The protective effect of dietary fl vonoid fraction from Acanthophora spicifera on streptozotocin induced oxidative stress in diabetic rats

Lvkumr VupplptiRvihnirn VelyumK.F.H.Nzeer AhmeSowmy CherukuriBhskr Rey Kesvn

a Center for Nanobiotechnology,Sri Venkateswara College of Pharmacy,RVS Nagar,Chittoor 517127,AP,India

b NIPER,Raja S.C.Mullick Road,Jadavpur,Kolkata 700032,India

c Center for Research and Innovation,Asia Metropolitan University,Cheras 43200,Malaysia

d Department of Pharmaceutics,RIPER,Anantapuram 515721,AP,India

Abstract The present investigation was considered in arraying of antidiabetic and antioxidant activity from dietary fl vonoid loaded fraction of Acanthophora spicifera (A. spicifera, Family: Rhodomelaceae) on streptozotocin (STZ) induced oxidative stress rats. The testings were acted upon male rats,which were alienated into fi e groups:control group,diabetic group(single dose of 65 mg/kg,streptozotocin(STZ)i.p.),diabetic with insulin(6 IU),and diabetic with fl vonoid rich fraction groups(FRF)at 50 and 100 mg/kg body weight,given orally for 21 days.The blood glucose level was determined at different week intermissions.The antioxidant consequences of FRF on STZ-induced diabetic rats were determined by the estimations of the oxidative stress marker like malonyldialdehyde and antioxidant enzymes such as superoxide dismutase,catalase and glutathione in tissue homogenates of heart, liver and kidney. FRF treatment of diabetic rats significantl (P<0.05) diminishes the blood glucose altitudes to normal in contrast with diabetic rats.However,FRF administration,significantl decreased the malonyldialdehyde(MDA)and increased the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione levels(GSH)in diabetic rats.The outcome designates that FRF fraction from red algae A.spicifera was potent anti diabetic and antioxidant asset against STZ induced diabetes and oxidative tissue breakups.

Keywords: Acanthophora spicifera;Red algae;Streptozotocin;Diabetes;Antioxidant?Corresponding author at:Center for Nanobiotechnology,Sri Venkateswara College of Pharmacy,RVS Nagar,Chittoor 517127,AP,India.Tel.:+919494804367.

1. Introduction

The seaweedAcanthophoraspicifera(Family:Rhodomelaceae, Ceramiales) is notorious red algae and widely distributed in the Gulf of Mannar,Rameshwaram coast,Tamilnadu, South India which is used as a food material,cosmetics, and fuel [11]. Besides, methanol extract ofA.spiciferahas been an evidence for anti-bacterial activity againstStaphylococcus aureusandBacillus subtilis[12].The sulphated agarans are isolated from the aqueous extract ofA.spiciferaand their anti-viral chattels with their structure activity relationship were recorded[13].Antioxidant properties ofA.spiciferawere deliberate in different types ofin vitrofree radical scavenging assays [14]. Hence, present study was designed to extend the current information on anti-diabetic folklore claim and antioxidant effect ofA.spiciferaand to determine its protective effects on rats having uncontrolled type I diabetes.

2. Materials and methods

2.1. Marine algae collection

The red algae,A.spicifera(Family:Rhodomelaceae,Ceramiales)was collected from Mandapam,during the month of March,2014 from Rameswaram coast, Tamil Nadu, India [15]. It was identifie and authenticated by Dr. Krishnamurthy, Institute of algology, Annanagar, Chennai. The voucher specimen(SVCOP/14-125)was deposited in the Department Museum.

