A New Diterpenoid of Indonesian Scoparia dulcis Linn: Isolation and Cytotoxic Activity against MCF-7 and T47D Cell Lines

Abstract

Scoparia dulcis Linn plays an important role in treatment because it contains active compounds that are proven to have a variety of activities, including cytotoxicity on various cancer cells. The objective of this study is to isolate and identify the cytotoxic compounds in the ethyl acetate fraction of Scoparia dulcis, observe cell cycle inhibition and induction of apoptosis in vitro, and carry out molecular studies using in silico studies. A new diterpene compound was isolated from the ethyl acetate fraction of Scoparia dulcis L. of Indonesian origin. Chromatographic methods were used to isolate the compound, spectroscopic methods were used to elucidate its structure, and these data were compared with those reported in the literature. The compound was tested for its cytotoxic activity against two breast cancer cells (MCF-7 and T47D). The results of the isolated compound showed a cytotoxic effect on MCF-7 and T47D breast cancer cells at IC₅₀ 70.56 ± 1.54 and <3.125 ± 0.43 µg/mL, respectively. The compound inhibited the growth of MCF-7 and T47D breast cancer cells and the accumulation of cells in the G1 phases, and it induced apoptosis. Based on a spectroscopic analysis, the isolated compound was identified as 2α-hydroxyscopadiol, which is a new diterpenoid. A docking study revealed that the isolate's hydroxyl groups are essential for interacting with crucial residues on the active sites of the ER and PR and caspase-9. The isolate inhibits ER and PR activity with binding energies of -8.2 kcal/mol and -7.3 kcal/mol, respectively. In addition, the isolate was also able to induce apoptosis through the activation of the caspase-9 pathway with an affinity of -9.0 kcal/mol. In conclusion, the isolated compound from S. dulcis demonstrated anticancer activity based on in vitro and in silico studies.

Keywords: 2α-hydroxyscopadiol; MCF-7; Scoparia dulcis; T47D; cytotoxic; molecular docking.

Figure 1

Chemical structures of 2α-hydroxyscopadiol (1), 7 α-hydroxyscopadiol (2) [22], 4-epi-7α-O-acetylscoparic acid A (3) [22], scopadiol (4) [23], and scopadiol decanoate (5) [21].

Figure 2

TLC profile with vanillin sulfate spray from the isolated 13.1 compound. Mobile phase: (A) n-hexane:ethyl acetate (2:3); (B) Chloroform:ethylacetate (1:3); and (C) n-hexane:ethanol (1:2).

Figure 3

The COSY and HMBC (A) and (B) correlations showing the NOESY interactions in compound (1).

Figure 4

The % viability value for compound (1) and doxorubicin. All data are presented as mean ± SD, n = 3 and VC 1: variation of concentration 1; VC 2: variation of concentration 2; and VC 3: variation of concentration 3.

Figure 5

Histogram profile of the cell cycle for compound (1) against MCF-7 and T47D breast cancer cells and the control.

Figure 6

Cell cycle distribution after compound (1) treatment using flow cytometry against MCF-7 and T47D breast cancer cells.

Figure 7

Histogram profile for apoptosis induction following treatment using compound (1) against MCF-7 and T47D breast cancer cell and cell control.

Figure 8

Effect of apoptosis induction after compound 1 treatment. Error bars represent the standard deviation (n = 3) and quadrant P2 indicates living cells. P3: early apoptosis, P4: late apoptosis, P5: necrosis. Different letters (a–d) indicate significant differences based on an ANOVA followed by a Tukey post hoc test (p < 0.05).

Figure 9

Superimpose and the interaction between the co-crystal ligand (green) and the docked pose (pink) of (A) TMX in the ER, (B) R18 in the PR, and (C) DTP in caspase-9.

Figure 10

Molecular interaction between (A) compound (1) and (B) doxorubicin and the estrogen receptor.

Figure 11

Molecular interaction between (A) compound (1) and (B) doxorubicin and the progesterone receptor.

Figure 12

Molecular interaction between (A) compound (1) and (B) doxorubicin and caspase-9.