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622 วารสารการแพทย์แผนไทยและการแพทย์ ทางเลือก ปีที่ 21 ฉบับที่ 3 กันยายน-ธันวาคม 2566

3. In vitro inhibitory effect on alpha- was calculated using the following equation,
glucosidase and the IC 50 of the extract was determined us-

The inhibitory activity of Kratom extracts ing curve fitting regression analysis with the
on alpha-glucosidase was investigated in vitro Prism program (GraphPad Prism 5.0, CA, USA).
using a method from Gowri et al. . The hydro- ODcontrol - ODsample
[22]
alcoholic extracts were prepared in DMSO at Inhibition (%) = OD control 5 100

a concentration of 20 mg/mL, the aqueous Where, OD control represented the optical
extract was prepared in water. Working sample density of the control sample and ODsample
solutions were prepared at concentrations of represented the optical density of the tested
100, 1,000, 2,000, 5,000, and 10,000 µg/mL by sample.
diluting the stock solution with phosphate buf-

fer pH 6.8. DMSO in buffer at concentrations at 4. In vitro inhibitory activity on alpha-
0.5, 5, 10, 25 and 50%v/v were utilized as con- amylase

trol solvent at their responsive concentration, The alpha-amylase inhibitory effect of
whereas water was utilized as the control for Kratom extracts was investigated using the
the aqueous extract. The enzymatic reaction method described by Ademiluyi and col-

was initiated by adding 80 µL of phosphate leagues. The stock solutions of hydro-alco-
[23]
buffer pH 6.8, 20 µL of sample solution, and 50 holic extracts were prepared in DMSO, while

µL of alpha-glucosidase (0.15 unit/mL) to each aqueous solutions were prepared in water at
well of a 96-well plate. After shaking, the plate a concentration of 10 mg/mL. Working sample
was incubated at 37˚C for 15 minutes. Next, solutions were then prepared by diluting the

50 µL of the substrate, 5 mM p-nitrophenyl-D- stock solution with phosphate buffer pH 6.9 to
glucopyranoside (p-NPG) in water, was added achieve concentrations of 2,500 and 5,000 µg/
to the wells and incubated at 37˚C for another mL. DMSO in buffer at a concentration of 50%

15 minutes. The final concentrations of the was used as control solvent for hydro-alcoholic
extracts in enzymatic reaction were 10, 100, extracts and water was used for aqueous ex-
200, 500, and 1,000 µg/mL. To terminate of tract. Next, in a reaction microtube, 50 µL of

the enzymatic reaction, 100 µL of 1M sodium the sample solution was mixed with 50 µL of
carbonate was added. The reaction product alpha-amylase (0.5 mg/mL in pH 6.9 phosphate

(p-nitrophenol) was determined by measuring buffer) and 100 µL of 0.02 M sodium phosphate
the absorbance at a wavelength of 405 nm us- buffer pH 6.9 containing 0.006 M sodium
ing a microplate reader. The inhibitory activity chloride. The mixture was then incubated in