2.2. Preparation of the ethanol extract and separation flavonoids rich fraction from Acanthophora spicifera

Dried, milledA. spicifera(1 kg) was extracted through 5 l of ethanol by means of soxhlet apparatus for 24 h.The extract was filtered and the filtrat was evaporated by a rotary vacuum evaporator.The gain in yield of the menthol extract was found to be 20.22% (w/w). The dried ethanol extract was suspended in water and assorted with n-hexane in a separating funnel and n-hexane portion was discarded after separation.To the aqueous portion, dichloromethane was added and the dichloromethane portion was discarded after separation and the aqueous portion was collected and further extracted with ethyl acetate.The ethyl acetate portion was collected and it was allowed to dry for complete removal of solvent by a rotary vacuum evaporator. The yield of the ethyl acetate fraction was 2.45%(w/w).The ethyl acetate fraction was subjected to qualitative chemical test and thin layer chromatography (TLC) studies and showed positive test for fl vonoids.

2.3. Estimation of total phenol content in FRF

Total phenolic content was estimated with Folin–Ciocalteu colorimetric method described previously [16,17] with a little modification Briefl ,the appropriate dilutions of the FRF were oxidized with 0.2 N Folin–Ciocalteu reagents and then the reaction was neutralized with saturated sodium carbonate (75 g/l).The absorbance of the resulting blue color was measured at 760 nm after incubation for 2 h at 23?C.Quantificatio was done on the basis of the standard curve of Gallic acid.Results were expressed as grams of gallic acid equivalent(GAE)per 100 g of dry weight(DW).

2.4. Animals

Healthy adult Wistar rats of either sex,weighing 200–250 g,were used.The animal room was maintained at 22±5?C with a daily light-dark cycle(06:00–18:00 light)and humidity about 50%–60%.Animals were given food and waterad libitum.All the studies were conducted in accordance with the Animal Ethical Committee(SVCOP/02/2015/SV0026).

2.5. Acute oral toxicity study

Acute toxicity was carried out according to the Organization for Economic Co-operation and Development guidelines(OECD 423).Two groups of control rats(n=3 in each group)were given FRF separately 2000 mg/kg p.o.as single dose.After oral administration animals were observed continuously for 2 h for beneath profile like alertness,restlessness,irritability,fearfulness spontaneous activity, reactivity, touch response, pain response, defecation and urination. After the stage of 24 and 72 h,animals were observed for signs of lethality or for death.

2.6. Evaluation of oral glucose tolerance test(OGTT)

Initial screening of fractions for hypoglycemic activity was carried out in normal healthy rats by conducting OGTT. The OGTT was performed for two different doses of FRF (50 and 100 mg/kg bodyweight per orally)and blood glucose level was measured by one touch glucometer (Accu-check, India). The glucose level was measured at the intervals of 0,30,60,90 and 120 min after the administration of test samples[18].

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2.7. Induction of diabetes in rats

Diabetes was induced by a single intra-peritoneal injection of newly prepared streptozotocin (65 mg/kg) in 0.1 mol/L citrate buffer(pH 4.5)to overnight fasted rats.In order to prevent initial drug induced hypoglycemia, STZ injected animals were given with 5%glucose water for 24 h.After three days of STZ administration,rats were divided according to their fasting blood glucose levels which showed>300 mg/dl.The animals did not show the above blood glucose range is excluded from the study[19].

2.8. Experimental design and drug administration

Rats were divided in to four groups of six rats in each group.Group I(normal animals)treated with only vehicle,0.3%CMC(v/v).Group II animals are diabetic rats treated with 0.3%CMC.Group III animals are diabetic animals treated with Insulin(6 IU)and Group IV–V rat are diabetic animals treated with FRF(50 and 100 mg/kg, per oral) on the 3rd day after the induction of diabetes. Rats were fasted overnight and blood samples were collected from the tail vein on the 3rd day of STZ treatment prior to and at 7th,14th and 21st day after the administration of the FRF.

2.9. Estimation of blood glucose and proteins

Blood glucose was determined using one touch instant glucometer by using glucose strips.At the end of the experiment,animals (n=3/group) are sacrifice by euthanasia, tissues like heart,liver and kidney were isolated and minced,homogenized with a polytron homogenizer using 20 mmol/L phosphate buffer solution(pH 7.4).The homogenate was centrifuged at 1000×gfor 8 min at 4?C. The supernatant obtained by centrifugation was used forin vitroprotein analysis[20].

