (1984) and feeding, streptozotocin (STZ) at a dose of 15 Maehly and Chance, (1954) respectively. The mg/kg (Zhang et al., 2003) in 0.1 M citrate activity of Glutathione peroxidise (GPx) was buffer (pH4.6) was injected intraperitoneally determined by the method of Lawrence and to induce pancreatic -cell damage. The Burk, (1976). Glutathione reductase (GRd) diabetic rats were treated with graded doses activity was determined by the method David and of PME viz, 150, 300, 450 and 600 mg/kg body Richard, (1983). Thiobarbituric Acid Reactive weight for 45 days. The groupings are as Substances (TBARS) were estimated according to the procedures of Ohkawa et al. (1979).
Statistical Analysis: The SPSS 17 statistical
: HCD + STZ (Diabetic control) programme was employed for analysis, and the : HCD + STZ + PME (150mg/kg results were evaluated using one way analysis of variance (ANOVA). The results were : HCD + STZ + PME (300mg/kg presented as mean value ± SD. Difference among the mean were assessed using Duncan's : HCD + STZ + PME (450mg/kg Multiple range test. P < 0.05 was considered signif icant.
: HCD + STZ + PME (600mg/kg RESULTS AND DISCUSSION
At the end of the experimental period the rats An ideal experimental model for human T2DM were fasted overnight and sacrificed. Blood and was developed following the procedures of Wang tissues were removed to ice cold containers for et al. (2007) and Zhang et al. (2003). In this the estimation of various biochemical model system, insulin resistance was created by feeding HCD for two months and the pancreatic Biochemical estimations: Blood glucose was
-cells were damaged by the intraperitoneal determined by glucometer (One Touch injection of streptozot.
Horizon). Glycated hemoglobin (HbA ) was Changes in body weight: In the present study
estimated according to the procedure of Trivelli we found that the high-energy feeding for two et al. (1971) using kit purchased from Beacon months increased the body weight of animals Diagonostics Pvt. Ltd. India. Serum glutamate (Table 1). But after the intra peritoneal injection pyruvate transaminase (SGPT) and serum of STZ, the body weight decreased significantly.
glutamate oxaloacetate transaminase (SGOT) This was similar to the observations of Zhang et were assayed using commercial kits purchased al. (2003). Diabetic rats showed marked reduction from Agappe diagonostics, India according IFCC in their body weights when compared to normal recommended procedure (Bergmeyer et al., rats, which could be due to poor glycemic control.
1976). Serum alkaline phosphatase (ALP) was The excessive catabolism of proteins to provide estimated by DGKC-SCE procedure using the aminoacids for gluconeogenesis during insulin kit from Agappe diagonostics, India. Serum deficiency resulted in muscle wasting and weight gamma glutamyl transferase (GGT) was assayed loss in diabetic untreated rats (Babu et al., 2010).
according to the procedure of Szasz, (1976) Treatment with graded doses of PME using commercial kits purchased from Reckon significantly increased the body weight of rats.

