Handling of diabetes mellitus can be done through inhibition of the α-glucosidase enzyme that catalyzes the final step of starch digestion and abundant disaccharides in the human diet on the border of the small intestine mucosal brush.1-2 Seagrasses are marine angiosperms growing abundantly in the sea that produce secondary metabolites as an antidiabetic,3 antioxidant3-4 and antibacterial5-6 activities with the chemical compound such as phenol, ﬂavonoids, tannin, terpene, sterol.6-7 The aim of the present work is to investigate the α-glucosidase inhibitory activities of three seagrasses in Banten Bay, Indonesia i.e. Enhalus acoroides, Thalassia hemprichii and Cymodocea rotundata extracts.
MATERIALS AND METHODS
The plant parts used in this study were all plant parts (leaves, stems, and roots) collected in October 2017 from the Bay of Banten, Indonesia and identified by the Department of Biology, FMIPA University of Indonesia. The chemical materials such as an α-glucosidase enzyme which was obtained from Saccharomyces cerevisae (Sigma Aldrich, USA), p-nitrophenyl α-D-glucopyranoside (pNPG) (Sigma Aldrich, USA), Acarbose (Huadong Medicine, China), Quercetin (Sigma Aldrich, German), bovine serum albumin (Merck, German). All reagents used in this research were analytical grade.
Preparation of the extract
All parts of the seagrass were washed with fresh water to remove epiphytes and other impurities. The sample was dried for 5 days in the shade at room temperature. The dried sample was homogenized to obtain the fine powder.
Each dried powdered of seagrasses (100 g) were homogenized by Ultraturrax with a rotation speed 15.000 rpm for 10 min and extracted by maceration for 2 days with 80% ethanol then evaporated.
The preliminary test of α-glucosidase
The preliminary test performed was to determine the maximum wavelength of pNPG, optimum incubation time and substrate concentration using microplate reader.
The α-glucosidase inhibition activity assay
The α-glucosidase inhibition of the extracts was assayed according to the published method with modification.2 One mg of α-glucosidase was dissolved in 10 mL of phosphate buffer (pH 6.8) containing bovine serum albumin (0.2% w/v). Sample (10 µL) was mixed with 66 µL phosphate buffer (pH 6.8) and 17 µL of 5mM pNPG then preincubated at 37°C for 5 min. 7 µL α-glucosidase (0.07 unit/mL) was added then incubated at 37°C for 15 min.
Then 100 μL Na2CO3 200 mM was added to stop the reaction. The α-glucosidase activity was read at 400 nm in the microplate reader by measuring p-nitrophenol released from pNPG. As a positive control of the α-glucosidase inhibitor used acarbose. An activity of α-glucosidase standard and extract could be defined in % inhibition which obtained using the following formula:
A = blank solution absorption
B = blank control solution absorption
C = sample solution absorption
D = sample control solution absorption
The inhibitory concentrations of standard and extracts required to inhibit the action of α-glucosidase by 50% (IC50) were calculated by linear regression.
Type of inhibition α-glucosidase
Inhibition types analysis were conducted only for the extract that has the best α-glucosidase inhibition activity. This a nalysis measured a correlation between the concentration of pNPG as a substrate in the existence or absence of extract at different concentrations. The type of inhibition was described by the Lineweaver-Burk data plot analysis, which was calculated from the Michaelis-Menten kinetics results.
The extract which has the best α-glucosidase inhibition activity were evaluated for its bioactive compounds by Thin Layer Chromatography (TLC). The developed TLC plates were air dried and observed under ultraviolet light at 254 nm and 366 nm. Then Retention factor (Rf) was calculated by dividing the distance of compound by the distance of solvent migration from the original position.
