Study On Synergistic Extraction Of Multiple Effective Constituents From Purple-heart Radish By Enzyme And Microwave Co-assisted Extraction

Purple-heart radish（Raphanus sativus.L）,also known as a fruit radish,an annual herb of the cruciferous plants which is used not only as vegetables and fruits but also as medicine in China.The main ingredients of Purple-heart radish are anthocyanins and polysaccharides which have been reported to be associated with a range of human health benefits,including antioxidant activity,anticancer activity,anti-inflammatory/bacteriostatic effects,preventing cardiovascular diseases,enhancing immunity and vision.Studies have recommended many technologies for extraction of anthocyanins and polysaccharides by using organic solvents,enzyme-assisted extraction methods and physical extraction processes using supercritical critical fluid,ultrasound assisted extraction or microwave assisted extraction resulting in high recovery of anthocyanins and polysaccharides as compared to the conventional method with the disadvantages of incomplete extraction,low extraction efficiency and solvent consumption.Enzyme-assisted extraction method,often used as auxiliary techniques,which can efficiently and specifically degrade and break down the plant cell wall thereby releasing the intracellular contents and thus enhancing the extraction of the bioactive compounds.Microwave-assisted extraction method,a promising extraction method with high extraction efficiency,low solvent consumption and easy control,is based on direct effect on molecules by ionic conduction and dipole rotation.Microwave irradiation induces the quickly elevating temperature of solvent and pressure inside cells to accelerate the diffusion of the targeted compound in plant matrix toward and release bioactive compounds inside plant cells.In order to improve the extraction efficiency,a novel extraction technology combined enzyme-assisted extraction and microwave-assisted extraction was established for extracting anthocyanins and polysaccharides.In view of the strong physiological activities of anthocyanins and polysaccharides,the spectral and chromatographic analysis are used to analysis the anthocyanins stability,monomer structure,polysaccharides composition,and antioxidant activity,respectively which can provide a basis for the development of pharmaceutical and food products.The specific research content are as follows:1.Enzyme-assisited extraction method and microwave-assisted extraction method were used and coupled in order to synergistically improve the extraction efficiency of anthocyanins.The effects of factors such as enzyme types,pH,extraction temperature,extraction time,liquid-to-material ratio,and granularity on the extraction yield of anthocyanins were studied and the extraction conditions were further optimized in combination with response surface design methods.A higher extraction rate can be achieved with the following conditions:24%cellulase,pH=4.00,60%ethanol as the extraction solvent,extraction temperature 50°C,extraction time 7.17 min,liquid-to-material ratio 60 mg/L and the particle size 80 mesh.The extraction yield of anthocyanins was 6.125±0.034 mg/g,which was basically consistent with the model prediction of 6.292 mg/g.In addition,the content of water,ash,protein,flavonoid and polyphenol of Purple-heart radish was measured to be 92.114±1.036%,6.065±0.025%,136±3 mg/g,38.01738.017±0.207 mg/g and 29.417±1.084 mg/g,respectively.Compared with the traditional single extraction technology,the experimentally established method could not only synergistically play the role of enzymes and microwave,but also overcome the shortcomings of time-consuming and complicated operation which could replace conventional extraction methods for anthocyanins extraction.2.The macroporous resin separation method was used to purify and isolate the anthocyanins extract in order to further study the stability and antioxidant activity of anthocyanins.The D101 macroporous resin was screened from four kinds of macroporous resins（D101,AB-8,HPD100,NKA-II）which exhibited excellent adsorption performance.The purification conditions of the anthocyanins extract were studied by using static test methods and the results showed that when the pH was 2.00,loading volume was 520 mL（concentration 0.15 mg/mL）,washing volume was 600 mL,80%ethanol（containing 0.1%HCl）was selected as the eluent and the volume was 150 mL,the purity of anthocyanins increased from0.794±0.026%to 19.538±0.522%（color value increased from 4.035±0.051 to 86.975±0.138）.The results of stability test showed that the degradation rate of anthocyanins was significantly affected by pH,temperature and light.Most metal ions and food additives had no significant adverse effects on anthocyanins such as precipitation or degradation.The antioxidant activity test showed that anthocyanins have strong scavenging ability against DPPH radicals,superoxide anions,ABTS radicals and hydroxyl radicals,which are 93.95±0.01%,87.40±0.14%,99.56±0.34%and 77.85±1.68%,IC₅₀ were 0.026 mg/mL,0.043 mg/mL,0.0080 mg/mL and 0.046 mg/mL.3.High-resolution HPLC-Q-Orbitrap-MS was used to identify anthocyanins after purification.11 kinds of anyhocyanin were separated and identified according to the chromatographic separation results,primary and secondary fragment ion mass spectra and the results was indicated that the basic chemical structure of 11 kinds anthocyanins was belong to pelargonidin derivatives.The mainly anthocyanins was pelaragonin-3-（p-coumaroyl）diglucoside-5-glucoside which the content was accounted for1.95 mg/g using HPLC with the cyanidin-3-O-glucoside as the external standard.4.On the basis of the above studies,papain was used for enzymatic hydrolysis and combined with microwave-assisted two-phase extraction method was established.The effects of various factors such as microwave field enhancement and enzyme-assisted extraction of polysaccharides.A novel method for synergistic extraction and obtaining different polysaccharides through two-phase extraction was developed aiming at improving the extraction efficiency of polysaccharides,The single-factor test and Box-Behnken Design were used to optimize the extraction process and the optimal extraction conditions were as follows:ethanol（26%）/ammonium sulfate（21%）as the extractant,papain concentration 24.50%,pH 5.00,particle size 80 mesh,extraction temperature 68℃,extraction time 8.40 min and liquid-to-material ratio 63 mL/g.PTP and PBP were9.107±0.391%（w/w）and 32.506±0.046%（w/w）,respectively.HPGPC results demonstrated that M_W and M_n were 15935 Da and 4648 Da for PTP and 27962 Da and 8239 Da for PBP,respectively.FT-IR showed that both PTP and PBP had absorption peaks at 3100 cm^-1,2900 cm^-1,1600 cm^-1,1400 cm^-1 and 1100-1000 cm^-1.HPLC showed that PTP was composed of mannose,glucuronic acid,aminogalactose,glucose,galactose and arabinose,whereas the PBP was composed of mannose,glucuronic acid,aminogalactose,glucose,galactose,arabinose and fucose.The antioxidant activities of PTP and PBP were evaluated in vitro,the results exhibited that both PTP and PBP exhibited strong inhibition activities in superoxide anion,DPPH,ABTS,hydroxyl radicals and lipid peroxidation with the scavenging rates were 39.12±1.89%,71.29±4.10%,57.89±0.16,49.73±0.17%,56.67±2.21%and 32.17±0.36%,40.80±1.11%,49.43±1.14%,39.51±1.60%,38.16±1.66%,respectively which indicated that polysaccharides of Purple-heart radish had the potential to be antioxidants and broadened the sources of new drug research and development.