New Anti-Inflammatory Aromatic Components from Antrodia camphorata

Three new benzenoids, 3-isopropenyl-2-methoxy-6-methyl-4,5-methylenedioxyphenol (1), 2-hydroxy-4,4′-dimethoxy-3,3′-dimethyl-5,6,5′,6′-bimethylenedioxybiphenyl (2), 4,4′-dihydroxy-3,3′-dimethoxy-2,2′-dimethyl-5,6,5′,6′-bimethylenedioxybiphenyl (3), together with two known benzenoids, 2,3,6-trimethoxy-5-methylphenol (4) and 2,3-methylenedioxy-4-methoxy-5-methylphenol (5), were isolated from Antrodia camphorata. Our results support that compounds 1–5 potently inhibited LPS (lipopolysaccharide)-induced nitric oxide (NO) production in a dose-dependent manner. The IC50 values of compounds 1, 3 and 5 were 1.8 ± 0.2, 18.8 ± 0.6 and 0.8 ± 0.3 μg/mL, respectively.

Compounds 5 is very potent (IC 50 = 0.8) for the inhibition of NO production. We will study the anti-inflammatory activities of compound 5 further. 14.3 ± 0.8 ** a The data were presented as the mean ± SD for three different experiments performed in triplicate. ### Compared with sample of the control group. * p < 0.05, ** p < 0.01 and *** p < 0.001 were compared with the LPS-alone group.

Plant Material
The solid cultural fruiting bodies of A. camphorata were identified and provided by Po-Zone Biotechnology Development, Taipei, Taiwan. A voucher specimen was deposited at Po-Zone Biotechnology Development Co. Ltd.

Extraction and Isolation
The fruiting bodies of wood culture A. camphorata (500 g) were extracted with MeOH (4 L) by maceration at room temperature (7 days × 3). After removal of MeOH under vacuum, the extract was partitioned into EtOAc (Fr. A, 113 g), n-BuOH (Fr. B, 15 g) and H 2 O-soluble (Fr. C, 27 g) fractions.

Cell Viability
Cells (2 × 10 5 ) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then, cells were cultured with compounds 1-5 in the presence of 100 ng/mL LPS (lipopolysaccharide) (Eschericha coli 026:B6; Sigma-Aldrich, St. Louis, Mo) for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was then discarded, and 100 μL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Measurement of Nitric Oxide/Nitrite
NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentrations of samples in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Then, cells were dispensed into 96-well plates, and 100 μL of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min; the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices).

Statistical Analysis
IC 50 values were estimated using a non-linear regression algorithm (Sigma Plot 8.0; SPSS Inc., Chicago, IL, USA). Statistical evaluation was carried out by one-way analysis of variance (ANOVA, followed by Scheffe's multiple range tests).