Chemical Constituents and Biological Studies of the Leaves of Grevillea robusta

Three new compounds: Graviquinone (1), cis-3-hydroxy-5-pentadecylcyclohexanone (2), and methyl 5-ethoxy-2-hydroxycinnamate (3), and thirty-eight known compounds were isolated and identified from the leaves of Grevillea robusta. The structures of these compounds were determined by spectroscopic and chemical transformation methods. Graviquinone (1) showed the strongest cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Methyl 2,5-dihydroxycinnamate (4), graviphane (13), and dehydrograviphane (14) exhibited very potent DPPH scavenging activity compared with α-tocopherol. Methyl 2,5-dihydroxycinnamate (4) and bis-norstriatol (17) demonstrated strong inhibition of L-DOPA oxidation by mushroom tyrosinase compared with kojic acid.


Introduction
Grevillea robusta A. CUNN (Proteaceae), commonly known as "silky oak", is native to Australia [1,2]. In Taiwan, it is cultivated as a shade tree. So far alkylresorcinols, macrocyclic phenols, and cinnamic acid derivatives have been reported to be constituents of this plant. In our preliminary screening, the

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MeOH extract of the leaves from G. robusta showed significant cytotoxicity against human breast carcinoma (MCF-7), lung carcinoma (NCI-H460), and central nervous system carcinoma (SF-268) cell lines. This information encouraged us to further investigate the chemical constituents of the MeOH extract. Fractionation of the extract led to the isolation of forty-one compounds, consisting of three new compounds, graviquinone (1), cis-3-hydroxy-5-pentadecylcyclohexanone (2), and methyl 5-ethoxy-2-hydroxycinnamate (3), as well as thirty-eight known compounds ( Figure 1). Herein, we discussed the isolation, structural elucidation, and cytotoxicity of these compounds.

Results and Discussion
Graviquinone (1) was isolated as a yellowish solid. The molecular formula was determined to be C 10 H 10 O 4 on the basis of the molecular ion at m/z 194.0577 in the HR-EIMS spectrum. The 1 H-NMR and COSY spectra showed the presence of symmetric o-protons at δ 6.13 (2H, d, J = 9.9 Hz, H-2 and -6) and 6.82 (2H, d, J = 9.9 Hz, H-3 and -5) together with an α,β-unsaturated carbonyl unit at δ 6.19 (1H, d, J = 15.7 Hz, H-8) and 6.63 (1H, d, J = 15.7 Hz, H-7). The 13 C-NMR, HMQC, and HMBC spectra indicated that both the proton signals at δ 6.13 and 6.82 exhibited 1 H-13 C long range correlations with a carboxyl carbon at δ 183.6 (C-1) and a quaternary carbon at δ 68.1 (C-4). Moreover, H-7 and H-8 showed HMBC correlations with the quaternary carbon and a carboxylic carbon at δ 164.8 (C-9). Furthermore, this downfield-shifted quaternary carbon correlated with a hydroxyl group (δ 5.49, br s) to which it was attached. A methyl group at δ 3.64 was assigned as forming an ester bond with the carboxylic acid because of the presence of the HMBC correlation between the methyl proton and the carboxylic carbon instead of the quaternary carbon. Consequently, the structure graviquinone (1) was assigned to be 4-hydroxy-4-(3-methoxycarbonyl)ethenylcyclohexadien-1-one.

General
Melting points were recorded on a Yanaco MP-3 melting point apparatus and were not corrected. Optical rotations were measured on a Jasco DIP-370 digital polarimeter. UV spectra were recorded on an Agilent 8453 spectrophotometer. IR spectra were recorded on a Nicolet Magna FT-IR spectrophotometer. NMR spectra were recorded on Bruker Avance 300 and AMX 400 FT-NMR spectrometers; all chemical shifts were given in ppm from tetramethylsilane as an internal standard. Mass spectra were obtained on a VG 70-250S spectrometer by a direct inlet system.

Plant Material
The leaves of G. robusta were collected on the campus of National Cheng Kung University, Tainan, Taiwan, in September of 2003. The samples were authenticated by Professor C.S. Kuoh of the Department of Life Sciences, National Cheng Kung University. A voucher specimen (No: PLW-0303) was deposited in the Herbarium of the same school.

Cytotoxicity Assay
The cytotoxicity assay was carried out according to procedures that have been previously described in the literature [14].

Free Radical Scavenging Activity Assay
The free radical scavenging activity of compounds 1, 4, 5, 7, 8, 11−18, and 20 was measured with DPPH • using the method of Chiu et al. [15]. Briefly, 0.1 mM solution of DPPH • in ethanol was prepared, and sample (20 μL) was added. The sample was thoroughly mixed and kept in the dark for 30 min. The absorbance was measured at 517 nm on a Quant universal microplate spectrophotometer. α-Tocopherol (Sigma Chemical Co.) was used as a standard agent. A lower absorbance of the reaction mixture indicated a higher free radical scavenging activity. The sample concentration that was used to provide the IC 50 data was calculated from a graph plotting inhibition percentage against sample concentration. Tests were performed in triplicate.

Tyrosinase Inhibitory Activity
The tyrosinase assay was carried out according to a procedure previously described in the literature [16]. The test substance was dissolved in 10% DMSO-H 2 O solution (0.1 mL) and then incubated with mushroom tyrosinase (0.1 mL, 135 U/mL, PBS pH 6.8) at 25 °C for 10 min. Next, L-dopa phosphate buffer solution (0.1 mL, 0.5 mM, pH 6.8) was added. The reaction mixture was incubated for 5 min. The amount of dopachrome in the mixture was determined by measuring the optical density (OD) at 475 nm using a Quant universal microplate spectrophotometer. Kojic acid (Sigma Chemical Co.) was used as a standard agent. The inhibitory percentage of tyrosinase was calculated as follows: