Chemical Analysis Of Maca And The Mechanism Of The Neuroprotective Effect On Its Active Ingredients

Lepidium meyenii Walp.(Brassicaceae)namely maca,known as "Peruvian ginseng",has been introduced into China successfully and vigorously been promoted the cultivation in Yunnan,Xinjiang and Tibet regions.Different maca products appeared in the market.Because of different origins and varieties of maca.a plenty of inferior products of Maca disturbed the marke,which will greatly affect the healthy growth of maca industry in the long run.On the other hand,the basic study of Maca could not meet the requirement of market.Previously,people mainly focused on the nutrient compositions in Maca,such as proteins,amino acids and fatty acids.However,the secondary metabolites were mainly responsible for its multiple functions.The alkaloids,glucosinolates and macaenes and their pharmacological effects should be deserved close attention.In the present paper,the chemical profiling of maca and its neuroprotective effect in vivo and in vivo were investigated.Furthermore,the mechanism of macamides was preliminarily clarified.1.Isolation of maca ingredientsFirstly,the chemical constituents of maca were systemically isolated and 23 were identified including 7 macamides,2 common amide alkaloids,2 macaridines,1?-carboline alkaloid,3 glucosinolates,4 acids and 4 other compounds.Meanwhile.6 compounds were obtained from maca for the first time.2.Analysis of maca ingredientsSecondly,the chemical analysis of secondary metabolites in maca based on UHPLC-ESI-Orbitrap MS has been established in the present study.The fragmentation patterns of macamides,macaridines,imidazole alkaloids,(3-carboline alkaloids,glucosinolates and macaenes were summaried(the fragmentation patterns of macaridines and ?-carboline alkaloids were firstly clarified).Then,the use of Octadecylsilyl gel(ODS gel)remarkably increased chromatographic peaks,and thus achieved the enrichment of minor constitutes.The unknown compounds and their MSn fragments were calculated by accurate high resolution mass measurements.In 6 enriched fractions,160 ingredients were observed and identified,including 64 macamides,3 macaridines,38 imidazole alkaloids,16 p-carboline alkaloids,14 glucosinolates,5 acids,11 macaenes and 9 other compoundsFinally,The validated UHPLC-ESI-QqQ MS method was employed for the quantification analysis of 14 constituents(m-methoxybenzylglucosinolate,benzylglucosinolate,p-hydroxybenzylglucosinolate,N-(3-methoxybenzyl)-hexadecana mide,N-benzylbenzamide,N-benzylhexadecanamide,LicochalconeA,3-benzyl-1,2-dih ydro-N-hydroxypyridine-4-carbaldehyde,3-methoxybenzyl)-N-pyridine-4-carbaldehy de,N-acetylbenzylamine,2-phenylacetamide,Nicotinic acid,Succinic acid,(1R,3S)-1-methyl-p-carboline-3-carbaldehyde)in 17 batches of Maca under the DMRM mode.Among them,glucosinolates(10.97?79.84 mg·g-1)and alkaloids(0.54?2.99 mg·g-1)were the predominant constituents.What's more,constituents in maca from different places,varieties and different parts of this plant were compared.The results indicated that the content of active ingredients in peru had some difference compared with those cultivated in China.The content of glucosinolates and P-carboline alkaloids cultivated in peru was higher than that cultivated in China.However,the content of macamides,macaridines,acids and common amide alkaloids were higher cultivated in China than that cultivated in cultivated in China,which provided evidence that Maca cultivated in Yunnan province could be as the potential substitute for Maca cultivated in peru.Among the Chinese samples,the contents of secondary metabolites in Yunnan province were higher than those from Xinjiang and Tibet.The contents of secondary metabolites in yellow Maca was in line with those in purple Maca generally.The quality discrepancy of them was not as significant as propagandized in market,which needed further research.The contents of active ingredients in the roots were higher than those in leaves,which were correlated with the biosynthetic pathways of macamides.Furthermore,the leaves of maca contained a certain amount of glucosinolates and macamides,which laid foundation for the potential resources of Maca exploitation.3.Neuroprotective effect of macaIn present study,the zebrafish embryos were treated with MPTP to form the DA neuronal loss model,which was used for the neuroprotective evaluation of different fractions and pure compounds of maca.As a result,the total Maca methanol extract displayed neuroprotective effect against MPTP-induced toxicity in zebrafish.Fr4-Fr6 could increase the amount of dopamine neurons in different degrees with dose-dependent effect,especially for 80%methanol elution fraction(Fr5).Combination with chemical profiling analysis,common alkaloids and macaridines were predominant constitutes in 50%methanol elution fraction(Fr4).80%methanol elution fraction(Fr5)mainly contained imidazole alkaloids,macamides and macaenes.Macamides were also observed in 100%methanol elution fraction(Fr6).Interestingly,both Fr5 and Fr6 contained N-benzylhexadecanamide(A54)with high abundance.A54 as the representative pure compound for the effective confirmatory experiment at the dose of 30 and 10 ?g·mL-1 could significantly prevented the MPTP-induced decrease in TH+ region area,and showed neuroprotective actions in a dose-dependent manner.Then,the in vitro AChE and BuChE inhibition assays were preliminarily conducted in this study.Thus,maca showed neuroprotective activity through the synergistic effect of dopaminergic system and cholinergic system.In conclusion,Fr5 and macamides produced the healing efficacy