Phenolic compounds from Eurycorymbus cavaleriei

Three new phenolic compounds, eurycorymboside A (1), eurycorymboside B (6), and eurycorymbic acid (8), were isolated from the stem part of Eurycorymbus cavaleriei (Sapindaceae) along with five known phenolic compounds, glucosyringic acid (2), vanillic acid 4-O-β-d-glucoside (3), koaburaside (4), tachioside (5), and 4-hydroxy-3,5-bis(3-methyl-2-butenyl)benzaldehyde (7). The structures were established on the basis of spectral analysis. The antioxidant activities of compounds 1–6 were evaluated by the 1,1-diphenyl-2-picrylhydrazyl-free radical scavenging assay. Compound 4 exhibited antioxidant activity with an IC50 value of 9.0 μM. Compound 4 also showed weak inhibitory activity against influenza A neuraminidase.


Introduction
Eurycorymbus cavaleriei (Levl.) Rehd. et Hand.-Mazz. is the only species of the genus Eurycorymbus (Sapindaceae). The plant is endemic in the southern regions of China including Hunan, Yunnan, Sichuan, Guangxi, and Guangdong Provinces [1]. Previous phytochemical investigations on this plant have resulted in the isolation of lignan, coumarin, coumarinolignoid, meroterpene, benzeneacetic acid derivative, and flavone compounds [2][3][4][5][6]. Our continuing phytochemical and pharmacological investigation of this plant led to the isolation of eight phenolic compounds, including three new compounds. In this paper, we describe the isolation and structure elucidation of three new phenolic compounds, 1, 6, and 8 ( Figure 1), and the evaluation of antioxidant and neuraminidase inhibitory activities of compounds 1-6.

Results and discussion
Compound 1 was obtained as a white amorphous powder. Its atmospheric pressure chemical ionization (APCI) mass spectrum displayed a quasi-molecular ion at m/z 344 [M þ NH 4 ] þ in positive mode, consistent with the formula C 15 H 18 O 8 , which was supported by HR-electrospray ionization (ESI)-MS (m/z 349.0900 [M þ Na] þ ; calculated for C 15 H 18 O 8 Na, 349.0894). The 1 H NMR signals at d 7.32 (2H, d, J ¼ 9.0 Hz, H-2 and H-6) and 7.02 (2H, d, J ¼ 9.0 Hz, H-3 and H-5) suggested the presence of a para-substituted phenyl moiety, whereas the 13 C NMR signal at d 170.3 (C-8) is attributed to a carbonyl group. The 1 H and 13 C NMR spectra also revealed the presence of a b-glucose moiety. The HMQC spectrum showed the correlations from two protons at d 6.19 and 5.81 to a carbon at d 125.8 (C-9), indicating that this carbon was an unsaturated secondary carbon. In the HMBC spectrum ( Figure 2), two methylene proton signals at d 6.19 and 5.81 (H-9a, 9b) displayed correlations with a quaternary carbon at d 142.7 (C-7), the carboxylic carbon at d 170.3 (C-8), and an aromatic carbon at d 132.4 (C-1), suggesting the presence of an acrylic acid moiety on the phenolic ring. On the other hand, the anomeric proton of glucose (d 4.87) exhibited long-range correlation with C-4 (d 159.0), suggesting that C-4 was glucosylated. On the basis of the above findings, the structure of 1 was established as depicted in Figure 1 and given a trivial name of eurycorymboside A. , and one saturated quaternary carbon (d C 70.7); these data suggested that compound 6 is composed of 5 and 3-hydroxy-3-methylglutaric acid (HMGA). In the HMBC spectrum ( Figure 2), the proton signals at d 4.43 and 4.15 (H-6 0 a, 6 0 b) displayed correlations with the carboxylic carbon at d 172.4 (C-1 00 ), suggesting that HMGA is  substituted at the C-6 0 position by an ester bond. Thus, the structure of compound 6 was determined as depicted in Figure 1 and given a trivial name eurycorymboside B. (12H, s, H-4 0 ,5 0 ,4 00 ,5 00 )], which revealed the symmetrical structure of 8. The 13 C NMR spectrum showed 13 carbon resonances including one carbonyl resonance at d C 173.6 (C-8), two olefinic carbon resonances at d C 121.7 (C-2 0 ,2 00 ) and 134.6 (C-3 0 ,3 00 ), and two methyl carbon resonances at d C 17.9 (C-4 0 ,4 00 ) and 25.8 (C-5 0 ,5 00 ).
The HMBC correlations ( Figure 2) were observed between 1 H signals at d 3.33 (H-1 0 ,1 00 ) and 13 C signals at d 126.9 (C-2,6), 153.4 (C-4), 121.7 (C-2 0 ,2 00 ), and 134.6 (C-3 0 ,3 00 ), indicating two isopentenyl groups symmetrically substituted to the benzene ring at the positions of C-3 and C-5. Furthermore, the 1 H signals at d 4.76 (H-7) displayed correlations with the 13 C signal at d 126.9 (C-2,6), suggesting that this carbon is attached to C-1 of the benzene ring. The long-range correlations between the 1 H signals at d 3.55 (H-9) and 13 C signals at d 80.1 (C-7) indicated that the ethoxy group was attached to C-7. Therefore, the structure of 8 was proposed as depicted in Figure 1 and given a trivial name of eurycorymbic acid.
Compounds 1 -6 were also evaluated for their anti-influenza virus activity by the neuraminidase inhibition assay. Compound 4 showed weak inhibitory activity against influenza A neuraminidase with 46.24% of inhibition at a concentration of 12.0 mM, but no obvious inhibitory activity was observed for other compounds.

