In vitro α-glucosidase and in vivo of anti-hyperglycemia activity extract of alginate from the brown marine algae Sargassum hystrix

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Research Article In vitro α-glucosidase and in vivo of anti-hyperglycemia activity extract of alginate from the brown marine algae Sargassum hystrix Agung Giri Samudra 1 *, K.

1 Research Article In vitro α-glucosidase and in vivo of anti-hyperglycemia activity extract of alginate from the brown marine algae Sargassum hystrix Agung Giri Samudra 1 *, K. Fathnur Sani 1, Amir Husni 2 ABSTRACT Background: Carbohydrate metabolism disorder can cause diabetes mellitus indicated by hyperglycemic. The carbohydrate in the intestine metabolizes into simple glucose, then it is absorbed into blood circulation, and it influences the glucose level in the blood. Brown algae (Sargassum hystrix) contain alginate as the anti-hyperglycemic agent. Methods: Alginate extract of S. hystrix was tested for their ability to inhibit carbohydrate hydrolysis enzymes α-glucosidase by in vitro methods. Oral glucose tolerance test in which the measurement was done in the minute of 15, 30, 60, 90 and 120 after giving the sucrose by in vivo methods. Results: The result showed that S. hystrix had the ability of lowering the blood glucose level in which the most influential was the alginate II 125 mg/kg rather than alginate I 62.5 mg/kg (P < 0.05). Conclusion: The carbohydrate metabolism resistance could lower the glucose level in the blood and indicate as the agent that was potential enough in overcoming the hyperglycemia. KEY WORDS: α-glucosidase, Blood glucose, Oral glucose tolerance, Sargassum hystrix INTRODUCTION The treatment of diabetes mellitus complications or that of diabetes mellitus symptoms like hyperglycemic is the chronic and lifelong medication. Globally, an estimated 422 million adults were living with diabetes in 2014, compared to 108 million in The global prevalence (age-standardized) of diabetes has nearly doubled since 1980, rising from 4.7 to 8.5% in the adult population. Diabetes caused 1.5 million deaths in Higher-than-optimal blood glucose caused an additional 2.2 million deaths, by increasing the risks of cardiovascular and other diseases. [1] There are three main types of diabetes, Types I and II and gestational diabetes. Type II diabetes is the most common, which is affecting 90-95% of the U.S. diabetes population. [2] One of the therapeutic approaches to treat the diabetes is to decrease the postprandial hyperglycemia by retarding absorption Access this article online Website: jprsolutions.info ISSN: of glucose. Inhibition of carbohydrate-hydrolyzing enzymes, such as α-glucosidase, is considered a possible pathway because the enzyme plays a key role in digesting carbohydrates. [3,4] Thus, treating diabetes has focused on preventing diabetic complications and/or suppressing further development of the disease by controlling the level of blood glucose. Because the ability to fundamentally cure diabetes using insulin or oral hypoglycemic agents is technically limited and because there are financial burdens as well as the side effects associated with the prolonged intake of medicines, the use of natural products with fewer side effects is strongly recommended. With advances in alternative medicine, there have been many studies on the effects of natural products on antidiabetic activity, and various functional foods for diabetes patients are under development. [5,6] Alginate is reported to play a key role as the fiber that can clean the digestive system, protect the membrane surface of the stomach and the intestines, lower the cholesterol concentration, give antihypertensive effect, and have the activity as the antioxidant. [7-9] In 1 Department of Pharmacology and Clinical Pharmacy, Akademi Farmasi Al-Fatah Bengkulu, Jalan Indragiri Gang Tiga Serangkai Padang Harapan Bengkulu, Indonesia, 2 Department of Fisheries Faculty of Agriculture Universitas Gadjah Mada, Jalan Flora Gedung A4 Bulaksumur Yogyakarta Indonesia *Corresponding author: Agung Giri Samudra,Department of Pharmacology and Clinical Pharmacy, Akademi Farmasi Al- Fatah Bengkulu, Jalan Indragiri Gang Tiga Serangkai Padang Harapan Bengkulu, Indonesia. Phone: agunggirisamudra@gmail.com Received on: ; Revised on: ; Accepted on: Journal of Pharmacy Research Vol 11 Issue

