Page 140 - Acrobat J Trad-21-3-2566

J Thai Trad Alt Med Vol. 21 No. 3 Sep-Dec 2023 623

an oven at 37˚C for 10 minutes. After that, 50 5. In vitro inhibitory activity on angio-
µL of 1% starch solution in pH 6.9 buffer was tensin-I converting enzyme

added to the reaction tube and the mixture The effect Kratom extracts on ACE inhibi-
was incubated again at 37˚C for 20 minutes. tion was investigated using method of Lahogue
The final concentrations of the extracts in the and co-workers with slight modification. [24]

enzymatic reaction were 500 and 1,000 µg/mL. Briefly, a reaction mixture containing 40 µL of
The enzyme-substrate reaction was stopped 0.1 M sodium borate buffer pH 8.3 was mixed
by adding 100 µL of DNS reagent (96 mM with 20 µL of 0.1 unit/mL of ACE in buffer and

3,5-dinitrosalicylic acid, 3.5 M sodium potas- incubated at 37˚C for 5 minutes. Next, 20 µL of
sium tartrate in 2 M NaOH) to the reaction the sample solution and 40 µL of the substrate
microtube. The reducing sugar produced dur- (7 mM HHL) were added into the reaction mix-

ing the reaction reacted with the DNS reagent ture, which was then incubated at 37˚C for 30
by placing the reaction tube in a heat box at a minutes. The reaction was stopped by adding

temperature of 90-100˚C for 5 minutes. After 100 µL of 0.1M HCl and determined the reac-
that, the reaction microtube was immediately tion product using HPLC. Stock sample solu-
placed on ice for 5 minutes and 650 µL of water tions of hydro-alcoholic and aqueous extracts

was added and mixed. The mixture solution were prepared at a concentration of 50 mg/
(200 µL) was transferred to a 96-well plate and mL in DMSO and water, respectively. Working

the optical density of the reaction was deter- solutions of sample were prepared by diluting
mined by a microplate reader at a wavelength the stock solution with buffer to produce solu-
of 540 nm. The percentage inhibition was cal- tion of 6 mg/mL (final concentration 1000 µg/

culated using the following equation. The IC 50 mL). For IC 50 evaluation, working solutions in
of the extract was calculated by curve-fitting buffer were prepared at concentration, 0.6, 3,
regression analysis using the Prism program. 6 and 12 mg/mL (final concentration 100, 500,

1000 and 2000 µg/mL). Captopril was utilized
ODcontrol - ODsample
Inhibition (%) = 5 100 as positive control and the HPLC analysis
OD control
was performed on a C18 column (4.6 mm i.d.
× 150 mm length), particle size 5 µm column
Where, OD control represented the optical using a Dionex Ultimate 3,000 HPLC system.
®
density of the control sample and ODsample The isocratic mobile phase consisted of 0.1%
represented the optical density of the tested trifluoroacetic acid and acetonitrile at ratio of
sample.