MATERIALS AND METHODS

Materials: α-glucosidase 0.15 U mL^–1 from Saccharomyces cerevisiae (Sigma Aldrich, Singapore), Potassium dihydrogen phosphate 2 M (Merck) pH 6.8, substrate 4-Nitrophenyl-α-D glucopyranoside (PNPG) 5 mM (Sigma Aldrich, Singapore), Sodium carbonate 200 mM (Merck), Acarbose (Sigma Aldrich, Singapore), α-amylase 1 U mL^–1 from Porcine kidney (Sigma Aldrich, Singapore), Soluble starch 1 and 2% (Merck), buffer Sodium dihydrogen phosphate 20 mM pH 6.9, Sodium chloride 6.7 mM, 3.5-Dinitro salicylic acid 96 mM (Sigma Aldrich, Singapore), Sodium hydroxide 2 M, Sodium potassium tartrate (Merck), standard of maltose 0.2%, Dipeptidyl peptidase IV (DPP-IV) from porcine kidney (Sigma Aldrich, Singapore), substrate Gly-Pro-P-Nitroanilide (GPPN) (Sigma Aldrich, Singapore), Diprotin-A (Ile-Pro-Ile) (Sigma Aldrich, Singapore).

METHODS

Phytochemistry screening: The viscous extracts (50 mg) were diluted using ethanol. Identification of the presence of alkaloids, flavonoids, tannins, saponins, glycosides, terpenoids and anthraquinone conducted in accordance with procedures of Indonesian Materia Medika and Harborne (MoH., 1995; Harborne, 1987).

α-glucosidase activity assay: A 36 μL of phosphate buffer solution, 30 mL sample solution with various concentrations (10, 25, 50, 100 and 150 μg mL^–1) and 17 μL PNPG substrate at concentration of 5 mM were put in 96 well microplate. This mixture was incubated at 37°C for 5 min. After 5 min, 17 μL of α-glucosidase solution 0.15 U mL^–1 was added in each well to obtain total volume of 100 mL. The mixture was incubated for 15 min to get the complete hydrolysis reaction. After 15 min, the reaction was stopped by adding 100 μL of Sodium Carbonate (Na₂CO₃) 200 mM. Absorbance was measured at 405 nm using a microplate reader. Each test was repeated twice (Sigma-Aldrich, 1995; Dewi et al., 2007).

Calculation of percent inhibition: The obtained data from the activity test was processed using the following formula in Eq. 1:

(1)

An experiment used 5 variation of sample concentration. Percent inhibition obtained in each sample was processed in form of graph and compared to the percent inhibition of acarbose as a positive control.

Calculation of IC₅₀: The IC₅₀ was calculated using linear regression equation in which the concentration of the sample as the x-axis and percent inhibition as the y-axis. From the equation y = a+bx, IC₅₀ values can be calculated using the following formula in Eq. 2:

(2)

α-amylase activity assay: A 250 μL of 500 μg mL^–1extract, 250 μL of starch 2.0% (w/v) and 250 μL of 1 U mL^–1 α-amylase solution were homogeneously mixed into a test tube. After incubated at 20°C for 3 min, 500 μL of color reagent (Dinitrosalicylic acid) was added to stop the enzymatic reaction. The mixture was put into boiled water and 250 μL α-amylase 1 U mL^–1 was added immediately. Heating process was continued for 15 min. Then the solution was removed from heating process and cooled at room temperature for 3 min. A 4500 μL aqua dest was added to obtain total volume of 6000 μL. The solution was homogenized using a vortex. The α-amylase activity was determined at 540 nm using spectrophotometry to measure product absorbance (maltose) which reduces DNS. The produced absorbance was compared with blank. Percent inhibition was calculated using the Eq. 1 (Sigma-Aldrich, 2014; Ali et al., 2006).

DPP IV activity assay: A 25 μL extract was added with 50 μL DPP-IV (500 μg mL^–1). The mixture was incubated at 37°C for 5 min. A 100 μL GPPN (2 mM) was added to the wells containing extract and enzyme. Incubation was continued for 15 min. The reaction was terminated by adding 25 μL acetic acid glacial 25%. The absorbance was measured at λ = 405 nm (Chakrabarti et al., 2011).

RESULTS

Phytochemistry screening: Phytochemistry screening conducted on 7 secondary metabolites presence: alkaloids, flavonoids, tannins, glycosides, saponin, terpene/steroids and anthraquinone. There was 42 samples tested, 40 plants contain alkaloids, flavonoids and glycosides (Table 1).

