Page 141 - Acrobat J Trad-21-3-2566

624 วารสารการแพทย์แผนไทยและการแพทย์ ทางเลือก ปีที่ 21 ฉบับที่ 3 กันยายน-ธันวาคม 2566

75 : 25 and the flow rate was set at 1.0 mL/ initiated by mixing 70 µL of buffer I with 20 µL
min. The reaction product (hippuric acid) and of substrate (1.5 mM ACTI for AChE or BuTI

the remained substrate (HHL) were detected for BuChE) in a well of a 96-well plate. Then, 20
at a wavelength of 228 nm. The peak area of µL of sample, 120 µL of freshly prepared 3 mM
hippuric acid was utilized to calculate the DNTB, and 20 µL of AChE (for AChE inhibitory

%inhibition as the following equation and the activity) or BuChE (for BuChE inhibitory activ-
IC 50 was calculate by Prism software. ity) were added to the mixture. The plate was
Acontrol - Asample incubated in room temperature for 30 minutes
Inhibition (%) = 5 100
Acontrol and the enzyme activity was measured using
Where, A control represented the peak area a microplate reader at 405 nm. Percentage of

of the control solvent and Asample represented inhibition was calculated using the following
the peak area of the tested sample. equation.
ODcontrol - ODsample
6. In vitro inhibitory activity on acetylcho- Inhibition (%) = ODcontrol 5 100

linesterase and butyrylcholinesterase Where, OD control represented the optical
The inhibitory activity of AChE/BuChE density of the control sample and ODsample

enzymes was evaluated using Ellman’s represented the optical density of the tested
method with slight modification. [25-26] Sample sample.

stock solutions of the extracts were prepared
at a concentration of 50 mg/mL in DMSO for 7. Content of mitragynine in the extracts
the hydro-alcoholic extract and in water for the Mitragynine, the major compound in

aqueous extract. A stock solution of donepezil Kratom leaves, was determined using a high-
in DMSO (2 mg/mL) was utilized as positive performance liquid chromatography (HPLC)

control. The working sample solutions at method following the procedure described by
concentrations of 12.5, 25, 125, 325, 1,250 and Sharma and colleagues with modification .
[27]
2,500 µg/mL were prepared by sequential dilu- The chromatographic system consisted of C18

tion of the stock sample solution with buffer column (4.6 mm i.d. × 150 mm length), particle
I (50 mM TRIS buffer pH 8.0). DMSO in buffer size 5 µm column using a Dionex Ultimate
®
I at concentration of 0.025, 0.25, 1.25, 2.5, and 3,000 HPLC system. The mobile phase con-

5%v/v were utilized as control solvent. The sisted of acetonitrile (A) and 0.1%v/v formic
final concentrations of sample in reaction were acid in 10 mM ammonium formate. The gradi-
1, 10, 50, 100, and 200 µg/mL. The assay was ent elution of mobile phase was programmed