A phlorotannin constituent of Ecklonia cava alleviates postprandial hyperglycemia in diabetic mice

Abstract Context: 2,7″-Phloroglucinol-6,6′-bieckol is a type of phlorotannin isolated from brown algae, Ecklonia cava Kjellman (Phaeophyceae; Laminareaceae). 2,7″-Phloroglucinol-6,6′-bieckol mediates antioxidant activities. However, there has been no research on improving postprandial hyperglycaemia using 2,7″-phloroglucinol-6,6′-bieckol. Objective: This study investigated the inhibitory effects of 2,7″-phloroglucinol-6,6′-bieckol on activities of α-glucosidase and α-amylase as well as its alleviating effect on postprandial hyperglycaemia in streptozotocin-induced diabetic mice. Materials and methods: α-Glucosidase and α-amylase inhibitory assays were carried out. The effect of 2,7″-phloroglucinol-6,6′-bieckol on hyperglycaemia after a meal was measured by postprandial blood glucose in streptozotocin-induced diabetic and normal mice. The mice were treated orally with soluble starch (2 g/kg BW) alone (control) or with 2,7″-phloroglucinol-6,6′-bieckol (10 mg/kg bw) or acarbose (10 mg/kg BW) dissolved in 0.2 mL water. Blood samples were taken from tail veins at 0, 30, 60, and 120 min and blood glucose was measured by a glucometer. Results: 2,7″-Phloroglucinol-6,6′-bieckol showed higher inhibitory activities than acarbose, a positive control against α-glucosidase and α-amylase. The IC50 values of 2,7″-phloroglucinol-6,6′-bieckol against α-glucosidase and α-amylase were 23.35 and 6.94 μM, respectively, which was found more effective than observed with acarbose (α-glucosidase IC50 of 130.04 μM; α-amylase IC50 of 165.12 μM). In normal mice, 2,7″-phloroglucinol-6,6′-bieckol significantly suppressed the postprandial hyperglycaemia caused by starch. The 2,7″-phloroglucinol-6,6′-bieckol administration group (2349.3 mmol·min/L) had a lower area under the curve (AUC) glucose response than the control group (2690.83 mmol·min/L) in diabetic mice. Discussion and conclusion: 2,7″-Phloroglucinol-6,6′-bieckol might be used as an inhibitor of α-glucosidase and α-amylase as well as to delay absorption of dietary carbohydrates.


Introduction
Diabetes mellitus is a progressive metabolic disorder characterized by high blood glucose levels (Sheetz 2002). Especially, a postprandial hyperglycaemia state is an important contributing factor to the development of type 2 diabetes mellitus as well as related complications, including atherosclerosis, diabetic nephours opathy and neuropathy. Therefore, control of hyperglycaemia is the most important factor for reducing risk of diabetic complications and is a major goal of diabetes treatment (Bonora & Muggeo 2001;Fujita et al. 2001). The most effective way to control postprandial blood glucose levels is medication in combination with dietary restriction and an exercise program (Yki-Jarvinen 1990). Available medications for diabetes include insulin and various oral hypoglycaemic agents such as sulfonylureas, biguanides, a-glucosidase inhibitors, etc. These drugs are used as monotherapies or in combination to achieve improved glycaemic control. However, each of the above oral antidiabetic agents is associated with a number of serious adverse effects. Hence, antidiabetic drugs have been recently screened and developed from natural sources with minimal side effects (Sels et al. 1999;Standl et al. 1999).
Ecklonia cava Kjellman (Phaeophyceae; Laminareaceae) is an edible marine brown alga species found in the oceans of Korea and Japan. Ecklonia cava has received attention recently due to its various biological activities, including radical scavenging, antiproliferative, antiallergic, antidiabetic and protease inhibitory effects (Ahn et al. 2004;Kang et al. 2005Kang et al. , 2010Kang et al. , 2013Kim et al. 2008b;Park et al. 2015). These effects are attributed to several compounds such as xanthophyll pigment, fucoxathin, phlorotannins and fucoidans. Especially, Ecklonia cava contains an abundance of biological polyphenolic compounds, referred to as phlorotannins. Phlorotannins are reported to possess antioxidant and anti-inflammatory activities but also metalloproteinase inhibitory activities (Li et al. 2009;Wijesekara et al. 2010).

