EFFECT OF ACTIVE FRACTIONS OF Gracilaria edulis EXTRACT IN INDUCING APOPTOSIS OF MCF-7

• Main Author: Rahadiani, Pratiwi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/36207
• Institution: Institut Teknologi Bandung

Seaweed is one of potential Indonesian natural resources. Its habitat spread throughout Indonesia Ocean and reach 1.2 million ha. In 2014, total product of seaweed reaches about 237.8 thousand tons production (56% of world total production). Bioactive compounds of seaweed have broad biological activities especially as an anticancer. In the last few decades, anticancer activity of seaweed extract and bioactive compounds has gained interest from researchers around the world. It happens because of the need for anticancer alternative candidate with more efficacious, specific, and has few side effects than currently available chemotherapy drugs. In addition, diets containing seaweed have been known to reduce cancer risk. The success of an anticancer drug candidate in inducing apoptosis is one of the determining of success in developing cancer therapy. There are still a few researches about the utilization of seaweed bioactive compounds in Indonesia especially as anticancer alternative candidates. Therefore the aim of this study was to examine effect of the active fractions of Gracilaria edulis extract to induce apoptosis of breast cancer cell lines (MCF-7). The research was conducted by determining the active fraction based on total phenolic content (TPC) and cytotoxicity assay. The obtained active fractions were tested for its effect in inducing apoptosis and the release of cytochrome c of MCF- 7 cell line. Separation method using thin layer chromatography (TLC) and further identification using GCMS were used to determine the bioactive compounds of active fraction of G. edulis extract. The extraction of G. edulis used maceration method resulting 1.29% (w/w) of crude extract and 91.06% of water content. From fractionation method using column chromatography, 8 fractions of G. edulis extract were obtained. The fractions with highest TPC were crude extract, fraction 8 (F8), and fraction 6 (F6), which were 134.11±2.78 mg GAE/g sample, 111.89±10.02 mg GAE g sample, and 103.60±10.06 mg GAE/g sample, respectively. According to the standard criteria of cytotoxicity activity for the crude extracts established by The U.S. National Cancer Institute (NCI), the most cytotoxic fraction of G. edulis extract was fraction 6 (F6), followed by fraction 8 (F8) and crude extract. The IC50 values were 11.25 µg/ml, 24.31 µg/ml, and 28.65 µg/ml, respectively. Based on the results of the cytochrome c release test on the MCF-7 cell line, the highest concentration of cytochrome c release from cells treated by fraction 6 was 0.98±0.25 mM (using IC50 concentration). While the highest concentration of cytochrome c release from cells treated by fraction 8 was 1.11±0.14 mM (using IC75 concentration). The results of the apoptosis assay using Annexin-V/FITC and PI staining analyzed by flow cytometry showed that the fraction 6 of G. edulis extract with the IC25, IC50, and IC75 concentration was able to induce early apoptosis of MCF-7 with the percentage of 62.10%, 58.70%, and 60.45%, respectively; induce late apoptosis with the percentage of 3.05%, 5.20%, and 2.20%, respectively; and induce necrosis with the percentage of 8.95%; 9.00%, and 14.80%, respectively. Fraction 8 of G. edulis extract with the IC25, IC50, and IC75 concentration was able to induce early apoptosis of MCF-7 with the percentage of 67.55%, 57.90%, and 74.45%, respectively, induce late apoptosis with the percentage of 4.45%, 1.05%, and 1.80%, respectively; and induce necrosis with the percentage of 8.15%; 10.20%, and 11.65%, respectively. Based on the separation method using TLC, fractions 6 was separated into 4 spots and fraction 8 was separated into 3 spots. First spot of fraction 6 have Rf value 0.550 and the rest of the spots of fraction 6 have the same Rf value as 3 spots obtained from fraction 8 which were 0.625; 0.688; and 0.750. The identification of bioactive compounds using GCMS showed that the conten t of fraction 6 was dominated with aldehyde unsaturated fatty acid compounds while the fraction 8 was dominated with ester of fatty acid compounds. In summary, the bioactive compounds contained in fractions 6 and 8 of G. edulis were able to induce apoptosis in breast cancer cell line (MCF-7) through the release of cytochrome c.