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Normal cell

RAW 264.7 (murine macrophage)HUVEC (human umbilical vein endothelial cells)HEK293T (human embryo kidney cells)

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Measurement of the nitric oxide producing activity of macrophages

5×105 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

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BDF1

0.2 ml of 1×105 B16F10

intraperitoneallyendo-polysaccharide

1 , 3, 10, 30 mg/kg/dayControl 0.85%NaCl

orallyendo-polysaccharide

30, 100, or 300 mg/kg/dayControl distilled water

Survival rate (%)=(MST of treated group/MST of control group)×100

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Cytotoxicity test

1×105 cells/ml 100 μl cell

10, 50, 100, and 200 μg/ml polysaccharide solutions

50 μl MTT

48h

4h 37℃

measurement at 540

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NMR spectroscopy

Bruker Advance 600 and Bruker Advance DPX-300

Trace amounts of water in the purified polysaccharide were eliminated using pure D2O

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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)

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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。

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30μL 1% purified endo-polysaccharide fraction

high performance gel permeation chromatography

188,000, 150,000, 75,000, 40,000, 17,500, and 10,000 Dawere used as standards