ANTICANCER ACTIVITY OF N-HEXANE EXTRACT FROM SPHAGNETICOLA TRILOBATA (L.) J.F PRUSKI AGAINST MCF-7 BREAST CANCER CELL

Sphagneticola trilobata (L.) J.F. Pruski is one of the perennial herbs that is widely used by the national and international community to treat various diseases including cancer. The objective of this study was to assessment the anticancer activity of n-hexane extract of S. trilobata leaves for inhibiting the growth of MCF-7 breast cancer cells in vitro by MTT (microculture tetrazolium salt) method. The n-hexane extract of sernai leaves was obtained from the maceration process of samples that were collected from the Langsa city, Aceh. The cytotoxicity test was carried out by incubating MCF-7 cells which had been exposed to several series of sample levels, viz. 1000; 500; 100; 50; 25; 10; 5 and 1 μg/mL. LC50 values are calculated using probit analysis. The results revealed that the n-hexane extract of S. trilobata leaves was cytotoxic against breast cancer cells (MCF-7) with an LC50 value of 0.037 μg /mL.


Introduction
Breast cancer is the most commonly diagnosed neoplasm with an incidence of death of 60% in developing countries (Wu et al., 2019). This type of cancer attacks women about 23% every year as a new case (Garbi et al., 2015) that occurs due to the abnormal cell growth in breast tissue. Handling and treatment are slow in patients causing cancer is in an advanced stage and difficult to control (Rahmawati et al., 2013).
Some treatments to overcome cancer have been performed viz. surgery, radiation, and chemotherapy. However, these efforts have a negative effect. Improper surgery causes the disease to become more severe if it can spread to other parts of the body. Radiation can be toxic to normal cells because it works not selectively. Chemotherapy or the administration of anticancer drugs such as taxol, bleomycin, doxorubicin, chlorambucil, thiotepa, and vincristine were used in high doses that could increase the risk of resistance or the normal cell to die (Rahmawati et al., 2013;Mahfur, 2016).
The current research encourages the development of novelty anticancer drugs with minimal side effects (Ala et al., 2018). The valuable research has been focused on new drugs that come from natural plant, particularly on herbs that have been trusted or used for generations (Garbi et al., 2015;Senthilraja and Katishresan, 2015;Antoney et al., 2016). One of them is Sphagneticola trilobata (L.) J.F Pruski (Husain and Kumar, 2017).
The research about S. trilobata as an anti-cancer agent is undeveloped in Indonesia, even though the available resources are very abundant predominantly in the Aceh region and its surroundings. Moreover, the geographical condition of this nation is very supportive of the spread of this plant. International research on S. trilobata is still limited to the potential of anticancer agents (Balekar et al., 2014). Further research on the utility of S. trilobata as a phytomedicine in terms of phytochemicals and pharmacology requires special studies that must be proven, specifically for the cases of breast cancer (Richard et al., 2015). Thomy dan Ginting (2011) have confirmed the potential of Wedelia biflora as an anti-cancer agent using the BSLT (Brim Shrimp Lethal Test) method. Kour (2014) reviewed potential plants as anti-cancer; one of them is Wedelia chinensis (Osbeck) Merr. Tsai et al. (2009) reported that Wedelia chinensis had cytotoxicity activity in prostate cancer cells (LNCaP / PC-3 / 22Rv1). Manjamalai and Grace (2013) have proven that oil extracts from Wedelia chinensis (Osbeck) had a chemotherapy effect on cases of lung cancer/lung cancer cells (C57BL / 6). Mardina et al. (2019) reported that S. trilobata has potential as a chemopreventive agent for tumors/breast cancer as evidenced by in vivo tests on mice.
The utility of sernai plants (S. trilobata) for traditional medicine had reported as the empirical evidence of the community without certain research/ information (Rahardhian and Utami, 2018). Thus, further research on the special effects of the S. trilobata plant is required. The objective of this study to evaluate the in vitro anticancer of n-hexane extract from S. trilobata leaves in term of cytotoxic and specific anti-proliferation of MCF-7 breast cancer cells.

Identification and Extraction of Sample
The main sample used in this study was the leaves of S. trilobata (L) J.F. Pruski which collected from the Langsa city, Aceh. Sample identification was carried out at Medanense Herbarium, Universitas Sumatra Utara, Indonesia. Samples were dried for ± 7-10 days and cut into small pieces (±0.3 cm). Samples were macerated using n-hexane for 3x24 hours with three repetitions. Each repetition was filtered with Whatman filter paper No.1. The maceration extract was concentrated using a rotary vacuum evaporator.

