| With the constantly increasing of pressure and the pace of modern life,people maybe accompany with different degree of fatigue symptoms.Moreover,long-term fatigue would occur immunity lessening et al,and further bring to sickness.Nowadays,as people are ongoing focusing on fatigue on behalf of sub-health status,government is vigorously promoting C hinese medicine modernizing,and exploring new medicinal substances with safety and low side effects from plants and microbes,which is current hot spot of scientific research and industrial development.maca(Lepidium Meyenii Walp.),containing rich nutrition and belonging to cruciferous plant,has been identified as new resources food in the early 2011.Amide compounds only found in maca,are widely considered as material basis for pharmacodynamics.According to literature,crude extract of maca in different solvent,both contain lipophilic active ingredients with the reputation of enhancement of sexual behavior and inhibiting fatty acid amide hydrolase(FAAH).Due to low content,varieties and structural similarities of macamides in maca,so the current study of maca has been focusing on discovery,quantitative or qualitative analysis and pharmacological efficacy of macamides.Even,the relative products attent to rough machining and crude extracts of maca.In order to further determine material basis and mechanism for maca pharmacodynamics,we personally think that traditional extraction and synthetic methods of macamides are only way of deep research and development of maca,this study came up with method about synthesizing macamides.In short,this project aimed to established the optimized extraction progress,and scheme out new method for synthesis and purification macamides.As well,setting up a vivo model evaluate N-benzyl-hexadecanamide about relieving physical fatigue,and providing new ideas with developing maca relative products.Objective: To establish the optimized experimental progress for extracting macamides from maca,and explore simple method for enriching macamides;to established the optimized synthetic and purification progress of five main macamides presented in maca;to established the mechanism of N-benzyl-hexadecanamide in relieving physical fatigue in vivo model.Methods In this study,macamides in maca powder extracted with ultrasonic,and firstly providing with acid-base reaction to rich macamides and reduce the main impurities(fatty acids).To optimize extraction progress through orthogonal experiment.Some extraction factors were solid-liquid ratio,ultrasonic time,solvent type,sodium hydroxide addition and reaction time et al.Traditional synthetic method of amide bond,calling carbodiimide condensation method,was employed to further explore the synthetic process of main macamides.A general method for amide synthesis involves activation of a carboxylic fatty acid moiety(linolenic acid,linoleic acid and stearic acid)with carbodiimide condensation agents(CCAs),followed by reaction with an amine to generate the corresponding amide.Acylation catalyst of amide bond and acid-binding agent were used to speed up synthetic reaction.And the method of acid-base reaction,recrystallization and silica gel column were utilized to increase purity of macamides.At last,synthetic product should be necessarily analysed using a combination of HPLC-DAD,MS,1H NMR,13 C NMR,and IR to identified structural information of macamides.To confirm macamides as materials base of maca pharmacology,C57 mice were given by gavage N-benzyl-hexadecanamide,which belongs to amide compounds.In the end,change of body weight and weight loading swim time of mice was observed at different time points,and detecting content changes of liver glycogen,urea nitrogen,lactate dehydrogenase,blood amine and blood lactic acid,to explore simply mechanism of N-benzyl-hexadecanamide.Results 1.Study on Optimization of Extraction Process of the Main Macamides from Maca Macamides was the active marker compounds of maca powders.Macamides was gathered from maca powders through acid-base reaction firstly,which could remove the main impurities such as fatty acid and so on.Then extraction process was optimized.The preliminary results presented that the optimum conditions of supersonic extraction of macamides as follows: the best extraction solvent was diethyl ether,ratio of material to solvent was 1: 30(g/m L)and time of extraction was 30 min;the optimal acid-base reaction conditions was that reaction temperature of 50 ?,solid-liquid ratio of 1: 5(g/m L)and reaction time of 50 min.Under optimized conditions,the content of the macamides in maca extract could reach to 20.79 %,compared with the extraction method in THE MACA DRY PRODUCTS QUALITY STANDARD(DBS 53/001—2015),it increased by 73.9 %.2.Synthesis and identification of the five main macamides present in maca(Lepidium meyenii Walpers)using a combination of HPLC,MS,1H NMR,13 C NMR,and IR HPLC-DAD,ESI-MS,IR,and NMR(1H and 13C)were also employed to synthsize the five macamides,whose names are N-benzyl-hexadecanamide,N-benzyl-9Z,12 Z,15Zoctadecatrienamide,N-(3-methoxybenzyl)-9Z,12Z-octadecadienamide,N-benzyl-9Z,12 Zoctadecadienamide,N-(3-methoxybenzyl)-9Z,12 Z,15Z-octadecatrienamide,with final purities of 97.63%,95.71%,96.21%,95.13%,and 95.92%,respectively.3.the effect of N-benzyl-hexadecanamide on relieving physical fatigue in mice C57 mice were given with N-benzyl-hexadecanamide by gavage once a day for 30 days.In the end,change of body weight and weight loading swim time of mice was observed at different time points,and detecting content changes of liver glycogen,urea nitrogen,lactate dehydrogenase,blood amine and blood lactic acid using sensitive detection kits.It appeared that experimental group compared with blank group,weight loading swim time and liver glycogen content were significantly higher,and lactate,urea nitrogen,ammonia et al were obviously lower.Conclusion Macamides presented in maca,as one of most weakly polar amide compounds,were widely identified as material base for pharmacology with broad market prospect.Due to special physicochemical property,diethyl ether was optimized extraction solvent,which could immensely rich five main macamides in maca.As well,acid-base reaction was utilized to purify products and reduce main impurities.Carbodiimide condensation method was effective method for synthetic macamides,which should need some purification progresses including acid-base reaction,recrystallization and silica gel column.In the pharmacology,N-benzyl-hexadecanamide is one of material base for maca pharmacology,which could significantly improve the time of mice loading swim ming and content of liver glycogen. |
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