might through increasing the acetylcholine and butyrylcholine level in brain.N-benzylhexadecanamide as the representative pure compound was confimed to possess neuroprotective effect in vitro and in vivo,which indicated that macamides were the main type of compounds in maca on neuroprotective effect.In order to investigate the protective effect of N-benzylhexadecanamide on MPTP-induced dopaminergic neurons in MN9D cells,the model of MPP + induced neuron loss in MN9D cells was eatablished.Though screening based on survival rate,the suitable concentration of MPP + was 500?M and the appropriate concentration range of N-benzylhexadecanamide was 50-100 ?M.After 500?M MPP+ on MN9D cells for 24 h,the cell survival rate decreased to about 50%.From the cell microscope,the changes in cell morphology could be also observed.the control group of MN9D cells mostly adhered to the wall and were enated,most of which also had protrusions gradually.After MN9D cells treated with 500?M MPP + for 24 h,the number of adherent cells decreased and protuberant disappeared.Thus,500?M was chosed as the model concentration.When the concentration of N-benzylhexadecanamide was 100 ?M,compared with the model group,the number of adherent cells was increased under inverted microscope,most of the cells had protrusive growth recovery and gradually extended outward.These findings suggested that N-benzylhexadecanamide possessed the neuroprotective effect on MPTP-induced dopaminergic neurons in MN9D cells.The content of neurotransmitters in MN9D cells and cell supernatant before and after N-benzylhexadecanamide on MN9D cells induced by MPP+ was measured by UHPLC-ESI-QqQ MS.The contents of neurotransmitters were relative quantified and absolute quantified 12 and 17 neurotransmitters in both cell supernatant and MN9D cells respectively.When N-benzylhexadecanamide was given to the MN9D cells for 48 h,the contents of neurotransmitters picked up,including mainly glutamine,taurine,norepinephrine,choline,acetylcholine,butyrylcholine in a dose-dependent manner in cell supernatant,mainly glutamate,norepinephrine,butyrylcholine,aspartic acid,homovanillic acid with dose-dependent relationship in MN9D cells.Serotonin and arginine in cell supernatant showed a decrease tendency after administration.For the rest of the neurotransmitters,N-benzylhexadecamide showed no obvious adjustment.Collectively,glutamate,norepinephrine and butyrylcholine both in cell supernatants and MN9D cells had significant differences in a dose-dependent manner after N-benzylhexadecamide was given to MN9D cells,especially the cholinergic neurotransmitters.The level of acetylcholine and butyrylcholine was associated with the results of the second part in this paper,and it was possible to regulate the dopaminergic system through the cholinergic syste as for the neuroprotective effect of N-benzylhexadecamide.The differential expression proteins of N-benzylhexadecanamide on MPP+injured MN9D cells were analyzed by LTQ-Orbitrap MS.The results showed that there were 142 differentially expressed proteins in a dose-dependent manner and increased after treatment with N-benzylhexadecanamide and there were 209 differentially expressed proteins in a dose-dependent manner and decreased after treatment with N-benzylhexadecanamide.Moreover,combined with ANOVA statistical test P less than 0.1 with Mev.4.9.0 software analysis,60 common differential proteins were determined in 4 groups.Furthermore,with the uniprot database about its functional annotation and according to the high abundance,34 different proteins were screened,involved in high percent of cellular processing,including autophagy,NF-?B signaling pathway,Toll-like receptor signaling pathway,MAPK signal pathway,EGFR tyrosinase inhibition Drug resistance,p53 signaling pathway and apoptosis.These finding suggested that the effect targets and signal pathways of N-benzylhexadecanamide was closely related to cellular processes and inflammation.Based on the UPLC-Q-TOF technology,the lipid metabolomics of N-benzylhexadecanamide on MPP+ induced MN9D cells was studied.The obtained data were introduced into the SIMCA-P13.0 software,then conducted orthogonal partial least squares discriminant analysis(OPLS-DA).The RSD values of each group were greater than 30%,Then the metabolite ions of VIP>1.5 were selected and P<0.05 were analyzed by t test,which were considered as significantly difference.Thus,differential lipid metabolites were identified when N-benzylhexadecanamide was given to MPP+ injured MN9D cells.111 differentially expressed lipids were identified in control group,the model group and N-benzylhexadecanamide group 114 and 115 respectively.108 common significant differential lipid molecules were analyzed after MPP+ injury and N-benzylhexadecanamide intervention in 3 groups.The results indicated that MPP+ nerve injury might be closely related to PC,PE,SM,PA,TG metabolism.In summary,the main secondary metabolites of maca were macamides,macaridines,imidazole alkaloids,?-carboline alkaloids,glucosinolates,macaenes and so on.The main type of compounds possessing neuroprotective effect in maca was macamides.On one hand,the mechanism of neuroprotective effect was through improving the levels of glutamate,acetylcholine,butyrylcholine,norepinephrine and other neurotransmitters in the brain.On the other hand,macamides regulated certain differences in lipid molecules and proteins.The differential proteins and lipid molecules with obvious biological significance still needed to be further verified to find out the metabolic pathways and targets of N-benzylhexadecanamide on neuroprotective effect.