Study On The Extraction And Structure Of Black Bean Polysaccharide And Its Protective Effect On Acute Liver Injury In Mice

Black soybeans have been widely used as a nutritionally rich foodstuff. They possess various bioactivities, such as antioxidant, anti-inflammatory, antiobesity, antidiabetic and antimutagenicity properties, as well as inhibition of low density lipoprotein oxidation and reduction of DNA damage. In this study, polysaccharides from black soybean (BSPS) were efficiently extracted by hot water technique. The optimal extraction conditions, purification, structural characterization, antioxidant activity and hepatoprotective effect of BSPS were investigated in details.(1) The extraction conditions for BSPS were investigated by single factor experiments and reponse surface method. The results of Box-Behnken design showed that the optimal extraction conditions for BSPS were as follows:ratio of water to material of 20 ml/g, extraction time of 6.4 h and extraction temperature of 92℃, with a corresponding yield of 2.56%.(2) The crude BSPS were further fractionated on DEAE-52 and Sepharose CL-4B chromatography to afford three purified fractions (BSPS-1, BSPS-2 and BSPS-3). The molecular weights of BSPS-1, BSPS-2 and BSPS-3 were determined as 1.95×105 Da,3.46×105 Da and 1.88 × 105 Da by high performance size-exclusion chromatography (HPSEC).(3) The structural characterizations of BSPS-1, BSPS-2 and BSPS-3 were carried out by various methods. UV spectra of the three purified BSPS showed no peak in the range of 260-280 nm, indicating that they did not contain any nucleic acid or protein. FT-IR spectra of BSPS-1 showed the absorptions at 916 and 762 cm-1 were typical pyranose form of glucose residues. For BSPS-2 and BSPS-3, the strong bands at 1078 and 1043 cm-1 were attributed to galactopyranose and arabinofuranose units, respectively. The monosaccharide compositions of BSPS-1, BSPS-2 and BSPS-3 were analyzed by GC. BSPS-1 was composed of arabinose, rhamnose, galactose, glucose and mannose in the molar ratio of 1.79:1.00:2.59:26.54:1.01. BSPS-2 was composed of arabinose, rhamnose, xylose, galactose and mannose in the molar ratio of 8.10:4.80:9.15:13.38:1.00. BSPS-3 was composed of arabinose, rhamnose, galactose and mannose in the molar ratio of 16.80:3.60:33.66:1.00. Methylation analysis and NMR spectra indicated that BSPS-1 was composed of 1,6-α-D-glucopyranosyl residues. By contrast, BSPS-3 was mainly composed of a 1,3-β-D-galactopyranosyl residue backbone with side chains substituted at the O-6 position consisting of large content of T-α-L-Araf-(1→ residues, and small contents of →5)-a-L-Araf-(1→,→2)-a-L-Rhap-(1→, and 4-O-Me-β-D-GlcAp-(1→ residues. 1D and 2D NMR suggested that BSPS-1 was a linear (1→6)-a-D-glucan, whereas BSPS-3 was a type Ⅱ arabinogalactan.(4) The antioxidant activity and hepatoprotective effect of BSPS against carbon tetrachloride (CCl4-induced acute liver injury were investigated. Antioxidant assays in vitro showed that crude BSPS and its purified fractions had potential superoxide anion and DPPH radical scavenging activities. The antioxidant activity decreased in the order of crude BSPS> BSPS-3> BSPS-2> BSPS-1. Administration of BSPS could significantly reduce the elevated levels of aspartate transaminase, alanine transaminase and alkaline phosphatase as compared to CCl4 treatment group. Moreover BSPS significantly increased the levels of hepatic superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, glutathione and total antioxidant capacity, whereas markedly decreased the malondialdehyde level when compared with CCl4 treatment group. In addition, the hepatoprotective effect of 400 mg/kg BSPS was comparable to positive standard of silymarin. Results suggested BSPS had potent antioxidant activity and potential effect against CCl4-induced acute liver injury.