Leaves of G. celebica were collected in the Pangandaran Beach Conservation Area of West Java (Indonesia). The plant species was identified by the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran (Jatinangor, Indonesia). The leaves were dried in the open air away from direct sunlight.

Dried G. celebica leaves were powdered and extracted with 95% ethanol (three times every 24 h) at room temperature and the solvent was evaporated under reduced pressure at 50°C to yield concentrated extracts. The extract was partitioned with a mixture of n-hexane-water (3:1) to generate hexane and water layers. The water layer was further extracted with ethyl acetate to yield ethyl acetate and water fractions. The n-hexane and ethyl acetate fractions had inhibitory activities against MCF-7 cell proliferation. Silica gel chromatography was used to elute the n-hexane fraction (23.32 g) into 5 fractions of n-hexane-ethyl acetate mixtures with increasing polarity (n-hexane:ethyl acetate, 9:1, 8:2, 7:3, 6:4, 5:5). Each fraction was tested for its toxicity against A. salina larvae (7), and the fraction with the highest toxicity was further chromatographed over silica gel with the same elution system to generate 11 fractions, of which fraction 6 contained the targeted compounds. Fraction 6 was subjected to preparative thin-layer chromatography using a mixture of chloroform-methanol (9.5:05) as a developing solvent system. The application of preparative chromatography resulted in a compound (50.20 mg, white amorphous solid) that showed high toxicity against A. salina larvae. The compound was identified by an analysis of its spectroscopic data [ultraviolet (UV), infrared (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR)].

MCF-7 human breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The cell lines were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cell proliferation was analyzed using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay in cells treated with various concentrations of the active compound isolated from leaves of G. celebica following the methods of Abdulah et al (8). Briefly, 2×10⁴ cells in 50 µl/well were plated in 96-well plates. Following the initial cell seeding, different concentrations of the active compound isolated from leaves of G. celebica were added and incubated for 24 h. WST-8 assay cell-counting solution (Dojindo Lab., Kumamoto, Japan) was added to each well (10 µl) and incubated at 37°C for 3 h. After the addition of 100 µl/well of 1 N HCl, the cell proliferation rate was subsequently determined by measuring the absorbance at a wavelength of 450 nm. The absorbance was read using a microtiter plate reader (Becton-Dickinson, Franklin Lakes, NJ, USA).

Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). In total, 40 µg protein was electrophoresed on a Mini-PROTEAN TGX Precast gel (4–20%; Bio-Rad Laboratories, Hercules, CA, USA) and electrotransferred to a 7×8 cm Hybond-enhanced chemiluminescence membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Apoptosis-associated proteins were analyzed by immunoblot analysis using poly(adenosine diphosphate-ribose) polymerase (PARP; #9542) and Akt (#4685) antibodies at a 1:1,000 dilution (both Cell Signaling Technology, Danvers, MA, USA). β-actin (#A5441; Sigma-Aldrich) served as the loading control.

An antiproliferative compound isolated from the leaves of G. celebica was amorphous and was white in color. It exhibited a molecular ion peak at m/z 484 in the electron ionization mass spectrum indicating that this compound had a molecular formula of C₃₁H₄₈O₄.

The UV spectrum indicated absorption bands at 275 and 439 nm and the IR spectrum revealed the presence of a hydroxyl group (3,500 cm⁻¹) and a carbonyl group of an α, β-unsaturated ester (1,700 cm⁻¹). The presence of the carbonyl functionality was further confirmed by a carbon signal appearing at δ 168.7 in the ¹³C NMR spectrum.

The ¹H and ¹³C NMR spectra of the compound exhibited that this compound was assumed to be a triterpenoid compound. The ¹H NMR spectrum indicated the presence of 5 tertiary methyls (δ 0.76, 0.89, 0.90, 0.98 and 1.01), 1 secondary methyl (δ 0.94, d, J=7.5 Hz), and 1 oxymethine proton (δ 3.45, br s). These signals were assumed due to a tetracyclic triterpene having a 3α-hydroxy group (9–11). Furthermore, signals due to 1 olefinic proton (δ 6.72, qd, J=7.5 and 1.5 Hz), 1 vinylic methyl (δ 1.87, d, J=1.5 Hz), 1 oxymethine proton (δ 4.57, ddd, J=10.7, 7.5 and 2.5 Hz), and methoxy protons (δ 3.75, s) were observed. These signals suggested that the compound had a side chain with a structure of [-CH(Me)CH₂CH(OH)CH=C(Me)COOCH₃]. The ¹³C NMR spectrum indicated the presence of 7 methyl carbons (δ 12.9, 15.5, 15.8, 17.3, 19.2, 22.4 and 28.2), 8 methylene carbons (δ 18.3, 22.9, 25.8, 26.9, 29.4, 30.3, 39.7 and 45.7), 6 methine carbons (δ 33.6, 44.6, 67.1, 76.0, 116.0 and 142.6), and 9 quartenary carbons (δ 37.8, 38.0, 48.2, 50.2, 123.1, 127.3, 144.6, 148.9 and 168.7). These NMR spectral data showed that the compound may be friedolanostane triterpenoid with one tetrasubstituted double bond and two trisubstituted double bonds. One of the two trisubstituted double bonds was located in the side chain and the two remaining double bonds in the tetracyclic system were indicated to be conjugated. The conjugated double bonds were assumed to be present at C-8/C-9 and C-14/C-15, therefore, the vinylic proton appearing at 5.26 (s) was at C-15. Thus, the structure of the compound was established as methyl-3α, 23-dihydroxy-17,14-friedolanstan-8,14,24-trien-26-oat (Fig. 1) which has been reported previously. The ¹H and ¹³C NMR data of the identified compound is shown in Table I. Confirmation of the structure was obtained by comparison of its spectral data with those reported in previous studies (11,12).

Methyl-3α,23-dihydroxy-17,14-friedolanstan-8, 14,24-trien-26-oat was evaluated for its effect on the proliferation of MCF-7 breast cancer cell lines by the MTT assay. The evaluation resulted in a time- and dose-dependent manner inhibition of the compound on the cell proliferation (Fig. 2). The compound strongly inhibited the MCF-7 cell line proliferation in 24 and 48 h treatments, with the IC₅₀ value of 82 and 70 µM in the 24 and 48 h measurements, respectively.

The MTT assay showed strong inhibitory activity of methyl-3α,23- dihydroxy-17,14-friedolanstan-8,14,24-trien-26-oat against the MCF-7 cell line proliferation, thus, the compound was further examined for its proapoptotic activity through PARP protein activation within 24 and 48 h of treatment. The inhibition of MCF-7 human breast cancer cells proliferation by the compound was mediated by the induction of apoptosis, marked by PARP protein activation, which is one of the most optimal biomarkers of apoptosis. Furthermore, the compound also inhibited the expression of Akt oncogene (Fig. 3).