Abstract: In the course of continuing research for finding bioactive compounds from Indonesian plants, the leaves of Artocarpus communis was extracted by ethanol. This extract partitioned with n-hexane-water (1:4) and then water extract was partitioned with dichloromethane. Dichloromethane extract was purified by column chromatography techniques on silica gel to afford yellow crystal (F-1). Based on LC-MS, 1H-NMR and 13C-NMR (1D and 2D) spectra and compared with previous spectral data, it was identified as prenylated flavonoid, 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl] 1-propanone. This compound showed significant cytotoxicity against murine P-388 leukemia cells.
INTRODUCTION
The genus Artocarpus (Moraceae), an exceptionally rich source of prenylated flavonoids, consists of approximately 50 species that are indigenous to the region of Southeast Asia, including Indonesia. Different compounds isolated from some species of Artocarpus have been shown to exhibit interesting biological properties (Lemmens et al., 1995; Nomura et al., 1998). Some of these compounds show interesting biological activities, such as cytotoxic (Nomura and Hano, 1998), antimalarial activity (Boonlaksiri et al., 2000), inhibition of tyrosinase and melanin biosynthesis (Likhitwitayawuid and Stritularak, 2001; Shimizu et al., 1998) and of 5α-reductase (Shimizu et al., 2000). Thus, in a continuation of our studies on the chemistry of Indonesian plants, the chemical constituents of A. communis have been investigated. In this paper, we report the isolation, structure elucidation and biological evaluation of prenylated flavonoid from dichloromethane extract of the leaves of this species. The structure of this compound was elucidated on the basis of spectroscopic data including 2-D NMR. The isolated compound exhibited cytotoxicity against P-388 cells.
MATERIALS AND METHODS
This research was conducted at Natural Product and Pharmaceutical Laboratory, Research Centre for Chemistry, Indonesian Institute of Sciences (LIPI) on 2006-2007.
General experimental procedures: 1H- and 13C-NMR spectra were recorded with JEOL JNM ECA-500 spectrometer, operating at 500 MHz (1H-) and 125.76 MHz (13C-), using TMS (Tetra Methyl Silane) as an internal standard. MS were obtained with Mariner Biospectrometry Spectrometer using ESI System (Electro Spray Ionization) and positive ion mode. Column chromatography was carried out using Merck Silica gel 60 (70-230 mesh ASTM) and TLC (Thin Layer Chromatography) analysis on precoated Silica gel plates (Merck Kieselgel 60 F 254, 0.25 mm).
Plant material: Sample of the leaves of Artocarpus communis was collected in March 2006, from plantation trees growing in Parung, Bogor, Indonesia. The plant was identified by staff at Biology Laboratory, Institute of Technology Bandung, West Java, Indonesia and a voucher specimen has been deposited at Biology Laboratory.
Extraction, isolation and identification: The dried leaves (4.95 kg) of A. communis were extracted exhaustively using macerator with ethanol 70%. The ethanol extracts (250 g) were concentrated using vacuum rotary evaporator and then partitioned with hexane-water (1:4). Water extracts added with dichloromethane and then dichloromethane extract was fractionated by column chromatography on silica gel using gradient elution (hexane-ethyl acetate), were resulted 80 fractions. Fractions 6-17 (F-1) were re-crystallized to give yellow crystal.
F-1 was identified using LC-MS and NMR (1H-NMR, 13C-NMR, DEPT 135, HMQC and HMBC) Spectrometer, to give prenylated flavonoid, named 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone.
Cytotoxicity assay (Alley et al., 1988): P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3x104 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 of samples were prepared using PBS (phosphoric buffer solution, pH 7.30-7.65). Control wells received only DMSO. The assay was terminated after an 48 h incubation period by adding MTT reagent [3-(4,5-dimethyl-thiazol-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 of incubation was conducted. Optical density was read by using a microplate reader at 550 nm. IC50 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 (FM). The IC50 value is the concentration required for 50% growth inhibition. Each assay and analysis was run in triplicate and averaged.
