Studies On Extraction, Purification, Isolation And Identification Of Flavonoids From Rough-peel Mandarin Peel

Rough-peel mandarin is the local varieties of Citrus. Flavonoids, which has the function of anti-inflammatory, decline blood pressure, antioxidant and anticancer etc, is one of the main active ingredients of Citrus. Extraction and isolation of flavonoids from rough-peel mandarin peel has great theoretical and practical significance for rough-peel mandarin application in deep-processing and pharmaceuticals, food industry and other fields. In this paper, the technological conditions of extraction and purification of flavonoids from the peel of rough-peel mandarin were studied. Then two flavonoids were isolated by high speed counter-current chromatography. The major flavonoids composition and its content in rough-peel mandarin peel were determined using HPLC. The main experimental results were as follows:1 Determination of total flavonoids content in rough-peel mandarin peelA pratical way of determining the content of flavonids using spectrophotometry was founded, which was depended on forming its complexes with certain metals. The method was simple and fast, it provided a rapid and effective evaluation method to explore the best exraction and purification process.2 Extraction of total flavonoids from the peel of rough-peel mandarinUse two ways of extracting flavonoids from rough-peel mandarin peel by single factor and variance experiment. The optimum technique conditions by alcohol soaking cycle extraction were determined as follows : the material size was 20 mu, the concentration of alcohol was 40%, the ratio of material and alcohol was 1:30, the temperature was 70℃, the time of extraction was 60 minutes, the extracted ratio was 1.54% under the technology parameters. The optimum technique conditions by ultrasonic ware extraction were determined as follows: the material size was 40 mu, the concentration of alcohol was 50%, the ratio of material and alcohol was 1:50, the temperature was 50℃, the time of extraction was 20 minutes, extracted twice under the technology parameters, then the extracted ratio was 1.52%.3 Purification of total flavonoids from the peel of rough-peel mandarinBy comparing static adsorption and desorption property of flavonoids crude extract from rough-peel manderin peel with 4 macroporous resins, AB-8, which had higher adsorption capacity and easier desorption, was the most appropriate resin.The purification of flavonoids crude extract was studied, the optimum conditions were as follows: the concentration of flavonoids crude extract was 2.9mg/mL, the time of static absorption was 30 minutes, the eluting solvent was 50% alcohol, the flow rate of eluting solvent was 1.5BV/h, the volume of alcohol was 3BV. Under this condition, the purity of total flavonoids was 15.45%, and AB-8 resin enrich mentamount were 2.41 multiple.4 Isolation and preparation of flavones from the peel of rough-peel mandarinHigh speed counter-current chromatography was successfully used for the isolation and purification of flavones from the peel of rough-peel mandarin. A two-phase solvent system composed of ethyl acetate-ethanol-water(4:1:5,v/v) was used, the upper phase was taken as the stationary phase, and the lower phase as the mobile phase. The mobile phase was operated at a flow rate of 2.0 mL/min, while the apparatus rotated at 700 r/min and the effluent was detected at 280 nm. From the crude extracts, neohesperidin and naringin were obtained with a purity of 98.95% and 98.67%, respectively, as determined by high performance liquid chromatography. The structures of the isolated compounds were identified by ¹H-nuclear magnatic resonance(NMR) and ¹³C-NMR.5 Analysis of major flavonoids composition and its content in rough-peel mandarin peelThe major flavonoids composition and its content in rough-peel mandarin peel were studied using high performance liquid chromatography, comparison with citrus reticulata bianco, citrus sinensis and citrus grandis. The results showed that rough-peel mandarin peel included the neohesperidin, naringin and hesperidin. The major flavonoid was neohesperidin, whose content was 10.79mg/g.DW, followed by naringin, hesperidin, whose contents were 1.32mg/g.DW, 0.43mg/g.DW, respectively. The neohesperidin was not detected in the peels of citrus reticulata bianco, citrus sinensis and citrus grandis.