Study On Separation And Purification And Stability Of Thearubigins

Thearubigins as reddish brown phenolic substances in the process of black tea fermentation,are the key factor in the color and taste of black tea.Thearubigins are produced by the oxidation of catechins under the action of polyphenol oxidase and peroxidase enzyme.Large differences in molecular weight,the component and structure is all not yet clear,has not prepared thearubigins monomers.Research on thearubigins separation also has been reported rarely,thearubigins detection system only remain in the Roberts’ s Systematic Analysis.The study of thearubigins is the focus of tea industry.And thearubigins as natural pigment and its biological activity,made thearubigins have a very good application prospects,So the research and utilization of thearubigins need to be resolved soonly.In this paper,the extraction of tea extract was optimized,thearubigins were prepared by fractional extraction,column chromatography and high-speed separation chromatography.And identified by infrared spectroscopy,mass spectrometry and nuclear magnetic resonance(NMR).Finally,the stability of thearubigins were studied under different parameters.The main contents are as follows:(1)CTC black tea as raw material,thearubigins were extracted from CTC black tea using hot water extraction,The optimal process of solid-liquid ratio,extraction temperature and extraction time were investigated.The results showed that solid-liquid ratio was 1:30,the extraction temperature was 80 ℃,and the extraction time was 30 min,the highest extraction rate was 10.57%.(2)Purification was carried out by reverse phase C18 column chromatography and further purified by Sephadex LH-20,the elution fraction was analyzed by HPLC.The results showed that: reversed phase C18 column purification,followed by 30%,50%,70% methanol gradient elution,can remove chlorophyll and other non-phenolic impurities,and a greater degree of removal of catechins and theaflavins from the crude,and can also completely remove the caffeine;While Sephadex LH-20 dextran gel was purified,followed by elution with methanol and acetone.The derived thearubigins,by HPLC analysis,high purity and no catechins and theaflavins and other impurities,The peak position of three parts thearubigins were 3-5min.(3)Separation of thearubigins by reversed phase C18 column chromatography combined with high speed countercurrent chromatography.The results showed that the solvent system was determined as ethyl acetate layer with ethyl acetate : n-hexane : methanol-water(1:3:1:5,v/v/v/v)quaternary solvent system,which is the distribution coefficient of 1.34,retention of stationary phase Rate of 42%.N-butanol layer and acidic n-butanol layer with ethyl acetate : n-butanol : water(3:2:5,v/v/v)ternary solvent system,which is the distribution coefficient of 1.65 and 1.71,retention of stationary phase Rate of 51% and 52%.(4)The structures of thearubigins were identified by infrared spectroscopy,mass spectrometry and nuclear magnetic resonance.The results showed that the three parts of thearubigins were found to exist-O-H between the phenolic molecules,-C=O,benzene ring,aromatic ether,and the position and number of substituents on the benzene ring are different.Mass spectrometry was performed by ESI electrospray in negative ion mode,results showed that the molecular weight of the ethyl acetate layer was 855,and the molecular weight of the n-butanol layer and the acid n-butanol layer was 742.And nuclear magnetic resonance spectrum analysis of three parts of thearubigins,because the material can not achieve the required purity,can only see 7 ppm for the H on the benzene ring and 9-10 ppm of the chemical shift,the H on the C position of C=O,unable accurately locate the location of each H and further deduce the structure.The basic structure of catechins are preserved in thearubigins.In summary,it is possible that thearubigins may be catechin trimer compounds.Through the retention rate and chromatic aberration a value as an indicator to explore the tea at different light,pH and temperature conditions,the stability of thearubigins.It was found that the shading treatment had better storage,and the high stability was at pH = 3,and below 60 ℃.High temperature and strong alkaline is not conducive to its stability,leading to oxidative browning of thearubigins.