2.10. Estimation of glycosylated hemoglobin

Glycosylated hemoglobin was estimated by the method of Eross et al. [21]. To the erythrocytes (0.5 mL) collected from whole EDTA blood,0.125 mL of distilled water and 0.125 mL of carbon tetrachloride were added,mixed well and centrifuged.The supernatant hemolysate was separated and its hemoglobin concentration was adjusted to 10%with distilled water.To 2 mL of hemolysate, 1 mL of 0.3 N oxalic acid was added into test tubes and heated at 100?C in a water bath for 60 min. After cooling, 1 mL of 40% TCA was added, shaken well and centrifuged.To 2 mL of supernatant was pipetted out into another set of test tubes,0.5 mL of 0.05 mol/L TBA was added and incubated at 37?C for 40 min.A blank with 2 mL of distilled water was treated similarly.The resulting yellowish color was read on a spectrophotometer at 443 nm. Concentration of HbA1c was calculated on the assumption that 1% HbA1c corresponds to an absorbency of 0.029 at 443 nm (experimentally determined millimolar extinction co-efficien for TBA-5 hydroxymethyl furfural adduct is 26 at 443 nm). Hemoglobin in RBC (g%) was estimated by cyanmethemoglobin method.

2.11. Biochemical or antioxidant analysis of heart,liver and kidney homogenates

After the collection of blood samples,the rats were sacrificed Heart,liver and kidney were excised,rinsed in ice cold normal saline followed by ice-cold 10% KCl solution, blotted, dried and weighed.10%(w/v)organs were homogenated separately in ice-cold KCl solution and centrifuged at 1500 r per minute for 15 min at 4?C. The supernatant thus obtained were used for the estimation of superoxide dismutase(SOD)[22],catalase(CAT)[23],glutathione(GSH)[24],thio-barbituric acid reactive substances(TBARS)[25].

3. Histopathology Studies

Subsequent to blood sample for the biochemical analysis,the animals were forfeit, quickly dissected. A tiny portion of pancreases was engaged and fi ed in 10%formalin.The specimen was dehydrated in ascending grades of ethanol, clean in xylene and entrenched in paraffi wax.Segments were of 6 μm in thickness stained with haematoxylin and eosin and subjected to microscopic inspections.

3.1. Statistical analysis

Data were expressed as mean±SEM.The data in each group analyzed by two way ANOVA followed by post hoc analysis– Dunnett’s comparison tests using PRISM PAD 5 statistical software, USA. Probability levelP<0.05 andP<0.01 were considered as significant

4. Results

4.1. Acute toxicity study

Inacutetoxicitytests,ratsarefedwithFRForally2000 mg/kg body weight, did not shown any behavioral abnormality and mortality.Animals are well tolerated and were not reported toxic to the dose of 2000 mg/kg body weight.The 1/20th(100 mg/kg)and 1/40th(50 mg/kg)dose was considered for further pharmacological evaluation.The experiments and dose fixation were carefully designed in order to minimize the animal’s usage.

4.2. Effect of FRF fraction on oral glucose tolerance test in experimental rats

In OGTT(Fig.1)investigation group I served as normal control, group II served as FRF 50 mg/kg and group III served as FRF 100 mg/kg body weight.The blood levels of glucose were demonstrated the significan change after oral administration of glucose solution.Dose dependent blood glucose reduction was observed in rats(group II and group III)after treatment of FRF 50 and 100 mg/kg at 60,90 and 120 min.

4.3. Effect of FRF on blood glucose levels in STZ treated diabetic rats

Fig.2 illustrates the levels of glucose in diabetic rats,which is measured at various time intervals after STZ treatment.The

Fig.1. Effect of FRF on blood serum glucose level(OGTT)on glucose loaded rats.*P<0.05,control vs group II(FRF 100 mg/kg);**P<0.05,group I(control)vs group II(FRF 50 mg/kg)and III(FRF 50 mg/kg)(one-way ANOVA–followed by Dunnett’s post-test).

