Effect of Sulfated Modification on the Molecular Characteristics and Biological Activities of Polysaccharides fromHypsizigus marmoreus

Molecular Characteristics of Water-Soluble Extracts fromHypsizigus marmoreusand Theirin VitroGrowth Inhibition of Various Cancer Cell Lines and Immunomodulatory Function in Raw 264.7 Cells

“…The total carbohydrate and protein contents of each sample were respectively determined by the phenol-sulfuric acid method, using glucose as a standard, and by the Lowry method using a DC protein assay kit (BioRad, Hercules, CA, USA) as described in our previous report. [11][12][13] The ash content was determined by heating the dried sample in a 48000 furnace (Barnstead-Thermolyne, Dubuque, IA, USA) at 600 C for 8 h.…”

“…The WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) colorimetric assay (Roche Diagnostics, Madison, WI, USA) was applied to determine the anticancer activity of the crude extract and fractions by using the human AGS gastric carcinoma cell line (ATCC, Rockville, MD, USA), human DLD-1 colon cancer cell line (ATCC, Rockville, MD, USA) and human HeLa cervix carcinoma cell line (ATCC, Rockville, MD, USA) as reported in our previous work. 11) Solutions of the sample at concentrations of 10, 100, 500, and 1000 mg/mL were added to previously incubated cells in triplicate, and the solution incubated for 48 h. A WST-1 solution was then added to each well, and the mixture incubated for 2 h at 37 C. The optical density was measured at 450 nm with an EL-800 microplate reader (BioTek instruments, Winooski, VT, USA), expressing the anticancer activity as a percentage of the cancer cell growth inhibition by the equation anticancer activity ð%Þ ¼ 100ð1 À ðA s =A c ÞÞ, where A s is the absorbance of the sample solution and A c is the absorbance of the control for which H 2 O was used instead of the sample solution.…”

“…Macrophage proliferation affected by the sample was determined by our previously reported method 11) using Raw 264.7 cells (ATCC, Rockville, MD, USA). Briefly, the cells in an RPMI-1640 medium containing 10% FBS were incubated with the sample at concentrations of 10, 25, 50, and 100 mg/mL in a 96-well microplate.…”

“…Control wells were treated only with the medium, and proliferation was quantified by the WST-1 assay already described. 11) The effect of the crude extract and fractions on the release of nitric oxide (NO) was determined by the NO level in the culture supernatant, using the Griess reaction according to our previously reported method. 11) The sample solution at a concentration of 3, 6, or 12 mg/mL was added to Raw 264.7 cells (1 Â 10 6 cells/mL) that had previously been incubated in an RPMI-1640 medium containing 10% FBS for 24 h at 37 C in the presence of 5% CO 2 .…”

“…11) The effect of the crude extract and fractions on the release of nitric oxide (NO) was determined by the NO level in the culture supernatant, using the Griess reaction according to our previously reported method. 11) The sample solution at a concentration of 3, 6, or 12 mg/mL was added to Raw 264.7 cells (1 Â 10 6 cells/mL) that had previously been incubated in an RPMI-1640 medium containing 10% FBS for 24 h at 37 C in the presence of 5% CO 2 . LPS (1 mg/mL) was used as a positive control in the presence or absence of arginine analogue N G -monomethyl-L-arginine (L-NMMA) (500 mM), and the cells were cultured in triplicate by incubating for 24 h at 37 C. After treating with the Griess reagent (1% (w/v) sulfanilamide and 0.1% (w/v) N-(1-naphthyl) ethylenediamine hydrochloride in 2.5% (v/v) phosphoric acid), the absorbance of the solution was measured at 540 nm with an EL-800 microplate reader (BioTek Instruments).…”