Studies On Extraction, Isolation And Purification Of Flavonoids From Citrus Peel

Citrus fruit is one of the biggest yield of fruit in the word and has important economic value,peel is their mainly byproduct and has 20％～40ï¼…output in the total citrus fruit. Citrus peel isrich in many biologically active compounds and good sources of flavonoids. Flavone drugs havethe effects of anti—cerebra ischemia, anti—myocardial ischemia, anti—cardiac amhyth,analgesic, protecting liver, protecting alimentary ulcer, anti—virus, anti—cancer, and so on.Otherwise, many researches indicate that flavone natural drugs have the pharmacologicals ofblood pressure depressing, lowering blood lipide, inhibiting platelet aggregation. So it was takeninto account by governments, pharmaceutical and food industries, and the researches of flavonenatural produce in food. The extraction of flavone natural produce was the key point of itsresearch and development. Extraction, purification, separation and identification of flavonoids incitrus peel were studied in this thesis for better developing this resource. The main results weredescribed as follows:1,Did establish a method which acted rutin as standard sample to measure determination oftotal flavonoids in citrus peel. Meanwhile, the contents of the total flavonoids in citrus peel thatdisposed through different conditions were measured and compared and analyzed by using theultraviolet—visible spectrophotometry with three methods. The results showed that, of the threemethods, the concentration of the total flavonoids was the highest by using the Al(NO₃)₃ (345nm), and the coloration method with direct measurement (270 nm) and AlCl₃ (415 nm) took thesecond and the third place. The method of assaying the total flavonoids was determined by theultraviolet—visible spectrophotometry with AlCl₃ (275 nm) system after being studied by HPLC.2,The influences of extraction time (X₂), extraction temperature (X₄), pH value (X₃) and theratio of cellulase to material (X₁, w/w) which affiliated with the enzyme—assisted extractiontechnique of total flavonoids from citrus peel on the yield (Y) were studied according to thecentral composite design in order to make sure the optimized extraction conditions. Onemathematical model (Y=-17.41531250+7.14583333X₁+0.11956250X₂+2.52125000X₃+0.25337500X₄-6.36458333X₁^*X₁-0.00064896X₂^*X₂+0.00862500X₂^*X₃-0.32958333X₃^*X₃-0.01275000X₃^*X₄-0.00204583X₄^*X₄) established and analyzed by response surface methodology (RSM)was adequate to describe the relationships between the studied factors and the response of totalflavonoids extraction yield. Based on the canonical analysis, the optimum conditions wereobtained as follows: the additived ratio of cellulose was 0.55ï¼…, the time was 123 minutes, pHvalue was 4.4 and the temperature was 48℃. With the technological conditions, the extractionrate was (3.51±0.13)ï¼…(n=3), which was close to the estimated value 3.621669ï¼…attained byusing regression model. This model can be used to predict the experiment results well.3,The total flavonoids in citrus peel were extracted by microwave treatment. The effects ofaverage microwave output power, liquid—solid ratio, irradiation time, pH value and extractionfrequency on the extraction yield were investigated. And the orthogonal test design L₁₆(4⁴) was used to optimize the conditions of microwave treatment. The optimum conditions of microwavetreatment were as follows: microwave output power 400 W, irradiation time 180 seconds, theliquid—solid ratio 50, pH value 5. Under the best extraction conditions whose frequency ofextraction was 2, the total content of flavonoids was 3.37%.4,Ultrasonic wave—assisted extraction technique of total flavonoids (Y) from citrus peel wasstudied according to a current, rotational and combinational design of quadratic regression. Then,one quadratic regression model (Y=-10.20174246+0.19594606X₁+1.32168206X₂+0.19143333X₃+0.00024257X₁^*X₄-0.00125581X₂^*X₄-0.00144333X₁^*X₁-0.10808333X₂^*X₂-0.00363833X₃^*X₃-0.00006954X₄^*X₄) for the total flavonoids yield was established, the extraction conditionswere optimized by using above model and the optimized results were verified by experiments.Results shows that the pH value had a very significant effect on the extraction rate of totalflavonoids. Ethanol concentration (X₁), the ratio of solution to material (X₃) and the interactionbetween time (X₄) and ethanol concentration, also time and pH value (X₂) had significant effectson the extraction rate. Extraction time had no significant effects on the extraction rate. Researchresults indicate that the optimized extraction conditions for obtaining maximum yield of totalflavonoids are pH value 5.6, ethanol concentration 75%, ratio of solution to material 26,extraction time 80 minutes. Under the optimized conditions, the experimental value of yield was(3.314+0.063)% (n=3), which was close to the estimated value 3.348449% attained by usingregression model. The model for the total flavonoids yield have better prediction effects.5,An RP—HPLC method was established for the determination of hesperidin and naringin inthe citrus peel under different extracted conditions. Hesperidin and naringin were separated on anODS C₁₈ column (150 mm×4.6 mm i.d.) with methanol—water (volume ratio of 40/60) as themobile phase and detected at 270 nm. The flow rate was 1.0 mL/min. The column temperaturewas set at 25℃. The calibration curves were linear in the ranges of 0.007168 g/L～0.28 g/L and0.012288 g/L～0.48 g/L for hesperidin and naringin respectively. Determination of hesperidinwas 17.5～21.9 mg/g (RSD=2.2～3.1%) and naringin's determination was 2.14～2.39 mg/g(RSD=2.8～3.7%). The average recoveries were 96.5～101.4% (RSD=2.3～3.3%) forhesperidin and 97.3～103.6% (RSD=2.8～3.6%) for naringin. The method is simple, rapid,accurate and reliable.6,The powder of citrus peel was extracted by 95% (V/V) and 70% (V/V) ethanol. Theextraction was successively partitioned with petroleum ehter, ethyl acetate, n—butyl alcohol.Then the three fractions were separated by silica, polyamide, Sephadex LH—20 columnchromatographies, thirty—six chemical constituents were isolated.