Extraction,Separation,Purification,Identification And Determination Of The Polyphenols From Garlic Peel

In order to make full use of garlic peel resources,the response surface method was used to optimize the extraction temperature,extraction time and other process parameters of garlic peel polyphenols.The solid phase extraction and polyamide column chromatography were used to separate and purify the crude garlic peel polyphenols.Using High resolution mass spectrometry(HRMS),High-performance liquid chromatography-High-resolution mass spectrometry(HPLCHRMS)methods,six phenolic substances in garlic peel were identified,and High-performance liquid chromatography(HPLC)quantitatively analyzed the six phenolic substances in garlic peel.The main findings are as follows:1.The response surface Box-Behnken test was used to optimize the extraction conditions of garlic peel polyphenols,and the optimal extraction conditions were 50% ethanol concentration,61 ? temperature,1.7 hour,and material-to-liquid ratio 1:15(g/m L).Under these conditions,the extraction amount of polyphenols from garlic peel is 81.5 mg/kg2.The solid phase extraction method was used to separate and purify the crude polyphenols from garlic peel.The optimized impurity removal conditions were: 6 m L of water and 6 m L of 10% methanol were used to wash the DPA-6S solid phase extraction cartridge with a volume of 4 m L;and with polyamide thin-layer chromatography,the optimal concentration of methanol in the eluent is 70%.3.Using polyamide column chromatography to separate and purify the crude garlic peel polyphenols,solid phase extraction method to further separate and purify the column chromatography products.Under the HPLC gradient elution method,obtain all the elution peaks from the eluate which collected by eluting with 40% and 50% methanol.Using HRMS to determine the elution peaks,and qualitatively determine the guaiacylglycerin-?-caffeic acid ether(GCAE)and guaiacylglycerin-?-ferulic acid ether(GFAE)in the garlic peel according to the molecular weight,secondary spectrum and references documents.4.Using HPLC-HRMS to qualitatively determine the phenolic substances in the garlic peel.The only molecular formula of an unknown substance is determined to be C9H8O4,and its retention time is consistent with the retention time of the standard sample of caffeic acid and mass spectrometry data.According to the analysis of biological metabolism mass spectrometry,it is confirmed that the substance is caffeic acid,this is the first time that caffeic acid has been detected in garlic peel.5.The HPLC-HRMS method combined with the corresponding standards was used to qualitatively determine the N-trans-coumaroyloctopamine(CO),p-coumaric acid,and ferulic acid in the garlic peel.6.The optimized HPLC gradient elution conditions for the determination of 6 phenolic substances are: mobile phase A: volume fraction of 0.1% formic acid aqueous solution;mobile phase B: acetonitrile;gradient elution program: 0 min,20% B;5 min,22%B;20 min,22%B;22 min,24%B;24 min,50%B;26 min,100%B;28 min,50%B;30 min,20%B;run after 10 min.flow rate: 0.5 m L/min;column temperature: 40?;injection volume: 5.0 ?L;UV detection wavelength 320 nm.7.Under optimized chromatographic conditions,the components of caffeic acid,GCAE,CO,p-coumaric acid,GFAE,and ferulic acid in garlic peel were well separated,the correlation coefficients were all 0.9999,and the limit of detection(LOD)was 0.33~0.66.mg/L,the limit of quantification(LOQ)is 1.26~2.32 mg/L,the recovery of standard addition is 82.79%~100.58%,the relative standard deviation(RSD)is less than 1%,and the retention time is 12.301,16.810,18.682,19.410,19.895,22.385 min respectively.8.The external standard method was used to quantitatively determine the caffeic acid,GCAE,CO,p-coumaric acid,GFAE,and ferulic acid in the garlic peel.Among them,caffeic acid and ferulic acid are used as references for GCAE and GFAE respectively,that is,they are quantified by the correction factor twice of caffeic acid and ferulic acid and the peak areas of GCAE and GFAE.After determination,the contents of caffeic acid,ferulic acid,p-coumaric acid,and CO in garlic peel were 4.99,7.27,2.96,9.15 mg/kg,and the contents of GCAE and GFAE were 32.89,8.11 mg/kg(calculated by caffeic acid and ferulic acid respectively),the total content of 6 phenolic substances is 65.37 mg/kg.