Fig.2. Effect of FRF and insulin on blood glucose level of STZ treated diabetic rats.**P<0.01,diabetic control vs control group; @@P<0.01,insulin treated group vs diabetic control; ##FRF treated group vs diabetic control (one-way ANOVA–followed by Dunnett’s post-test).

blood glucose level was increased several fold to more than 300 mg/dl in STZ treated rats which are compared to non diabetic control rats (average 101.8 mg/dl) indicating diabetes in STZ treated animal group.By administration of insulin,it was observed that appreciably(P<0.05)decrease in blood glucose level measured at 3rd,7th,14th and 21st day to STZ treatment animals.Oral administration of FRF(50 and 100 mg/kg)to diabetic animals significantl lower elevated blood glucose levels from 7th to 21st day as analyzed by Tukey’s multiple comparison with the diabetic group alone.

4.4. Effect of FRF on HbA1c levels

Fig. 3. Effect of FRF on Glycosilated hemoglobin. ##P<0.0001, control vs diabetic control; **P<0.0001, insulin treated and FRF treated diabetic group vs diabetic control(one-way ANOVA–followed by Dunnett’s post-test).

Fig.4. Effect of FRF and insulin on SOD levels in heart,liver and kidney of STZ treated diabetic rats. ##P<0.01,diabetic control vs control group; @@P<0.01,FRF treated group vs diabetic control(one-way ANOVA–followed by Dunnett’s post-test).

As shown in Fig. 3, there was a significan increase in the levels of HbA1c in diabetic animals due to the state of disorder.However,after administration of FRF(50 and 100 mg/kg)significantl reversed the HbA1c levels to a normal control group.It was worth noticing that,at the end of the study all the results were close to insulin treated group.

4.5. Effect of FRF on SOD levels in Heart,liver and kidneys of STZ treated rats

The effect of insulin and FRF on SOD levels in rat heart,liver and kidneys were shown in Fig. 4. In the diabetic group,depletion of SOD level was observed in all organs compared to that of the control animal group.Administration of FRF 50 and 100 mg/kg significantl (P<0.05)elevated the SOD levels in all organs tested in diabetic rats as compared to the diabetic group alone. In insulin treated group, an increase in SOD levels was observed,which is statistically insignificant

4.6. Effect of FRF on catalase levels in heart,liver and kidneys of STZ treated rats

Catalase(CAT)activity in the heart,liver and kidney of diabetic rat was shown in Fig.5.Significan decrease(P<0.05)in catalase activity was noted in STZ treated diabetic rats as compared to that of control non-diabetic animals. Administration of FRF 50 and 100 mg/kg and insulin,significantl reverse the decreased catalase activity in liver and kidney tissues. Moreover,there is no significan effect was observed in heart tissue as compared to STZ treated diabetic group alone.

4.7. Effect of FRF on LPO levels in heart,liver and kidneys of STZ treated rats

The effect of insulin and FRF in TBARS levels was represented in Fig. 6. In diabetic rats, an increase in the level of LPO marker malonyldialdehyde (MDA) was observed in vital

Fig. 5. Effect of FRF and insulin on catalase (CAT) levels in heart, liver and kidney of STZ treated diabetic rats.*P<0.05,diabetic control vs control group;@,#P<0.05,FRF treated group vs diabetic control(one-way ANOVA–followed by Dunnett’s post-test).

Fig.6. Effect of FRF and insulin on malonylaldehyde(TBARS)levels in heart,liver and kidney of STZ treated diabetic rats.*P<0.05,diabetic control vs control group;#P<0.05,insulin treateddiabeticgroupvs diabetic control;#P<0.05,FRF treated diabetic group vs diabetic control(one-way ANOVA–followed by Dunnett’s post-test).

tissues. The amount of MDA formed inside liver is very high(830±35 μmol/mg of protein)was higher as compared to control group liver(210±45 μmol/mg of protein).Treatment with FRF(50 and 100 mg/kg)and insulin was significantl (P<0.05)lowered the MDA levels in all organs as compared to diabetic group.

