{"id":6576,"date":"2016-04-28T08:10:22","date_gmt":"2016-04-28T08:10:22","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=6576"},"modified":"2020-04-24T06:55:21","modified_gmt":"2020-04-24T06:55:21","slug":"evaluation-of-the-antioxidant-hypoglycaemic-and-anti-diabetic-activities-of-some-seaweed-collected-from-the-east-coast-of-india","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol9no1\/evaluation-of-the-antioxidant-hypoglycaemic-and-anti-diabetic-activities-of-some-seaweed-collected-from-the-east-coast-of-india\/","title":{"rendered":"Evaluation of the Antioxidant, Hypoglycaemic and Anti-diabetic Activities of Some Seaweed Collected From the East Coast of India."},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Diabetes mellitus is a cluster of never-ending diseases, which is recognized by its most common characteristic, hyperglycaemia. It is usually classified in to two main categoriesi.e. Type I diabetes mellitus (T1<\/sub>DM), caused due to complete absence of insulin production and Type II diabetes mellitus (T2<\/sub>DM), due to the relative deficiency of insulin secretion and tissue resistant to the insulin action1<\/sup>.Majority of this disease all over the world may be correlated to modern diet, obesity and modern sedentary lifestyle. The mortality associated with diabetes is mainly due to the increased risk of several complications of this disease. Very often the most common life threatening complications includes hypertension, retinopathy, nephropathy and other cardio vascular diseases2<\/sup>. These incidences necessitate and warrant the search for new preventive measures as well as healing strategy of this disease.<\/p>\n

Traditionally, several seaweeds have been consumed as food in many parts of the world especially among Asiancoastal communities. According to some report, Japanese consume approximately 5.3 g seaweed in their daily diet3<\/sup>. In addition to this, seaweed has also been reported to be used against numerous disease causing ailments in different Asian traditional medical systems4<\/sup>. Some studies have revealed that, regular consumption of seaweed lowers menace of several diseases like cardiovascular disease, hyperlipidaemia and breast cancer5,6<\/sup>. Furthermore some studies have also established the fact that, reduction of seaweed consumption in some Asian societies that regularly used to consume seaweed due to an enhanced yearning for Westernised diet, augmented occurrence of unending lifestyle diseases6<\/sup>. These studies highlight the promising health benefits offered by seaweed. It\u2019s true that despite of these proven facts, this generous treasure of the ocean has been ignoredsince long.Even today they are the same under-utilised and untapped resources of diversified secondary bio molecules having potential therapeutic applications, normallyabsent in terrestrial plants.<\/p>\n

In this study, we aim to communicate the beneficial utility of some seaweed obtained from Indian east coast in preventing and managing diabetes via various pharmacologically relevant attestations.Seaweed flora is represented by three major classes\u2019 namely green, brown and red algae which differ from one another in their morphology, life cycle, distribution, pigment and secondary metabolite composition. It was decided by us to take one or two species from each class to assess their antidiabetic potential. During the course of study one species of red algae Gracilariaverrucosa<\/em>(Huds.) Papenfuss (Family: Gracilariaceae), two species of green algae Eenteromorphacompressa<\/em>(L.) Grev. (Family: Ulvaceae) and Ulvafasciata<\/em>Delile (Family: Ulvaceae) were collected from the Chilika Lake, Odisha, Indian east coast. Due to non-availability of brown seaweed along the Odisha coast, one of the species from this variety Turbinariaconoides<\/em>(J. Agardh) Kutzing (Family: Sargassaceae) was obtained from the Palk Bay and Gulf of Mannar, Tamilnadu, Indian east coast. All seaweeds were properly identified and authenticated by Prof. R.C. Panigrahy, marine taxonomist, Berhampur University, Berhampur, Odisha, India.<\/p>\n

From some of the studies it is confirmed that a sufficient use of antioxidant may prevent or delay \u03b2-cells dysfunction in diabetes and prevent the development of complications associated with this disease7,8<\/sup>. Hence, initially all of these seaweed extracts were investigated for their probable antioxidant property. Those extracts showing goodresultsin this preliminary investigation, were further evaluated for their in vivo<\/em> hypoglycaemic and antidiabetic properties in normal and diabetes induced animals respectively.<\/p>\n

Materials And Methods <\/strong><\/p>\n

Materials<\/strong><\/p>\n

Only analytical grade chemicals were used for this investigation. Streptozotocin (STZ), Ethylene diamine tetra acetic acid (EDTA) and haematoxylin\u2013eosin (H&E) dye were purchased from Himedia Lab. and Loba Chemie Pvt. Ltd., India. All other solvents and chemicalswere procured from Merck Specialties Pvt. Ltd., India. Biochemical parameters were investigated by using glucose kit (GOD\/POD method), triglycerides kit (GPO\/PAP method) and cholesterol kit (CHOD\/PAP method)which were obtained fromCrest Biosystems, India.<\/p>\n