Table 1. Changes in body weight
The ability of the extract to restore body weight for acquiring good glycemic control. The seems to be a result of its ability to reduce antihyperglycemic effect of PME can be attributed to the presence of phenolic compounds present in the extract. The phenolic Fasting blood glucose and glycated compounds stimulates insulin secretion by the
haemoglobin: Two months of high energy closure of K+-ATP channels, membrane
feeding resulted fasting blood glucose around depolarization and stimulation of Ca2+ influx, 150mg/dL and upon STZ injection, the values an initial key step in insulin secretion from the shoot up to 300-400mg/dL. This may be due to remanent -cells or from regenerated -cells the concerted action of insulin resistance and (Sunil et al., 2012).
pancreatic -cell damage. It was observed that treatment with graded doses of PME for 45 days Serum toxicity markers: The activities of
reduced the fasting glucose to normal level hepatic toxicity markers like GOT, GPT, ALP and (Table 2). The percentage of glycated GGT were increased in serum of diabetic groups haemoglobin also showed a similar pattern compared to control group (Table 3). The (Table 2). There was no statistically significant activities decreased signif icantly in PME treated difference in the fasting blood glucose and groups when compared to diabetic group in a glycated haemoglobin between the groups dose dependent manner. The concentration of treated with 450mg/kg and 600 mg/kg PME, renal toxicity markers urea and creatinine were suggesting that 450mg/kg is the optimum dose increased in serum of diabetic groups as compared to control group (Table 4). Their Table 2. Fasting blood glucose and glycated
concentration decreased signif icantly in PME treated groups when compared to diabetic group in a dose dependent manner.
Liver is the vital organ of metabolism, detoxif i- cation, storage and excretion of xenobiotics and their metabolites (Rej, 1978). Serum GOT, GPT, ALP and GGT are reliable markers of liver func- tion. In STZ induced diabetic rats the liver was necrotized. An increase in the activities of these toxicity markers in serum might be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (Navarro et al., 1993) which gives an indication of the hepato- toxic effect of STZ. Treatment of the diabetic rats with graded doses of PME reduced the

Table 3. Activity of hepatic toxicity markers
Table 4. Concentration of renal toxicity markers production of reactive oxygen species (ROS),
which can lead to increased lipid peroxidation, alter antioxidant defense and further impair glucose metabolism in biological system (Balasubashini et al., 2004). SOD has been postulated as the one of the most important enzymes in the enzymatic antioxidant defense system which catalyses the dismutation of superoxide radicals to produce H O and molecular oxygen. Catalase is a hemoprotein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals. Previous studies have reported that the activity of SOD is low in activity of these enzymes in serum when com- diabetes mellitus (Coudary et al., 1999). This is pared to the diabetic untreated group and con- in agreement with our results of reduced sequently alleviated liver damage caused by STZ- activities of SOD and CAT in the liver and kidney induced diabetes. Diabetic rats showed signif i- of diabetic rats. Whereas, treatment with PME cantly increased levels of urea and creatinine in showed a significant increase in the hepatic the serum, which are considered as significant renal SOD and CAT. GPx plays a primary role in markers of renal dysfunction (Bethesda, 2001). minimizing oxidative damage. Glutathione In the present study, the significant reduction peroxidase (GPx) is an enzyme which in the levels of serum urea and creatinine in decomposes H O or other organic the treated diabetic rats indicated that PME hydroperoxides to non toxic products at the prevented the progression of renal damage in expense of reduced glutathione. Glutathione diabetic rats.
reductase is the major enzyme responsible for Activity of Antioxidant Enzymes
Administration of PME signif icantly restored Table 5 and Table 6 show the activity of various the activities of GPx and GRd in the liver and antioxidant enzymes in liver and kidney kidney of diabetic rats when compared to respectively. Activity of SOD, CAT, GPx and GRd diabetic control. Thus PME improved the showed a similar trend with decreased activity activities of all the antioxidant enzymes in in diabetic group. Treatment with PME diabetic rats which may be due to the free radical signif icantly increased the activity of the scavenging property of the extract.
scavenging enzymes in all groups in a dose dependent manner.
Effect of PME on Lipid peroxidation: The level
of lipid peroxidation products is measured as thio Hyperglycemia is a well-known cause for barbituric acid reactive substances (TBARS). The elevated free radical levels, followed by concentration of lipid peroxidation products in

serum, liver, kidney and pancreas were increased lipid peroxidation marker, TBARS, in the diabetic in the diabetic control group. However there is a control rats is a reflection of insuff iciency of marked decrease in TBARS level in PME treated antioxidant defenses in combating ROS- groups in a dose dependent manner (Table 7).
mediated damage. The present data also demonstrated that the treatment with PME Lipid peroxidation of unsaturated fatty acids is reduced the concentration of TBARS in the liver, a frequently used indicator of increased kidney, pancreas and serum of diabetic animals.
oxidative stress and subsequent oxidative This activity may be due to the free radical damage (Hauggard, 1968). The high level of scavenging effect of PME.
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