Cytotoxicity Study of Ethanol Extract of the Leaves of Asam Kandis (Garcinia cowa Roxb.) on T47D Breast Cancer Cell line

Objective: To investigate the cytotoxic effect of ethanolic extract of the leaves of asam kandis (Garcinia cowa Roxb.) against T47D breast cancer cells. Methods: The cytotoxicity of ethanol extract was carried out by measuring the activity of mitochondrial dehydrogenase in living cells that have ability to convert dissolved MTT pale yellow to purple formazan product. The extract was added at various concentrations (0.1, 1, 10 and 100 μg/ mL). The level of cytotoxicity was determined by calculating the IC50 value that was based on the percentage of the cell death after 24 hours treatment with the extract. Cell morphological changes were observed by using inverted microscope. Results: The IC50 value showed that ethanol extract of leaves of asam kandis could resist T47D breast cancer cells with IC50 6.13 ± 3.51 μg/mL. The statistic results proved that ethanol extract of the leaves of asam kandis could inhibit the growth of T47D breast cancer cells significantly at concentrations of 10 μg/mL and 100 μg/mL. Conclusion: The results suggest that ethanol extract of the leaves of asam kandis was potential source of herbal medicine for cancer-related ailments.


Plant materials and extraction
The leaves of G. cowa were collected in Padang.The plant material was air dried in the green house for 3 days at room temperature, followed by oven drying at 40°C for one day.The dry leaves were grinded to powder form using a laboratory blender.The powdered sample was kept in an airtight container until required.About 2 kg of the powdered the leaves of G. cowa was macerated in 15 L of ethanol 70% for 3 days.This process was repeated 3 times.Rotary evaporator was used to evaporate and concentrated ethanol extract at 40 o C. The resulting extract was kept in the refrigerator.

Methods
All procedures were described in our previous study. 13

Cytotoxic effect toward T47D
The cytotoxicity effect of ethanol extract of leaves of asam kandis on T47D was evaluated by MTT assay.The extract from asam kandis in multiple concentrations was used.The effective concentration was calculated from concentration-response curve.The percentage of viability of each plate was shown in Table 1.Based on the MTT assay (Figure 1), it was found that the ethanol extract of the leaves of asam kandis had IC 50 value of 6.13 ± 3.51 µg/ mL.The criteria of cytotoxicity for the crude extract, as established by the U.S. National Cancer Institute (NCI), are an IC 50 <20 µg/mL in the preliminary assay. 14

Evaluation on morphological changes upon treatment with extracts
The difference in concentration gives a different picture when viewed with a microscope inverted when compared to control.Figure 2 showed that many cancer cells grow and attached at the base of the flask at a concentration of 0.1 µg/mL and 1 µg/mL.Cells grow very dense and very little distance between one cell to another cell so it almost looks no difference with control.While at a concentration of 10 µg/mL, the cells look less than the concentration of 0.1 µg/mL and 1 µg/mL.Cells are visible only in small groups so that the distance a group of cells with other cells look far.It is also evident from the low absorbance value.Another case in a concentration of 100 µg/mL, the cells look less, there is no longer even visible cell growth.Dead cells will not be visible cell nucleus (white) and opaque.Absorbance also suggests that little living cells.

DISCUSSION
MTT assay is a colorimetric cytotoxic test method to determine the number of living cells based on changes in a solution of 3-(4,5-dimetilthiazol-2-il)-2,5-difeniltetrazolium bromide were colored yellow to purple formazan crystals by active mitochondria in living cells.MTT is absorbed into living cells and broken through the oxidation reaction by nicotinamide adenine dinucleotide (NAD + ) as enzyme in the mitochondrial respiratory chain into formazan that was not soluble in water.
Purple color intensity is directly proportional to the amount of the active cell metabolism.Formed the darker color, the higher the absorbance value, and the more living cells. 15reast cancer cell used in this research was T47D cell.T47D breast cancer cell is sensitive to chemotherapeutic agents and have a fast replication capability that is well suited for the cytotoxic test.The multiple concentrations of ethanol extract from asam kandis were used and effective concentration was calculated from concentration-response curve.The results of the cytotoxicity evaluation against T47D cell lines of the kandis extract were shown in Figure 1.The ethanol extract of the leaves of asam kandis exhibited significant activity against T47D cell lines with IC 50 6.13 ± 3.51 µg/mL.Data processing using One Way ANOVA followed by Duncan Advanced Test.Results of One Way ANOVA test (α=0.05)suggesting that the leaves extract asam kandis gave significantly different barriers to the growth of T47D breast cancer cells.The results obtained on a One Way ANOVA test were significantly different, then followed by Duncan test at 5% significance level (Table 1).Duncan further test the data obtained that the concentration of 100 µg/mL significantly different from the concentration of 10 µg/mL, a concentration of 10 µg/mL significantly different from the concentration of 1 µg/mL.However the concentration of 1 µg/ mL and 0.1 µg/mL was not significantly different.Based on the relationship curve between the concentration of the test compound with the % cell viability can be observed that the leaves of asam  kandis extract at a concentration of 0.1 µg/mL and 1 µg/mL did not affect the amount of reduction in the number of cells that survive, but at higher concentrations i.e. at a concentration of 10 µg/mL and 100 µg/mL can be observed a decrease in % cell viability, significantly.Based on the statistical results, it can be concluded that the average percentage of cell viability at a concentration test showed a highly significant difference (p<0.01).

CONCLUSION
The results of this research suggest that ethanol extract of the leaves of asam kandis was potential source of herbal medicine for cancer-related ailments.

Figure 1 :Figure 2 :
Figure 1: Dose-response relationship curve of the leaves extract of G. cowa against breast cancer T47D cells (24 Hours)