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332 วารสารการแพทย์แผนไทยและการแพทย์ ทางเลือก ปีที่ 21 ฉบับที่ 2 พฤษภาคม-สิงหาคม 2566

dissolve the purple formazan product. The 2.4 ACE2 inhibition assay
absorbance of the formazan product of viable Angiotensin II converting enzyme (ACE2)

cells was read using the microplate reader at activity was determined using the angioten-
570 nm. The background absorbance was sin II converting enzyme (ACE2) activity kit
reduced by the blank and % viability was (Abcam, MA, USA) and conducted at the

calculated compared to the control. Toxicology Laboratory, Department of Medical
2.3 Anti‑SARS‑CoV‑2 activity using Sciences. The enzyme solution and reagents
plaque reduction assay were prepared following the procedure de-

Vero cells were seeded at 3.5x105 cells scribed in the manufacturer’s protocol. The
in 3 mL medium per well of 6–well plates and plant extracts were diluted with water at the
left in the incubator for 24 hours to present in highest concentration, which was not toxic

monolayer. The extracts were pre-incubated to Calu-3 cells. A total of 48 µL of ACE2 assay
with SARS-CoV-2 at 37˚C for 1 hour before buffer was mixed with 2 µL diluted ACE2 en-

transferring 200 µL of the solution with virus zyme solution and then the solution was added
particles onto the monolayer of vero E6 cells. to the diluted extracts or MLN4760 standard
Viral adsorption was allowed for 1 hour in the inhibitor (the background and enzyme controls

CO 2 incubator. The cells were then washed were also tested). The mixed solutions were
with fresh medium to remove both the un- placed at room temperature for 15 min. A To-

bound viral particles and the extract/com- tal of 40 µL of ACE2 substrate mix was then
pound. Total 3 mL of medium was then added added to the wells and immediately placed in
to the wells. The semi-solid medium was al- spectrofluorometer for measurement (Ex/Em =

lowed to be set and all plates were placed in 320/420 nm) in kinetic mode for 1 hour. The
the incubator for 7 days. The overlaid medium relative inhibition activity (%) of the sample
was discarded and cells were fixed with 5% was calculated using a relative fluorescence

formaldehyde and then stained with 0.5% (w/v) unit (RFU) compared to enzyme control.
crystal violet. The excess colour was washed 2.5 TMPRSS2 Fluorogenic inhibition
with tap water. The plaques were counted assay

and % inhibition was calculated compared to The activity of the TMPRSS2 enzymes
[19]
the controls (without the compound). All was determine using the TMPRSS2 Fluoro-
extracts were used at previously determined genic Assay Kit (BPS Bioscience, CA, USA)
non-toxic concentrations. and conducted at the Toxicology Laboratory,
Department of Medical Sciences. The enzyme