| Maca（Lepidium meyenii Walp.）is a new resource food,which possesses immunomodulatory,antioxidant,antifatigue,anticancer,improving sexual performance and fertility and alleviating menopausal syndrome activities.Polysaccharides and alkaloids are important active ingredients of maca.Studies on the structural characterization of maca purified polysaccharide have indicated that its water elution fraction is a kind of heteropolysaccharide,mainly consisting of glucose residues as backbone.Salt elution fraction is a kind of heteropolysaccharide,mainly consisting of galactose,arabinose,glucose and rhamnose,however,little attention has been devoted to the complete primary and advanced structure of salt elution fraction;the antioxidant activity against oxidative stress-induced RAW264.7 cell injury,immunomodulatory against immunosuppression and molecule mechanism mediated by MAPKs signaling pathway of maca purified polysaccharide have not been reported.Alkaloid content is low in maca and usually extracted by traditional methods which are low efficient,the study on alkaloid purification is rarely reported,so it is significant to develop an effective method in maca alkaloids enrichment and purification.In view of the above two points,the complete primary and advanced structure of maca polysaccharide MP1 were studied by modern analytical method,its antioxidant activity,immunomodulatory effects and the potential mechanism were systematically studied;maca alkaloids were extracted and purified by using ultrasonic-circulating extraction and macroporous resins,and the purified fraction was identified.This study could provide a basis for the application of polysaccharides and alkaloids in maca.The main experimental procedures and results were as follows:（1）Study on extraction,purification and structural characterization of maca polysaccharide.Crude maca polysaccharide MP was purified by DEAE-52 to obtained five fractions,0.05 M Nacl fraction was the major fraction and further separated by SephacrylTM S-500 column,one major purified polysaccharide fraction was collected as MP1.The molecular weight was estimated to be 4.67×105 Da by HPLC analysis.Monosaccharide composition was determined to be arabinose and galactose in a molar ratio of 2:1 by GC-MS analysis.Methylation combined with 1D and 2D NMR spectrum analysis revealed that MP1 had a chain consisting of T-Araf,1,3-Araf,1,5-Araf,1,4-Galp and 1,2,3,5-Araf residues,the predicted primary structure of MP1 was established as below:（?）SEM analysis indicated MP1 was easily curled to form various irregular shapes and had an amorphous structure.AFM analysis indicated that MP1 polysaccharide chains entangled with each other and easy to aggregate in aqueous solution with the height in a range of 1-3 nm.Congo red analysis indicated that MP1 was not triple-helical structure.（2）Study on antioxidant activity of MP1.Chemical antioxidant experimental results indicated that MP1 possessed a moderate antioxidant activity in DPPH·,ABTS+·,O2-·and·OH radicals scavenging,Fe2+chelating,lipid peroxidation inhibition and reducing power assays.H2O2-induced injury cell model results indicated that MP1 could significantly improve RAW264.7 cells viability and CAT activity from 400 to 1000μg/mL,enhance SOD and GSH-Px activity from 200 to1000μg/mL,decrease ROS and LDH level from 100 to 1000μg/mL and reduce MDA level from 200 to 1000μg/mL.MP1 possessed good antioxidant activity and exhibited significant protective effects against H2O2-induced injury RAW264.7 cell.（3）StudyonimmunomodulatoryeffectofMP1against cyclophosphamide-induced immunosuppression in Balb/c mice.MP1 could remarkably increase the weight growth rate and organ indexs,stimulate the phagocytosis of phagocyte and DTH reactions,up-regulate IL-2 level and activate leukocyte proliferation at 50,100 and 150 mg/kg.MP1 could significantly stimulate hemolysin formation,up-regulate INF-γ,IgA,IgG and IgM levels and activate erythrocytes and platelets proliferation at 100 and 150 mg/kg.MP1 showed a potential immunomodulatory effect against cyclophosphamide-induced immunosuppression in Balb/c mice.（4）Study on the immunomodulatory effect and molecular mechanisms of MP1on RAW264.7 cells.MP1 could notably enhance the phagocytic capacity,TNF-αand IL-1βsecretion from 250 to 1000μg/mL and improve NO level at 1000μg/mL.RT-PCR analysis indicated that MP1 could significantly promote the expression of iNOS mRNA from 250 to 1000μg/mL and TNF-αand IFN-γmRNA from 125 to1000μg/mL.Western blot analysis revealed that MP1 could significantly up-regulate phosphorylation JNK expression level at 500 and 1000μg/mL,phosphorylation ERK and CREB expression level from 62.5 to 1000μg/mL and phosphorylation p38 and EIK-1 expression level from 250 to 1000μg/mL.Antibody blocking results indicated that MP1-induced phosphorylation of MAPKs（p-JNK,p-ERK and p-p38）was inhibited by pretreatment with SP600125,PD98059 and SB203580,which were specific inhibitors of JNK,ERK and p38,respectively.MP1 could activate macrophages through the MAPKs signaling pathway,and promote the expression of cytokines and genes,thus improving the body immune function,which was one of the mechanisms of its immunomodulatory effect.（5）Extraction,purification and preliminary identification of maca alkaloids.Maca alkaloids were extracted and purified by using ultrasonic-circulating extraction and macroporous resins,and identified by HPLC-MS/MS.The results showed that two site model gave satisfactory data fit of ultrasonic-circulating extraction.Adsorption data of AB-8 resin fitted best to the pseudo-second order kinetics model and Langmuir isotherm model,under optimal adsorption condition,the content of total alkaloids in the purified product was 175.6 mg/g with recovery yield of 70.34%.11 alkaloids were identified by HPLC-MS/MS:（1S,3S）-1-metyltetrahydro-β-5,6-hydride carboline-3-carboxylic acid,（1R,3S）-1-butanetetrahydro-β-5,6-hydride carboline-3-carboxylicacid,（1R,3S）-1-pentanetetrahydro-β-5,6-hydride carboline-3-carboxylic acid,1,3-dibenzyl-4,5-dimethylimidazolium,1,3-dibenzyl-2,4,5-trimethylimidazolium,1,3-dibenzyl-2-phenyl-4,5-dimethylimidazolium,1,3-dibenzyl-2-butyl-4,5-dimethylimidazolium,1,3-dibenzyl-2-pentyl-4,5-dimethylimidazolium,N-benzyl-（9Z,12Z,15Z）-octadecatrienamide,N-benzyl-（9Z,12Z）-octadecadienamide and N-benzylhexadecanamide,respectively. |