2 this research, the activity of the extract of alginate as the anti-hyperglycemia would be tested. MATERIALS AND METHODS Materials The sample used in this research was brown algae (Sargassum sp.) hydrochloric acid (E. Merck), sodium carbonate (E. Merck), hydrogen peroxide (E. Merck), sodium hydroxide (E. Merck), alginate extraction of brown algae (Sargassum hystrix), acarbose, sucrose, and methanol, 3,5-dinitrosalicylic acid (Sigma-Aldrich D 0550), α-glucosidase from Saccharomyces cerevisiae Type I (Sigma-Aldrich G 5003), 4-nitrophenyl-α-d-glucopyranoside (Sigma- Aldrich N 1377). Extraction of Alginate Sargassum sp. was soaked in 5% hydrochloric acid solution, washed with distilled water. Sample was added to 4% sodium carbonate solution (1:30 w/v) left for 1 h, stirred frequently until it becomes a paste. Paste discoloration was formed by adding 25% hydrogen peroxide solution into the filtrate. The precipitate that formed was added to 5% hydrochloric acid solution. The viscous mixture was separated from its residue, added 10% sodium hydroxide solution into the residue. It was then precipitated with filter paper which has been known to weigh and dried in an oven at 60 C. Fourier Transform Infrared Spectroscopy (FTIR) Alginate The sample powder that had been prepared was mixed with KBr to make pellets. The spectrum measurement was conducted using FTIR spectrometer. The measurement was done by means of the transmission of the wavenumber of cm 1, with the scan speed 0.20 cm/s and 30 accumulations on the resolution of 4 cm 1. [10] In Vitro α-glucosidase Inhibition Study Various concentrations extract or acarbose (0.125; 0.25; 0.5; 1; 2 mg/ml) was made. The reaction mixture contained 10 μl of test sample at various concentrations extract or acarbose, 25 µl α-glucosidase (0.2 Unit/mL) in 20 mm phosphate buffer (ph 7), and 25 µl of 0.5 mm 4-nitrophenyl α-d-glucopyranoside as substrate. This reaction mixture was then incubated at 37 C for 30 min. The reaction was terminated by adding 100 μl of 0.2 M sodium carbonate solution. The absorbance was read at 540 nm using microplate Oral Glucose Tolerance Test In this research, 12 male white mice used were divided into several groups, namely, negative control (aquadest), positive control (acarbose 6.5 mg/kg), extract of alginate I (62.5 mgkg 1 ), and extract of alginate II (125 mg/kg) in which each group consisted of 3 male, white mice chosen randomly. Previously, all the mice fasted for 10 h, but they were still given water, and then the blood glucose level of their fast was measured. Next, the mice were induced sucrose with the dose of 9 grkg 1, and 30 min later its blood glucose level was measured. After giving the supply, its blood glucose level was measured in the minute of 15, 30, 60, 90, and 120. [14] RESULTS AND DISCUSSION Extraction Extraction of alginate acquired from the seaweed S. hystrix consecutively was 46.84%. Alginate Extract Identification Alginate FTIR spectrum was the first band of spectra of standard alginate and its extract containing the frequency vibration of area and cm 1 carboxylate anion group (COO-). The second band was and cm 1 carboxylate anion group (C-OH). The third band was and cm 1 anion group C-O-C. The band between standard alginate and alginate extract contained the similar groups of -COO-, -OH, C-O-C that it can be concluded that its extract compound was alginate. FTIR spectrophotometer can be used to determine the approximate comparison mannuronic/guluronic (ratio of M/G). Result FTIR spectrophotometer showed the presence of local wave number 1030 cm 1 (mannuronic), alginate standard wavenumber cm 1 absorbance value 0.375, alginate extract wavenumber cm 1 absorbance value of 0.514, showed no local wave number 1080 cm 1 (guluronic). It is estimated standard compiler, and extract alginate a ( A Control 540 A Extract 540 ( A Control 540) ) reader. [11-13] % Inhibition = 100 b Figure 1: Infrared spectra of Na-alginate standard (a) and Na-alginate of Sargassum hystrix (b) 928 Journal of Pharmacy Research Vol 11 Issue

3 Table 1: Signals assigned in the FTIR of sodium alginate of S. hystrix and sodium alginate standard Band Wave number (cm 1 ) Assignments Na alginate standard Na alginate of S. hystrix ν asym O C O [16,17] δ C O H, νsym COO (carboxylate ion) [16] ν C O [16] ν C O [16] δ C1 H (β mannuronic residues) [16] FTIR: Fourier Transform Infrared Spectroscopy, S. hystrix: Sargassum hystrix has a mannuronic ratio is high. The higher the content of guluronic blocks in alginate gel then progressively generate more powerful with a more rigid texture (Figure 1 and Table 1). [15] In Vitro α-glucosidase Inhibition Study The in vitro α-glucosidase inhibitory studies demonstrated that both alginate extract α-glucosidase inhibitory activity. The percentage inhibition at 0.125; 0.25; 0.5; 1; 2 mg/ml contractions of alginate extract showed a concentration-dependent reduction in percentage inhibition. The inhibition activity of α-glucosidase showed that acarbose has the greatest ability to inhibit α-glucosidase activity, followed by alginate standard and alginate extract. Figure 2 showed that increasing on the concentration of S. hystrix, the level of α-glucosidase inhibitory activity also increased. The high inhibitory activity was found in acarbose (91,87%) followed by alginate standard (60%), alginate extract (58,75%). The IC 50 acarbose, alginate standard, alginate extract was 0.75, 1.54, and 1.55 mgml 1, respectively (Table 2). IC 50 values of the three compounds that have ability to inhibit the activity from the largest to the smallest were acarbose, alginate standard, and alginate extract. Oral Glucose Tolerance Test To confirm the in vivo relevance of our in vitro findings that alginate extract exhibited α-glucosidases inhibitory activities, we performed a sucrose loading test in mice, which is a model more relevant toward Type 2 diabetes prevention with normal or prediabetic individuals, rather than Type 2 diabetes treatments. The result of the testing of the effect of extracts of alginate S. hystrix. From the data of the glucose level value (Table 3), it showed that the provision of alginate from Sargassum sp. had an effect on the glucose level in the blood. The calculation result of the glucose level decrease (Figure 3) showed that the order of the percentage of the highest value to the lowest value was acarbose, alginate II, and alginate I. The alginates are widely utilized as gelling agents in pharmaceutical and food applications. Alginates do not have any nutritional value; however, they are often employed as additives to change and stabilize Table 2: Inhibitory activity (IC 50 ) α glucosidase Inhibitory IC 50 of α glucosidase (mg/ml) Acarbose 0.75 Alginate standard 1.54 Alginate extract 1.55 Figure 2: Effect sample concentration ( acarbose, : alginate standard alginate extract) on inhibition activity of α-glucosidase Figure 3: Decrease of the percentage AUC of blood glucose level the texture of foods. The potentially beneficial role of alginate in the gastrointestinal tract makes it a commercially valuable product texture. Alginate belonged to polysaccharides having fibers that were easily soluble in the water. [18] This polysaccharide could form grids like a net that was able to strongly bind many molecules of water. The net-like grids formed allowed to ensnare either substrate or watersoluble enzyme. [19,20] Substrate and enzyme ensnared by the grids and enclosed in the polysaccharide did not react each other. If the substrate did not react with Journal of Pharmacy Research Vol 11 Issue

