Cytotoxic Effects of Ethyl Acetate and Hexane Extracts of Capsicum annuum L. on Cal 27 Oral Cancer Cells

This study aims to compare the cytotoxic effects of ethyl acetate and n-hexane extracts of Capsicum annuum L. on Cal27 oral cancer cell lines by examining caspase-3 expression.

This study used an in vitro experimental design with a post-test-only control group. Capsaicin in the ethyl acetate extract of Capsicum annuum was identified using Thin Layer Chromatography (TLC) with detection at UV 254 nm. Cytotoxicity was evaluated using the MTT assay, morphological assessment, and caspase-3 expression. Caspase-3 expression was quantified using qPCR with GAPDH, and relative expression was calculated by the 2^–ΔΔCt method.

Statistical Analysis: Differences in caspase-3 expression between selected treatment groups were examined using paired two-sample t-tests, whereas overall variation among all groups was analyzed with one-way ANOVA. Bonferroni post hoc tests were applied in multiple comparisons. Statistical significance was defined as p < 0.05 and adjusted to p < 0.005 for multiple testing.

Phytochemical testing confirmed the presence of capsaicin in the extract. TLC analysis on silica gel GF 254 using a toluene–ethyl acetate–formic acid (5:4:1 v/v/v) mobile phase showed a single blue-violet spot (Rf = 0.71) under UV 254 nm, consistent with the capsaicin standard. Both fractions reduced the viability of Cal 27 cells in a concentration-dependent manner. The n-hexane fraction showed stronger cytotoxic activity, with an IC₅₀ of 218.10 µg/mL, compared with 317.10 µg/mL for the ethyl acetate fraction. Morphological examination revealed features consistent with apoptosis, such as apoptotic body formation. Under certain conditions (medium-only, 2% DMSO, and 1 × IC₅₀), caspase-3 expression appeared slightly higher in the ethyl acetate fraction; however, these differences were not statistically significant (p > 0.05). Although ANOVA indicated overall significance within the hexane-treated group (p = 0.005), Bonferroni-corrected post hoc analysis did not find any significant pairwise differences.

The stronger cytotoxic effect observed with the n-hexane fraction suggests that non-polar constituents may play a major role in suppressing Cal 27 cell proliferation. While both extracts produced morphological evidence of apoptosis, the lack of significant variation in caspase-3 expression shows that additional mechanisms may be involved. Variations in solvent polarity likely influenced the phytochemical profiles of the extracts and their biological activity.

Both n-hexane and ethyl acetate fractions of C. annuum exhibited cytotoxic effects on Cal 27 oral cancer cells and produced morphological changes consistent with apoptotic features; however, further studies are required to confirm the involvement of apoptotic pathways.