Plant material
The stem part of E. cavaleriei was collected from the Nanyue Arboretum (Hunan, China) in May 2008 and was identified by Prof. Ji Zhang, National Institutes for Food and Drug Control. A voucher specimen (No. 2008-016) has been deposited at the School of Chinese Medicine, The Chinese University of Hong Kong.
The petroleum ether soluble part of the ethanol extract (21.7 g) was subjected to CC on silica gel and eluted with stepwise gradients of petroleum ether (BP 60 -808C)-ethyl acetate to give 20 sub-fractions (P1 -P20). Fraction P2 was subjected to CC on MCI Gel CHP20P and eluted with gradients of water -methanol to yield compound 7 (20 mg). Fraction P15 was subjected to CC on MCI Gel CHP20P and eluted with gradients of water -methanol to provide 10 sub-fractions. The fourth subfraction was purified by semi-preparative HPLC with MeOH-0.1% TFA (75:25) as mobile phase to yield compound 8 (10 mg).

Antioxidant activity
The DPPH assay was carried out according to the modified method of Aquino et al. [12]: the test sample (10 ml) at different concentrations of DMSO was added to 190 ml of freshly prepared DPPH solution (6.5 £ 10 25 M, in MeOH). The absorbance of DPPH at 517 nm was measured on a Zenyth 200 UV -vis spectrophotometer after 30 min. The percentage of scavenging was calculated, and IC 50 values were expressed as the concentration of sample required to scavenage 50% DPPH-free radicals. Vitamin C was used as positive control, showing an IC 50 21.1 mM.

Neuraminidase inhibition assay
The in vitro assay is based on the method reported previously [13,14]. The influenza viruses A/PR/8/34 (H1N1) were used as source of neuraminidase. The enzyme reaction system consists of 30 ml of the enzyme in 33 mM MES buffer (pH 3.5), 10 ml of 4 mM CaCl 2 , 20 ml of 20 mM 2 0 -(4methylumbelliferyl)-a-D-acetylneuraminic acid, 30 ml water, and 10 ml test solvent in a 96-well microplate. The final volume was 100 ml. After 10 min of incubation at 378C, 150 ml of 14 mM NaOH in 83% ethanol was added to the reaction mixture to terminate the reaction. The intensity of fluorescence was quantitated in Fluostar Galaxy (excitation, 360 nm and emission, 450 nm), and substrate blanks were subtracted from the sample readings. The IC 50 was calculated by plotting percent inhibition versus the inhibitor concentration, and determination of each data point was performed in duplicate. Zanamivir was used as positive control, showing an IC 50 of 0.72 nm.