4 Table 3: Value of mice blood glucose level Group Blood glucose level (mg/dl) AUC Baseline Minutes 15 Minutes 30 Minutes 60 Minutes 90 Minutes 120 Negative control 56± ± ± ± ± ± ±756 a Acarbose 6.5 mg/kg 57.7± ± ± ± ± ± ±437 c Alginate 62.5 mg/kg 60.3± ± ± ± ± ± ±295 b Alginate 125 mg/kg 48.3± ± ± ± ± ± ±717 bc Values are means±sem (n=3). Values followed by the same superscript symbol (s) in each column are not significantly different (P>0.05). AUC: Area under curve the enzyme, there was no formation of simple glucose. The alginate also showed the ability in decreasing or obstructing the rate of blood glucose level increase in vivo. The cation binding alginate would change intestinal ph by affecting the secretion of acids and bases through the influence of hormone and enzyme. [21] This would influence the process of carbohydrate breakdown (disaccharide) inside intestine and, in the end, the process of monosaccharide absorption too so that it can hold the rate of postprandial blood glucose level increase. The polysaccharides effect in decreasing the glucose level was also caused by its ability in massively absorbing water by forming gel or condensed solution. [22] Thus, the absorption of glucose became obstructed. In vivo and in vitro experiments have shown that administering these polysaccharides has hypoglycemic effects and alleviates β-cell dysfunction in addition to eliciting other antidiabetic activities. [9,23] The most important hormone in controlling sugar metabolism is insulin, which is produced by β-cells. In addition, in diabetes, the mrna available for α-amylase production in the exocrine acini of the colon decreases, thereby decreasing the synthesis of α-amylase; it has been reported that injection of insulin into diabetic mice increases the colonic excretion of α-amylase. [24] Decreasing postprandial hyperglycemia peak is crucial in the treatment of diabetes. [25] We, therefore, hypothesize that possible inhibition of carbohydrate-hydrolyzing enzymes α-amylase, α-glucosidase could represent part of the biochemical rationale in which plant phenols exert their therapeutic potentials in the management treatment of diabetes mellitus. CONCLUSION Sargassum sp. had the ability of lowering the blood glucose level in which the influential primary metabolite (alginate). Future work will be to investigate the changes in plasma insulin concentrations following treatment with the alginate and polyphenol extracts to determine the mechanisms of hyperglycemia. REFERENCES 1. World Health Organization (WHO). Global Report on Diabetes. Geneva: WHO; Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: Estimates for the year 2000 and projections for Diabetes Care. 2004;27(5): Bhandari MR, Nilubon JA, Gao H, Kawabata J. α-glucosidase and amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata Haw.). Food Chem. 2008;106: Krentz AJ, Bailey CJ. Oral antidiabetic agents: Current role in Type 2 diabetes mellitus. Drugs. 2005;65(3): Oh WK, Lee CH, Lee MS, Bae EY, Sohn CB, Oh H, et al. Antidiabetic effects of extracts from Psidium guajava. J Ethnopharmacol. 2005;96(3): Han HK, Je HS, Kim GH. Effect of Cirsium japonicum powder on plasma glucose and lipid level in streptozotocin induced diabetic rats. Korean J Food Sci Technol. 