Chemical Structure And Immunomodulatory Of Ultrasonic-assisted Extraction Of Polysaccharides From Hovenia Dulcis Peduncles

Hovenia dulcis (Rhamnaceae), commonly known as Guai Zao, is a kind of plant using as both medicine and fruit. Polysaccharides from Hovenia dulcis have been reported to have biological functions such as hepatoprotective effect on acute alcoholic liver damage, antioxidant, immunomodulatory and antitumor activities. In this paper, the peduncles of H. dulcis were used as raw material, and polysaccharides from H. dulcis were obtained under the optimal extraction conditions. The homogeneity component of the polysaccharide was isolated by separation and purification. Meanwhile, the physicochemical property, chemical structure and immunomodulatory activities were investigated. The main research methods and results are as following:(1) The optimization of ultrasonic-assisted extraction conditions of polysaccharides from H. dulcis:Response surface methodology was employed to optimize and model the extraction conditions of HDPs with a Box-Behnken design based on single-factor experiments to obtain higher yield, namely extraction temperature, ultrasonic power and extraction time. The optimal conditions were extraction temperature 60℃, ultrasonic power 362W and extraction time 65min. Under these conditions, the maximal yield of crude HDPs was 25.12 mg/gDW.(2) Grading of crude polysaccharides and researching of their preliminary characterization and antioxidant activity in vitro:The crude polysaccharides of HDPs were graded by the ethanol precipitation method at different final concentrations (40%、60% and 80%) and three fractions HDPs1、HDPs2 and HDPs3 were harvested. The physicochemical property, FT-IR spectra, monosaccharide composition and antioxidant activity in vitro were investigated. The contents of uronic acid in HDPs1, HDPs2 and HDPs3 were 2.35±0.03%, 11.1±.05% and 2.15±0.08%, respectively. The contents of protein were 2.37±0.05%,5.98±0.16% and 11.77±0.35%, respectively. And they all exhibited strong scavenging activities against DPPH and ABTS radicals in vitro, but there are somewhat different. The results indicated that the FT-IR spectra of the fractions exhibited an almost similar characteristic absorption peak, and the monosaccharides of HDPsl, HDPs2 and HDPs3 were mainly composed of Ara, Rha, Glu and Gal by GC. However, the contents were different and result the different antioxidant activities. HDPs2 and HDPs3 displayed relatively higher antioxidant activity than HDPs1.(3) The separation and purification of HDPs and the structural characterization and physicochemical property of the polysaccharide homogeneity:Three fractions of HDP1, HDP2 and HDP3 were isolated by column chromatography using cellulose DEAE-52. HDP3 was concentrated, dialyzed by distilled water and lyophilized, and further purified by Sephacryl S-300 filtration chromatography, the homogeneity component HDP3A was obtained. Analysis of physicochemical property indicated that HDP3A was an acidic polysaccharide with uronic acid content of 45.9% and no protein. The structural characterization of HDP3A was obtained by IR, UV, monosaccharide composition, HPLC, GC-MS and NMR. The analysis of monosaccharide composition indicated that HDP3 A was composed of Ara, Rha, Glu Gal, Xyl, Man and Fuc. The average molecular weight was estimated to be 3.65×105Da by HPLC. By analysis of NMR (13C-NMR、1H-NMR、HSQC and HMBC), methylation and GC-MS, the glycosidic linkage of HDP3A were 1,4-linked Rhap,1,3,6-linked Manp,1-linked Xylp,1,4-linked GalA,1,6-linked Fucp, 1,4,6-linked Glcp and 1-linked Araf in the molar ratio of 1.0:0.7:0.4:11.7:0.5:0.5:0.8. And the 1,4-linked GalA was a backbone.(4) In vitro immunomodulatory activities of HDP3A、HDP3A-0.1、HDP3A-0.5 and HDP3A-R:The immunomodulatory assays of the fours fractions at different concentrations (20-500ug/mL) on macrophage were studied. The results indicated that the four fractions have different proliferation activity, they all can increase the cell proliferation and HDP3A is the best. The proliferation rate of HDP3A was 2.38 at the low concentration of 20ug/mL, exceed that of LPS. And the four fractions can enhance neutral red phagocytic activity, and the phagocytosis ability of HDP3A-0.1 and HDP3A-0.5 was very strong at low-middle concentration.