Cytotoxic Activity of Pentacyclic Triterpene-3-Heptadecanoate Ester against Hela Cell Line and Its Docking Study

Pentacyclic triterpene-3-heptadecanoate (12,13-dihydro-α-amyrin-20,30-en-3-heptadeca no ate) ester refers to the compound as a result of reaction between pentacyclic triterpene-3-ol (12,13-dihydro-α-amyrin-20,30-en-3-ol) and heptadecanoic acid. This research was aimed to conduct cytotoxicity test of pentacyclic triterpene-3-heptadecanoate; 12,13-dihydro-α-amyrin-20,30-en-3-ol and 12,13-dihydro-α-amyrin-20,30-en-3-acetate esters against Hela cell line. The activity assay was carried out using MTT method. The results indicated that IC50 value of 12,13-dihydro-α-amyrin-3heptadecanoate; 12,13-dihydro-α-amyrin-20,30-en-3-acetate and 12,13-dihydro-α-amyrin-20,30-en-3ol were 111.0, 151.1 and 944.4 μg/ml, respectively. The compound of 12,13-dihydro-α-amyrin-20,30en-3-heptadecanoate had the lowest IC50 value, suggesting it has a potency to be synthesized as an anticancer drug.


Introduction
Cancer is a disease that cause the loss of cell control towards both the regulation of cell cycle and homeostatic function leading to fast and uncontrolled proliferation [1]. A number of re searchs have been conducted to create new anti cancer compound. Isolation of natural substances known to have anticancer activity have also been conducted. By finding the lead compound hav ing some activities as anticancer, therefore, the chemi cal groups playing a role of having corres ponding activities can be determined. This, as a result, offers an opportunity to modify a lead compound in order to find a new more potential compound. The natural compounds useful as the anticancer are 12,13-dihydro-α-amyrin-20,30en-3-ol and 12,13-dihydro-α-amyrin-20,30-en-3acetate that are categorized as the pentacyclic triterpenes.
Some problems emerging from this research included how the cytotoxic activity of the triter pene pentacyclic3heptadecanoate ester (C16) and whether it is more effective compared to the pentacyclic triterpene3acetate ester (C1). Thus, the objective of this research is to find out the cy totoxic activity of triterpene pentacyclic3hep tadecanoate ester as the anticancer towards Hela cell line. Hela cell line is cervix cancer cell that can be used for cancer research [6].

Cytotoxicity test
Stock solution of 12,13-dihydro-α-amyrin-20,30-en-3-heptadecanoate was prepared by dis solving in dimethyl sulfoxide (DMSO) and media culture of RPMI 1640 was initially done in order to obtain a series of concentration from 15.6 to 500 μg/ml. The 12,13-dihydro-α-amyrin-20,30-en-3acetate and 12,13-dihydro-amyrin-20,30-en-3-ol was also prepared in the series of concentration from 15.6 to 500 μg/ml. The Hela cell line with concentration of 20,000 cells/well were placed into 96-well plates containing RPMI 1640 with the volume of 100 μl/well were incubated within 1 day. A series of concentrations of pentacyclic triterpene3heptadecanoate ester were placed into the 100 μl wells. Here, triterpene pentacyclic triterpene 3heptadecanoate ester had a series of concentrations of 15.6 , 31.3, 62.5, 250 and 500 μg/ml. Then, it was incubated within 24 hours in an incubator with 5% CO 2 at the temperature of 37 o C. MTT with the concentration of 5 mg/ml in PBS was added to this compound before the compound was placed into each of 10-μl well and incubated for 4 hours in an incubator with 5% CO 2 at 37 o C. The stop solution of 10% SDS in hy drochloride acid 0.01 N at 100 μl, subsequently, was added and incubated at room temperature. In this case, the absorbance of it was read using Elisa reader with the wavelength of 550 nm.
The IC 50 value was obtained from the analysis of regression from the result of absorbance ob tained. Based on the equation of y = Bx + A with x = the level of pentacyclic triterpene-3-heptadecanoate ester, pentacyclic triterpene3acetate ester, pentacyclic triterpene-3-ol and y = mean of the cell viability (%). Thus, the IC 50 could be found by correlating percentage of cell viability at 50% at the equation. In this case, the IC 50 value can be calculated as μg/ml and then divided with the molecular weight. The calculation of cell via bility is as follows: sample Ab−media control Ab viability = x 100% cell control of Ab−media control Ab Remark: Ab = absorbance

Results and discussion
The results of cytotoxicity test of pentacyclic triterpene3heptadecanoate ester, pentacyclic triterpene3acetate ester and triterpene pen tacyclic triterpene3ol against the Hela cell line ( Table 1,  The results of the cytotoxicity test of three pentacyclic triterpene compounds showed effect on the inhibition of the Hela cells' growth. The IC 50 value of pentacyclic triterpene3heptadeca noate ester, pentacyclic triterpene3acetate es ter and pentacyclic triterpene-3-ol were 110.0, 151.1 and 944.40 μg/ml, respectively. This shows that pentacyclic triterpene3heptadecanoate es ter as the result of modification had the lowest IC 50 value. In other words, esterification of pen tacyclic triterpene3ol with heptadecanoic acid increased the activity.
The highest concentration of compound se lected was 500 μg/ml. The relationship between percentage of viability of Hela cell line and the concentration of each compound are shown in Figure 2.