2010;42: Murata M, Nakazoe J. Production and use of marine algae in Japan. Jpn Agric Res Q. 2001;35: Nishide E, Uchida H. Effects of ulva powder on the ingestion and excretion of cholesterol in rats. In: Chapman AR, Anderson RJ, Vreeland VJ, Davison IR, editors. Proceedings of the 17 th International Seaweed Symposium. Oxford: Oxford University Press; p Sokolova EV, Barabanova AO, Homenko VA, Solov eva TF, Bogdanovich RN, Yermak IM. In vitro and ex vivo studies of antioxidant activity of carrageenans, sulfated polysaccharides from red algae. Biophys Biochem. 2011;150(4): Gomez E, Ruperez P. FTIR-ATR spectroscopy as a tool for polysaccharide identification in edible brown and red seaweeds. Food Hydrocoll. 2011;2: Subramanian R, Asmawi MZ, Sadikun A. In vitro α-glucosidase and α-amylase enzyme inhibitory effects of Andrographis paniculata extract and andrographolide. Acta Biochim Pol. 2008;55(2): Mayur B, Sandesh S, Shruti S, Sung-Yum S. Antioxidant and α-glucosidase inhibitory properties of Carpesium abrotanoides L. Med Plants Res. 2010;4(15): Apostolidis E, Karayannakidis PD, Kwon YI, Chong ML, Navindra PS. Seasonal variation of phenolic antioxidantmediated α-glucosidase inhibition of Ascophyllum nodosum. Plant Foods Hum Nutr. 2011;66: Sornalakshmi V, Tresina SP, Paulpriya K, Packia LM, Mohan VR. Oral glucose tolerance test (OGTT) in normal control and glucose induced hyperglycemic rats with Hedyotis leschenaultiana DC. Int J Toxicol Pharmacol Res. 2016;8(1): Subramanian V, Ganapathy K, Dakshinamoorthy B. FT-IR, 1 H-NMR and 13 C-NMR spectroscopy of alginate extracted from Turbinaria decurrens. World J Pharm Pharm Sci. 2015;4(12): Leal D, Matsuhiro B, Rossi M, Caruso F. FT-IR spectra of alginic acid block fractions in three species of brown seaweeds. Carbohydr Res. 2008;343(2): Campos-Vallette MM, Chandía NP, Clavijo E, Leal D, Matsuhiro B, Osorio-Roman IO, et al. Characterization of sodium alginate and its block fractions by surface-enhanced raman spectroscopy. J Raman Spectrosc. 2010;41: Holdt SL, Kraan S. Bioactive compounds in seaweed: Functional food applications and legislation. J Appl Phycol. 2011;23: Sankalia MG, Mashru RC, Sankalia JM, Sutariya VB. Stability 930 Journal of Pharmacy Research Vol 11 Issue

5 improvement of alpha-amylase entrapped in kappa-carrageenan beads: Physicochemical characterization and optimization using composite index. Int J Pharm. 2006;312(1-2): Sankalia MG, Mashru RC, Sankalia JM, Sutariya VB. Reversed chitosan alginate polyelectrolyte complex for stability improvement of alpha-amylase: Optimization and physicochemical characterization. J Pharm Biopharm. 2007;65: Zhang CY, Wu WH, Wang J, Lan MB. Antioxidant properties of polysaccharide from the brown seaweed Sargassum graminifolium (Turn.) and its effects on calcium oxalate crystallization. Mar Drugs. 2012;10: Truss K, Vaher M, Taure I. Algal biomass from Fucus vesiculosus (Phaeophyta): Investigation of the mineral and alginate components. Proc Estonian Acad Sci Chem. 2001;50(2): Wu J, Shi S, Wang H, Wang S. Mechanisms underlying the effect of polysaccharides in the treatment of Type 2 diabetes: A review. Carbohydr Polym. 2016;144: Jang SH, Park J, Kim SH, Choi KM, Ko ES, Cha JD, et al. Red ginseng powder fermented with probiotics exerts antidiabetic effects in the streptozotocin-induced mouse diabetes model. Pharm Biol. 2017;55: Aguilar-Santamaría L, Ramírez G, Nicasio P, Alegría-Reyes C, Herrera-Arellano A. Antidiabetic activities of Tecoma stans (L.) Juss. ex Kunth. J Ethnopharmacol. 2009;124(2): Journal of Pharmacy Research Vol 11 Issue

International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: , ISSN(Online): Vol.9, No.12, pp , 2016

International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.12, pp 531-535, 2016 Inhobitory Potential of Poligala paniculata L. Against α-glucosidase