Seaweed extraction<\/strong><\/p>\n

Seaweeds collected from different places were carefullywashed with sea water and subsequently in distilled water to get rid of all debris and impurities. These seaweeds were then air dried and stored in air tight polyethylene bags for further use. <\/strong>Required amount of each of the seaweed was cut into small pieces with the help of sharp knife and powdered with the help of electronic grinder separately. These powders were sievedby using No.16 mesh. Exactly, 1000g of each seaweed powder was extracted separately in soxhlet apparatus in the increasing order of polarity using three solvents i.e petroleum ether (pet. ether), ethyl acetate and methanol9,10<\/sup>. All the extracts obtained were encoded as following:<\/p>\n

PEG, EAG & MEG: pet. ether, ethyl acetate & methanol extract of Gracilaria<\/em>respectively.<\/em><\/p>\n

PEE, EAE & MEE: pet.ether, ethyl acetate & methanol extract of Enteromorpha<\/em>respectively.<\/em><\/p>\n

PEU, EAU & MEU: pet. ether, ethyl acetate & methanol extract of Ulva<\/em>respectively.<\/p>\n

PET, EAT & MET: pet. ether, ethyl acetate & methanol extract of Turbinaria<\/em> respectively.<\/p>\n

Phytochemical analysis of extracts<\/strong><\/p>\n

The extracts obtained were analyzed for presence of different phyto constituents using established validated qualitative methods9,10<\/sup>.<\/p>\n

Table 1:<\/strong> Phytochemical composition of petroleum ether, ethyl acetate and methanol extracts of different seaweeds.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Constituents<\/strong><\/td>\nE. compressa<\/em><\/strong><\/td>\nU. faciata<\/em><\/strong><\/td>\nT. conoides<\/em><\/strong><\/td>\nG. verrucosa<\/em><\/strong><\/td>\n<\/tr>\n
PE<\/strong><\/td>\nEA<\/strong><\/td>\nME<\/strong><\/td>\nPE<\/strong><\/td>\nEA<\/strong><\/td>\nME<\/strong><\/td>\nPE<\/strong><\/td>\nEA<\/strong><\/td>\nME<\/strong><\/td>\nPE<\/strong><\/td>\nEA<\/strong><\/td>\nME<\/strong><\/td>\n<\/tr>\n
Alkaloids<\/strong><\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Carbohydrates<\/strong><\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Gum & mucilage<\/strong><\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Protein & amino acid<\/strong><\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n<\/tr>\n
Terpenoids<\/strong><\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n<\/tr>\n
Saponins<\/strong><\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Flavonoids<\/strong><\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n<\/tr>\n
Tannins<\/strong><\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n<\/tr>\n
Glycosides<\/strong><\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n<\/tr>\n
Steroids<\/strong><\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n+<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
Phenolic compounds<\/strong><\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n–<\/td>\n+<\/td>\n+<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

PE, petroleum ether extract; EA, ethyl acetate extract; ME, methanol extract; (+): present; (-): absent.<\/p>\n

In vitro<\/strong> antioxidant evaluation of extracts<\/strong><\/p>\n

The antioxidant property of each extract was evaluated following the standard methods described below. The chemical tests were performed in triplicate for each seaweed extract.<\/p>\n

Determination of total phenol content:<\/em><\/p>\n

Total phenolic content was determined by Folin-ciocalteu reagent method using Gallic acid as the standard for phenolic compound11<\/sup>. In this method, initially 5 ml of the reagent was mixed with 1 ml of Gallic acid at different concentrations (50, 100, 150, 200 \u03bcg\/mL) and to which 4 ml of 2% Sodium carbonate was added after 3 minutes. Thirty minutes later, the absorbance of the blue colour was measured at 760 nm. The concentrations of total phenols were expressed as mg\/g of dry extract12<\/sup>.<\/p>\n

Total reducing power:<\/p>\n

The seaweed extracts (50, 100, 150 and 200 \u03bcg\/ml) prepared in distilled water and 1% potassium ferric cyanide were mixed with phosphate buffer (0.2 M, pH 6.6) and incubated at 50\u00b0C for 20 min. Then, 2.5 ml of 10% TCA was added to this reaction mixture and centrifuged at 1000 \u00d7 g for 10 min. The upper layer of the solution (2.5 ml) was mixed with 2.5 ml of distilled water and FeCl3<\/sub> (0.5 mL, 0.1%). The absorbance was measured at 700 nm. BHT (50 to 200\u03bcg\/ml) was used as positive control. The higher the absorbance of the reaction mixture, the greater is its reducing power. Total reducing capacity of seaweed extracts was determined adopting a most widely used method13<\/sup>.<\/p>\n

