Study On The Major Water-soluble Anti-fatigue Components Of Maca (Lepidium Meyenii Walp.)

Maca(Lepidium meyenii Walp.)is an annual or biennial herb belonging to the Brassicaceae family,which originates from the Andes region(more than 3500 meters above sea level)in Peru.In tradition,it is considered that maca has the activities in anti-fatigue,increasing fertility,improving sexual function and alleviating menopausal syndrome.Since maca introduced to China in 2002,maca has been the leading role in the function food market.However,the market is glutted with the low-level and repeated maca products,and the product quality is usually advertised by concentrating times.The nominal ingredient,the type and content of functional component in maca,the effect of processing on maca,and the lowest effective dose of functional component are still unclear.This thesis focuses on the determination of benzylglucosinolate(BG),the effect of processing on BG content,and the anti-fatigue activity of water-soluble components(BG and maca polysaccharides)in the maca,which cultivated in Lijiang,Yunnan.1.Study on the determination and extraction process of benzylglucosinolate.BG was extracted by 70%methanol from maca,and purified on acidic alumina column and semi-preparative Cosmosil cholester column.The purified sample was verified as BG by LC-MS and NMR with the purity of 98.3%,which could be used as standard in follow-up research.The determination of BG content was performed on the analytical Cosmosil cholester column(4.6 mm×250 mm)at 235 nm.The mobile phase consisted of HPLC-grade water and methanol(90:10 v/v,containing 0.05%TFA)was run in isocratic mode;the flow rate was 1 m L/min;the injection volume was 20μL.The linear range of the method was0.04-0.2μmol/mL,linear correlation coefficiency(R~2):0.99906,LOD:0.003μmol/mL,LOQ:0.012μmol/m L,precision:1.28%,accuracy(recovery):98.52%.The method validation proved that this method is precise,repeatable and sensitive for the analysis and determination of BG in maca.The extraction process of BG was optimized by single factor and orthogonal experiments.The optimum extraction condition was as follows:methanol concentration was70%,extraction temperature was 75~°C,extraction time was 60 min,and extracting twice.2.Study on the change of BG content and its mechanism in Maca process.Effects of three methods of enzyme inactivation on the myrosinase activity and BG content were investigated.The myrosinase was respectively inactivated by boiling(100~°C),microwave(700W),steam treatment(ordinary pressure,100~°C),while the retention rate of BG was 66%,85%,95%,respectively.BG content of steamed maca presented no significant difference after shredding,however,raw maca lost 57%of BG over 24 h.Steamed maca showed no significant loss of BG after natural air drying and vacuum drying at the temperature from 20to 80~°C in 24 h.However,the BG content in raw Maca decreased by 56%after natural air drying;significant decrease of BG content were observed in raw maca after vacuum drying at different temperature below 80~°C.BG content decreased the most(64%)at 40~°C.The thermal degradation of BG was studied by the first order kinetic model,and the activation energy(E_a)was 136.3 kJ/mol;it indicated that no significant decrease in BG when heating at 90~°C within80 min,or heating at 100~°C within 10 min.During the 90 d of storage,all the steamed maca pieces,steamed maca powder and the raw maca pieces showed no significant loss in BG.However,the BG content in raw maca powder decreased 35.2%,21.9%and 30.5%,respectively at-18~°C,-4~°C and 25~°C in 90 d.The results indicated that the myrosinase activity was the main factor affecting the change of BG in Maca during processing and storage.The author hereby suggested that the raw maca should be processed by 5min of steaming at 100~°C before shredding,drying,grinding,etc.This treatment could protect BG from degradation during processing and storage.3.Extraction,purification and characterization of maca polysaccharides.The maca polysaccharide(MPS)was extracted from hot water,and the content of MPS in maca was 6.9%determined by the phenol-sulfuric acid method.The water-soluble proteins could be totally removed by deproteinization for 5 times.After graded and stepwise ethanol precipitation,the total deposition rate of polysaccharides was 72.9%,and the total sugar of polysaccharides was62.1%.The two fractions of polysaccharide MPS-1 and MPS-2 were purified from the crude MPS by using a DEAE-52 and a Sephadex G-100 column.The total sugar of MPS-1,MPS-2was 93.2%,91.5%,respectively.The two polysaccharides were homogeneous macromolecules.The molecular weight(M_W)of MPS-1 was 7.6 kDa,and the M_W of MPS-2was 6.7 kDa.The MPS-1 was composed of xylose,arabinose,galactose,glucose,and the mole ratio was 1:1.7:3.3:30.5;the MPS-2 was composed of arabinose,galactose,glucose,and the mole ratio was 1:1.3:36.8.The IR spectrum implied that onlyα-pyranose existed in MPS-1,and bothα-pyranose andβ-pyranose existed in MPS-2.4.Study on the anti-fatigue activity of BG and MPS.This study also investigated the anti-fatigue activity of the water-soluble components,benzylglucosinolate(BG)and polysaccharide(MPS)from maca,in forced swimming mice.In this work,the anti-fatigue activity in mice was measured using the forced swimming test,along with the determination of blood lactate(BLA),urea nitrogen(BUN),malonaldehyde(MDA),lactic dehydrogenase(LDH)activity,superoxide dismutase(SOD)activity and liver glycogen(LG).The mice were divided into 8 groups:high dose of MPS group,low dose of MPS group,aqueous extract of maca group,high dose of BG group,low dose of BG group,MPS+BG group,control group and positive control group.Both BG supplementation and MPS supplementation dose-dependently extended the swimming time;compared with the control group,the exhausting time of MPS high dose group and BG high dose group was increased by 140%and121%,respectively.Both BG supplementation and MPS supplementation dose-dependently decreased the BLA,BUN,and LDH activity level in mice.Additionally,the BLA level of high dose of BG group was same as that of the positive control group.MPS supplementation dose-dependently increased the LG level.The LG level of high dose of MPS group was 25%higher than that of positive control group.There was no significant difference in SOD activity and MDA content between each group,which implied that anti-fatigue activity of the water-soluble components from maca may not attribute to the antioxidant effect.Compared the maca aqueous extract group with BG+MPS group,no significant difference was observed in all the physical and biochemical parameters.Thus it indicates that the predominant anti-fatigue active components in maca aqueous extract are BG and MPS.The resuls indicated that the anti-fatigue activity of maca mainly attributed to the dreasing of BLA level and increasing of LG level;both MPS and BG could decrease the BLA level in vivo;MPS was the predominate substance in maca anquous extract to increase the LG level,however,BG showed no significant effect on increasing the LG level in forced swimming mice.5.Study on the anti-fatigue activity of MPS-1 and MPS-2.The anti-fatigue activity of MPS-1 and MPS-2 was measured by the forced swimming test,along with the determination of BLA,BUN,LDH activity and LG.The results indicated that both MPS-1 and MPS-2presented a dose-dependently positive effect on the fatigue related parameters.Additionally,the anti-fatigue activity of MPS-2 is better than that of MPS-1.