A Cytotoxic Rocaglate Compound from The Stembark of Aglaia argentea ( Meliaceae )

The Aglaia genus belong to Meliceae family is unique plant species because the presence of rocaglate and rocaglamide which is so far isolated only from Aglaia genus, indicate that type of this compound as a chemical marker for the genus of Aglaia. This type of compound known to have strong activity, such as insecticide and cytotoxic. This study describe the isolation, structure elucidation, and cytotoxic activity of an isolated rocaglate compound. Dried stembark of A. argentea extracted with methanol and partition between n-hexane, ethyl acetate, and n-butanol, respectively The extracts were tested against P-388 murine leukemia cells and the ethyl acetat showed strongest activity with IC50 value of 15.5 g/mL. The ethyl acetate then was separated and purified with chromatography technique to obtain isolated compound 1. The chemical structure of isolated compounds were elucidated by spectroscopic methods including one and two-dimensional NMR as well as high-resolution mass spectrometric analysis and identified as a methyl rocaglate. Compound 1 showed strong cytotoxic activity with an IC50 value of < 0.1 μg/mL.

Members of the Aglaia genus have recently received considerable attention due to the presence of a structurally unique group of cyclopentabenzofurans, the so-called rocaglamide derivatives, which occur exclusively in this genus (Ishibashi et al., 1993;Wu et al., 1997;Nugroho et al., 1999).The parent compound rocaglamide and many of its congeners are potent natural insecticides comparable in activity to azadirachtin (Nugroho et al., 1999;Chaidir et al., 1999;2001) In addition to their insecticidal activity, the rocaglamides exhibit pronounced antiproliferative activity against human cancer cells in vitro, comparable to that of vinblastine sulfate (Chaidir et al., 2001;Bohnenstengel et al., 1999), thus making these compounds an even more fascinating group of potently bioactive plant secondary metabolites.
During the course of our continuing search for anticancer candidate compounds from Indonesia Aglaia plants, we isolated and described cytotoxic triterpenoids from the bark of A. smithii and A. eximia (Harneti et al., 2012;2014) as well as a lignan and bisamides from the bark of A. eximia (Sianturi et al., 2015;2016).In the further screening for cytotoxic compounds from Indonesia Aglaia species, we found that ethyl acetate extracts of the bark of A. argentea exhibited a significant cytotoxic activity against P-388 murine leukemia cells with IC 50 value of 15.48 ± 0.03 g/mL.A. argentea, also known as langsat hutan in Indonesia is a higher plant traditionally used to moisturizing the lungs, reducing fever and for treating contused wound, coughs and skin diaseases (Mabberley, Pannel, & Sing., 1995;Muellner et al., 2010).
Previous phytochemical studies of A. argentea has revealed the presence of a compounds with cytotoxic activity against KB cells, including cycloartane-type triterpenoids (Omobuwajo et al., 1995) and 3,4-secoapo tirucallane-type triterpenoids (Mohamad et al., 1999).Although secondary metabolites of other Aglaia species have been investigated previously, the rocaglamide derivatives of A.
argentea is yet to be reported.We report herein the isolation and structure identification of rocaglate compound along with its cytotoxic activity against P-388 murine leukimia cells that not yet invertigated previously.

Plant Material
The stembark of A. argentea were collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia in June 2015.The plant was identified by the staff of the Bogoriense Herbarium, Research Center for Biology, Indonesian Institute of Science, Bogor, Indonesia and a voucher specimen (No.Bo-1288718) has been deposited at the herbarium.

General Experimental Procedure
The IR spectra were recorded on a Perkin-Elmer spectrum-100 FT-IR (Waltwam, MA, USA) in KBr.Mass spectra were obtained with a Synapt G2 mass spectrometer instrument (Waters, Milford, MA, USA). 1 H, 13 C, DEPT NMR spectral data and 1 H-1 H COSY, HMQC and HMBC experiments were performed on a JEOL ECZ-600 spectrometer at 600 MHz (Tokyo, Japan) with CDCl 3 as a solvent, chemical shifts are given on a  (ppm) scale and tetramethylsilane (TMS) as an internal standard.Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan) and (Merck, Darmstadt, Germany).Preparative MPLC using a Buchi Pump Controller C-610, Buchi Pump Modules C-605 with FLH-R10030B SiliCycle column-ISO04 Siliasep TM (Buchi, Swizerland).TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% H 2 SO 4 in EtOH, followed by heating and under ultra-violet light at wavenumber 254 and 367 nm.