Nitric oxide radical scavenging activity:<\/p>\n

In this test, 3 ml of the reaction mixture containing10 mM sodium nitroprusside and different seaweed extracts (50, 100, 150, 200 \u03bcg\/ml) in phosphate buffer were incubated at 25\u00b0Cfor 150 min. Then, 0.5 ml of each incubated reaction mixture was mixed with 1 ml of sulfanilic acid reagent (0.33%in 20% glacial acetic acid) and allowed to stand for 5 minfor complete diazotization. Then 1 ml of naphthylethylenediaminedihydrochloride (0.1%) was added and thesolution was mixed thoroughly. The mixture was allowed to stand for 30 min at 25\u00b0C.A pink colored chromophore is formed in diffused light.The absorbance of pink colour solution was measured at 540nm against the corresponding blank solutions. BHT (50-200\u03bcg\/ml) was used as positive control. The nitric oxide scavengingactivity of the seaweed extracts is reported as percentage inhibition and was calculated as per established method14<\/sup>.<\/p>\n

Nitric oxide scavenging effect = (Acont<\/sub> \u2013 Atest<\/sub>)\/Acont<\/sub> \u00d7 100\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 (Eq. 1)<\/p>\n

Where Acont<\/sub>was the absorbance of the control reaction and Atest<\/sub> was the absorbance in the presence of the seaweeds extracts.<\/p>\n

Hydrogen peroxide (H2<\/sub>O2<\/sub>) radical scavenging activity:<\/p>\n

H2<\/sub>O2<\/sub> scavenging activity of different seaweeds extracts was determined adopting standard procedures15<\/sup>. Initially, 40mM of H2<\/sub>O2<\/sub> was prepared in phosphate buffer (pH 7.4) and the concentration of H2<\/sub>O2 <\/sub>in it was determined spectrophotometrically at 230 nm. The seaweed extracts of 50, 100, 150, 200\u03bcg\/ml concentrations in distilled water were prepared. Ascorbic acid of same concentrations was also prepared separately. Then 0.6 ml of 40 mM H2<\/sub>O2<\/sub> solution was added after the lapse of 10 min. Absorbance of the mixture was determined at 230 nm against a blank solution containing phosphate buffer without H2<\/sub>O2<\/sub>. The percentage scavenging of H2<\/sub>O2<\/sub> was calculated as per Eq. 1.<\/p>\n

In vivo <\/strong>hypoglycaemic and antidiabetic study<\/strong><\/p>\n

Experimental animals:<\/p>\n

Animal experiments were designed and conducted in accordance with the policies of committee for the purpose of control and supervision of experiments in animal (CPCSEA), India. The experimental protocol was approved (no. 66, dated 7\/06\/2012) by institutional animal ethics committee (IAEC), Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, India. Swiss albino mice (22-27 g) were housed in standard cages (48\u00d735\u00d722 cm) at room temperature (20 \u00b1 2\u00b0C), relative humidity (55-60%) and a 12 h light\/dark cycle. Theywere fed with normal chow pellet, and water ad libitum<\/em>.<\/p>\n

Toxicity study:<\/strong><\/p>\n

The extracts i.e. EAU, EAT and EAG were selected based on their potential antioxidant activity. The extracts of the seaweed Enteromorpha compressa<\/em> were excluded from further in vivo<\/em> evaluation due to their poor antioxidant property. Toxicity study of these selected extracts on female Swiss albino mice was carried out as per OECD regulations 42316<\/sup>. The highest dose (i.e. 2000 mg\/kg) of each extract was administered orally to the overnight fasted mice. The signs of possible toxicity were observed at every 3 hours for the first 24 hours and every day for 14 days. Individual animal weight was noted down daily and any signs or symptoms of toxicity and mortality was observed for 14 days as described in previous studies17<\/sup>.<\/p>\n

Hypoglycemicactivity in normal mice:<\/strong><\/p>\n

Animals were grouped as follow and administered the respective treatment intraperitoneally (i.p.). Group 1: 1% sodium CMC; Group 2: EAU (200 mg\/kg); Group 3: EAT (200 mg\/kg) and Group 4: EAG (200 mg\/kg). Each group consists of fiveanimals. Animals were fasted overnight for a period of 12 h. After the single i.p. injection of the respective extracts, blood sample (0.1 ml) was collected in micro tubes previously filledwith 10% EDTA solution (20 \u03bcl of 10% EDTA\/ ml of blood). Blood was collected through retro orbital route under mild ether anesthesia at 0, 0.5, 1, 2, 4, 6, 10 and 24 h. The micro tubes were centrifuged at 4000 rpm at 4\u00b0C for 20 min to obtain clear plasma. The plasma was thenanalyzed for glucose in the auto analyzer (3000 Evolution, BSI Italy) using commercially available glucose estimation kits. The data is expressed as the percentage change of blood glucose obtained at different time points18<\/sup>.<\/p>\n

