Extraction And Purification Of Flavonoids From Three Chinese Herbs Based On UPLC Fingerprinting

Flavonoids,as one of the main components in Chinese herbal medicines,have multiple effects,such as anti-inflammatory,antioxidant and anti-tumor.Due to the complex chemical composition of Chinese herbal medicines,the flavonoid content in the crude extracts obtained by traditional extraction methods is low,and it is difficult to accurately derive the content of each flavonoid,which seriously affects the deep research and development of Chinese herbal medicines.In this study,20 flavonoids were selected as reference compounds,and UPLC chromatographic conditions were optimized to establish flavonoid UPLC fingerprint profiles.Three Chinese herbal medicines rich in flavonoids were used as the research objects,in which the total flavonoids were extracted and separated and purified.The qualitative and quantitative analysis of the characteristic flavonoids of the herbs was carried out using the established UPLC fingerprint profiles of flavonoids,with a view to providing reference for the future development and comprehensive utilization of flavonoids in Chinese herbs.The experimental results were as follows:(1)Twenty flavonoid compounds were selected as reference materials,and the column,detection wavelength,column temperature and mobile phase were investigated by UPLC method to establish the UPLC fingerprint profile of flavonoids.The optimized chromatographic conditions were as follows: Agilent EC-C18 2.7 μm(3×150 mm)column;mobile phase A: 0.1% phosphoric acid aqueous solution,mobile phase B: acetonitrile with gradient elution;flow rate of 0.6 m L/min;detection wavelength of 215 nm;column temperature of 35 ℃.The method was also investigated for stability,precision,linearity and spiked recovery.The results showed that the method is stable and reliable,and can provide a basis for the quality control of flavonoids in Chinese herbal medicines.(2)The effects of extraction solvent type,extraction time,extraction temperature and material-to-liquid ratio on the extraction rate of Sophorae tonkinensis total flavonoids were investigated by single-factor test using ultrasonic extraction method.The optimized extraction process conditions were: acetone extraction,extraction temperature of 50 ℃,extraction time of 1 h,material-liquid ratio of 1:15 g/ml,and the total flavonoid content reached 5.82 mg/g.By comparing the adsorption and resolution rates of six types of macroporous resins,AB-8macroporous resin was selected for purification,and the purity of total flavonoids ranged from 17.32% to 45.32% after the single-factor test with the resolution solvent of 70% ethanol and the optimal pH of 4.0.After further purification by polyamide resin,the purity of the Sophorae tonkinensis total flavonoids of santoprene reached 61.42%.The purified total flavonoids were analyzed by UPLC,and the total of five flavonoids were trifolirhizin,formononetin,maackiain 4’,7-dihydroxy-6,8-diprenylflavone,and sophoranone,at 15.97mg/g,22.53 mg/g,100.32 mg/g,13.98 mg/g,and 183.41 mg/g,respectively.(3)The effects of extraction solvent type,extraction time,extraction temperature and material-to-liquid ratio on the extraction rate of Belamcanda chinensis total flavonoids were investigated by single-factor test using ultrasonic extraction method.The optimized extraction process conditions were: 70% ethanol extraction,extraction temperature 60 ℃,extraction time 1.5 h,material-liquid ratio 1:20 g/ml,and the total flavonoid content reached 7.64 mg/g.By comparing the static adsorption and resolution parameters of six types of macroporous resins,D-101 macroporous resin was selected,and the purity of total flavonoids ranged from22.32% to 42.32% after the single-factor test with the resolution solvent of 70% ethanol and pH size of 5.0.After further purification by polyamide resin,the final purity of the Belamcanda chinensis total flavonoids of eugenol reached 62.96%.The purified total flavonoids were analyzed by UPLC and a total of five flavonoid compounds were tectoridin,iridin,tectorigenin,irigenin,and irisflorentin,at 106.3 mg/g,30.84 mg/g,23.36 mg/g,27.45mg/g,and 10.32 mg/g,respectively.(4)The effects of extraction solvent type,extraction time,extraction temperature and material-to-liquid ratio on the extraction rate of Desmodium styracifolium total flavonoids were investigated by single-factor test using ultrasonic extraction method.The optimized extraction conditions were: 80% ethanol extraction,60 ℃,1.5 h ultrasonic time,1:15 g/ml material-liquid ratio,and the total flavonoid content reached 2.32 mg/g.By comparing the static adsorption and resolution parameters of six types of macroporous resins,D-101 macroporous resin was selected,and the total flavonoid purity was increased from 11.32% to20.47% after the single-factor test with the resolution solvent of 80% ethanol and pH size of6.0.After further purification by polyamide resin,the purity of Desmodium styracifolium total flavonoids of eugenol reached 28.28%.The purified total flavonoids were analyzed by UPLC,and four flavonoids were Isovitexin,schaftoside,Isoschaftoside,and luteolin-6-C-glucoside,with the contents of 52.56 mg/g,41.25 mg/g,20.33 mg/g,and 10.72 mg/g,respectively.