4.8. Effect of FRF on GSH levels in Heart,liver and kidneys of STZ treated rats

The effect of insulin and FRF in GSH levels was represented in Fig.7.In diabetic rats,significan (P<0.001)decrease in the level of GSH was observed in all tissues.Treatment with FRF(50 and 100 mg/kg)was significantl (P<0.01)increased GSH levels in all organs as compared to diabetic group.

Fig.7. Effect of FRF and insulin on GSH levels in heart,liver and kidney of STZ treated diabetic rats.***P<0.001,diabetic control vs control group;##P<0.05,FRF treated diabetic group vs diabetic control(one-way ANOVA-followed by Dunnett’s post-test).

5. Histopathology Reports

FRF fraction showed the marked results on the pancreas which was compared to that of insulin treated groups. In the control group, Islets of Langerhans were scattered in the pancreatic tissue by varying in size at the same lobule. It was observed that no evidence of inflammator cells with less intracellular spaces(Fig.8A).But in case of diabetic control group(Fig. 8B) elicits disarray of cell into surrounding exocrine tissues.All these conditions were seemed to return back to normal(Fig. 8D, E) by reducing the intracellular space and returning of islets in to exact location after the treatment of FRF fractions.

6. Discussion

The present study highlights the defensive outcome of fl vonoid rich fraction (FRF) from the marine sourceA. spicifera,red algae in STZ induced oxidative stress rats.The effect of FRF on blood glucose,tissue antioxidants and oxidative stress markers were considered in STZ induced rat model.This meticulous model of STZ-induced hyperglycemia has been described as a useful experimental model to study the activity of antidiabetic agents with or without insulin [26,27]. Our results have been an evidence for intraperitoneal administration of STZ(65 mg/kg)effectively induced hyperglycemia in normal fasted rats as compared with control saline treated rats. The elevated level of blood glucose could be due to destruction of pancreatic β-cells by STZ [28]. The capacity of FRF to decrease the elevated blood sugar to normal glycemic level is an essential trigger for the liver to revert to its normal homeostasis during experimental diabetes.In OGTT results,FRF 50 and 100 mg/kg B.W. exhibited significan effect on serum blood glucose levels at 30, 60, 90, 120 min which is compared to control. The test results revealed that FRF 100 mg/kg has shown maximum antihyperglycemic activity in all time intervals than 50 mg/kg.Further,results confirme that fl vonoid fractions from the seaweed ofA.spiciferacontrolled hyperglycemia by significantl reducing blood glucose level in diabetic rats.FRF extracts(50 and 100 mg/kg)fall short to show anti-diabetic effect after diabetic onset at 48th hrs. However FRF significantl decreased blood glucose levels at 7th and 21st day after onset of diabetes.In earlier reports,red algaeA.Spiciferacontains various phytochemicals like steroids, fl vonoids, and proteins [29] which posses antiviral and antibacterial activities[12,13].In additional,amplifie heights of HbA1c in diabetic rats indicated the incidence of glycosylation due to hyperglycemia.Administration of FRF extracts(50 and 100 mg/kg)and insulin to the diabetic rats significantl reduced HbA1c levels.The rationalization behind this was assumed to be an improvement in insulin secretion.This suggested that FRF extracts(50 and 100 mg/kg)might have the potential to reorganize the progress of diabetes with connected hitches.

Fig. 8. Photomicrograph of pancreatic tissues stained by hematoxylin and eosin of (A) control; (B) STZ treated diabetic rats; (C) insulin treated group; (D) FRF 50 mg/kg treated group;(E)FRF 100 mg/kg treated group.

