Composition Analysis And Hypoglycemic Mechanism Of Polysaccharides From Acanthopanax Trifoliatus

Acanthopanax trifoliatus(Linn.)Merr.is a climbing shrub of Araliaceae,which has the effects of clearing away heat and detoxifying,expelling wind and removing dampness,eliminating blood stasis and relieving pain,invigorating spleen and replenishing qi.In our previous study,A.trifoliatus polysaccharide(ATP)was prepared by water extracting and alcohol sedimenting technique.The neutral polysaccharide ATP1 and the acidic polysaccharides(ATP2 and ATP3)were obtained after separation of ATP by DEAE-52 cellulose column chromatography.Then ATP1 was purified repeatedly by Dextran gel Sephadex G-75,and ATP1-1 with homogeneous molecular weight was obtained.Pharmacological experiments confirmed that ATP1-1 can reduce the blood glucose of streptozotocin(STZ)-induced diabetic mice,and its hypoglycemic mechanism was related to the hepatic glucose metabolism.In this study,the decolorization technology of ATP was optimized and the hypoglycemic mechanism of ATP1-1 related to apoptosis was investigated.The acidic polysaccharides ATP2 and ATP3 were separated and purified to obtain polysaccharides with homogeneous molecular weight by Dextran gel Sephadex G-75.The monosaccharide compositionsare analyzed by HPLC pre-column derivatization.The research content provides a scientific basis for evaluating the hypoglycemic mechanism and composition analysis of ATP and for making full use of the resources of A.trifoliatus.The main research contents and results are as follows:1.The decolorization technology of ATP was systematically investigated.The decolorization effects of six resins,including NKA-9,S-8,HPD450,AB8,D101,HPD600,were evaluated.The effects of polysaccharide concentration,resin dosage,shaking speed and shaking time on the decolorization were investigated.The results showed that the decolorization effects of S-8 and HPD450 resins were better.The optimal decolorization process: 30 mL of polysaccharide solution(15 mg/mL)was mixed with 5 g of resin and shaken at 85 r/min for 8 h.The decolorization rate and polysaccharide retention rate of S-8 were 88.89% and 61.86%,respectively;the decolorization rate and polysaccharide retention rate of HPD450 were 87.20% and 64.62%,respectively.The decolorization effect of the two resins after mixing was investigated.The results showed that the decolorization effect of the mixed resin was better.When S-8 and HPD450 were mixed in a ratio of 1:4,the decolorization rate and polysaccharide retention rate were 89.78% and 69.35%.2.Diabetic mice model was established by multiple low-dose injection of streptozotocin(STZ).Blood glucose was measured weeklyand the body weight,food intake and water intake of the mice were measured daily.It displayed that ATP1-1 can increase the body weight of diabetic mice,reduce water intake?food intake and blood glucose and improve glucose tolerance.Hematoxylin-eosin staining(HE)was used to study the changes of pancreatic tissue structure.Quantitative Real-time PCR(qPCR)and Western Blotting techniques were used to detect the expression of apoptosis factors for elucidating the mechanism of hypoglycemic activity of ATP1-1.The results showed that ATP1-1 can decrease the expression of Fas ? FasL and pro-apoptotic protein Bax,increase the expression of anti-apoptotic protein Bcl-2 and Bcl-2/Bax.ATP1-1 can inhibit Fas/FasL interaction,reduce cytotoxicity,inhibit apoptosis cascade,reduce islet B cell death,and enhance cell anti-apoptosis ability by maintaining Bcl-2 and Bax homeostasis.The results of Western Blotting technique were consistent with those of qPCR,which indicated that ATP1-1 could reduce blood glucose and protect islet B cells by regulating Fas/FasL and Bcl-2/Bax.3.After repeated purification of ATP2 and ATP3 by Dextran gel Sephadex G-75,ATP2-1 and ATP3-1 with uniform molecular weight were obtained.Ultraviolet spectroscopy confirmed that the two polysaccharides were free of proteins and nucleic acids.High performance infiltration gel chromatography results showed that the number-average molecular weight(Mn)of ATP2-1 was 4370 and theweight-average molecular weight(Mw)of ATP2-1 was 8990.The number-average molecular weight(Mn)of ATP3-1 was 68700 and the weight-average molecular weight(Mw)of ATP3-1 was 75200.The monosaccharide composition analysis of ATP2-1 and ATP3-1 was conducted by HPLC pre-column derivatization.The results show that ATP2-1 was composed of mannose,rhamnose,glucose,galactose and arabinose,with the ratio of 1.0:1.2:5.8:7.3:2.8.ATP3-1 consists of rhamnose,galacturonic acid,galactose,and arabinose with the ratio of1.0:4.1:2.6:2.9.In addition,the hydrolysis time,derivatization time and temperature of the polysaccharide were optimized.The optimum reaction conditions: the hydrolysis time of the polysaccharide was 6 h;the derivatization time was 40 min;and the derivatization temperature was60 ?.Periodate oxidation results indicated that ATP3-1 contained1-6-bonded glycosyl or non-reducing terminal glycosyl group,and a small amount of 1?2 or 1?2,6 or 1?4 or 1?4,6 glycosidic bonds.