Studies On The Determinantion And Extraction Of The Polymethexylated Flavones From Orange Peels

At 2005,China had become the second biggest producer of citrus in the world,whose annual output exceeded 20.00 million tons in 2007.On the other hand,every year 500 thousand tons citrus peel might be brought from processing procedures in China,of which little amount could be reused for essential oil and feeds,but the most might pollute environment as waste without full utilization.Some studies confirm that:the polymethoxylated flavones(PMFs)is a sort of flavonoids only from citrus genus mainly existing in citrus peel,which owns many physiological functions such as anti-cancer,tumor inhibition,anti inflammation,anti-platelet aggregation,reducing cholesterol,antibacterial activity and so on,and is able to be regarded as marker material for variety identification,hybrid primary screening and checking adulteration.According to the important functions above,PMFs has been developed as functional food in US,having a good future.Therefore,study on content and distribution of PMFs in citrus peels and how to enhance extraction and purification technology of PMFs may result in important social and economic effects.In this experiment,the testing methods of NOB,TAN and SIN in orange peel by HPLC was established firstly;on the basis of testing methods,PMFs contents of 67 orange samples were analyzed from 10 countries and districts in Three Gorges reservoir area of Chongqing;the extraction technique of PMFs was optimized by response surface analysis,and cation exchange and macroporous resin were applied to study primary purification of crude extracts.The main results are listed as follows.1.Testing methods of NOB,TAN and SIN by HPLC.Firstly,samples were extracted by petroleum ether;the chromatogram conditions includes HPLC(Shimazu LC-6AD),C₁₈column(YWG,250×4.6mm, 10μm),the solution of 0.2%acetic acid and 80%acetonitrile as the mobile phase,gradient elution(speed: 1.0mL/min),diode array detector(DAD,330nm),10μL injection volume and 30℃column temperature. The results showed that:retention time of NOB,TAN and SIN ranged from 10.90min to 14.30min,the average recoveries were 96.07%-98.79%,the relative standard deviations were 1.91%～2.27%and the precision reached 1.27%-1.55%.Hence,this simple and fast method can satisfy the requirements of testing PMFs and quality control. 2.From the results of PMFs content analysis of Three Gorges reservoir area of Chongqing,NOB (0.219mg/g)was the main part(60.23%)in PMFs,the second was SIN(0.09mg/g)and the content of TAN(0.05mg/g)was the lowest.Effects of varieties on PMFs contents showed that:Trovita sweet orange had the highest PMFs level (0.60mg/g),which was 2.73 times of the lowest(Cara Cam navel orange),and the second highest content of PMFs existed in both Hamlin Orange and TongShui72-1 Jincheng(C.sinensis(L.)Osbeck) (0.44mg/g);besides,Trovita sweet orange owned the highest levels of NOB,SIN and TAN(0.38,0.12 and 0.10 mg/g,respectively)among all varieties,accounting for 63.33%,20.00%and 16.67%of the total PMFs respectively;the varieties of the lowest NOB and SIN contents were Cara Cara,Fengjie navel orange and Fuling orange(C,sinensis(L.)Osbeck).In ten sampling areas,NOB and TAN contents(0.26 and 0.07mg/g,respectively)in orange peels from Courntry Zhong were the highest,and for SIN(0.11mg/g)was from District WanZhou;the contents of PMFs in orange peels from Courntry Zhong(0.43 mg/g)and District WanZhou(0.41mg/g)were the highest.From different districts and counties in the same species,the results showed that the highest PMFs content in sweet orange(C.sinensis(L.)Osbeck)and Bonanza navel orange peels came from Courntry Zhong and the highest in Newhall Navel Orange came from District WanZhou,whose contents were 0.46, 0,40 and 0.37mg/g,respectively.3.Applying petroleum ether as extracting solvent,effects of solid-liquid ratio,extraction temperature,time and frequency on extracting rate of PMFs were investigated.According to response surface analysis,the optimum extraction processes of solid-liquid ratio(W/V),extraction temperature, time and frequency were obtained:1:13.35,57℃,5.3h and 3 respectively,and the theoretic extracting rate was 1.05‰(dried peel);on the optimum extraction processes,the yield rate of crude PMFs extracted from peel was 24.48g/kg(dried peel),and the factual extracting rate was 1.04‰,which was just lower 1.00%than the theoretic value.The regression equation was as follow: Y=0.097682-0.003013^*X₁+0.004683^*X₂+0.004180^*X₃-0.002062^*X₁^*X₁+0.001398^*X₂^*X₁-0.0067 70^*X₂^*X₂-0.002257^*X₃^*X₁+0.003725^*X₃^*X₂-0.002945^*X₃^*X₃4.The primary resin experiment showed that:Dowex cation exchange resin had the best results, whose adsorption amount and desorption rate reached 0.33mg/g and 92.11%respectively.The optimized conditions and process route of purifying PMFs by Dowex cation exchange resin were listed as follows: injecting concentration and flow speed were set as 0.30mg/mL,2.0mL/min,respectively;the eluent was 80%ethanol and the amount used was 400mL,purity was 92%.