Aim: The purposes of this study were to determine the anticancer activity of Xestospongia testudinaria sponge isolate and identify the responsible compounds.

Materials and Methods: The metabolites were extracted using methanol maceration at room temperature. The separation and purification of metabolites were performed using fractionation and column chromatography. The toxicity was examined using the brine shrimp lethality assay, and the toxic isolates were tested for anticancer activity against HeLa cells. Gas chromatography-mass spectrometry analysis was used to identify the compounds in the isolate.

Results: When the methanol extract was partitioned with n-hexane, chloroform, and n-butanol, the chloroform fraction was the most toxic, with a concentration that results in 50% lethality (LC₅₀) value of 39.81 ppm. After separation of the chloroform extract, fraction B (FB) was the most toxic, with an LC₅₀ value of 44.67 ppm. The isolate from FB showed anticancer activity with a concentration at which 50% of growth was inhibited (IC₅₀) of 2.273 ppm. In total, 21 compounds were identified in anticancer isolates: Nonanedioic acid; tetradecanoic acid; trans-phytol; 2-pentadecanone- 6,10,14-trimethyl; pentadecanoic acid; 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-; pentadecanoic acid; 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-; 2,3,7-trimethyloctanal; palmitic acid; docosanoic acid, ethyl ester; 1,E-11,Z-13-octadecatriene; chloromethyl 4-chlorododecanoate; 1-tricosene; 9,12-octadecadienoic acid; 4,8,12,16-tetramethylheptadecan-4-olide; 1-docosene; heneicosane; phosphonic acid, dioctadecyl ester; dodecane,4,6-dimethyl-; n-tetratriacontane; 1-iodohexadecane; and n-heneicosane.

Conclusion: These findings indicate that the isolate of X. testudinaria can be used as a natural anticancer toward HeLa cell.

Keywords: anticancer activity, HeLa cell, Xestospongia testudinaria.