Extraction And Purification Of Lycium Barbarum Polysaccharide And Its Effects On The Production Of Aβ_1-42 In N2a/APP695 Cells

Lycium barbarum is one of the traditional Chinese medicinal materials.As recorded in Compendium of Materia Medica,“Lycium barbarum,improving kidney and sperm,nourishing liver,improving eyesight,strengthening essence,relieving fatigue,changing complexion,whitening,improving eyesight and calming nerves,and helping people to live longer”.Lycium barbarum polysaccharide（LBP）is the one of the main effective components in Lycium barbarum which has various biological activities such as anti-tumor,anti-oxidation,and immune regulation.Researches on Lycium barbarum polysaccharide have become hotspots in recent years due to its great development value.This article mainly studied on the extraction,purification,physical and chemical properties of Lycium barbarum polysaccharide and its inhibitory activity on the producing of Aβ_1-42 in N2a/APP695 cells.Polysaccharide extracted by hot water will maintain the natural structure best compared to other extraction methods.It is economy and safety.Lycium barbarum from Jingyuan County,Gansu province was used as raw material for this study and LBP was extracted by hot water.The effects of extracting time,water-material ratio and temperature on the yield of LBP were studied by single-factor test.Furthermore,Design Expert software and Box-Benhnken test were used to optimize the extracting conditions according to the principle of response surface methodology.Analyzing the physical and chemical properties of LBP:Establishing a method for measuring the monosaccharides composition of LBP and examining the methodology;Studying on the monosaccharides composition,molecular weight and distribution,sugar content,uronic acid content and protein content of LBP.Isolation and purification of crude polysaccharide from Lycium barbarum and determination of the physical and chemical properties:LBP was deproteinized by Sevag method,and separated by DEAE-Crystarose Fast Flow column chromatography,so as to obtain purified polysaccharide LBP-3.Physicochemical properties including monosaccharides composition,molecular weight and distribution,sugar content,uronic acid content and protein content of LBP-3 were measured as described previously.LBP-3 was used to study the effects on the producing ofβ-amyloid protein 1-42(Aβ_1-42)in N2a/APP695 cells.And the mRNA and protein expression levels ofβ-secretase 1（BACE1）and amyloid precursor protein（APP）in N2a/APP695 cells was determined by RT-PCR and Western-blot.The results showed that the optimal extracting conditions of LBP were:extraction time 3.9 h,material-to-liquid ratio 1:36.6,and temperature 93.2℃.On this condition,the yield of LBP was 4.28%.PMP pre-column derivatization High Performance Liquid Chromatography（HPLC）method was used to establish a method for determining the monosaccharides composition of LBP.The methodology examining results showed that this method was stable and reliable,and it can be used to determine the monosaccharides composition of LBP.HPLC analysis showed that LBP was basically comprised of mannose,rhamnose,galacturonic acid,glucose,galactose and arabinose with percentage of 1.00:059:1.68:4.51:5.10:7.21.The result of High-performance Gel Permeation Chromatography（HPGPC）showed that LBP was a mixture of different polysaccharide fragments with different molecular weights.The molecular weights of 2 main fragments were 1.11×10⁵ Da and 1.94×10⁴ Da,and the Mw/Mn values were 1.85 and 1.62.The sugar content,uronic acid content,and protein content of LBP were 51.37%,6.47%,and 6.80%.LBP was purified by Sevag method and DEAE-Crystarose Fast Flow column chromatography to obtain the homogeneous component LBP-3.The determination results of physicochemical properties showed that the sugar content,uronic acid content,and protein content of LBP-3 were 90.12%,1.02%and 1.55%.LBP-3 was comprised of galactose and arabinose with percentage of 1:3.17.The molecular weight of LBP-3 was 8.02×10⁴Da,and the Mw/Mn value was 1.35.After treated with LBP-3 for 24 h,the production level of Aβ_1-42 in N2a/APP695 cells was significantly reduced（P<0.05）compared with the control group,and it is concentration-dependent.The RT-PCR and Western-blot results showed that the mRNA and protein levels of APP and BACE1 in N2a/APP695 cells decreased significantly（P<0.05）after treated with LBP-3 for 24hours compared with the control group.These results indicated that LBP-3 can reduce the expression levels of APP and BACE1 and inhibit the production of Aβ_1-42 in N2a/APP695 cells.This study provides strong basis for the further development and utilization of LBP.