Antidiabetic activity:<\/strong><\/p>\n

Antidiabetic study was carried out for EAU, which showed the best hypoglycaemic activity as compared to the other extracts.<\/p>\n

Diabetes induction and grouping of animals:<\/strong><\/p>\n

Alloxan monohydrate (150 mg\/kg, i.p.) was injected to the overnight fasted male Swissalbino mice19<\/sup>. Hyperglycaemia was confirmed by the elevated plasma glucose levels (>180 mg\/dL) after 3 days of the induction. The diabetic mice were randomized into 5 different groups and the treatment was administered orally for six consecutive days as follows. However, one group was kept as non-diabetic control without drug treatment (Group 1); Group 2: diabetic control, 1% sodium CMC; Group 3: EAU (100 mg\/kg); Group 4: EAU (200mg\/kg); and Group 5: Gliclazide (GLI) (10 mg\/kg). Each group consists of five animals. At the end of sixth day, blood sample was collected from the retro orbital route of each mouse and biochemical parameters such as plasma glucose, triglyceride and total cholesterol was estimated.<\/p>\n

Oral glucose tolerance test (OGTT):<\/strong><\/p>\n

OGTT was performed in overnight (18 h) fasted mice with water ad libitum<\/em>20,21<\/sup>. On 6th<\/sup> day of treatment, glucose (2 g\/kg) was fed to each animal 10 min after collecting blood sample which was taken as reference (0 min). Exactly 0.1 mL of blood was withdrawn from the retro orbital route of each mouse under mild ether anaesthesia at 30, 60 and 120 min after the glucose load. The blood samples were centrifuged to obtain clear plasma which was then analyzed for glucose by using commercially available biochemical kits.<\/p>\n

In vitro \u03b1-amylase enzyme inhibition assay<\/strong><\/p>\n

The \u03b1-amylase inhibition activity of EAU was determined by following a established method22<\/sup>. Starch azure (2mg) was suspended in each of tubes containing 0.2ml of 0.5M Tris-HCL buffer (pH 6.9) and 0.01M CaCl2<\/sub>. The tubes containing substrate solution were boiled for 5 min and were then incubated at 37\u1d52C for 5 min. Seaweed extract (0.2ml) was taken in each tube containing different concentrations (20, 40, 60, 80 and 100 \u00b5g\/ml). Porcine Pancreatic amylase (PPA) was dissolved in Tris-HCL buffer to form a concentration of 2units\/ml and 0.1ml of this enzyme solution were added to each of the above mentioned tubes. The reaction was carried out at 37\u1d52C for 10 min and was stopped by adding 0.5ml of 50% acetic acid in each tube. The reaction mixture was centrifuged at 3000 rpm for 5 min at 4\u1d52C. The absorbance of the resulting supernatant was measured at 595 nm. The \u03b1-amylase inhibition activity was calculated as follows:<\/p>\n

Alpha-amylase inhibition activity = [(Ac<\/sub>+)-(Ac<\/sub>-)]-[(As<\/sub>-Ab<\/sub>)\/ [(Ac<\/sub>+)-(Ac<\/sub>-)]\u00d7100 ——–Eq. 2<\/p>\n

Where Ac<\/sub>+, Ac<\/sub>-, As <\/sub>are the absorbance of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme activity), a test sample (with enzyme) and a blank (a test sample without enzyme), respectively.<\/p>\n

Statistical analysis<\/strong><\/p>\n

Results were expressed as mean \u00b1 SEM. Data were analyzed with One-way Analysis of Variance (ANOVA), followed by Tukey\u2019s multiple comparison tests. The level of significance was set at p< 0.05.<\/p>\n

Results<\/strong><\/p>\n

Preliminary phytochemical analysis of different extracts<\/p>\n

The detail results of qualitative preliminary phytochemical assays of different seaweed extracts are illustrated in Table 1. Presence of alkaloid was detected in PEU. Gum and mucilage was detected in two green seaweed extracts (MEU and MEE). Tannins, protein and amino acids were detected in methanol extracts of all seaweeds. Phenolic compounds were detected in ethyl acetate and methanol extracts of all seaweeds. Whereas, carbohydrate was absent in all the seaweed extracts.<\/p>\n

In vitro <\/em><\/strong>antioxidant evaluation of extracts<\/strong><\/p>\n

Table 2:<\/strong> Total phenolic content of ethyl acetate and methanol extracts obtained from\u00a0seaweeds.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Seaweed extracts<\/strong><\/td>\n\n