RESULTS AND DISCUSSION
The dried leaves of A. communis were macerated with ethanol and the ethanol extract was fractionated by using hexane-water (1:4) and then dichloromethane. The product of dichloromethane extract was fractionated by column chromatography to give a number of fractions that contained a major compound. The LC-MS spectrum of compound F-1 gave an [M]+ ion at 409.546 with a molecular formula of C25H28O5 (Fig. 1). The analysis of its NMR data and comparison with reference showed in Table 1. From the 13C-NMR spectrum, F-1 indicated 25 carbons, including 8 sp2 methine carbons, 4 methylene carbons, 3 methyl groups and 10 quartenery carbons.
The 1H-NMR spectra suggested the existence of two aromatic rings, ring A and B. At ring A have three proton aromatics, revealed a clear AMX system as indicated by the proton resonances at δH 6.38 (H-2, d, J 2.45 Hz) and 6.37 (H-4, dd, J 2.45 and 8.5 Hz) and at 7.54 (H-5, d, J 8.5 Hz) and 6.37 (1 H, dd, J 2.45 and 8.5 Hz). The down field chemical shift at 12.85 (s) indicated the presence of hydroxyl group formed a hydrogen bond with a carbonyl group (C-7) at 204.14 ppm. The multiplicity of carbons were assigned by the DEPT-135 experiment and correlation of the chemical H and C shift for all protonated carbons was determined based on the HMQC spectrum. The presences of the functional groups above were suggested by the long range coupling HMBC experiment as summarized in the Fig. 2. Partial structure A was confirmed by the presence of the long range coupling (HMBC correlation) of the aromatic proton H-2 at δH 6.38 to C-1, C-2, C-3, C-4 and C-6. The other aromatics proton H-4 at δH 6.37 and H-5 at δH 7.54 also indicated the presence of long range coupling between C-2, C-6 and C-1, C-7.
At ring B, there are ortho-coupled aromatic protons at δH
6.60 (H-11, d, J 8.5 Hz) and 6.72 (H-12, d, J
8.5 Hz). The presence of a methyl group in a singlet at δH
1.38 (CH3) and two cis-olefinic protons in doublets at δH
5.63 and 6.54 (each, J 9.8 Hz) and a quaternary carbon at δC
78.7 (s) implied the presence of the dimethylchromene group (ring
C). The presence of two methylene group in the spectrum at δC
3.1 and 2.9 and the presence of long range coupling to carbonyl group
C-7 at 204.14, indicated the presence of propanone group, which connected
to ring A and B.
| Fig. 1: | LC-MS spectrum of compound F-1 from dichloromethane fraction of A. communis leaves |
| Table 1: | Comparison of 13C and 1H NMR assignment between compound F-1 and reference |
| Spectra compound F-1 recorded at 500 MHz for 1H spectrum and 125 MHz for 13C spectrum in CDCl3 and reference`s spectra recorded at 400 MHz. The values are in ppm and J values (Hz) in parentheses. Abbreviations for NMR signal are as follows: s = singlet, d = doublet, t = triplet. Correlation of chemical shift H and C were assigned, based on the HMQC spectra | |
| Fig. 2: | HMBC experiment of compound F-1 |
| Fig. 3: | Molecular structure compound F-1 [1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1- benzopyran-5-yl]-1-propanone] |
The 1H-NMR spectrum data also indicated the presence of isoprenyl group at δH 5.08 (1H, t, H-21), 2.09 (2H, m, H-20), 1.7 (2H, m, H-19), 1.66 (3H, s, H-23), 1.57 (3H, s, H-24), 1.38 (3H, s, H-25). This isoprenyl was located in ring C based on the presence of long range coupling between H-19 at δH 1.7 to C-18 at δC 78.9 (s) and C-17 at δC 130.30 (d).
From 1D- and 2D- NMR spectrum, MS data and comparison with previous spectral data (Koshihara et al., 1988; McLean et al., 1996), compound F-1 was identified as prenylated flavonoid, named 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (Fig. 3).
The cytotoxicity of this compound was evaluated according to the method previously described (Alley et al., 1988). This compound exhibited significant active in the murine P388 leukemia cells bioassay with IC50 6.7 μg mL-1.
ACKNOWLEDGMENTS
This study has been supported by the Indonesian Government budget for the Research Center for Chemistry, Indonesian Institute of Sciences (LIPI). We also thanks to Dr. lenny Sutedja for suggesting this research; the staff of Biology Laboratory, Institute of Technology Bandung for identification of the plant speciment and the staff of Natural Product Organic Chemistry, Chemistry Department, Institute of Technology Bandung for murine P388 leukemia cells bioassay.