Research On The Limonoids And Flavonoids In Ponkan-pit

This article takes Ponkan-pit as the raw material and establishes spectrophotometry as the rapid detection method of Limonoids and Flavonoids.Based on the detection technology,the extraction condition and macroporous resin’s separation and purification process of the two ingredients are explored.Finally,through high performance liquid chromatography-mass spectrometry technology,qualitative analysis of the two purifications are investigated.The main results are as follows:1. The spectrophotometry, as the content determination of limonoids and flavonoids in Ponkan-pit, is established. Taking Ehrlich reagent as the chromogenic agent to confirm that the limonin standard liquid’s maximum absorption wavelength is490nm, the establishment of the linear regression equation is Y=0.0037X-0.0370, correlation coefficient R2=0.9931; NaNO2-Al (NO2)3-NaOH is the flavone Chromogenic system, at the wavelength of510nm, rutin standard substance solution’s standard curve is Y=0.0121X+0.0147, R2=0.9995.2. The extraction, isolation, purification condition of limonoids in Ponkan-pit are investigated in this study.With soldium salicylate as the extraction solvent and limonoids yield as the inspection index, the extraction conditions are optimized through single factor test and Lp(34) orthogonal test. The optimal extractant concentration, extraction time, temperature, ratio of material to extractant are2.5mol/L sodium salicylate,6h,40℃,1:20(g/mL) respectively. The optimum rate of extraction of Limonoids is4.21%under the conditions above. The range value shows that the significant order of four factors is:ratio of material to extractant> extractant concentration> time> temperature; ratio of material to extractant is verified that has significant influence on the whole test under variance analysis.In the purification process, four kinds of macroporous resin:AB-8, NKA-9, D101, D4020were selected to compare adsorption rate, adsorption amount and desorption rate of limonoids. The results show that the limonoids adsorption rate (46.52%) and desorption rate (96.11%) of D4020macroporous resin are higher than that of the other three kinds of resin. D4020macroporous resin will be much more suitable for limonoids’ separation and purification. Through the D4020macroporous resin dynamic adsorption and desorption test, the optimal purification condition for limonoids were:adsorption velocity1.0mL/min, sample liquid concentration1.481mg/mL,5BV70%ethanol solution as eluent, desorption velocity1.0mL/min.3. The extraction, isolation, purification condition of flavonoids in Ponkan-pit are investigated in this study. With ethanol as the extraction solvent and flavonoids yield as the inspection index, the extraction conditions are optimized through single factor test and L9(34) orthogonal test. The optimal ratio of material to extractant, extraction time, ethanol concentration, temperature are1:25(g/mL),2.5h,80%,70℃, respectively. The optimum rate of extraction of flavonoids is5.81%under the conditions above. The range value shows that the significant order of the four factors is:ethanol concentration> ratio of material to extractant> time> temperature. Ethanol concentration is verified that has significant influence on the whole test under variance analysis.In the purification process, four kinds of macroporous resin:AB-8, NKA-9, D101, HP-20were selected to compare adsorption rate, adsorption amount and desorption rate of flavonoids. The results show that the flavonoids adsorption rate (58.29%) and desorption rate (79.71%) of AB-8macroporous resin are higher than that of the other three kinds of resin. AB-8macroporous resin will be much more suitable for flavonoids’ separation and purification. The dynamic adsorption and desorption test of AB-8macroporous resin indicate that the optimal purification condition for flavonoids are:adsorption velocity1.0mL/min, sample liquid concentration0.256mg/mL,5BV80%ethanol solution as eluent, desorption velocity1.0mL/min.4. Using Agilent1100Series LC/MS liquid instrument to qualitatively analyse the purifications of limonoids and flavonoids. In the limonoids’ analysis process, limonin and nomilin are selected as standard samples, through the chromatographic retention time and the comparison of standard and sample by mass spectrum ultimately, limonin and nomilin are found in the extracted limonoids sample. The retention time of them were27.0min,31.7min, respectively, the peak area of them are2754.19mAu,1634mAu, respectively. Moreover, the content of limonin is higher than that of nomilin.In the flavonoids’ analysis process, naringin, new hesperidin, hesperidin, hesperetin were selected as standard samples, through the chromatographic retention time and the comparison of standard and sample by mass spectrum ultimately, high amount of hesperidin was found in the extracted flavonoids sample, its retention time was21.8min, but the others were not detected. According to the literature, the molecular mass of nobiletin is580.53, so, nobiletin will be in the sample.