Total phenolic content\/ gm<\/strong><\/p>\n

(mg gallic acid equivalent\/ gm of extract)<\/strong><\/p>\n<\/td>\n<\/tr>\n

EAU<\/td>\n207.23 \u00b1 2.41<\/td>\n<\/tr>\n
EAT<\/td>\n55.71 \u00b1 3.65<\/td>\n<\/tr>\n
EAG<\/td>\n35.86 \u00b1 3.77<\/td>\n<\/tr>\n
EAE<\/td>\n31.77 \u00b1 4.23<\/td>\n<\/tr>\n
MEU<\/td>\n38.82 \u00b1 2.21<\/td>\n<\/tr>\n
MET<\/td>\n36.77 \u00b1 3.17<\/td>\n<\/tr>\n
MEG<\/td>\n29.80 \u00b1 3.77<\/td>\n<\/tr>\n
MEE<\/td>\n25.03 \u00b1 4.91<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Each experiment was performed in triplicte and the results are expressed as mean \u00b1 S.E.M.<\/p>\n

EAE: \u00a0ethyl acetate extract of Enteromorpha<\/em>; MEE: methanol extract of Enteromorpha<\/em>; EAU: ethyl acetate extract of Ulva<\/em>; MEU: methanol extract of Ulva<\/em>; EAT: ethyl acetate extract of Turbinaria<\/em>; MET: methanol extract of Turbinaria<\/em>; EAG: ethyl acetate extract of Gracilaria<\/em>; MEG: methanol extract of Gracilaria<\/em>.<\/p>\n

Total phenol content:<\/strong><\/p>\n

The total phenol content expressed as gallic acid equivalents was found to be highest for EAU (207.23 \u00b12.41mg\/g) compared to the other extracts as represented in Table 2. The lowest phenol content was found in MEE (25.03 \u00b1 4.91 mg\/g). In general, phenolic compounds were found in all the three groups\u2019 i.e. brown, green and red seaweeds taken in the present study.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: <\/strong>Total reducing activity of seaweed extracts (50- 200 \u00b5g\/mL) and BHT (50\u2013200 \u00b5g\/mL).<\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Each experiment was performed in triplicte and the results are expressed as mean \u00b1 S.E.M.<\/p>\n

EAE: \u00a0ethyl acetate extract of Enteromorpha<\/em>; MEE: methanol extract of Enteromorpha<\/em>; EAU: ethyl acetate extract of Ulva<\/em>; MEU: methanol extract of Ulva<\/em>; EAT: ethyl acetate extract of Turbinaria<\/em>; MET: methanol extract of Turbinaria<\/em>; EAG: ethyl acetate extract of Gracilaria<\/em>; MEG: methanol extract of Gracilaria<\/em>.<\/p>\n

Total reducing power:<\/strong><\/p>\n

Reducingcapacity of seaweed extracts and BHT (50-200 \u00b5g\/mL) is given in Figure 1. EAU showed higher reducing ability at all concentrations compared to other seaweed extracts. At 200 \u00b5g\/mL the reducing activity of EAU was found to be (absorbance, 1.18 \u00b1 0.006), which was almost nearer to the value recorded for standard BHT (absorbance, 1.48). The reducing ability of methanol extracts of E.compressa<\/em> and G. Verrucosa<\/em>was found to be the lowest as compared to the other seaweed extracts.<\/p>\n

IC50<\/sub> value of seaweeds extracts in H2<\/sub>O2<\/sub> and nitric oxide scavenging activity:<\/strong><\/p>\n

The IC50 <\/sub>value of hydrogen peroxide and nitric oxide free radical scavenging activity is presented in Table 3. The lowest IC50 <\/sub>values for both H2<\/sub>O2<\/sub> and nitric oxide scavenging activitywas encountered with EAU suggesting thereby that it has potent antioxidant activity compared to other seaweed extracts. The rank order of IC50<\/sub> value of H2<\/sub>O2<\/sub> and Nitric oxide scavenging activity of seaweeds are EAU> EAT> MEU> MET> EAG> EAE> MEG> MEE.<\/p>\n

