Cytotoxicity of the Most Active Fraction of the Seeds of Swietenia macrophylla using Human Breast Cancer MCF-7 Cells H )

Ethyl acetate fraction from Swietenia macrophylla was reported to have toxicity against the larvae of Artemia salina shrimp larvae. However , there are no reports about S. macrophylla,which can inhibit human breast cancer cells MCF-7.Therefore, this study aims to evaluate S. macrophylla extract's cytotoxicity using human breast cancer MCF-7 cells assay, followed by confirmation of its toxicity using brine shrimp lethality assay.The most active fraction obtained from the ethyl acetate extract of S. macrophylla showed 76.49% inhibition at 50 pg/mL (IC50=34.n pg/mL). At the same time, the most active fraction may contain a mixture of limonoid compounds after LCMS analysis. The most active fraction obtained from ethyl acetate extract of S. macrophylla showed 76.49% Article history:

seeds are rich for saponin, alkaloid, steroid , triterpenoid , and tannin [ 7 , 8, 9 , 10]. The major compounds that show antineoplastic are triterpenoid and limonoids [11]. Based on the literature search, S. macrophylla seeds have never been extensively examined before. Therefore, the need to investigate either biological activities and chemical contents of the plant is urgent. We began with the work of isolating active compounds against human breast cancer MCF-7 cells, which is the subject of this report.

Methodology
The research methods consisted of collecting S. macrophylla seed sample, sample preparation, determination of water content, extraction, phytochemical analysis, fractionation using vacuum liquid chromatography (VLC), and further purification using open column chromatography. Each fraction obtained from the fractionation was tested for toxicity against A. salina larvae. Cytotoxicity tests on MCF-7 cancer cells were carried out in the most active fraction on brine shrimp lethality assay. Finally, the chemical entities

Introduction
Swietenia macrophylla belongs to the Meliaceae ' s family, which has been used as a source of traditional medicine [1]. Almost all parts of the plants possess biological activities such as antimicrobial, antiinflammatory, antioxidant effects, antimutagenic, antidiabetic, antitumor, and anticancer [ 2,3 ]. Moreover, the natural products of the species have not explored much. Some of them are reported as a folk medicine for the treatment of hypertension, diabetes, malaria [ 4 ], antibacterial [ 5 ].
There is increasing interest in research on natural products based on antineoplastic activity with low nonspecific toxicity. This can be exemplified by the plant S. mahogany. A previous report [ 5 ] showed that the CHC 13 and ethyl acetate extracts could kill brine shrimp at the LC 5o of 13.75 and 11.64 g / mL. Another report from the ethyl acetate extract of S. macrophylla seed, which was evaluated using human colon colorectal HCT116 cells, was able to kill the cancer cells with IC 50 of 3535 ± 0.50 pg / mL [6]. Regardless of its biological activity, the mahogany

Material and Equipment
The material used in this research was S. macrophylla obtained from the Surabaya region. The chemicals used were a variety of organic solvent in analytical grade and several consumable materials for separation. Brine amount of ethyl acetate extract obtained is 8 g (1.6% w / w of the dried sample). Evaluation of the ethyl acetate extract against brine shrimp lethality assay shows LC 50 of 156 g / mL. The extract is positive, containing triterpenoids through a phytochemical test using Liebermann-Burchard reagent. The number of triterpenoid molecules from S. macrophylla is still limited and encouraged us to investigate chemically the Indonesian S. macrophylla. Purification of the active ethyl acetate using VLC resulted in 10 fractions, which the seventh fraction showed the most active against brine shrimp lethality assay LC 50 43 -94 Pg/mL ( 3 -6 g). Further purification of the seventh fraction using column chromatography with a mixture of chloroform and ethyl acetate gave ten subfraction 7 a 7 j. The result of the brine shrimp assay for subfraction 7 a 7 j is depicted in Figure1 .

Extraction and Isolation
Before extraction, S. macrophylla powder samples were measured for their water content (loss of drying) [12]. Some portions of the sample ( 500 g) were dissolved with n -hexane for 6 hours, followed by ethanol three times for 24 hours to obtain the ethanol extract. The ethanol extract was then partitioned by ethyl acetatewater solvent (1:1). The ethyl acetate extract was identified for its phytochemical content (limonoid). The ethyl acetate extract was fractionated by VLC (n -hexaneethyl acetate) to give ten fractions. The most active fraction against the brine shrimp lethality assay (fraction 7 ) was purified by column chromatography (chloroformethyl acetate).

. Determination of cytotoxic characteristics
The cytotoxic characteristic of the most active fractions (fraction 7 a, 7 b, and 7 i), after the second fractionation, were tested for the MCF-7 breast cancer cells by following the MTT method ( 3 -( 4 -5dimethyliazo-2-il) 2.5 -diphenyltetrazolium bromide) [ 13 ]. The principle of the method is a color change of MTT from yellow to blue. The raised color is due to the presence of remaining living MCF-7 cells. In this method , the cytotoxic activity is expressed as a percentage of inhibition. The test was conducted by adding each isolation material to various concentrations in triplicate to the MCF-7 cells. After incubating for 48 hours, the MTT was added into the sample and incubated the plate for 4 hours. The absorption after being treated with MTT was measured using the enzyme-linked immunosorbent assay (ELISA) reader device at 595 nm after the addition of a solvent to stop the reaction.

. Result and Discussion
The moisture content of the sample is 4.34 %, which can be kept for long time storage. After extraction, the 19.93 pg / mL, respectively. These results are more toxic than the most active fraction reported by Hajianto [ 14 ], which was 35 -46 pg / mL. Further confirmation of their antitumor activity was carried out using MCF-7 cells. The result of the MCF-7 cytotoxicity assay can be seen in Table 1and Figure 2.