Extraction,Separation,Purification,Identification And Anti-Oxidative Activity Of Flavonoids In Jackfruit Peel

Jackfruit, known as "rare fruit", is one of typical tropical fruits in southern China due to it's rich carbohydrate, protein, vitamins, minerals and other essential nutrients of human body. Jackfruit is huge in volume but inedible part of peel and fruit tendon accounted for almost half of the fruit quality.By-products like wasted peel can not be fully and effectively utilized in the process of production and resulting in a great waste of resources.Therefore, studying the deep processing and utilization of jackfruit's Peel is an urgently solution for improving the jackfruit's comprehensive production capacity and economic benefit.Flavonoids are secondary metabolites of plants which are widely distributed in nature.Lots of studies have showed that flavonoids have antioxidant, anti-bacterial anti-inflammatory properties and improving blood circulation as well.Meanwhile flavonoids have been widely used in food industry, medicine, cosmetics and other industries as a kind of natural pigment, natural antioxidant and antibacterial agent.In this study, the extraction, separation, purification and functional activity of flavonoids from jackfruit peel were discussed so as to provide a broader development and application of jackfruit research and lay a theoretical foundation for the application of natural antioxidants based on jackfruit peel.This paper takes the jackfruit peel as raw material and ultrasonic assisted extraction methods.The extraction process was optimized by response surface method on the basis of single factor in order to get a higher yield of flavonoids crude extract from jackfruit peel followed with extraction by ethyl acetate water solution and enrichment was carried out later.Macroporous resin column chromatography and silica gel column chromatography were used to separate and purify the flavonoids in the two phases and identify the purified product's structure by use of UV, IR, MS, NMR and other methods, and vitro antioxidant activity was evaluated by DPPH, OH free radical scavenging rate and reducing power, research results are as follows:(1) Flavonoids weree extracted from jackfruit peel by ultrasonic assisted solvent extraction.The effects of factors such as solvent concentration, solid-liquid ratio, extraction temperature, ultrasonic time and ultrasonic power on the extraction rate of flavonoids were investigated and the extraction process was optimized by Box-Behnken combination experiment design.Results showed that: effect of various factors on the extraction rate of flavonoids were presented in sequence: concentration of solvent> ratio of material to liquid>extraction temperature> ultrasonic power.The optimum extraction condition was as follows: 61% solvent concentration, solid-liquid ratio 1:25, extraction time 24 min, extraction temperature of 45?, ultrasonic power 300 W.Under these conditions, the extraction rate of flavonoids was 6.9213% and the relative error between the theoretical prediction and the experimental results was 1.55%.(2) Macroporous resin was used to purify the total flavonoids in the aqueous phase of jackfruit skin extraction and chose NKA macroporous resin as the best separation and purification resin from 6 different polarity of macroporous adsorption. The purification process conditions were studied based on further investigation on the adsorption properties of NKA resin.Results showed that:NKA resin was fast balance type resin and the best sample concentration of column chromatography was 0.9mg/m L, the volume was 200 m L, the dosage of elution agent was 120 m L, and the amount of washing agent was 200 m L. The optimum purification process was obtained when the sample flow rate and elution flow rate were 4m/min, sample p H was 4, the elution agent was 70% ethanol through single factor and orthogonal test.Under these conditions, the recovery rate of total flavonoids from jackfruit peel could reach up to 96.02%.The sample purity before and after the column was compared and analyzed.Results showed that the purity of total flavonoids from the peel of jackfruit increased from 16.74% to 84.42%, increased 5.04 times.Therefore, the use of NKA resin for the enrichment and purification of total flavonoids in the jackfruit peel aqueous phase has a significant effect.(3) Separation and purification of ethyl acetate phase total flavonoids from jackfruit fruit peel extracts by silica gel column chromatography.TLC thin layer chromatography was used to screen out the petroleum ether ethyl acetate as the silica gel column chromatography elution agent system.The A, B, C, D, E five series elution fractions were separated by silica gel dry-packing column and petroleum ether ethyl acetate 10:1~5:1~2:1~1:1~1:0 gradient elution.By TLC point plate and liquid phase analysis, the low polar fraction of ethyl acetate phase mainly existed in A,B and C flow,however, the content of flavonoids was less and in various forms which were dispersed in D and E flow.Through the preparation and collection of the liquid phase, I and II were isolated and purified from the A,B and C elution series,III?IV?V?VI were isolated and purified from D and E elution series.(4) The purified product in the aqueous phase of jackfruit was analyzed by the methods of specific color reaction, UV, IR, HPLC-MS and NMR.Results showed that the molecular framework of flavonoids substances in aqueous solution may be constructed by 7,4 ', 5'- trihydroxy flavanone alcohol as a unit of aggregation and connected with oligomeric flavanol of ?-D- galactose sugar- O-?-L-rhamnoside.The relative molecular weight was 866.1966, the possible molecular formula was C45H38O18 and the degree of saturation was 27.Further studies should be conducted to analyze the specific structure due to large molecular weight, complex structure, product polarity and poor of NMR spectrum.The structure of the purified product from the ethyl acetate phase of jackfruit peel was found that I and II purified by MS and NMR analysis were palmitic acid(C16H32O2)and dibutyl phthalate(C16H22O4)which was the main component of low polar substance in ethyl acetate phase.The purified products III, IV, V and VI were flavonoid detected by TLC, however, monomer purification product yield was microscale in the process of repeatedly passing through the column and purification, due to the low content, complex types and other reasons can't get their accurate structure. In order to obtain enough purified product,separation and purification methods should be further optimized.(5) The effect of extraction conditions on the antioxidant activity of flavonoids from jackfruit skin was studied in vitro.Results showed that when the concentration of the extraction solvent was 60% acetone, extraction material liquid ratio of 1:25, the extraction temperature was 45?, ultrasonic time 20 min, the ultrasonic power was 300 W, jackfruit peel flavonoids extraction liquid presented good antioxidant capacity.The IC50 value and reduction ability of DPPH and OH free radical scavenging rate were compared under the optimum extraction conditions of crude extract, with contrast by two kinds of common antioxidants Vc and BHT.The results showed that the antioxidant capacity of them was as follows: the crude extract >Vc solution >BHT.Comparative study of NKA purified in vitro antioxidant activity of total flavonoids in aqueous solution before and after, results showed that with the improvement of the purity of flavonoids, the in vitro antioxidant activity of samples increased as well. NKA purification was beneficial to increase the antioxidant activity of flavonoids.In this study, the in vitro antioxidant capacity of crude extract was also compared to study preliminary on the distribution of antioxidant active substances in jackfruit peel.The results showed that DPPH, OH radical scavenging capacity and reducing capacity of the aqueous phase were much higher than that of ethyl acetate.Therefore, the antioxidant activity of the extract of jackfruit fruit peel is mainly distributed in the water phase.