Table 3<\/strong>: IC50 <\/sub>value of hydrogen peroxide and nitric oxide free radical scavenging activity of ethyl acetate and methanol extracts obtained from seaweeds.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
Seaweed\u00a0 extracts<\/strong><\/td>\nIC50 <\/sub>of H2<\/sub>O2<\/sub> scavenging activity (\u00b5g\/mL)<\/strong><\/td>\nIC50 <\/sub>of NO radical scavenging activity (\u00b5g\/mL)<\/strong><\/td>\n<\/tr>\n
EAU<\/td>\n123.39<\/td>\n127.65<\/td>\n<\/tr>\n
EAT<\/td>\n141.35<\/td>\n164.99<\/td>\n<\/tr>\n
EAG<\/td>\n168.96<\/td>\n258.06<\/td>\n<\/tr>\n
EAE<\/td>\n181.12<\/td>\n283.69<\/td>\n<\/tr>\n
MET<\/td>\n163.31<\/td>\n249.65<\/td>\n<\/tr>\n
MEU<\/td>\n157.79<\/td>\n240.94<\/td>\n<\/tr>\n
MEG<\/td>\n195.30<\/td>\n306.39<\/td>\n<\/tr>\n
MEE<\/td>\n211.01<\/td>\n313.97<\/td>\n<\/tr>\n
Ascorbic Acid<\/td>\n109.30<\/td>\n97.33<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Each experiment was performed in triplicte and the results are expressed as mean \u00b1 S.E.M.<\/p>\n

EAE: \u00a0ethyl acetate extract of Enteromorpha<\/em>; MEE: methanol extract of Enteromorpha<\/em>; EAU: ethyl acetate extract of Ulva<\/em>; MEU: methanol extract of Ulva<\/em>; EAT: ethyl acetate extract of Turbinaria<\/em>; MET: methanol extract of Turbinaria<\/em>; EAG: ethyl acetate extract of Gracilaria<\/em>; MEG: methanol extract of Gracilaria<\/em>.<\/p>\n

Toxicity study of the seaweed extracts as per OECD guidelines 423:<\/strong><\/p>\n

We observed no significant toxic signs or death during the 14 day observation period. None of the mice showed clinical toxic signs such as anorexia, depression, lethargy and also no mortality happened throughout the examination.<\/p>\n

Table 4:<\/strong> Effect of EAU on fasting plasma glucose, triglyceride and total cholesterol in alloxan induced diabetic mice.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Treatments<\/strong><\/td>\nGlucose (mg\/dl)<\/strong><\/td>\nTriglyceride (mg\/dl)<\/strong><\/td>\nTotal Cholesterol (mg\/dl)<\/strong><\/td>\n<\/tr>\n
Before<\/strong><\/td>\nAfter<\/strong><\/td>\nBefore<\/strong><\/td>\nafter<\/strong><\/td>\nBefore<\/strong><\/td>\nAfter<\/strong><\/td>\n<\/tr>\n
Group 1(N)<\/strong><\/td>\n92.0 \u00b1 4.4<\/td>\n91.4 \u00b1 3.9<\/td>\n35.8 \u00b1 8.3<\/td>\n36.0 \u00b1 5.1<\/td>\n34.2 \u00b1 6.0<\/td>\n36.8 \u00b1 7.6<\/td>\n<\/tr>\n
Group 2(D)<\/strong><\/td>\n211.4 \u00b1 20.4#<\/td>\n226.6 \u00b1 19.8#<\/td>\n57.0 \u00b1 12.9<\/td>\n56.2 \u00b1 14.8<\/td>\n58.0 \u00b1 11.0<\/td>\n78.0 \u00b1 8.5#<\/td>\n<\/tr>\n
Group 3 <\/strong>(EAU1)<\/strong><\/td>\n218.2 \u00b1 18.3<\/td>\n211.4 \u00b1 16.3<\/td>\n46.2 \u00b1 11.8<\/td>\n40.4 \u00b1 3.8<\/td>\n69.8 \u00b1 6.8<\/td>\n57.2 \u00b1 7.3<\/td>\n<\/tr>\n
Group 4<\/strong> (EAU 2)<\/strong><\/td>\n235.2 \u00b1 14.7<\/td>\n214.0 \u00b1 10.8\u00a3<\/td>\n51.4 \u00b1 9.2<\/td>\n36.2 \u00b1 4.8<\/td>\n64.0 \u00b1 4.2<\/td>\n44.4 \u00b1 5.9\u00a3<\/td>\n<\/tr>\n
Group 5 <\/strong>(G)<\/strong><\/td>\n222.6 \u00b1 18.7<\/td>\n176.0 \u00b1 18.4\u20ac<\/td>\n31.8 \u00b1 6.0<\/td>\n29.6 \u00b1 2.8<\/td>\n61.6 \u00b1 16.8<\/td>\n56.2 \u00b1 10.3<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

N: Normal control, D: Diabetic control, 200 mg\/kg, EAU1: Ethyl acetate extract of Ulva sp<\/em>. 100 mg\/kg, EAU2: Ethyl acetate extract of Ulva sp<\/em>. 200 mg\/kg, G: Gliclazide 10 mg\/kg. #: p<0.05w.r.t Gr.1; \u20ac: p<0.01w.r.t Gr.2; \u00a3: p<0.05w.r.t Gr.2. n= 5 in each group.<\/p>\n

