LOGO Normal cell  RAW (murine macrophage)  HUVEC (human umbilical vein endothelial cells)  HEK293T (human embryo kidney cells)

LOGO Measurement of the nitric oxide producing activity of macrophages 5×10 5 cells/ml of RAW 264.7cell on 24-well plates containing either endo-polysaccharide or lipopolysaccharide 24 h at 37 °C equal volume of Griess reagent absorbance at 540 nm

LOGO BDF1 0.2 ml of 1×10 5 B16F10 intraperitoneally endo-polysaccharide 1, 3, 10, 30 mg/kg/day Control 0.85%NaCl orally endo-polysaccharide 30, 100, or 300 mg/kg/day Control distilled water Survival rate (%)=(MST of treated group/MST of control group)×100

LOGO Cytotoxicity test 1×10 5 cells/ml 100 μ l cell 10, 50, 100, and 200 μ g/ml polysaccharide solutions 50 μl MTT 48h 4h 37 ℃ measurement at 540

LOGO NMR spectroscopy  Bruker Advance 600 and Bruker Advance DPX-300  Trace amounts of water in the purified polysaccharide were eliminated using pure D 2 O

LOGO Determination of the sugar content and molecular weight 10 μl of 1% purified endo-polysaccharide 2 M trifluoroacetic acid (TFA) or 6 N HCl for 4 h at 100 °C centrifugation for 30 min at 32,600×g high pH anion exchange chromatograph (HPAEC-PAD)

LOGO HPAEC-PAD  醣類在高鹼性溶液中 (pH 12-14) 會易於離子化 (ionization) ，利用每一種單醣分子有各自離子化的 pH 範圍，調整溶液的的 pH 質，我們即可利用陰離 子交換樹酯來分離不同的單醣分子。  再利用 pulse amperometric detector (PAD) 來偵測醣 類因離子化而造成的氧化電位，並判斷流析物成分。 也因此特性， HPAEC-PAD 除了可分析單醣，亦可 分析寡醣及多醣類。

LOGO 30μL 1% purified endo-polysaccharide fraction high performance gel permeation chromatography 188,000, 150,000, 75,000, 40,000, 17,500, and 10,000 Da were used as standards