The Study Of Extraction, Isolation, Fingerprints Of Flavonoids From Citrus Maxima (BURM.) Merr. CV. Wentan

Citrus maxima (Burm.) Merr. cv. Wentan contains a variety of essential nutrients for human health and non-nutritional active ingredients. It's of health effects such as lowering blood pressure, lowering cholesterol, preventing atherosclerosis,antioxidant, promoting gastrointestinal motility, purgation and so on. There is also a wide range of content-rich flavonoids existed in the pericarp which has a large variety of physiological functions and medical value. In China, a few of pericarp produced by processing is used to take essential oils and used in Chinese medicine, but most as a waste, hadn't been rational used. This text is about the study of extraction, isolation, fingerprints of flavonoids from Citrus maxima (Burm.) Merr. CV. Wentan and differences among flavonoids from different kinds of Citrus maxima. It is to establish a scientific and reliable research method for Citrus flavonoids and provide more detailed information and evidence for the study of citrus flavonoids. Through the studying of fingerprints of Citrus maxima, different varieties of elementary flavonoids components from Citrus maxima can be obtained from the fingerprints, provide scientific and reliable data. The main contents and results now have been concluded as follows:(1) Exert microwave assisted technology in extraction of flavonoids, study the influence of the ratio of water to sample, ethanol concertraction, extraction temperature, extraction time, extraction power on the effect of extraction. Through response surface optimization method, obtain the most excellent conditions: extraction time 35 min, extraction temperature 55℃, ratio of water to sample 34.74, ethanol concertraction 70%, extraction power 500 W, extraction times 2. Flavonoids extraction rate measured is consistented with the predicted rate, extraction rate acheives to 0.805%.(2) With the liquid chromatograph, obtain the HPLC elution conditions of flavonoids from Citrus maxima (Burm.) Merr. cv. Wentan: mobile phase A- methanol, B- 0.1% solution of acetic acid (0-80min, A 0-100%, B 100-0%), rate of flow 1mL/min, detection wavelength 254 nm, volumn of sample injection: 5μL, Waters C18 column. The chromatograms of flavonoids from ten speices of Citrus maxima were analysed by software of analysis of similarity for traditional Chinese medicine. The contrasting fingerprint was built by that software. Eleven chromatogram peaks were mutual fingerprint peaks of ten speices of Citrus maxima, and also one of necessary condition for identification of Citrus maxima flavonoids. They can be identified as the basic components of flavonoids.(3) Crude extraction of flavonoids from Citrus pericarp in the butanol layer was separated and purified. The condition of isolation of flavonoids by HSCCC was as follows, n-hexane: butanol: methanol: 0.5% acetic acid = 1:3:1:4 (v/v), stationary phase was upper phase, the rate of rotation 850 rpm/min, the rate of flow of mobile phase was 1.5mL/min, and the injection ,volume was 500mg. Semi-preparative liquid chromatography was used to further separation,and the condition was: mobile phase A- acetonitrile, B- 0.5% acetic acid (0-5 min A 5-22% ,6-21 min A 22-60%, 21 -30min A 60-100%), running time 30 min, the rate of flow 5 mL / min, UV detected at 254 nm, Waters C18 column. An unknown compound was obtained , and its purity is 98%.(4) Unknown compound isolated was chemical identificated and structural analysed. Through the colour reaction and UV scanning, the unknown compounds could be determined as flavonoids. Through analysis by LC-MS, molecular weight of this unknown compound was 377, and determine initially the mass fragmentation pathway of the compound. By hydrogen nuclear magnetic resonance method, initially identifie the compound is a flavonol, and then further analysis of its structure was done.