| Macamides and macaenes are special substances to Maca,which are considered to be its unique iconic ingredient.Their content is used to evaluate the quality of Maca.In recent years,ultra–high performance liquid chromatography mass spectrometry(UPLC–MS)has been widely used in the analysis of macamides and macaenes.Macamides has a variety of activities,such as improving fertility,improving sexual function,promoting cell proliferation,anti–osteoporosis,nerve protection,antioxidant,anti–cancer and other biological activities;while as far as we know,the activity of macaenes has not been independently reported.Therefore,it is important to separate macamides and macaenes and separately study their biological activities.In this thesis,we report the qualitative and quantitative analysis of macamides and macaenes from Maca by UPLC–MS;the separation and analysis of crude extracts from Maca;the evaluation of their biological activities;the study of tannic acid(TA)as a new calibrator suitable for electrospray ionization mass spectrometry(ESI–MS).The detail contents of the thesis were as follows:(1)UPLC–MS makes a good combination of separation,qualitative and quantitative analysis.In this thesis,a novel UPLC–MS method for equal degree elution was established to detect of macamides and macaenes in Maca.The feasibility of this method was evaluated by precision test,stability test of sample solution,linear relationship,detection limit,quantitative limit,repeatability test and spike recovery test.The result suggests that the precision of UPLC–MS met the requirement of this experiment,sample solutions indicated good stability,this method established has a good linear relationship between the peak area and the concentration,and this method showed a good repeatability and accuracy.This method was used to quantitatively analyze macamides and macaenes in Maca with different color and from different origins.(2)The crude maca extract(MCE)was extracted in order with the solvent of mineral ether and ethyl acetate,separated and purified with eluent of mineral ether—ethyl acetate by silica gel column chromatography.Total macamides(TMM)and total macaenes(TME)were characterized by UPLC–MS,infrared absorption spectroscopy(FT–IR),and ultraviolet–visible absorption spectroscopy(UV–Vis).The results show that TMM and TME are well separated by silica gel column chromatography.(3)The antioxidant activity of MCE,TMM and TME was evaluated by DPPH radical scavenging,ABTS radical scavenging and reducing power.The ability of MCE,TMM and TME against multiple cancer cell lines(leukemia HL-60,lung cancer A594,liver cancer SMMC-7721,breast cancer MCF-7,and colon cancer SW480)were used to evaluate their anticancer activity by MTS method(3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazoliu m).The results show that TMM has the highest free radical scavenging ability and reducing power,and TME has the weakest antioxidant activity among them;TMM also has a good inhibitory effect on five cancer cells(leukemia HL–60,lung A549,liver cancer SMMC–7721,breast cancer MCF–7 and colon cancer SW480).N-benzyl–(9Z,12 Z,15Z)–octadecatrienamide(NBOT)was the most cytotoxicity inhibitory activity among those of them.(4)The ESI mass spectrum of TA in the positive ion mode shows a normal distribution in the range of mass-to-charge ratio(m/z)371.0368–1739.1169.Based on this fact,using TA as the ESI–MS calibrator,the m/z of 4 macamides and 2 β–cyclodextrin were detected.Compared the relative error of them with TA calibrator with that with sodium formate(SF)and Prod#88323 calibrator,the relative error of molecular weight by TA are no more than 3.00 ppm,which is less than that by SF and Prod#88323 calibrators.The discovery of this calibrator provides a scientific basis for the accurate detection of compounds with m/z<2000. |
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