Potential cytotoxic and antioxidant effects of chloroform extract obtained from Holothuria tubulosa

Natural products have been used for the treatment of various diseases since ancient times. The marine environment is an important resource for new and leading bioactive compounds with its rich biodiversity. In this study, the potential cytotoxic effect of the chloroform extract obtained from H. tubulosa (HTK) collected from Muğla/Gökova Gulf of our country, as well as the potential antioxidant activity and phenolic compound contents of the extract were investigated. The effects of HTK extract on cell viability depending on concentration (1-0.0078 mg/mL) and time (24, 48 and 72 hours) on A549, HeLa, MCF-7, PC-3 human cancer cell lines and HEK-293 cell line were evaluated with MTT test. According to the results obtained from this experiment, the IC50 value of the extract in each tested cell was calculated separately for each incubation time. Annexin V/FITC method was used to determine the apoptotic effect of HTK extract on A549 and HeLa cell lines for 48 hours. In addition, cell cycle analysis of the extract in A549 and HeLa cells was performed by flow cytometry. In order to determine the antioxidant activity of HTK extract, DPPH free radical scavenging activity, ABTS radical cation scavenging activity, β-carotene/linoleic acid total antioxidant activity, phosphomolybdenum antioxidant activity and iron ion reduction power antioxidant activity of the extract were investigated, and total flavonoid and total tannin amount of the extract were analyzed. Phenolic compounds in the content of HTK extract were determined by HPLC analysis. Considering the calculated IC50 values for cell lines, the lowest IC50 value (35.62 µg/mL) among cancer cells was determined for the A549 cell line at 72 h. It was determined that the total apoptotic cell percentage increased due to the increase in the extract concentration in A549 and HeLa cells. The total apoptotic cell percentage was determined as %73.7 in A549 cells treated with at 1 mg/mL, while it was %87.5 in HeLa cells. The HTK extract at 0.5 mg/mL was found to cause an increase in apoptotic cell percentage by about %21.6 and %40.9 in A549 and HeLa cells compared to control cells, respectively. In A549 cells treated with 0.125 mg/mL extract, the percentage of cells in the G1 stage increased from %53 to %62 in HeLa cells, while the percentage of cells in the G1 stage increased from %52 to %82. DPPH free radical scavenging activity, ABTS radical cation scavenging activity and total antioxidant activity determined by β-carotene/linoleic acid method of the extract were found to be lower than the antioxidant capacity of BHA, which was used as a positive control. The antioxidant capacity of the extract determined by the phosphomolybdenium method was 32.511 mg AAE/g extract equivalent to ascorbic acid, while the iron ion reduction power antioxidant capacity was found to be 30.406 mg TE/g extract equivalent to Trolox. The total flavonoid and total tannin amount of HTK extract was determined as 8.5 mg QE/g extract equivalent to quercetin and 9.668 mg CE/g extract equivalent to catechin, respectively. 15 phenolic compounds were used as standard in HPLC analysis. The amount of 7 phenolic compounds was determined in the content of the extract. Among these compounds, the highest amount of compound was found as elagic acid (482,293 µg/g extract) and the lowest amount of compound was 4-hydroxybenzoic acid (0.411 µg/g extract). Considering all the data, it can be said that H. tubulosa may be a potential agent for cancer treatment. Keyword: Anticancer, Holothuria tubulosa, Apoptosis, Antioxidant, HPLC