Total phenolic content
The total phenolic content of the extracts was measured by Folin-Ciocalteu method according to Vanitha et al. (2017)7 with modification. An aliquot of the extract (0.5 mL) was mixed with 1 mL of Folin-Ciocalteu reagent (10% v/v). The tubes were vortexed for 20 sec and allowed to stand for 5 min. Two mL of sodium carbonate (7.5% w/v) was added. The tubes were vortexed again for 20 sec and allowed to stand for 75 min. Absorbance was then measured at 750 nm using the spectrophotometer UV-Vis. The total phenolic content was expressed as mg/g Gallic acid equivalent.
The percentages of each extract yield were shown below in Table 1.
The preliminary test of α-glucoside activity
The determination of maximum wavelength, incubation time and also substrate optimization was performed in enzyme solutions 0.07 U/mL. Temperature and pH used in the product information from Sigma Aldrich was 37°C and pH 6.8. Then the substrate concentration was optimized to determine the appropriate substrate concentration to react with enzyme unit used. Substrate concentration was said optimum if all active site in the enzyme had been bound to the substrate so that there were no free enzyme which would produce products. In substrate optimization, enzyme unit was used 0.07 U/mL with substrate variations were 1,25; 2,5; 5; 10; 15; 20; and 30 mM. The result showed that the optimum concentration of pNPG was 5 mM. The optimum conditions for the enzyme were 400 nm, 15 min incubation time and 5mM substrate concentration.
α-glucosidase inhibition activity assay
Inhibition α-glucosidase activity of extracts was determined using pNPG as a substrate and compared with acarbose (Figure 1). The IC50values of the three types of seagrass extract and acarbose as controls have been obtained. The sequence inhibition α-glucosidase activity from the strong potential to the weak is the extracts of Enhalus acoroides (168.15 ± 2.71 µg/mL), acarbose (197.27 ± 3.07 µg/mL), Thalassia hemprichii (425.86 ± 5.15 µg/mL) and Cymodocea rotundata (429.28 ± 8.89 µg/mL). The type of inhibition of 80% ethanol extract of Enhalus acoroides was investigated and showed a noncompetitive inhibition (Figure 2).
(+) : present.
The 80% ethanol extract Enhalus acoroides had the highest α-glucosidase inhibition activity compare with Thalassia hemprichii and Cymodocea rotundata and more potential than acarbose. The α-glucosidase inhibition activity of the three types of seagrass extracts is more likely due to more than one compounds containing in the extracts such as phenolic and terpenoid compounds in each extract. Their chemical compounds may have a synergic effect to inhibit the enzyme α-glucosidase.8 The type of enzyme inhibition mechanism of 80% ethanol extract Enhalus acoroides was a noncompetitive inhibitor. Noncompetitive inhibition may have been due to the chemical compound extract as an inhibitor binds to the enzyme at a location other than the active site.2 The phytochemical compound of the extract is phenols, flavonoid, tannin and terpene. While the total phenolic content of the 80% ethanol extract Enhalus acoroides was 28.76 ± 2.46 mgGAE/g. Phenolics compounds are reported as antioxidant, anti-inflammatory, antidiabetic etc.3,5,10 Phenolic compounds affect responses that are related to diabetes by inhibiting α-glucosidase enzyme thereby decreasing starch digestion.11-13
The 80% ethanol extract Enhalus acoroides (IC50 values :168.15 ± 2.71 µg/mL) had the highest α-glucosidase inhibition activity compare with Thalassia hemprichii, and Cymodocea rotundata (IC50 values : 425.86 ± 5.15 µg/mL; 429.28 ± 8.89 µg/mL) and more potential than acarbose (IC50 values :197.27 ± 3.07 µg/mL). While the type of enzyme inhibition mechanism of 80% ethanol extract Enhalus acoroides was a noncompetitive inhibitor. The phytochemical compound of the extract is phenols, flavonoid, tannin and terpene. The total phenolic content of the 80% ethanol extract Enhalus acoroides was 28.76 ± 2.46 mgGAE/g. Enhalus acoroides potential inhibits the α-glucosidase enzyme and further research about the isolation of active compounds is possible.