Extraction and Isolation
The crushed and dried bark (2.5 kg) of A. argentea was extracted with methanol (12 L) at room temperature for 5 days.The methanol extract was evaporated under reduce pressure to give concentrated of MeOH extract (133.5 g).The concentrated MeOH extract was first suspended in H 2 O and then partitioned successively with n-hexane (4 L), EtOAc (4 L) and n-butanol (4 L).The EtOAc layer was subsequently dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.The EtOAc soluble fraction (12.1 g) was fractionated by column chromatography on silica gel using a gradient n-hexane-EtOAc to give eight fractions (A-H).Fraction D (1.12 g) was chromatographed on a Medium Presure Liquid Chromatography (MPLC) column of silica gel, eluted with CHCl 3 -EtOAc (50:1), to give eight subfractions (D01-D08).Subfraction D05 was separated on preparative TLC silica gel GF 254 , eluted with nhexane:Me 2 CO (7:3), to give 1 (10 mg).

Determination of Cytotoxic Activity
The cytotoxicity assay was conducted according to the method described by Sahidin et al., 2005 andAlley et al., 1998.P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3 x 10 4 cells cm - 3 .After 24 h of incubation for cell attachment and growth, varying concentrations of samples were added.The compounds added were first dissolved in DMSO at the required concentration.Subsequent six desirable concentrations were prepared using PBS (phosphoric buffer solution, 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, in which the MTT-stop solution containing SDS (sodium dodecyl sulphate) was added and another 24 h incubation was conducted.Optical density was read by using a micro plate reader at 550 nm.IC 50 values were taken from the plotted graph of percentage live cells compared to control (%), receiving only PBS and DMSO, versus the tested concentration of compounds (µg/mL).The IC 50 value is the concentration required for 50% growth inhibition.Each assay and analysis was run in triplicate and averaged.

RESULT AND DISCUSSION
The dried bark of A. eximia were grounded and successively extracted with n-hexane, ethyl acetate and methanol.All of the extracts were evaluated cytotoxic activity against P-388 murine leukimia cells and the ethyl acetate showed strongest cytotoxic activity.Subsequent phytochemical analysis was therefore focused on the ethyl acetate extract.By using the cytotoxic activity to follow the separations, the ethyl acetate extract was chromatographed over a vacuum-liquid chromatographed (VLC) column packed with silica gel 60 by gradient elution.The VLC fractions were repeatedly subjected to normalphase column chromatography, medium presure liquid chromatography and preparative thin layer chromatography (PTLC) on silica gel GF 254 to afford a cytotoxic compounds 1 (Figure 1).
The stereochemistry of 1 was determined by comparation to those coupling constant in 1 H NMR spectra ( 1,2 Jand 2,3 J) and chemical shift of 13 C NMR spectra with previously literature data along with biogenetic point of view the occurance of cyclopenta[b]benzofuran-type rocaglate in Aglaia genus (Othman et al., 2016).The full assignments of compound 1 are shown in Table 1.Comparison of the NMR data of 1 with those of methyl rocaglate previously reported (An et al., 2016) showed high similarity, consequently compound 1 was identified as a methyl rocaglate and showed in this plant for the first time.The cytotoxicity effects of the isolated compound 1 against P-388 murine leukemia cells were investigated according to the method described in previous papers (Harneti et al., 2012;Sianturi et al., 2015) and artonin E (IC 50 0.3 µg/mL) was used as a positive control (Hakim et al., 2007).The cytotoxic activity of isolated compound 1 showed very strong activity with in IC 50 value of < 0.1 µg/mL.
Rocaglate compounds known as cytotoxic agent.Since rocaglate and its derivatives have been found to have anticancer activities in experimental animal models, efforts have been taken to explore the molecular mechanisms of their actions.In several early publications, rocaglate derivatives were reported to function mainly in a cytostatic rather than a directly cytotoxic manner (Bohnenstengel et al., 1999;Lee, Cui, Mehta, Kinghorn & Pezzuto, 1998).It was shown that treatment of the human lung carcinoma cells Lu1 with 40-demethoxy-30,40-methylenedioxy-methyl rocaglate resulted in inhibition of tumor cell proliferation with cell accumulation in the G1/G0 phase of the cell cycle with only marginal cell death.Thereafter, didesmethyl-rocaglamide, aglaroxin D (aglaiastatin), and silvestrol were shown to inhibit cell proliferation of different human malignant cell lines with the cell cycle blocked at the G2/M phase with negligible death.Based on these observations, it was thought that inhibition of translation is the key mode of action by which cyclopenta[b]benzofurans exert their antitumor activities.Induction of apoptosis is now known to be also an important mechanism of rocaglamide mediated anticancer activity (Ebada et al., 2011).

CONCLUSION
A rocaglate-type compound was identified as a methyl rocaglate (1) has been isolated from the stembark of Aglaia argentea for first time.This investigation supported the occurance of rocaglate-type compound in Aglaia genus.Methyl rocaglate 1, was evaluated for its cytotoxic activity against P-388 murine leukemia cells, in vitro and showed strong cytotoxic activity with IC 50 value of < 0.1 µg/mL.A rocaglate is a unique compound only isolated from Aglaia genus.This type of compound known as a cytotoxic agent to inhibit cell proliferation and cell translation.