Cytotoxic Triterpenoid from the Stembark of Chisocheton celebicus (Meliaceae)

Plants belonging to the Chisocheton genus are a rich source of tetracylic triterpenoids with diverse biological activities. Two triterpenoid compounds, dammar-20,24-dien-3-one (1) and 3β-hydroxy-tirucall-7-en (2) were isolated from the stembark of Chisocheton celebicus. The chemical structures of compounds 1 and 2 were identified by spectroscopic data, including IR, NMR (H, C, DEPT 135, HMQC, HMBC, H-H COSY), and MS, and they were compared with previously reported spectral data. Compounds 1 and 2 were evaluated for their cytotoxic effects against P-388 murine leukemia cells. The compounds showed cytotoxicity against P-388 murine leukemia cells, with IC50 values of 30.2 and 4.3 μg/mL, respectively.

Although secondary metabolites from other Chisocheton species have been reported previously, the chemical constituents of C. celebicus have yet to be reported.C. celebicus is a higher plant that is widely distributed in the nothern part of Sulawesi island in Indonesia [14].Its bark has been used as an Indonesian folk medicine for reducing fever, treating contused wounds, and for skin diseases [14,15].The isolation, structure elucidation, and cytotoxic evaluation of these isolated compounds are described herein.9 internal standard.Chromatographic separations were carried out on silica gel 60 (Merck) and octa desyl silane (ODS) (Fuji Silysia).Thin layer chromatography (TLC) plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and ODS, and detection was achieved by spraying with 10% H 2 SO 4 in ethanol, followed by heating.
Plant material.The stem bark of C. celebicus was collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia, in April 2012.The plant was identified by the staff of the Bogoriense Herbarium, Bogor, Indonesia, and a voucher specimen was deposited at the herbarium.
Determination of cytotoxic activities.The P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3 x 10 4 cells cm -3 , supplemented with a growth medium containing various concentrations of NABV or vinblastine in 0.5% dimethyl sulfoxide (DMSO) for 72 h.After 24 h of incubation for cell attachment and growth, sample with various concentration was added.The added compounds were first dissolved in DMSO at the required concentration.Subsequently, six desirable concentrations were prepared using PBS (phosphate buffered saline, pH = 7.30-7.65).Control wells received only DMSO.The assay was terminated after a 48 h incubation period by adding MTT reagent [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; also named as thiazol blue], and the incubation was continued for another 4 h, after which the MTT-stop solution containing SDS (sodium dodecyl sulphate) was added and another 24 h incubation was conducted.Optical density was measured using a microplate reader at 550 nm.IC 50 values were taken from the plotted graph of the percentage of live cells compared to the control (%), which received only PBS and DMSO, versus the tested concentrations of the compounds (g/mL).The IC 50 value is the concentration required for 50% growth inhibition of cells.All analyses were carried out in triplicate, and the results were expressed as mean ± standard deviation (SD) and compared using the Waller-Duncan test.A value of p < 0.05 was considered statistically significant.

Result and Discussion
The stembark of C. celebicus was ground and successively extracted with n-hexane, ethyl acetate, and methanol.All the extracts were evaluated for their cytotoxic activity against P-388 murine leukimia cells, with the ethyl acetate extract showing the strongest cytotoxic activity.Therefore, the subsequent phytochemical analysis was focused on the ethyl acetate extract, which was chromatographed over a vacuum-liquid chromatographed (VLC) column packed with silica gel 60 with gradient elution.The VLC fractions were repeatedly subjected to normal-phase and reverse-phase column chromatography, yielding two cytotoxic triterpenoids, 1 and 2 (Figure 1).There was one olefinic methine group, resonating at δ H 5.11 (1H, t, J = 2.6 Hz, H-24), and one methylene group, resonating at δ H 4.71 (1H, d, J = 1.9 Hz) and 4.75 (1H, d, J = 1.9 Hz, H-21), which indicates that the olefinic protons were in the geminal position.The proton pairing was also confirmed with the 1 H-1 H COSY spectrum (Figure 2).The 13 C-NMR (CDCl 3 125 MHz) and DEPT 135°spectra showed the presence of seven methyl groups, exhibiting the characteristics of triterpenoid compounds [16], one olefinic methine, one olefinic methylene, two olefinic quaternary carbons, and a ketone group, resonating at δ C 218.4.The HMBC crosspeaks (Figure 2) from H-28 ( H 1.05) and H-29 ( H 1.05) and the methylene protons from H-2 (δ H 1.44 and 1.93) to the quaternary carbon at  C 218.4 indicated the presence of a ketone group at C-3.These functionalities accounted for three of seven total degrees of unsaturation, and the remaining four degrees of unsaturation were consistent with the triterpenoid skeleton.A comparison of the NMR data of 1 with dammar-20,24-dien-3-one [17] revealed that the structures of the two compounds were very similar; consequently, compound 1 was identified as dammar-20,24-dien-3-one and was shown in this species for the first time.

Dammar-20,24-dien-3-one
Compound 2 was obtained as a white amorphous powder.The HR-TOFMS spectrum showed  [17].One olefinic methine group, resonating at δ H 5.30 (1H, t, J = 3.2 Hz, H-7), and one oxymethine group, resonating at δ H 3.20 (1H, t, J = 5.8 Hz, H-3), indicating that the configuration was 3β-OH.The proton pairing was also confirmed with the 1 H-1 H COSY spectrum (Figure 2).The 13 C-NMR (CDCl 3 125 MHz) and 135°spectra showed the presence of eight methyl groups, one olefinic methine, and one oxymethine, resonating at δ C 79.3, supporting the presence of triterpenoid compound in 1 [16].The HMBC crosspeaks (Figure 2) from H-28 ( H 0.  7,8 ).These functionalities accounted for one of the five total degrees of unsaturation, and the remaining four degrees of unsaturation were consistent with the triterpenoid skeleton.A comparison of the NMR data of 2 with the data for 3β-hydroxy-tirucall-7-en [18] revealed that the structures of the two compounds were very similar, and thus compound 2 was identified as 3β-hydroxy-tirucall-7-en and shown in this species for the first time.
The cytotoxicity effects of the two isolated compounds (1 and 2) against P-388 murine leukemia cells were investigated according to the method described in previous papers [16], and artonin E (IC 50 0.3 µg/mL) was used as a positive control [19].
The cytotoxicity activity of isolated compounds 1 and 2 in terms of IC 50 values was 30.5 ± 0.24 and 4.3 ± 0.08 µg/mL, respectively.These results suggested that the activity of 3β-hydroxy-tirucall-7-en (2) was influenced by the hydroxy group at C-3, the position of the methyl group at C-18, and the position of olefinic carbon.In dammar-20,24-dien-3-one (1), the presence of a ketone group at C-3, the position of C-18 (which is different from compound 2), and the position of an olefinic group at the side chain can decrease cytotoxic activity.The cytotoxic activity of both compounds (1 and 2) was weaker than that of the control (artonin E), so neither can be used as a model compound for anticancer directly; their partial structures need to be modified to increase cytotoxic activity.

Conclusions
Two known triterpenoid compounds, 1 and 2, have been isolated from the stembark of Chisocheton celebicus.These compounds were evaluated for their cytotoxic activity against P-388 murine leukemia cells in vitro.
The result indicated that the presence of a hydroxyl group at C-3 and the location of the methyl and olefinic groups can increase cytotoxic activity.Both compounds require modification of their partial structures to increase cytotoxic activity.