Effects Of Extraction Methods On The Structure And Anti-cancer Activity Of Goji (Lycium Barbarum) Polysaccharides

Lycium barbarum is a traditionally medicinal and edible plant in China.Lycium barbarum polysaccharide(LBP)is considered to be one of the most important active components in Lycium barbarum berries and has many biological effects.At present,the extraction of polysaccharides from Lycium barbarum is mainly by water at high temperature and in some of the cases assisted by modern novel approaches such as microwave and ultrasound.A large amount of literatures reported that different extraction methods will release different parts of the cell wall and result in differences in the structure of the extracted polysaccharides,making the bioactivities differ from each other.However,there is no relatively comprehensive research on LBP,and the relationship between extraction methods and physicochemical properties is still unclear.Therefore,taking Lycium barbarum as the raw material,this study extracted LBP by 9different methods such as water extraction,acid extraction,alkali extraction and continuous extraction.Moreover,the chemical structure and bioactivity of the LBP were studied.The aim is to establish the relationship between extraction methods,physicochemical properties and bioactivities.The main research contents and results are as follows:(1)Preparation and physicochemical properties of water-soluble LBPTaking Lycium barbarum as raw material,hot water extraction,low temperature and high temperature acid extraction,low temperature and high temperature alkaline extraction were used to prepare hot water extracted Lycium barbarum polysaccharide(W_H),low-temperature acid-extracted Lycium barbarum polysaccharide(A_L),high temperature acid extracted Lycium barbarum polysaccharide(A_H),low-temperature alkali-extracted Lycium barbarum polysaccharide(AL_L)and high temperature alkali extracted Lycium barbarum polysaccharide(AL_H).At the same time,in order to explore the limitations of the single extraction,continuous extraction methods were designed to prepare hot water and high-temperature alkali continuous extraction of Lycium barbarum polysaccharide(W_HAL_H),hot water and low-temperature alkali continuous extraction of Lycium barbarum polysaccharide(W_HAL_L),high-temperature acid and alkaline continuous extraction of Lycium barbarum polysaccharide(A_HAL_H)and low-temperature acid and alkaline continuous extraction of Lycium barbarum polysaccharide(A_LAL_L).The monosaccharide analysis and GPC-MALLS were performed on all the samples.Considering that the yield of continuous extraction was relatively low,just took the five single-extracted-samples into analysis by FT-IR,atomic force microscopy(AFM)and ~1H-NMR.The results indicated that the physicochemical properties of LBP were significantly different from different extraction methods.The polysaccharide extracted by hot water is a kind of heteropolysaccharides.The low-temperature alkaline extracted polysaccharide is dominated by RG-I,while the acid-extracted polysaccharide contains more HG domains than alkali-or water-extracted polysaccharides.The acid extraction can significantly remove the neutral sugars in side chains,and the main part was Ara.No matter which p H condition,high temperature condition releases more neutral sugars in side chains than low temperature extraction.(2)Effects of extraction methods on the anti-cancer activity of LBPThe results of surface plasmon resonance(SPR)experiment showed that AL_L,A_L,W_H and A_H had the activity of binding to galectin-3,which was the cancer cell target protein,and the activity decreased in turn.The AL_L screened out by SPR was used to conduct anti-metastasis test and anti-proliferation test to A549 cells.The W_H,whose bioactivities had been studied wildest,was used as a control group.The results indicated that AL_L has a higher anti-metastasis and anti-proliferation effects.Therefore,AL_L,dominated by RG-I structure and had more galactan side chains,may have a potential anticancer effect by binding with Gal-3 to inhibit the metastasis of cancer cells.(3)Isolation and purification of ALL and physicochemical properties,bioactivity identification of its componentsAL_L was isolated and purified with Q-Sepharose fast flow(QFF)packing,and two components(AL_L-1 and AL_L-2)were obtained.AL_L-1 was a neutral sugar and AL_L-2was an acid polysaccharide.The AL_L-1 was mainly composed of Ara and Gal,while AL_L-2 contained 22.6%of Gal A.The average molecular weight of AL_L-2(316 k Da)was significantly larger than that of AL_L-1(183.9 k Da).Both components have no degree of esterification.The results of SPR experiments showed that only AL_L-2 had the activity of binding to Gal-3(K_D=9.67?M).Based on the systematic extraction method,this study explored the effects of different extraction methods on the physicochemical properties and anti-cancer activity of LBP,and established the relationship between extraction method and the type of polysaccharide,hoping to promote the process of targeted extraction of polysaccharides.