Optimization of acid hydrolysis conditions for an adjuvant polysaccharide PCP-Ⅱ from Poria cocos,isolation of hydrolysis products,and determination of their physic-chemical properties
Objective:To investigate three optimal conditions of acid hydrolysis for an adjuvant polysaccharide(PCP-Ⅱ)from Poria Poria to produce three objective polysaccharide fragments with different molecular weights(close to 1.0 × 104,5.0 × 103 and 2.5 × 103),and further to isolate these fragments and determinate their physic-chemical properties.Methods:The factors including concentration of trifluoroacetic acid(TFA),hydrolysis temperature and hydrolysis time were firstly and respectively investigated.Secondly,a L9(34)orthogonal design of three factors and three levels was used to study acid hydrolysis of PCP-Ⅱ before the optimal hydrolysis conditions were determined.Sephadex gel column was used to isolate and purify these hydrolysates to obtain three fragments of homogeneous molecular weight(PCP-Ⅱ-hy-1,PCP-Ⅱ-hy-2 and PCP-Ⅱ-hy-3).The changes of monosaccharides in the hydrolysates were analyzed by thin layer chromatography(TLC),the molecular weight distribution or molecular weight of these hydrolysates or fragments were determined with high performance gel filtration chromatography(HPGPC)and their monosaccharide compositions were analyzed with capillary electrophoresis(CE)after acid hydrolysis and 1-phenyl-3-methyl-5-pyrazolone(PMP)derivation.Results:The three acid hydrolysis conditions of 0.25 mol·L-1 TFA,80 ℃,2h,0.25 mol·L-1 TFA,80 ℃,3 h and 0.25 mol·L-1 TFA,80 ℃,6 h were suitable to produce three objective fragments.The Mw of PCP-Ⅱ-hy-1 was 9.51 × 103,and its monosaccharide composition was Fuc∶Man∶Gal=1.00∶1.54∶9.05.The Mw of PCP-Ⅱ-hy-2 was 6.39 × 103,and its monosaccharide composition was Fuc∶Man∶Gal=1.00∶2.04∶14.36;The Mw of PCP-Ⅱ-hy-3 was 1.80 × 103,and its monosaccharide composition was Fuc∶Man∶Gal=1.00∶4.55∶27.59.Conclusion:The three acid hydrolysis conditions are suitable to obtain three target polysaccharide fragments.The ratio of mannose and galactose increases along with the decrease of the molecular weight of the fragments,and PCP-Ⅱ-hy-3 is mainly composed of galactose.These results suggested that the backbone of PCP-Ⅱ might be a galactan.
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