| Shiitake mushroom (Lentinus edodes), known in China as Xiang-gu, is one of the most valuable medicinal mushrooms, and has been used for thousands of years both as food and medicine. Shiitake mushroom extracts have also been found to have antioxidant properties and their antioxidant ability is positively correlated with their phenolic content. However, thus far, investigation of the antioxidant ability of shiitake mushroom has mainly focused on the fruiting body, and the antioxidant properties of its mycelial exudates are rarely reported.;In this study, exudates (DE) secreted from two shiitake mushroom mycelia (strains 1358 and L5458) were evaluated for their antioxidative properties and phenolic content. 1358DE and L5458DE showed distinct antioxidant activity in different in vitro assays, including scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical, superoxide anions and hydrogen peroxide; the ability to chelate ferrous ions; reducing power; hemolysis inhibition activity in rat erythrocyte; and lipid peroxidation inhibition (IC50 values of 1358DE and L5458DE were 3.3 and 132.6; 44.5 and > 1000; 26.9 and 53.7; 153.6 and >175.0; 176.0 and 521.0; 26.7 and 746.4; 47.8 and 736.9; and 3.1 and > 1000 microg/mL, respectively). Their total phenolic content was 237.33 and 24.08 mg gallic acid equivalent (GAE)/g of dry DE, respectively. Overall, these results show that 1358DE generally possesses better antioxidant properties than L5458DE, possibly due to its larger total phenolic content.;1358DE were selected to further investigate for its better antioxidant effect. 1358DE was fractionated using water-solvent partition and two fractions [water soluble fraction (WF) and ethyl acetate fraction (EF)] were obtained. The antioxidant effects of WF were similar to those of the original 1358DE, while EF did not possess any antioxidant activities. The WF was further isolated with polyamide column, which can separated the polyphenols from other components, and two sub-fraction (P-1 and P-2) were obtained. After the WF passing through the polyamide column, carbohydrate content in the sub-fraction 1 (P-1) was significantly increased, while its total phenolic content reduced dramatically, and its antioxidant activity decreased. However, the sub-fraction 2 (P-2) was the opposite. Carbohydrate content in P-2 was significantly reduced, while its total phenolic content increased dramatically, and its antioxidant activity increased. Apparently, carbohydrate contributed little to the antioxidant effect than that of the phenolic compounds as shown from this investigation. These results suggest that the antioxidant effect in 1358DE was contributed by the presence of polyphenols. Besides, results from Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ferric trichloride reaction suggested that P-2 was oligomers of water soluble polyphenols (WSP) with the molecular weight of about 600∼1200 Da.;The water soluble polyphones (WSP) were the potent antioxidant components in 1358DE and further study its protective effect against the hydrogen peroxide which induced cytotoxicity in V79-4 cells. In the cell viability experiments, pretreatment of WSP at the concentrations of 6.25∼50 microg/ml increased the cell viability significantly more than at the presence of H2O2 only. Besides, the pretreatment of cells with WSP significantly inhibited the increase of Malondialdehyde (MDA, which is a by-product of lipid peroxidation) production and the decrease of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) activities induced by H2O2.;Quite a few antioxidant compounds have been reported that a causative relationship may exist between the anti-angiogenic activity and antioxidant effect. Therefore, a zebrafish model was using to investigate the anti-angiogenic activity of the WSP because of its excellent antioxidant activity. In quantitative of endogenous alkaline phosphatase (EAP) assay, after the embryos treated with WSP at final concentrations of 50, 100, 150, 200, 250 microg/ml, and the vessel formation were significantly (p < 0.05) reduced to 87.2, 85.6, 74.8, 69.4, and 62.8% of the control value, respectively. Moreover, from the microscope, compare to the control, WSP at the concentration of 250 microg/ml also showed potent inhibition on the intersegmental blood vessels (ISVs) formation in Tg(fli1a:EGFP)y1 zebrafish embryos. Thus, the finding indicated that WSP could inhibit vessel formation in zebrafish.;Overall, this study revealed that water soluble polyphenols (WSP) was the active components of 1358DE. Besides of the antioxidant effect, the WSP could inhibit vessel formation significantly in zebrafish. The findings indicate that exudates of shiitake mushroom mycelia have good potential as a source of natural antioxidants. |
