Extraction,Enrichment And Preparative Separation Of Polymethoxyflavones From Citrus Fruit

Four citrus cultivars(Newhall sweet orange,Eureka lemon,Liangping pummelo and Dahongpao tangerine)were selected as meterials in this study to analyze their flavonoid compounds using ultra performance liquid chromatography-time of flight mass spectrometry(UPLC-Q-TOF-MS/MS).The peel of Dahongpao tangerine was used as material to extract polymethoxyflavones(PMFs)for its highest content of total polymethoxyflavones(TPMF)comparing with other three cultivars.The method of extraction,enrichment and purification of PMFs from the peel of tangerine were established.Five PMF monomers were simultaneously isolated by MS-directed preparative HPLC.The main contents are as following:(1)The main flavonoid components in the four citrus fruits were determined by UPLC-Q-TOF-MS/MS.16 major flavonoids,including seven flavanones and nine PMFs were identified.Validation of the UPLC-PDA method was carried out.The limits of detection and limits of quantitation were less than 0.7167 and 1.7917 μg/mL,respectively.The intra-day and inter-day precisions were 0.70%-1.07% and 0.38%-1.67%,respectively.The recovery rate was 92.95%-105.71%,which showed that the method is accurate and reliable.Based on this method,the distribution of flavonoids in different citrus cultivars,different sampling sites and different parts of fruits were analyzed.The results showed that the kinds of PMFs and TPMF content in Dahongpao tangerine were higher than other three cultivars.Moreover,PMFs were only detected in peels.(2)The extraction condition of TPMF was optimized by single-factor experiment and reponse surface methodology.Ultrasound-assisted extraction is more efficient than maceration method.The optimized condition of ultrasound-assisted extraction were ethanol concentration of 89.1%,solid-liquid ratio of 1:20,extraction temperature of 40.9℃,extraction time of 34.1 min,and extraction times of 2.Under this condition,the average extraction yield of TPMF in tangerine peel was 11.09 mg/g DW.(3)Eight macroporous resins with different physical properties(HPD100,HPD300,HPD400,HPD600,NKA-9,D101,DM130 and AB-8)were employed for enrichment and purification of TPMF in crude extracts.Static adsorption and desorption tests were performed,and the results showed that HPD-300 resin had better adsorption/desorption properties for TPMF among the test resins.The best purification parameters were as follow: the resin column was loaded with PMFs solution at a concentration of 8.70 mg TPMF/mL;the loading flow rate was 4.0 BV/h and the loading volume was 11 BV;then 90% aqueous ethanol was used as eluent,the elution flow rate was 4.0 BV/h and the elution volume was 4 BV.After purified by HPD300 resin,the content of TPMF in the crude extract increased from 31.720 ± 1.255 mg/g to 594.57 ± 28.05 mg/g.The adsorption kinetics and thermodynamics on HPD300 resin for TPMF were also investigated: the kinetics fitted the pseudo-second-order rate equation;its equilibrium adsorption data for TPMF was accordance with Langmuir adsorption isothermal equation,and the adsorption process is an exothermic process.(4)MS-directed preparative HPLC was used to separate and isolate different kinds of PMF monomer.The optimal liquid phase separation conditions established was as follows: XBridge Prep RP18 preparative column(19 × 250 mm,5 μm)was chosen;the mobile phase were 0.1% formic acid/acetonitrile(A)and 0.1% formic acid/water(B);the flow rate was 20 mL/min and elution gradient was 0-6 min,18%-30% A;6-35 min,30%-42% A;35-45 min,2%-60% A;45-50 min,60%-18% A.The structures of the isolated monomers were identified by UPLC-Q-TOF-MS/MS and nuclear magnetic resonance(NMR).The five PMF compounds were 5,6,7,4’-tetramethoxyflavone,nobiletin,tangeretin,sinensetin and 5-hydroxy-6,7,8,3’,4’-pentamethoxyflavone,and their purities were all above 95.3%.