Materials
Brown alga, Ecklonia cava, was collected along the coast of Jeju Island, Korea, between February and May of 2012. Verification of vouchers or living alga was performed by department of Faculty of Marine Biomedical Sciences of Jeju National University. Samples were washed three times with water to remove any attached salt, epiphytes, and sand, then rinsed carefully with fresh distilled water, and stored in a medical refrigerator at À20 C. Thereafter, frozen samples were lyophilized and homogenized using a grinder prior to extraction.

Inhibition assay for a-glucosidase activity in vitro
The a-glucosidase inhibitory assay was carried out by the chromogenic method developed by Watanabe et al. (1997) using a readily available yeast enzyme. Briefly, yeast a-glucosidase (0.7 U, Sigma, St. Louis, MO) was dissolved in 100 mM phosphate buffer (pH 7.0) containing 2 g/L of bovine serum albumin and 0.2 g/L of NaN 3 and used as an enzyme solution. p-Nitrophenyl-a-D-glucopyranoside (5 mM) in the same buffer (pH 7.0) was used as a substrate solution. Enzyme solution (50 lL) and 10 lL of sample dissolved in dimethylsulfoxide at a concentration of 5 mg/mL were mixed in a well, and absorbance at 405 nm was measured using a microplate reader. After incubation for 5 min, substrate solution (50 lL) was added and incubated for another 5 min at room temperature. The increase in absorbance from zero time was measured. Inhibitory activity was expressed as 100 minus the relative absorbance difference (%) of the test compounds compared to the absorbance change of the control where the test solution is replaced by carrier solvent. Measurements were performed in triplicate, and IC 50 value, i.e., concentration of extracts resulting in 50% inhibition of maximal activity, was determined.

Inhibition assay for a-amylase activity in vitro
The a-amylase inhibitory activity was assayed in the same way (Watanabe et al. 1997) as described for the a-glucosidase inhibitory assay, except that porcine pancreatic amylase (100 U, Sigma, St. Louis, MO) and blocked. p-Nitrophenyl-a-D-glucopyranoside (Sigma, St Louis, MO, USA) were used as enzyme and substrate, respectively.

Experimental animals
Four-week-old male mice (ICR, Orient, Inc., Seoul, Korea) were kept under a 12 h light/dark cycle at room temperature. The animals were provided pelleted food every day, whereas tap water was provided ad libitum. After an adjustment period of 2 weeks, diabetes was induced in the fasted (18 h) animals by intraperitoneal injection of STZ (60 mg/kg) freshly dissolved in citrate buffer (0.1 M, pH 4.5). Although STZ-induced diabetic mice were an animal model of type 1 diabetes, it was generally used in the study on the effect of short-term intake such as alleviating effect of postprandial hyperglycaemia. After 7 days, tail bleeds were performed and animals with a blood glucose concentration above 250 mg/dL (14 mM) were considered to be diabetic.

Measurement of blood glucose level
Both normal and STZ-induced diabetic mice fasted overnight were randomly divided into four groups. Fasted animals were deprived of food for at least 12 h but allowed free access to water. After overnight fasting, mice were orally administered either soluble starch (2 g/kg body weight) alone (control) or starch with 2,7 00 -phloroglucinol-6,6 0 -bieckol (10 mg/kg body weight). Blood samples were taken from tail veins at 0, 30, 60, and 120 min. Blood glucose was measured using a glucometer (Roche Diagnostics GmbH, Germany). Areas under the curve (AUC) were calculated using the trapezoidal rule (Kim 2004). All procedures were approved by the animal ethics committee of our university.

