METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells. Colony forming assay was conducted to quantify the colony inhibition in HCT 15 and HT 29 colon cancer cells after p-Coumaric acid treatment. Propidium Iodide staining of the HCT 15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells. Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15 cells after exposing to p-Coumaric acid. Levels of reactive oxygen species (ROS) of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2', 7'-dichlorfluorescein-diacetate. Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry. Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540. Apoptosis was confirmed and quantified using flow cytometric analysis of HCT 15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1.
RESULTS: Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC₅₀ (concentration for 50% inhibition) value of 1400 and 1600 μmol/L respectively. Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment. Propidium iodide staining of treated HCT 15 cells showed increasing accumulation of apoptotic cells (37.45 ± 1.98 vs 1.07 ± 1.01) at sub-G1 phase of the cell cycle after p-Coumaric acid treatment. HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure. Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid. A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells. Further apoptosis evaluated by YO-PRO-1 staining also showed the time-dependent increase of apoptotic cells after treatment.
CONCLUSION: These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway.
OBJECTIVES: GST inhibition activity and characterization of Kanji and methanol extract of D. carota roots, and oral absorption pattern of ferulic acid from Kanji in rats.
MATERIALS AND METHODS: GST inhibition activity of Kanji and methanol extract of D. carota roots in concentration range 0.001-100.00 mg/mL was determined using Sprague Dawley rat liver cytosolic fraction. Methanol extract upon column chromatography gave ferulic acid, which was used to characterize Kanji and determine its oral absorption pattern in Wistar rats.
RESULTS: The GST inhibition activity of Kanji (100.00 μg/mL), methanol extract of D. carota roots (100.00 μg/mL) and tannic acid (10.00 μg/mL, positive control) was found to be 0.162 ± 0.016, 0.106 ± 0.013 and 0.073 ± 0.004 μM/min/mg, respectively. Different Kanji samples and methanol extract contained ferulic acid (0.222-0.316 mg/g) and 0.77 mg/g, respectively. Ferulic acid did not appear in plasma after oral administration of Kanji.
DISCUSSION: Kanji having solid contents 80.0 μg/mL, equivalent to 0.0025 μg/mL ferulic acid, does not inhibit the activity of GST. The oral administration of Kanji, in human equivalent dose (528 mg/kg, 16.67 μg ferulic acid), to rats indicated poor absorption of ferulic acid.
CONCLUSION: Kanji having solid contents 14-36 mg/mL does not inhibit GST activity, hence may not interfere with drugs that are the substrates of GST, if taken concomitantly.