Physicochemical characteristics and anti-inflammatory activities of antrodan, a novel glycoprotein isolated from Antrodia cinnamomea mycelia

Abstract

Antrodia cinnamomea (AC) is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named "antrodan" was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6±0.8 mg/g). The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%), and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which β-glucan (14.20%) was prominently higher than α-glucan (1.45%). Its FTIR confirmed the presence of β-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and β-glucans, which had (1→4)-linked α-Glcp and (1→3)-linked β-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 μg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 μg/mL.

Figure 1

Gel filtration chromatogram of polysaccharides AC-2 prepared from AC mycelia using a Sepharose CL-6B column (3.0 × 82 cm). Fractions (5.0 mL/fraction) were collected and assayed for the contents of sugars (at 490 nm) and proteins (at 280 nm). The vertical dashed lines indicated the fractions collected for preparation of antrodan.

Figure 2

High performance size exclusion chromatograms of antrodan detected at UV 280 nm and ELSD absorption and the molecular weight estimation. The regression equation was obtained from the retention time vs. The MWs of pullunan reference set: 788, 404, 212, 112, 47.3, 22.8, 11.8, and 5.9 kDa. log Da = −0.466X + 10.009, R² = 0.9937, where X is the retention time of the target polymer. Samples: antrodan, biobran, and yeast β-glucan.

Figure 3

Total ion chromatograms of GC/MS analysis of monosaccharides. (a) authentic monosaccharides and (b) monosaccharides obtained from the hydrolyzed antrodan. Ara: arabinose, Rha: rhamnose, Fuc: fucose, Xyl: xylose, I.S.: internal standard arabitol, Man: mannose, Gal: galactose, Glc: glucose. *: Mannitol.

Figure 4

Comparison of the FTIR spectra. (a) antrodan, (b) biobran, and (c) yeast β-glucan. 2 mg of pure dry sample and 300 mg of pure dry KBr were mixed and pressed into a disc. The whole IR spectrum (400 to 4000 cm⁻¹) was recorded on a FTIR spectrophotometer. Biobran is a reference of pure polysaccharide and yeast β-glucan acts as the β-glucan reference.

Figure 5

(a) ¹H-NMR spectra of antrodan and (b) a standard β-d-(1→3, 1→6)-linked glucan of curdlan.

Figure 6

Effect of (a) antrodan and biobran, and (b) LPS on the cell viability of RAW 264.7 mouse macrophages. Cells were incubated with antrodan or biobran for 24 h (a) and incubated with lipopolysaccharide (LPS; 0.05~5 μg/mL) for 24, 48 and 72 h, respectively. Cell proliferation was evaluated by the MTT assay. Values are expressed as mean ± SEM of three independent experiments. * p < 0.05 vs. Control; ** p < 0.01; *** p < 0.001.

Figure 7

Effect of LPS on (a) NO production in RAW 264.7 mouse macrophages, (b) 24 h treatment, (c) 48 h treatment and (d) 72 h treatment with antrodan and biobran. Cells were treated with various concentrations of LPS for 24, 48 and 72 h, respectively. Nitrite content in the cultured medium was determined by a Griess reaction assay. Values are expressed as mean ± SEM of three independent experiments. The confidence levels * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control were determined by the Student t-test.