New and Cytotoxic Components from Antrodia camphorata

The solid-state cultured products of Antrodia camphorata as health foods has been blooming for the past few decades in Taiwan. In continuing our studies on the chemical constituents of the solid-state cultured products of this fungus, 6-methoxy-4-methyl-2,3-(methylenedioxy)phenol (1) and 4,4'-(ethane-1,2-diyl)bis(2,3,6-trimethoxyphenol)(2) together with 2,3,6-trimethoxy-4-methylphenol (3), 1(10→6)abeo-ergosta-5,7,9,22-tetraen-3α-ol (4), citreoanthrasteroid B (5) and dankasterones A (6) and B (7) were purified by a series of column chromatography. Their structures were elucidated by spectral data analysis. For bioactivity assay, compounds 4–7 showed significant cytotoxicity toward murine colorectal CT26 and human leukemia K562 cancer cell lines with IC50 values ranging from 6.7 to 15.3 µM and from 12.5 to 23.1 µM, respectively.

For the anticancer activity, Table 1 shows the IC50 values of compounds 1-7 against murine colorectal cancer CT26 cells and human leukemia K562 cells. Compounds 1-3 exhibited no obvious effect toward CT26 and K562 cells with their IC50 values higher than 20 μM, and 4-7 showed significant cytotoxicity toward murine colorectal CT26 and human leukemia K562 cancer cell lines with IC50 values ranging from 6.7 to 18.2 μM and from 12.5 to 23.1 μM, respectively. At the same condition, the IC50 value of staurosporine against K562 leukemia cells was 16.7 nM.  The IC 50 value of staurosporine, the positive control, against K562 cells was 16.7 nM.

General
Optical rotations were measured on a JASCO DIP-1000 polarimeter (Tokyo, Japan). 1 H and 13 C-NMR were acquired on a Bruker DMX-500 (Ettlingen, Germany). Low resolution and high resolution mass spectra were obtained using an API4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) and a Synapt High Definition Mass Spectrometry system with an ESI interface and a TOF analyzer (Waters Corp., Manchester, UK), respectively. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). TLC was performed using silica gel 60 F254 plates (200 µm, Merck, Taipei, Taiwan).

Extraction and Isolation
The dried powder (2.83 kg) of solid-state cultured Antrodia camphorata was extracted with 12 L methanol for three times, and the concentrated residues (323.8 g) were suspended in H2O and further partitioned three times with equal volumes of ethyl acetate, then concentrated in vacuum to dryness (131.5 g). The ethyl acetate extract was applied onto an open column with silica gel. The column was eluted with mixtures of n-hexane, ethyl acetate and methanol, and each 1 L was collected as one fraction. Fractions 16-33 were combined and evaporated to dryness (1.4 g), which were further purified by HPLC on a semi-preparative Phenomenex Luna Si column (5 µm, 10 × 250 mm) with n-hexane-ethyl acetate (90:10, v/v) as the eluent, 2 mL/min, obtaining 1 (4.6 mg), 2 (6.4 mg) and 3 (8.9 mg). Fractions 92-124 were combined and evaporated to dryness (33.1 g), which were further purified by HPLC on the same column with n-hexane-ethyl acetate (50:50, v/v) as the eluent, 2 mL/min, obtaining 4 (5.6 mg), 5 (7.4 mg), 6 (10.3) and 7 (9.2 mg).

Cells and Viability Assay
Murine colorectal cancer CT26 cells were cultured in RPMI1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT) and 2 mM L-glutamine at 37 °C in a humidified 5% CO2 incubator. The viability of CT26 cells was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) colorimetric assay. Human chronic myeloid leukemia K562 cells were cultured in RPMI 1640 medium, 10% FBS and 2 mM L-glutamine at 37 °C in an incubator. The viability of K562 cells with compound treatments for one day were measured using the Trypan blue dye exclusion test. The IC50 values of compounds in CT26 and K562 cells were determined by using SigmaStat software (Jandel Scientific, San Rafael, CA, USA).

Conclusions
In this study, three C6-C1 derivatives, as well as four phytosteroids with rearranged skeletons were isolated from the industrial fermented products of Antrodia camphorata. Of the compounds isolated, four phytosteroids exhibited significant cytotoxicity against cancer cell lines, which may, to some extent, account for the traditional uses of this fungal product as a health food to treat cancer. More experiments should be performed to deduce the action modes of these compounds.