Data statistical analysis
Data were represented as mean ± SD. Statistical analysis was performed using SAS software (SAS Institute, Inc., Cary, NC). Student's t-test was used for comparisons between control and sample groups. Values were evaluated by one-way analysis of variance (ANOVA), followed by post hoc Duncan's multiple range tests.

Discussion
Elevated postprandial hyperglycaemia is caused by consumption of high-carbohydrate diets, which can progress to full symptomatic type 2 diabetes. One therapeutic method to decrease postprandial hyperglycaemia is reduction of absorption of glucose through inhibition of carbohydrate-digesting enzymes such as a-glucosidase and a-amylase in the digestive organs (Abesundara et al. 2004;You et al. 2004). a-Glucosidases hydrolyze disaccharides to monosaccharides while a-amylase hydrolyzes alpha-1,4-glycocidic bonds and splits up starch components such as amylose and amylopectin into smaller oligosaccharides and disaccharides (Casirola & Ferraris 2006;Kwon et al. 2008). In this regard, a-glucosidases and a-amylase play critical roles in carbohydrate digestion and absorption. Therefore, inhibition of a-glucosidases provides an effective antidiabetic option by targeting postprandial hyperglycaemia.
Studies have reported that the hydroxyl groups in polyphenolic compounds could play important roles in inhibiting enzyme activities (Stern et al. 1996;Piparo et al. 2008;Xiao et al. 2013). Thus, the hydroxyl group in 2,7 00 -phloroglucinol-6,6 0 -bieckol may bind to enzyme active sites and inhibit enzyme activities.
Control of postprandial hyperglycaemia level is important not only in diabetic patients but also individuals with impaired glucose tolerance. Various epidemiological studies have suggested that postprandial hyperglycaemia might be more strongly correlated with cardiovascular morbidity and mortality than fasting hyperglycaemia (Bonora & Muggeo 2001;Chiasson et al. 2003). Therefore, alleviation of postprandial hyperglycaemia indeed plays an important role in controlling diabetes and preventing cardiovascular complications.
Acarbose decreases the requirement for insulin by controlling postprandial hyperglycaemia and can reduce the blood glucose level after meals. However, this hypoglycaemic agent has limitations and side effects such as flatulence, abdominal discomfort and diarrhoea (Clissold & Edwards 1988). Thus, there has been increased research into more efficacious agents presenting lesser side effects. Phlorotannin from marine algae become good source of natural anti-diabetic materials (Kim et al. 2008a). Several phlorotannins isolated from marine algae have the potential to prevent diabetes mellitus because of their high a-glucosidase and a-amylase inhibitory activities (Heo et al. 2009;Lee et al. 2014). These studies have shown similar results with our research that phlorotannin from brown algae may have a beneficial effect on controlling postprandial glucose levels. Therefore, this study suggest that 2,7 00 -phloroglucinol-6,6 0 -bieckol might be useful as a natural compound for treating postprandial hyperglycaemia.

Disclosure statement
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. ,7"-Phloroglucinol-6,6 0 -bieckol (10 mg/kg), acarbose (10 mg/kg), and control (distilled water) were orally co-administered starch (2 g/kg). Each value is expressed as the mean ± S.D. of seven mice (n ¼ 42). a,b Values with different letters are significantly different at p < 0.05 in Duncan's multiple range tests. . Blood glucose levels after administration of 2,7 00 -phloroglucinol-6,6 0bieckol in streptozotocin-induced diabetic mice (A) and normal mice (B). 2,7 00 -Phloroglucinol-6,6 0 -bieckol (10 mg/kg), acarbose (10 mg/kg), and control (distilled water) were orally co-administered starch (2 g/kg). Each value is expressed as the mean ± S.D. of seven mice (n ¼ 42). Values with different symbols ( Ã , ÃÃ ) are significantly different at p < 0.05 in Duncan's multiple range tests.