The Extraction, Purification And Structural Identification Of Flavonoids In Raspberry Leaves Of Litte Berry

The raspberry, which is planted more and more now, is an important plant resource and existedin mountainous area in north of our country. It is not only one delicious fruit but also widely usedfor the process of cosmetic and health care food, because the root, stem and leaves of raspberry arealso be used for medication. It is reported that the flavonoids in the raspberry fruits and leaves havethe ability of antioxidant and scavenging hydroxyl radicals. This experiment used the leaves ofraspberry as material, in order to explored the process of extraction, purification and structuralidentification of the flavonoids. All this done is aimed for providing theoretical basis and somescientific message for developing and utilizing raspberry resource.The two methods, the dual wavelength spectrophotometry and HPLC, were established for thequantitative determination of the flanovoids in the raspberry leaves. It is made certain by spectrumScanning that the experimental conditions of dual wavelength spectrophotometry were asfollow, detection wavelength 506nm, reference wavelength 572nm. It is proved that this method wasable to overcome the disturbance of the strong absorbance of solvent. HPLC was applied toquantitative determination of trigonelline in pumpkin flesh. The experimental conditions were asfollows, the Agilent-1100 C18 column, mobile phase methanol: 0.4% phosphoric acid(55:45), flowrate 1.0ml/min, and detection Agilent-G1314A uv detector.In this experiment the process of flavonoids extraction is explored. The flavonoids inraspberry leaves was extracted by enzymatic hydrolysis of Cellulase and alcohol extraction. Thebest method of alcohol extraction were obtained, namely extracted for 2h by using 65% ethanolsolution at 75℃with the material ratio of 1:20g/mL and 0.35mg/mL enzyme solution concentration.The content of flavonoids was 3.46%.In this experiment, the macroporous adsorption resin and chromatographic methods wereapplied to the process of purification. The technologies were established by the choose ofmacroporous adsorption resin, the research of effects on adsorption and elution. The appropriateconditions were as follows, AB-8 macroporous adsorption resin, pH=6, the concentration1.5834mg/mL, elution solvent 70% alcohol solution, elution flow rate 1.5mL/min. This method wasproved to be with purification result and elution efficiency of 45.62%.The purified extraction was extracted by ethyl acetate, the ethyl acetate extraction was mixedwith silica gel, dried in vacuum and encased in column. The chloroform-methanol (100:0-80:20) was chose as elution solvent. The elution solution was examined by paper chromatography, inwhich normal butanol-water-acetic acid(4:5:1) was chose as the spread solvent. The hydrolysis partof the elution purified by silica gel was detected by HPLC. The results of HPLC showed that themain components of the hydrolysis part were quercetin and kaempferol and the contents wererespectively 186.4μg/g and 52.9μg/gThe Sephadex LH-20 column was used for purification of the hydrolysis part, 50%～90%alcohol was chose as elution solvent, and elution flow rate was 1.0ml/min. At last we got twocompounds by the detection of TLC and HPLC. Lastly, we determined the purity of the twocompounds by melting point test, TLC and HPLC. The two compounds were identified by UV andNMR methods. It is made certain that they were respectively quercetin and kaempferol.