| Naturally sourced polysaccharides have received extensive attention due to their biocompatibility,easy structural modification,low toxicity,biodegradability and dual mediation properties.Maca(Lepidium meyenii.Walp,Spanish name: maca)was originally from Peru,and was later introduced into China and planted in large quantities in Yunnan Province.It is a natural medicinal material for both medicine and food in Yunnan Province.Maca Polysaccharide is one of the ingredients of Maca.The current research is mainly to study the extraction method,polysaccharide composition and in vitro physiological activity of Lijiang maca.The lack of specific structure and in vivo physiological activity of maca polysaccharide limits the application of maca.Therefore,this article uses Lijiang black maca as raw material to study maca polysaccharide extraction,separation,purification and structural identification.The maca polysaccharide is cationically modified to enhance its immune activity.Finally,the two polysaccharides are combined with dox to study the mechanism of its synergistic antitumor activity,and the specific research contents are as follows:First,we extracted the crude maca polysaccharide from Lijiang black maca,and purified the crude maca polysaccharide through depigmentation,deproteinization,DEAE cellulose column and Sephadex G-50 column chromatography.A neutral maca polysaccharide,namely MPW,was obtained.Molecular weight identification by HPGPC showed that the molecular weight of MPW was 8-9k Da.Through monosaccharide composition,molar ratio analysis and methylation analysis,it was concluded that MPW was mainly composed of glucose and contained three glycosidic linkages,namely: D-Glcp(1→,→4)-D-Glcp–(1→,→4,6)-D-Glcp(1→,the molar ratio is 0.050:0.875:0.075.Through the structure analysis of infrared,one-dimensional and two-dimensional NMR,MPW consisting of(1→4)α-D-Glcp linkage as the main chain with the terminal α-D-Glcp branches occurred at the O-6 bond.Therefore,MPW is a polysaccharide compound with concentrated molecular weight,uniform structure and high purity.Secondly,MPW and ethylenediamine was reacted to modify the cationized structure(C-MPW),we conducted an experimental design on the amount of MPW,ethylenediamine and condensing agent,taking substitution degree andζ potential as evaluation indicators,and finally selected maximum substitution(53.37%)with potential was +17.92 m V,that is,the mass ratio of CDI to MPW was 1:2,and the molar ratio of ethylenediamine to CDI was 2:1,and the subsequent experiments were carried out.Thirdly,the author conducted an anti-tumor study on 4T1 tumor-bearing mice by combining MPW and C-MPW with the chemotherapeutic Dox.It found that after the combined administration of MPW/C-MPW with Dox,the body weight of the mice showed a trend of “fall-rise,”,While the body weight of the Dox group mice dropped immediately during the experiment.At the end of the experiment,the body weight of the Dox group mice was lower than the body weight of the two polysaccharide groups,indicating that the MPW/C-MPW combined with Dox administration can significantly improve the immunosuppressive effect caused by chemotherapy drugs.Among them,when Dox was used alone,the tumor suppression rate(TSR)can reach 60%,but when combined with two polysaccharides,the TSR could reach 80%.H&E staining of tissues and lung tissues showed that MPW and C-MPW not only have good anti-tumor activity,but also can inhibit tumor metastasis to the lung,and MPW had a stronger anti-tumor effect after cationized modification.Finally,the authors studied the regulation mechanism of the tumor microenvironment after treatment of 4T1 tumor-bearing mice with MPW and C-MPW combined with Dox.The polarization of TAMs was analyzed by immunofluorescence staining and flow cytometry.The expression of two polysaccharide groups of CD206 in TAMs decreased,while the expression of CCR7 increased,indicating that TAMs was re-educated from M2 to M1;the levels of secreted cytokines produced by M1 and M2 were checked by immunohistochemistry,ELISA and WB.It was verified that MPW and C-MPW can bind and act on M2 type Pattern recognition receptors(PRR)such as Toll-like receptors(TLR2,TLR4)and mannose receptor(MR)on the surface of macrophages activate downstream signaling pathways such as NF-κB and Jun,thereby upregulating the secretion of inflammatory factors(TNF-α,IL-6 and i NOs),which could induce the transformation of M2 type TAMs into M1 type in a targeted manner.Compared with MPW,C-MPW had a stronger binding capacity with TLR4 and showed a stronger polarization effect.The above studies show that MPW is a new structure of neutral maca polysaccharide,which has immunomodulatory activity and anti-tumor activity on tumors.This anti-tumor effect is further enhanced by cationic modification.These studies laid foundation of Maca polysaccharide for wide application... |
