New compounds of pregnanone from Calotropis gigantea roots actively against colon cancer cell WiDr through cell cycle inhibition

Calotropis gigantea (L.) W. Aiton (C. gigantea) is a medicinal plant that has been empirically proven to have anticancer activity. In a previous study, it showed that the fraction of ethyl acetate from the root part of C. gigantea had higher anticancer activity than the other fractions. It suspected that the ethyl acetate fraction of C. gigantea root contained active compounds that has anticancer properties. This study aimed to determine the anticancer activity of active compounds from the ethyl acetate fraction of C. gigantea root regarding induction of apoptosis, cell cycle arrest, and expression of caspase-8 colon cancer cell WiDr. Isolation of the active compounds from the ethyl acetate fraction of C. gigantea root was carried out using Bioassay-guided Isolation method. Identification of active compounds was using NMR-1H, NMR-13C, HMBC, HMQC and UPLCMS/MS methods. The anticancer activity test of the identified compounds performed by using MTT method. The induction of apoptotic and cell cycle arrest evaluated by a flow cytometry method. The result of this study showed two active compounds were identified namely (1) (Pregnanon-5-en, 3,14,17 trihydroxy-12- (4'-cyclohexyl benzoate) -, (3β, 12β, 14β) - (9CI), (2) Pregn-5-en-20-one, 3,8,14-trihydroxy-12 - [(4'-hydroxy benzoyl) oxy] -, (3β, 12β, 14β, 17α) - (9CI). Both compounds inhibited the growth of colon cancer cell WiDr with IC₅₀ values respectively were 15.89 μg/mL and 0.77 μg/mL. Both compounds increased the induction of apoptotic by increasing sub-G1, S, and G2-M following depletion of G0-G1 phase accumulation.