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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.52 n.4 Concepción  2007 


J. Chil. Chem. Soc, 52, N° 4 (2007), págs: 1338-1340





Food Industry Research and Development Institute, P.O. Box 246, Hsinchu, Taiwan 300, Republic of China


The mycelia of Antrodia cinnamomea (BCRC 36799) were extracted and four compounds were purified: one new benzoquinone, 5-methyl-benzo[1,3]dioxole-4,7-dione (1), one new benzenoid, isobutylphenol (2), along with two known benzoquinone derivatives, 2,3-dimethoxy-5-methyl[1,4]benzoquinone (3), 2-methoxy-5-methyl[1,4]benzoquinone (4). Their structures were elucidated by intensive MS and 2D NMR study. These compounds were evaluated for their antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and supero xide radical scavenging assay. Preliminary antioxidant activity data revealed that compound 4 showed a poor radical scavenging activity.

Key words: Antrodia cinnamomea, Polyporaceae, mycelium, antioxidant, DPPH


The fruiting body of Antrodia cinnamomea (Synonymous A. camphorata, Polyporaceae, Aphyllophorales) is a very rare and expensive mushroom. The fungus is known only from Taiwan and is restricted to the endemic species Cinnamomum kanehirai. It slowly grows on the inner cavity of heart wood wall of the C. kanehirai. [1]. Antrodia cinnamomea has long been used as herbal medicine in Taiwan. The natural produced fruiting body of A. cinnamomea contains polysaccharides, sesquiterpene lactone, steroids and triterpenoid constituents. It was reported that limited amount of antcin B triterpenoid which isolated from A. cinnamomea have anti-cholinergic and anti-serotonin activities [2-5]. There are several studies on A. cinnamomea mycelia and showed pharmacological effects such as anti-inflammatory [6], antioxidant [7], anticancer [8,9] and anti-hepatitis B virus activities [10]. In this paper, we present the isolation and structure elucidation of one new benzoquinone, and one new benzenoid, along with two benzoquinone from the mycelia of A. cinnamomea. The elucidation of structures (1-4) was established by using spectroscopic data analysis, including ID and 2D NMR techniques (COSY, NOESY, HSQC and HMBC). Most of the isolates were evaluated their antioxidant activities with DPPH and superoxide radical scavenging assay. The isolation and the antioxidant properties of the isolates are described herein.


General experimental procedures

Melting points were determined with a YANACO micro-melting point apparatus and were uncorrected. IR spectra were taken on a Hitachi 260-30 spectrophotometer. UV spectra were obtained on a JASCO UV-240 spectrophotometer. EIMS spectra were recorded on a VG Biotech Quattro 5022 spectrometer. HREIMS were recorded on a JEOL JMX-HX 110 mass spectrometer. 1H NMR and 13C NMR spectra were measured on a Varian Gemini 200 and Varian Unity Plus 400 spectrometers and are given in parts per million (δ) downfield from internal TMS. Si gel 60 (Merck 70-230 mesh, 230-400 mesh) was used for column chromatography and Si gel 60 F254 (Merck) for TLC

Fungal Material

Antrodia cinnamomea BCRC 36799 was maintained on PDA (potato dextrose agar) plates and incubated at 28 °C Mycelia harvested from a 14-day-old PDA plate were homogenized with 50-ml sterile water and used as the inocµLum for submerged cµLtivation in flasks. The seed cµLture for fermentar was prepared in 500 ml flasks containing 300 ml medium on a rotary shaker at 100 rpm and 30 °C The 5-day-old cµLture broth was inocµLated into a 150-L fermentar (B. Braun, Germany) operated at 100 rpm and 30 °C with the aeration rate of 0.3 vvm. After 12 days of cµLtivation, the pellet mycelia harvested from the cµLture broth were freeze dried and stored in -80 °C and used as sample for further extraction.

DPPH Radical Scavenging Assay

The effect of crude fractions and pure compounds on the DPPH radical was monitored according to the method of Hatano et al. [11]. 190 µL of various concentrations of compounds were mixed with 10 µL methanolic solution of DPPH radical (2.5 mM). The mixture was shaken vigorously and left standing at room temperature for 10 min; the absorbance was measured spectrophotometrically at 517 run. Butylated hydroxyanisole was used as the reference antioxidant.

Scavenging activity (%): (1—A sample/ A control)*100% A: absorbance of 517 nm

Superoxide Radical Scavenging Assay

Superoxide radicals were generated via a non-enzymatic reaction. The method described by Robak and Gryglewski [12] was used to measure the ability to scavenge superoxide radicals. The reaction mixture contained 50 µL of 120 uM phenazine methosµLfate, 50 µL of 937 uM of NADH, 50 µL of 300 uM nitro blue tetrazolium and 50 µL of various concentrations of the compounds. Gallic acid was used as the reference antioxidant. All solutions were prepared in a 100 mM phosphate buffer (pH 7.4) solution. The absorbance values of the mixtures were read at 560 nm after 5 min.