In vivo <\/strong>hypoglycaemic and antidiabetic study<\/strong><\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2: Hypoglycaemic effect of ethyl acetate extracts of different seaweeds (EAU, EAT and EAG) in male Swiss albino mice.<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*p< 0.05 and **p< 0.01 vs control; EAU: ethyl acetate extract of Ulva<\/em>; EAT: ethyl acetate extract of Turbinaria<\/em>; EAG: ethyl acetate extract of Gracilaria.<\/em><\/p>\n

Hypoglycaemic activity in normal mice:<\/p>\n

Hypoglycaemic effect of EAU, EAT and EAG ate 200 mg\/kg is presented in Figure 2. It has been observed that EAU showed the highest hypoglycaemic effect in comparison to other seaweed extracts. Hypoglycaemic effect of seaweeds extracts follows as EAU>EAT>EAG. Maximum effect was observed in between 4 – 6 h of extract administration. EAU treated mice showed -20% and -25% change in plasma glucose at 4 and 6 h respectively. The least change (-10%) in plasma glucose was observed in case of EAG treated mice at 4 h.<\/p>\n

Plasma glucose, triglyceride and total cholesterol:<\/p>\n

Fasting plasma glucose, triglyceride and total cholesterol was estimated before and after the drug treatment. EAU (200 mg\/kg) treated animals showed significant (p < 0.05) reduction in their plasma glucose level after the treatment. However, GLI (10 mg\/kg) treated mice showed the maximum reduction (p < 0.01) in plasma glucose as compared to other treatment groups. Fasting plasma triglyceride level in all control and treated animals did not showed significant changes before and after treatment. Plasma total cholesterol was measured before and after the drug treatment for all the groups. EAU at 200 mg\/kg showed significant (p < 0.05) reduction in total cholesterol level after treatment. The results are summarized in Table 4.<\/p>\n

Oral glucose tolerance test (OGTT):<\/p>\n

The extent of reduction in AUC of OGTT was higher in Gliclazide (50 mg\/kg) treated mice (p<0.01). The EAU (100 and 200 mg\/kg) treated animals though showed decreased trend in the AUC as compared to the diabetic groups but, it was not found to be significant. The AUC of the plasma glucose disappearance curve is shown in Figure 3.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: Effect of EAU on Area under curve (AUC) of plasma glucoselevelin OGTT.<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

DB control, diabetic control; EAU: ethyl acetate extract of Ulva<\/em>; GLI: Gliclazide; #p< 0.05 and ##p < 0.01 vs. normal control; *p < 0.05 and **p < 0.01 vs. diabetic control.<\/p>\n

In vitro \u03b1-amylase inhibitory assay<\/strong><\/p>\n

In the present study, the results of in vitro \u03b1-amylase inhibitory assay showed that Ulva sp.<\/em>exhibited a potent inhibitory activity on \u03b1-amylase enzyme (IC 50<\/sub> = 69.122\u00b5g\/ml)compared with the positive standard, acarbose (IC 50<\/sub> = 49.344 \u00b5g\/ml) (Table 5). A concentration dependent inhibition was observed for various concentrations of these algal extracts. The percentage inhibitory activity for EAU was found to be 60.191 \u00b1 2.235 (at 100\u00b5g\/mL). The percentage inhibitory activity for acarbose at the same concentration was observed as 75.733 \u00b1 0.068.<\/p>\n

Table 5:<\/strong>\u00a0 IC50 <\/sub>(50 % inhibition) value of alpha amylase inhibiting activity of EAU.<\/strong><\/p>\n\n\n\n\n\n
Seaweed\u00a0 extracts<\/strong><\/td>\nIC50 <\/sub>of\u00a0 alpha amylase inhibiting activity (\u00b5g mL–<\/strong>1<\/sup>)<\/strong><\/td>\n<\/tr>\n
EAU<\/td>\n69.122<\/td>\n<\/tr>\n
Acarbose<\/td>\n49.344<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

EAU: ethyl acetate extract of Ulva<\/em><\/p>\n

Discussion<\/strong><\/p>\n

Marine macro algae are recognized as potential source of several important secondary metabolites including alkaloids, phenols, flavonoids, saponins, steroids and other related active metabolites.These secondary metabolites participate extensively in their defence against diseases, microorganism, stress and interspecies protections.They have been extensively studied for their importance in furnishing good health and defend against diseases and thus seaweeds could definitely behave as budding hypoglycemic and antidiabetic agents23<\/sup>.Therefore, priliminary phytochemical screening serves as the opening step in predicting the types of probable active compounds present in these seaweed extracts so that the best ever extracts among them could be selected for further studies.The results of the preliminary photochemical study revealed that ethyl acetate and methanol extracts of almost all the seaweeds showed the presence of some of the most important secondary metabolites like terpenoid, flavonoids and phenolics. These metabolites have been proven to provide encouraging antioxidant and anti-diabetic properties24,25<\/sup>.<\/p>\n

