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262 วารสารการแพทย์แผนไทยและการแพทย์ ทางเลือก ปีที่ 18 ฉบับที่ 2 พฤษภาคม-สิงหาคม 2563

Methods extracts were added into the wells containing

1. Plant extraction cells. The treated cells were re-incubated for 24
Leaves of T. triquetrum were chopped hours. Fifty microliters of MTT reagent ([3-(4,

into tiny pieces and then macerated with one 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-] tet-
of the following solvents: 50% ethanol, 95% razolium bromide) in DMEM free serum (1 mg/
ethanol, or hexane. Each of extract solutions mL) was then added into each well. The plates

was filtered. Then the organic solvent was were incubated in a humidified atmosphere of
evaporated by rotary evaporator. To create the 5% of CO at 37 °C for 4 hours. After removing
2
water extract, the leaves of T. triquetrum were the medium, formazan crystals were dissolved
boiled with hot water for 15 minutes. The water by 100 mL of DMSO, and the absorbance was
extract was filtered. The water was removed measured at 595 nm.

from the extract by freeze drying. All extracts 3. Antioxidant activity study by DPPH
were kept at -20 C and protected from light. (1,1-diphenyl-2-picrylhydrazyl) assay
o
2. Cytotoxicity testing One hundred and ninety micro-liters

Fibroblast cells were isolated from the of 0.12 mM DPPH solution was mixed with
foreskin of male infants who underwent neona- 10 mL of T. triquetrum extract solution in 96-
tal circumcision in the hospital. The protocol well plates. The extracts were dissolved in

of sample collection was approved by Ethics ethanol with 0.0001-5 mg/mL of concentration.
Committee for Research in Human Subject The mixture was then mixed vigorously, and

Naresuan University with approval number maintained at room temperature in the dark
140/62. The fibroblast cells were isolated from for 30 minutes. The decrease of absorbance
the dermis layer and cultured with 10% fetal was measured at 515 nm using a microplate

bovine serum in Dulbecco’s Modified Eagle reader. The positive control was trolox.
Medium (DMEM). The culture was incubated 4. Antioxidant activity study by ABTS

in a humidified atmosphere of 5% of CO at (2,2′-azino-bis-3-ethylbenzthiazoline-6-sul-
2 phonic acid) assay
37 °C to allow cell attachment. Cell culture
medium was changed every 3 days. The stock solution was performed by

The cytotoxicity of T. triquetrum extracts dissolving 7.4 mM ABTS into 2.6 mM potas-
was evaluated using MTT assay. The cells sium persulfate. ABTS radical cation was
were seeded into 96-well plates at 1x10 cells prepared by mixing the two stock solutions
4
per well. Cell attachment was allowed for 24 in equal quantities. The mixture was allowed
hours. Various concentrations of T. triquetrum to stand for 12 hours in the dark place. ABTS