Cytotoxic Activity of Secondary Metabolites from the Bark of Dipterocarpus Confertus

1) Faculty of Pharmacy, Universitas Muhammadiyah Surakarta, Jalan A. Yani Tromol Pos I Pabelan Kartasura, Surakarta 57102, Indonesia *muhtadi@ums.ac.id 2) Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) 40450 Shah Alam, Selangor, Malaysia nico4orgchem@yahoo.com 3) School of Chemistry and Environtmental Studies, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia. khonghy@sarawak.uitm.edu.my ABSTRACT


INTRODUCTION
Dipterocarpus confertus, which known with local name "Keruing Pungguh" in Indonesia is a species of genus Dipterocarpus (Dipterocarpaceae). Dipterocarpus is the third largest genus in the family of Dipterocarpaceae with 75 species [1,2]. As other genus in Dipterocarpaceae, Dipterocarpus has been known as a rich source of oligostilbenoid type of compounds, as well as other type of phenolic compounds [3,4,5,6,7]. The secondary metabolites of Dipterocarpaceae have withdrawn much attention of many scientists due to the structural complexity, and their biological activities such as antifungal [8,9], anti-HIV [10], cytotoxic [10,11,3,4], anti-inflammatory [12,13] and antibacterial [14,15,7]. Our previous study reported the isolation of phenolic compounds from Dipterocarpus as well as their cytotoxic activity [4,16]. In continuation of our interest in the phytochemical study of phenolic compounds in the new species Dipterocarpus plant, we report here the cytotoxicity of four compounds isolated from the bark of Dipterocarpus confertus.

EXPERIMENTAL General experimental procedure
The following instruments were used: UV and IR spectrum were measured with a Varian Conc. 100 instruments and a Perkin Elmer Spectrum One FTIR spectrometer, respectively.

Plant material
The bark of D. confertus was collected from Bukit Bengkirai, East Kalimantan, Indonesia, and a voucher specimen has been deposited at the Herbarium Bogoriensis, Research Center for Biology, Indonesian Institute of Science, Bogor, Indonesia (collection number SR-027).

Cytotoxic Activity
The cytotoxicity was determined by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against P388 and Vero cell lines. The assay was performed as described by Mosmann (1983) [17], but with slight modifications. MTT was first prepared are a stock solution of 5 mg/ml in phosphate buffer (PBS, pH 7.2) and filtered. Samples of different concentration of the extracts were prepared in triplicates. At the end of the treatment period (72h), 20 l of MTT solution was added to each sample and incubated for 3 hours. After 72 h, supernatants were discarded and 50 μl of MTT stock solution (5 mg/ml) were added to each well and the plates were further incubated for four hours. After that, supernatant was removed, and the formazan crystals were dissolved with 100 μl DMSO. The amount of MTT-formazan was directly proportional to the number of living cells, and was determined by measuring the optical density (OD) at 570 nm using microplate reader (Quant Universal Microplate Spectrophotometer, BIOTEK Instrument, Inc.). The IC50 values represented the concentration that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells [18].

RESULTS AND DISCUSSION
The methanol extract of the stem bark of D. confertus was separated by combination of vacuum liquid and flash column chromatographies have resulted the isolation and characterization of compounds 1-4. The structure of these compounds were identified on the basis of spectroscopic data evidence as steroid; β-sitosterol (1) [19], pentacyclic triterpene; betulinic acid (2) [20], cinnamic acid (3) [21], and oligomeric resveratrol; α-viniferin (4) [12] (Fig. 1). All compounds were isolated for the first time from this plant.

Fig. 1. The structure of isolated compounds
The cytotoxicity of all isolated compounds from the bark of D. confertus was tested against P-388 mice leukemia and Vero normal kidney monkey cell lines. From the assay, cinnamic acid (3) and betulinic acid (2) demonstrated potent activity against P-388 cell lines with IC50 value of 2.25 and 5.10 μg/ml, respectively. However, they showed different response against normal vero cell, in which betulinic acid (2) was toxic to the cell (IC50 6.19 μg/ml), while cinnamic acid (3) was not toxic (IC50 69823.24 μg/ml). On the other hand, other compounds were only in the range of the lowest and not active against both cell lines (Table 1). This fact indicated that only 3 displays high potency as anticancer agent, as it is only toxic to the cancer cell and not to the host cell. In this study, compound 3 is more active compared to 2 even though they contain similar acid group. This may be due to the existence of conjugate system in the skeleton of 3, which activated its aromatic ring. This suggestion was supported by Otero et al. (2013) [22], which found that the cytotoxicity of chalcone and coumarin increased with the presence of conjugated double bond in the side chain. The similar case was displayed by compound 2, which showed stronger activity compared to 1. Several literatures reported that compound 2 is a pentacyclic triterpenoid that commonly studied, and the studies have been focused on the modifications of carbons C-3 and C-28. Ding et al. (2013) [23] reported that electrondonating groups at the C-28 and C-3 of 2 would improve the activity.