Isolation, Purification, Pharmacological Activity And Structural Characterization Of Polysaccharides From Apple

Apple is one of the most widely cultivated tree fruit in China. Fruit thinning is to remove excessive fruitlets from apple trees, which is an effective measure to improve the quality of fruit in apple production. Thinned-young apple contains biologically active compounds and is an available resource with additional values. At present. polyphenol in thinned-young apple has been thoroughly investigated. However, little work has been done regarding the water-soluble polysaccharides from thinned-young apple. Herein, thinned-young apple was taken as main material in the study and the extraction process, pathological activities, purification and structure characterization of polysaccharides were studied. The results were as follows:1 The single-factor experiment and orthogonal experiment design were adopted to study the effects of different factors on the extraction yield and determine the optimum parameters of polysaccharides from thinned-young apple (TYAP). The results indicated that the effect sequences of main factors were as follows:exteraction temperature, extraction time and solid-liquid ratio. According to the orthogonal experiment design, the optimum extracting technique parameters were as follows:exteraction temperature was 88℃, extraction time was 132min, solid-liquid ratio was 1:41g/mL and the theoretical yield was 10.35%.2 High intake of dietary fructose exerts a number of adverse metabolic effects. The present study was to investigate the preventive effects of TYAP on insulin resistance and oxidative stress in mice fed with a high-fructose (HF) diet. TYAP was identified as the heteropolysaccharides by HPLC which was mainly composed of D-glucose (10.32%), D-galactose (25.55%) and L-arabinose (46.72%). Mice fed with 20% fructose in drinking water for 6 weeks significantly displayed hyperglycemia, hyperinsulinemia, dyslipidemia and liver oxidative stress with impaired insulin sensitivity (P<0.05). The administration of TYAP at 400 and 800 mg/kg·bw significantly reduced the fasting serum glucose and insulin concentrations and lipid deposition in HF-fed mice, and caused the reduction of liver lipid peroxidation and the elevation of hepatic antioxidant system. These results suggest that thinned-young apple is a potential source of polysaccharides, and might be regarded as the novel preventive and therapeutic products for the mitigation of insulin resistance, oxidative stress and liver dysfunction.3 Cellulose DEAE-52 and Sephadex G-150 were adopted to purify TYAP into three purified fractions named TYAP-1, TYAP-2 and TYAP-3. The structure characterizations were discussed by HPLC, UV, FT-IR and ESEM and the antioxidant activities were further studied.The results indicated that the total carbohydrate contents measured by the phenol-H2SO4 assay for TYAP-1, TYAP-2 and TYAP-3 were 99.76,99.24 and 96.38% and the molecular weights of three were 115 kDa,479 kDa and 403 kDa, respectively. Meanwhile, TYAP fractions of three all had no absorptions at 260 nm and 280 nm in UV spectrum, indicating the absence of protein and nucleic acid. FT-IR spectra of them all had characteristic absorption peaks of carbohydrate in 400-4000cm-1 with some small differences. Moreover, the monosaccharide compositions of TYAP-1, TYAP-2 and TYAP-3 were determined by a validated HPLC-UV technique, and the results showed that TYAP-1 was composed of glucose, xylose, galactose and arabinose in the mole percentages of 5.74%,4.68%,10.60% and 78.98% of all quantitative monosaccharides, respectively. TYAP-2 was mainly composed of galacturonic acid, glucose, xylose, galactose and arabinose in the mole percentages of 2.18%,9.14%, 5.27%,23.32% and 59.45%, while rhamnose and glucuronic acid were presented in trace amounts. Surprisingly, the monosaccharide profile of TYAP-3 was highly similar to that of TYAP-2, and it was consisted of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose, with their corresponding mole percentages of 7.19%,4.42%,0.90%,6.57%,6.55%,7.97%,20.58% and 45.82%, respectively. Finally, the results of ESEM showed that the structure of TYAP-1 were almost irregular curls, flake and rods morphologies were observed in TYAP-2 and the structure of TYAP-3 were more complicated which consisted of spherical, rods and flake morphologies.4 In vitro antioxidant activities of three fractions were characterized by DPPH·2 HO·, O2·- and ferric-reducing antioxidant power systems. The results indicated that TYAP-1, TYAP-2 and TYAP-3 possessed significant antioxidant effects in a concentration-dependant manner in the tested concentration range of 0.25-5.0 mg/mL. Among them, the antioxidant effect of TYAP-3 was highest followed by TYAP-2, while that of TYAP-1 was the lowest.