Some studies reveal the fact that ROS leads to cell and tissue dysfunction and injury caused by glucolipotoxicity in diabetes26,27<\/sup>. In addition to that studies have also proven that, antioxidants can bring to bearvaluable effects on pancreatic \u03b2-cells function in diabetes by providing security against glucose toxicity8<\/sup>. Thus, anadequateuse of antioxidants may be valuable in counteracting diabetic complications and managing the disease. In this study, the extracts containing suitable metabolites (Terpenoid, flavonoids, amino acids and phenolics) were evaluated for potential antioxidant activity using established in vitro<\/em> assays. The results of the present study revealed that ethyl acetate extracts of all the seaweeds exhibited considerable free radical scavenging activity. The reason behind this may be due to the presence of considerable amount of phenolics in them as some studies have claimedthat there exist a positive correlation between total phenolic content and antioxidant property28,29<\/sup>. Based on this fact, EAU, EAT and EAG were studied for hypoglycaemic effect in normal Swiss albino mice as they showed good total phenolic content and prior study has reported that phenolic components present in seaweeds exert potential hypoglycaemic effect30<\/sup>. The finding of this study leads to infer that EAU has potent hypoglycemic effect in normal mice and hence this extract was selected for further antidiabetic study as compounds with hypoglycemic principle have got more direct role in antihyperglycemic activity31<\/sup>.<\/p>\n

The chiefobjective in the management of diabetes mellitus is to preserve normal plasma glucose levels in both the fasting and postprandial state. In the present study, EAU has effectively lowered and normalised the blood glucose level at the higher dose. Notably, this effect was significantly prominent and glucose level was pragmatic to be low even on the sixth day of the treatment. However, promising effects were not reflected by AUC of OGTT curve. OGTT reflects the competence of the body to dispose of glucose after an oral glucose load or meal. This test closely mimics the glucose and insulin dynamics of physiological conditions more closely. Impaired glucose tolerance is reflected in a larger incremental AUC of the plasma glucose disappearance curve. Results of OGTT revealed that AUC significantly (p<0.01) increased in diabetic control compared to non diabetic control. Whereas, AUC was lowered in the other treatment groups as compared to diabetic control. EAU (100 and 200 mg\/kg) treated animals though showed a decrease in the AUC as compared to the diabetic groups, it was not significant. The important salutary approach to reduce postprandial glucose elevation is to repress the production and\/or assimilation of glucose from the gastrointestinal tract. The latter effect can be achieved through inhibition of some enzymes like \u03b1-amylase or \u03b1-glucosidase, those play important role in glucose absorption form the g.i.t.32<\/sup>.In our investigation we found that ethyl acetate extract Ulva sp.<\/em> moderately inhibited \u03b1-amylase. Hence, the prevailing antidiabetic and hypoglycaemic effect of this extract may be due \u03b1-amylase inhibiting property of this extract. In order to establish the real mechanism behind these activities and to find out the actual active compounds responsible in imparting such activities, an extensive study is required.<\/p>\n

Conclusion<\/strong><\/p>\n

In conclusion the ethyl acetate extract of the marine macro algaU. Fasciata<\/em>showed considerable antidiabetic activity compared to other seaweed extracts. The underlying reason behind this marked activity may be related to its significant antioxidant, hypoglycaemic and in vitro alpha amylase inhibiting activities. However, more comprehensive in vitro <\/em>and in vivo <\/em>studies are needed to elucidate the exactmechanism of its antidiabetic activity. Further, the active principle responsible for such activities is required to be isolated andstudied. Acknowledgements<\/strong><\/p>\n

The corresponding author expresses deepest sense of gratitude to the Centre of Scientific and Industrial Research (CSIR), Govt. of India, for providing financial support in the form of senior research fellowship to carry out this work. We express our sincere thanks to the Principal, Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, India, for providing necessary facilities to carry out the animal studies.<\/p>\n

Authors\u2019 Contribution<\/strong><\/p>\n

LM, SKB and RCP have conceived and designed this experimental protocol. LM and SP performed the experiment. SKB and RCP analysed the final data.\u00a0 LM and SP wrote the manuscript.<\/p>\n

Conflict of interest disclosure: <\/strong>The authors declare that there is no conflict of interest.<\/p>\n

References<\/strong><\/p>\n

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    Introduction Diabetes mellitus is a cluster of never-ending diseases, which […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[30],"tags":[],"class_list":["post-6576","post","type-post","status-publish","format-standard","hentry","category-vol9no1"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/6576","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=6576"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/6576\/revisions"}],"predecessor-version":[{"id":32642,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/6576\/revisions\/32642"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=6576"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=6576"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=6576"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}