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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
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