Superoxide Radical scavenging capacity (%): (1—A sample / A control)*100%
A: absorbance of 560 nm

Extraction and separation of compounds

The dried mycelia of the Antrodia cinnamomea BCRC 36799 (1205 g) were extracted three times with MeOH (1.5 L) at room temperature. After the crude methanol syrup was evaporated in vacuo and partitioned with «-hexane-H20 (1:1) three times to give «-hexane and H20 layers. The filtered samples were combined and then the solvents were evaporated to yield crude «-hexane (15.2 g) crude extract. The «-hexane layer extract was chromatographed on a silica gel column with a stepwise gradient of «-hexane-EtOAc [15:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, EA, Acetone and Methanol, each 1L] to give 15 fractions. Fraction 5 was further purified by column chromatography on silica gel eluting with «-hexane-CH2Cl2 [8:1, 6:1, 4:1, 2:1, 1:1, and CH2C12 (each 500 niL)] to yield 6 fractions (fr. 5.1-5.6). The compound 2 (1.2 mg) furnished from fraction 5.3 by preparative TLC Fraction 7 was chromatographed on a silica gel column with «-hexane-EtOAc [6:1, 4:1, 2:1, 1:1, EtOAc] to give 6 fractions and further purified with preparative TLC to yield Compound 3 (15.4 mg) and 4(13.2 mg). The compound 1 (12.4 mg) separated from fraction 10 by preparative TLC with n-hexane-EtOAc (2.5:1).

Spectroscopic data

5-methyl-benzo[l,3]dioxole-4,7-dione (1): C8H604; amethyst solids; mp 121-124 °C; IR (KBr) Vmax= 1665 (conjugated C=0), 1147 and 940 (OCH20) cm1; EI-MS m/z 166 (reí. intensity) ([M]+, 8); high resolution HR-EI-MS m/z 166.0263 [M+] (Caled for C8H604:166.0266).; 1H and 13C NMR data are showed in Table 1.

4-isobutylphenol (2): C10Hls4O; yellow oil; IR (Neat) vmax= 3424 (OH), 1604 and 1510 (aromatic) cm-1; EI-MS m/z 150 (rel. intensity) ([M]+, 100); high resolution HR-EI-MS m/z 150.1234 [M]+(Caled for C10H14O: 150.1045).; 1H and 13C NMR data are showed in Table 1.


The DPPH free-radical scavenging activity of the crude Antrodia cinnamomea (BCRC 36799) mycelia fraction 1 to 15 were 10.3, 11.5, 11.7, 21.2, 50.2, 36.4, 59.5, 37.5, 20.3, 50.3, 27.2, 17.5, 13.2, 10.3 and 10.4 %, respectively. The over 50% fractions (5, 7 and 10) were further fractionation and resµLted in the isolation of benzoquinone type derivatives 1-3 and one benzenoid 4.

5-Methyl-benzo[l,3]dioxole-4,7-dione (1) was isolated as a amethyst solids and with the molecular formula, C8H6O4, was established by EIMS ([M]+, m/z 166) and HR-EI mass spectrometry. Infrared absorptions at 1147 and 940 cm-1 showed that the compound was a methylenedioxy and conjugated carbonyl at 1665 cm-1. The 1H-NMR spectrum showed signals for one methylenedioxy [δH6.10 (2H, s, H-2)], one methyl group [δH2.07 (3H, d, J = 1.6 Hz)] and a single aromatic proton [δH6.32 (IH, d, J= 1.6 Hz)]. In addition, two conjugated ketone group (8 177.4 and 177.5) was found in the 13C-NMR spectrum. According to the above data, the structure of 1 was elucidated as 5-methyl-benzo[l,3]dioxole-4,7-dione, which was further confirmed by COSY, NOESY, 13C NMR, DEPT, HSQC and HMBC (Fig. 1) experiments.

4-Isobutylphenol (2) was isolated as yellow oil and the molecular formula, C10H14O was determined by EIMS ([M]+, m/z 150) and HREI mass spectrometry. Infrared spectrum showed characteristic absorption for a hydroxyl group and benzene ring at 3400, 1604 and 1510 cm-1, respectively. The 1H and 13C NMR spectrum of 2 displayed signals that coµLd be attributed to apara-disubstituted benzene ring [δH 7.57 (2H, d,J= 8.8 Hz, H-3 and 5), 6.95 (2H, d, J= 8.8 Hz, H-2 and 6); δc 131.4 (C-3, 5), 116.0 (C-2, 6)] and a hydroxyl group [δH 5.60 (IH, br s, OH-1), D20 exchangeable]. In addition, the signal of a isobutyl moiety at [δH 0.93 (6H, d, J = 6.9 Hz, CH3-9, 10), δH 2.12 (IH, sep, 7 = 6.9 Hz, H-8) and δH 2.58 (2H, d, J = 6.9 Hz, H-7); δc22.7 (CH3-9, 10), 27.9 (C-8), 33.6 (C-7)] were also found in the 1H and 13C NMR spectrum. According to the above data, the structure of 2 was elucidated as 4-isobutylphenol. This is the first reported from nature source although it has been synthesized [13]. The known isolates including 2,3-dimethoxy-5-methyl[1,4]benzoquinone (3) [14] and 2-methoxy-6-methyl-1,4-benzoquinone (4) [15] were identified by comparison of physical and spectroscopic data (UV, IR, 1H-NMR, [a]D and mass spectroscopic data) to corresponding authentic samples or literature values. These two compounds (3 and 4) were first isolated from Antrodia cinnamomea.

The compounds 1-4 isolated from active «-hexane layer were evaluated for radical scavenging effect on the 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and superoxide radical (Table 2). The DPPH radical scavenging data indicated that compound 4 displayed scavenging effect activity at 54.83 %. The ED50 value of 4 was 437.06 µM.


This work was supported by a grant (04A011) from the Food Industry Research and Development Institute. The author thank Senior Technical Specialist Ms. Chyi-Jia Wang of Instrument Center of Kaohsiung Medical University for measuring the 2D-NMR data.



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