Study Of Opuntia Dillenii Haw. Polysaccharides On The Preliminary Structure And The Promoting Cholesterol Efflux From Foam Cell Function

Opunti adillenii Haw.is a cactus belonging to the Cactaceae family,widely grown in tropical and subtropical regions,including the south of China and Taiwan.The resource of Opuntia dillenii Haw(cactaceae)is rather abundant and may be further exploited rationally.The main component of Opuntia dillenii Haw polysaccharide(ODP-Ia)has been proved to be a potential cholesterol-reducing substances by lowering lipidemia and retarding atherosclerosis.In order to explore the molecular mechanism of the cholesterol-reducing activity,we will study the cholesterol removal from the classic pathway “reverse cholesterol transport”(RCT).Furthermore,in order to improve the extraction yield of ODP,the ODPs were extracted using the hot water extraction method and extraction conditions were optimized.The purification,the structure features,and the antioxidant activity of ODP,which may play a very important role in the characterization of polysaccharides,were studied.The results are as follows.1.The optimal conditions exhibited a liquid – materal ratio of 62 to1,extraction time of 3.5 h and exraction temperature of 93? by optimized using an RSM(Response Surface Method)with a three-level,three-variable Box-Benhnken.Under these conditions,the actual extraction rate of ODPs was 25.05%,while the predicted value was 25.14%.The exraction temperature was one of the major factor affecting the exraction rate of ODPs with a very low p-values(P<0.001).The polysaccharides and protein content of ODPs and the DPPH· scavenging of ODPs were the highest under the 95? extraction temperature.The paired comparison by SNK method demonstrated that,there was significant differences(P<0.05)existed among three groups(coarse powder,60 and 100 mesh granularity,200 and 300 mesh granularity).Compared with coarse powder,the extraction rate,purity,and the scavenging ability against free radicals of ODPs were increased respectively by 100%,25% and 150% in 300 mesh granularity.These results suggested that the grinding mechanism was better for enhanced the extraction yield,quality and antioxidant activity of ODPs.2.The extracted ODP was purifiedby DEAE sepharose fast flow anion exchange and Sephacryl S-400 chromatography with four fractions obtained(ODP-Ia,ODP-Ib,ODP-IIa and ODP-IIb).Analysis with UV-vis chromatography indicated that ODP-Ia and ODP-IIa were relatively homogeneous molecules with a molecular weight of 339 kD and 943 kD,respectively.Results of infrared spectroscopy indicated that ODP,ODP-Ia,and ODP-IIa were acidic polysaccharides.Further,the antioxidant activity against DPPH(1,1-diphenyl-2-picrylhydrazyl)radical,hydroxyl radicals,and superoxide radical in vitro demonstrated that the purified fraction(ODP-Ia)was superior tothe ODP.Results of FT-IR Spectroscopy indicating that ODP contained mainly ?-type glycosidiclinkages,ODP-Ia and ODP-IIa contained mainly ?-type glycosidiclinkages.These results will offer a theoretical basis for further research on the structure-function relationship of ODP and the rational utilization of Opuntia dillenii Haw.3.The THP–1 macrophage derived foam cell model was built.The lipid content and the cholesterol content were increased significantly after the THP–1 cells were induced into the THP–1 macrophage derived foam cell,and then were significantly decreased after the treatment with ODP-Ia concentration-dependently.These results indicated that ODP–Ia promoted “reverse cholesterol transport”(RCT)by accelerating cholesterol efflux of THP–1 macrophage derived foam cell.4.The cholesterol efflux was significantly enhanced after 24 h incubation by apoA-I,and the highest cholesterol efflux were reached by 20 mg/mL apoA-I.Futhermore,these increase of cholesterol efflux were enhanced more dramatically if incubated 2 h by ODP-Ia previously.5.ODP-Ia promoted cholesterol efflux of THP-1 macrophage derived foam cell significantly by up-regulation of PPAR?,PPAR?,LXR?,ABCA1,ABCG1 and SR-BI.6.Both the PPAR? antagonist GW9662 and LXRa antagonist GGPP dramatically inhibited the upregulating effect of ODP-Ia on the expression of PPAR??LXR??ABCA1?ABCG1 at both protein and mRNA levels(P<0.05).It means that ODP-Ia promotes cholesterol efflux in THP-1 macrophages through PPAR?-LXR? pathway.These conclusion offer the reasonable,normative and scientific data for industrialized production of ODPs.Futhermore,these results will provide new ideas for the mechanism research of the polysaccharides hypolipidemic biological activity,and provide the scientific basis for further research and utilization of polysaccharides.