This is the firs biochemical investigation that the organ protective effect of FRF from seaweed ofA.spiciferain streptozotocin induced oxidative stress diabetic animals.There is an understandable relationship between hyperglycemia and active oxygen/nitrogen species in experimental and clinical types of diabetes[30].Amassing of reactive oxygen species(ROS)due to oxidative stress is also instrumental in the expression of cell death, as ROS can easily react and oxidize vital cellular components such as lipids, proteins and DNA [31]. The vital organs like heart, liver, and kidney are particularly susceptible to the effects of ROS due to its poly-unsaturated integrity and modest antioxidant defense[32].Experimental studies have indicated the potential usefulness of exogenous antioxidants for prevention and treatment of diabetes mellitus. A plant derived anti-oxidant treatment has been reported to reduce the development of diabetic complication such as, retinopathy, cataract formation,neuropathy,vascular complication and nephropathy by increasing the antioxidant status in insulin dependent and non-insulin dependent tissues[33].

In the present study, there is apparently no compensatory mechanism in visceral organs in order to overcome STZ induced oxidativestress.ReducedactivitiesofSODandCATinliver,kidney and heart have been observed during diabetes.SOD is vital defence enzyme which catalyses the dismutation of superoxide radicals[34].CAT is a hemeprotein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals[35].Therefore,fall in the activity of these enzymes (SOD, CAT) may consequence in a number of deleterious effects due to the accumulation of superoxide anion radicals and hydrogen peroxides. Administration of FRF significantl increased SOD levels in all the vital organs tested.However,increased in CAT level was only observed in liver and kidneybutnotinhearttissueofthediabeticrats.Fromthepresent study,it was quite interesting to observe that,increased lipid peroxidation in vital organs of rats exposed to hyperglycemia and its attenuation by the control of hyperglycemia with FRF treatment.It strongly suggests the protective role of the FRF,which could be due to the antioxidative effect of fl vonoids present in the red algae, which acts as strong superoxide radical scavengers and singlet oxygen quenchers.Earlier report on STZ treated animals with insulin treatment significantl reverse SOD and CAT levels in the heart and kidneys of the diabetic rats[36].The effect of insulin in SOD, CAT and TBARS level was in accordance with the earlier reports[37,38].It is interesting to observe that high dose of FRF significantl increases the GSH level in STZ treated rat vital organs.Cellular Glutathione pool enzymes have a key role in enzymatic defense systems and acts on peroxides (H2O2, lipid or organic peroxides) to remove them. In the present investigation,it was observed that FRF treatment could effectively increase the activity of GSH and other antioxidant enzymes. The cellular GSH pool is mainly maintained by the oxidation of GSH and regeneration of reduced GSH by GSHperoxidase and GSSG-reductase enzymes respectively. Under oxidative stress condition,increased oxidation of GSH by GSHPx and decreased regeneration of GSH by GSSG-reductase have been implicated as the main reason for the reduced level of GSH.Histopathology study also props up our output.STZ was supposed to annihilate pancreas partially.Diabetic rats showed condensed(or)reduced islet cells,which were restored to normal upon treatment with the FRF (50 and 100 mg/kg) ofA.spicifera. No such variation was found in normal rats. These results indicate that the FRF fraction of red algaeA. spiciferashowed significan pro protection against the oxidative damage induced by STZ in liver and kidney of rats.FRF may act as protective agents against STZ induced liver and kidney damage and free radical scavenger agent.

7. Conclusion

Our study confirme that FRF extracts had an outstanding antidiabetic effect,which could be elucidated,at least in part,by its antihyperglycemic and antioxidant activities in STZ induced diabetic rats.FRF also had the potential to ameliorate diabetic problems. However, FRF had no effect on normal rats, so it cannot lead to hypoglycemia in normal rats.All these finding provide the scientifi basis for the use of algae as a promising medicine in the therapy of diabetes mellitus and its complications.However,there is a fragmentary research stepping ahead to purify and identify the bioactive compound(s) responsible for the anti-diabetic/anti-oxidative action in this fl vonoid rich fraction.

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgement

The authors express their sincere thanks to the principal and college management for their constant support in the fulfillmen of the work.