Preparation of the Extract for MTT Assay
The n-hexane extract as a sample test was prepared with a concentration of 1000; 500; 200; 100; 50; 25; 10; 5 and 1µg/mL that was dissolved in RPMI 1640 media. Each concentration series was performed in triplicates.

Cytotoxicity Test
The MTT method was used in this cytotoxicity test. A total of 100 µL of MCF-7 cell suspension (5x104 cells / ml) were distributed into 96 microplate wells, then 100 µl of the test preparation solution was set with a predetermined concentration (1000; 500; 100; 50; 25; 10; 5 and 1 µg / mL). After incubation for 24 h at 37 o C, MTT reagent was added to the cell suspension and then reincubated for 4 h. 10% SDS solution in 0.01 N HCl was given immediately as a stopper reagent and re-incubated for 24 hours at room temperature. Living cells would react with MTT to form formazan salt and were marked in purple. Absorbance readings were performed at a wavelength of 595 nm (Garbi et al., 2015).

Data Analysis
The data of cytotoxic test were analyzed in order to calculate LC 50 (Lethal Concentration that caused the 50% of MCF-7 cancer cells tested was die) by performing a linear regression equation of log concentration versus probit % mortality. The percentage of cell death is calculated by the following formula: (Rahardhian and Utami, 2018). Samples were dried for 7-10 days and cut into small pieces (±0.3 cm) and macerated with n-hexane, then evaporated to obtain the viscous extract. In vitro cytotoxicity test of the sample was carried out against MCF-7 breast cancer cells with a density of 5x10 4 cell/mL. MTT assay was chosen in the cytotoxicity test due to several advantages, namely rapid, simple, inexpensive and recognized as the qualitative method (Arisanty, 2013) to measure growth, survival and cell proliferation (Antoney et al., 2016).
The calorimetry test using 3-4,5-dimethylthiazol2-yl-2,5-diphenyl tetrazolium bromide (MTT) was first introduced by Mossman to promote the viability of mammalian cells. Cells that react with the help of the dehydrogenase enzyme convert the tetrazolium salt in yellow MTT into insoluble formazan (Figure 1). MTT testing is based on the principle that the amount of formazan produced is directly proportional to the number of living cells (Abate et al., 1998). Figure 2 showed the formazan crystals under a microscope. The concentration of formazan formed was measured using ELISA reader (multiwell scanning spectrophotometer) at a measured wavelength of 595 nm and calculated as the amount of optical absorbance/density. The greater the absorbance value obtained (the higher purple intensity) reflected an increase in cell viability. Thus it can be used to calculate the acquisition of death (Antoney et al., 2016).  The test results in Table 1 exhibited the treatment of n-hexane extract could induce MCF-7 cell death. The higher concentration of n-hexane extract from S.trilobata toward MCF-7 cells would produce a greater mortality percentage of MCF-7. This might be influenced by the presence of secondary metabolites in S. trilobata extract as explained by Ahmed et al. (2019) that Wedelia trilobata (L.) Hitchc, consisting of. 3α-tigloyloxypterokaurene L3, ent-17-hydroxy-kaura-9 (11), 15-dien-19-oic acid, wedelobatins A and wedelobatins B which have toxic effects on cancer cells. Research Venkatesh et al. (2016) found that methanol extract of Wedelia trilobata was toxic to MEG-01 cancer cells with an LC 50 value of 80 μg / mL for 48 hours incubation. Based on the graph in Figure 2, it could be calculated the LC 50 which levels cause the death of 50% of MCF-7 cells using probit analysis method. The regression equation obtained was y = 15.593x + 27.34 with the r-value of 0.8697. The LC 50 was obtained by substituting probit 5 into the linear equation. Then the x was obtained. Antilog of x was LC 50 . The LC 50 calculation results obtained were 0.037 μg/mL. This means that at the level of 0.037 μg/mL of n-hexane extract from S. trilobata leaves could cause breast cancer cell death (MCF-7) by 50% of the number of cells tried. The smaller the concentration required to kill 50% of the viability cells means compound in the sample was more toxic.

Conclusions
The sample used in this study was Sphagneticola trilobata (L.) J.F Pruski which was macerated with n-hexane. In vitro cytotoxicity assay of crude extract was conducted on MCF-7 breast cancer cells with an LC 50 value of 0.037 µg/mL.