Enzymatic activity assay: The test results of α-glucosidase presented with IC₅₀ values of samples using Eq. 2, while for the testing of α-amylase and DPP-IV test, data is shown only through the percent inhibition values were calculated based on Eq. 1.

Table 1:	Phytochemistry screening result

This was done because the α-glucosidase inhibition was linear (extract concentration is proportional to the percent inhibition produced), while the α-amylase and DPP-IV inhibition properties are non-linear, the increase of the extract concentration does not increase the percent inhibition. Therefore, the inhibition of α-amylase and the DPP-IV can not be analyzed by using linear regression equation (Eq. 2).

DISCUSSION

In vitro α-glucosidase inhibitory studies demonstrated that all samples had inhibitory activity (Table 2). The percentage inhibition at 10, 25, 50, 100 and 150 μg mL^–1 concentrations of samples showed a concentration dependent on percentage inhibition. The highest inhibitory activity of samples was obtained from Camelia sinensis (white) extracts with IC₅₀ value 10.54 μg mL^–1. The IC₅₀ values of Camelia sinensis (white) were much smaller than the IC₅₀ of Acarbose as a positive control (IC₅₀ Acarbose = 36.98 μg mL^–1). It shows that Camelia sinensis (white) inhibits the activity of α-glucosidase better than acarbose in a smaller concentration. Eight other samples also showed better results than Acarbose, which are Camelia sinensis (white tea), Camelia sinensis (green tea), Camelia sinensis(with fermentation), Pometia pinnata, Syzygium polyanthum, Artocarpus heterophyllus, Lagerstroemia speciosa and Persea Americana.

Table 2:	Assay of enzyme inhibition activity

All of them contain tannin. In recent years, tannin has been reported as non-specific inhibitors for several hydrolytic enzymes such as lipases, α-glucosidases, α-amylases and invertase (Barret et al., 2013; Goncalves et al., 2011; Grussu et al., 2011; Ikarashi et al., 2011; Kusano et al., 2011; Yilmazer-Musa et al., 2012; Zajacz et al., 2007). This hypothesis can be proved by further experiments such as tannin isolation and activity assay of isolate that have been separated from other compounds.

In α-amylase in vitro study, all extracts showed inhibition value between 3-99%. There were 14 samples that have inhibition values more than 80%. All of them contain glycosides. The glycosides present in the crude extracts acts as a substrate for the α-glucosidase enzyme and may be responsible for the inhibitory activity (Elya et al., 2012). Amylases degrade starch by cleaving glycosidic bonds. Glycoside was found from the sample has glycosidic bonds so it’s changing the role of starch as a substrate. With this mechanism, the starch in the body was not changed to form a disaccharide. It could help the work of glucosidase which converts disaccharide into monosaccharides (glucose) and the level of glucose can be controlled.

In DPP-IV in vitro activity assay, inhibition activity of extracts were relatively lower compared to Diprotin as positive control. In total concentration of 62.5 mg mL^–1, diprotin inhibited DPP-IV with percent inhibition is 96.40±0.03%, while the plant extracts inhibited DPP-IV with percent inhibition 10-20%. Camelia sinensis (white tea) showed the highest inhibition of all extracts in this study with percent inhibition is 30.57±0.08%.

Different from alpha glucosidase, inhibition activity of alpha amylase and DPP-IV were not dependent on concentration. IC₅₀ calculations on the inhibition of α-amylase and DPP-IV are not done because the extract has a non-linear inhibition profile.

Our in vitro studies demonstrated an appreciable α-glucosidase, α-amylase and DPP-IV inhibitory activity present in 42 samples, where further experiments can be performed on animal models to confirm the hypoglycemic activity.

CONCLUSION

All of the tested extracts show inhibition activity of α-glucosidase, α-amylase and DPP-IV. Camelia sinensis (white), Camelia sinensis (green), Camelia sinensis (with fermentation), Pometia pinnata, Syzygium polyanthum, Artocarpus heterophyllus, Lagerstroemia speciosa, dan Persea Americana have better inhibition activity of α-glucosidase than Acarbose in concentration 62.5 μg mL^–1. Plant extract which contain tannin (polyphenol) and glycoside have better inhibition activity of α-amylase in concentration 62.5 μg mL^–1. All of the tested extracts showed inhibition activity, but they have shown relatively lower inhibition of DPP-IV than Diprotin in concentration 62.5 μg mL^–1.

ACKNOWLEDGMENT

This work was supported by Hibah Grant Cluster University of Indonesia.