| Epigallocatechin gallate(EGCG),a major active ingredient of green tea catechin,has a wide application prospect in the fields of food,medicine and other fields.However,there is no good simple,rapid,low-cost,environmentally friendly method to prepare high-purity EGCG.In addition,the low storage stability and gastrointestinal stability of EGCG limit its use in production.Therefore,the purification process of EGCG monomer from tea polyphenols by domestic macroporous resin was studied and optimized.The storage stability,gastrointestinal stability and bioavailability of EGCG encapsulated by liposomes,niosomes and bilosomes were evaluated.Finally,the changes of EGCG functional activity before and after encapsulation in bilosomes were explored through the mouse colitis model.The main research contents and results were as follows:The NKHC-2 resin and LX-20B resin with high adsorption capacity and desorption rate were screened out through the static adsorption/desorption experiment,and the LX-20B resin with good separation effect on EGCG was further screened out through the dynamic ethanol gradient elution experiment to study the purification process of EGCG.The dynamic adsorption/elution optimization experiment confirmed that the optimal process conditions were as follows:after loading the samples with 0.2 BV 250 mg/m L tea polyphenol solution with the flow rate of 1 BV/h,Fraction W was eluted with 2 BV deionized water first,then fraction E30-1 was eluted with 1 BV 30%(v/v)ethanol,then fraction E30-2 was eluted with 1 BV 30%(v/v)ethanol,and finally fraction E80 was eluted with 3 BV 80%(v/v)ethanol.Among them,the purity of EGCG in fraction E30-2 could reach 70.08±2.55%,and the recovery rate of EGCG could reach 68.07±2.43%.There was no significant change in the separation efficiency after the resin was reused for 6 times.EGCG purity of 95.87±0.89%was achieved after 7 d of crystallization of fraction E30-2 in a 40%(w/w)aqueous solution.The purified substance was confirmed to be EGCG by ultraviolet spectrum,mass spectrum,infrared spectrum and ~1H nuclear magnetic resonance spectrum.The crystal structure of purified EGCG obtained in the present study was confirmed to be Form I by X-ray diffraction,and the crystalline form of EGCG was observed to be a slender fragment structure by scanning electron microscopy.Using the encapsulation efficiency,particle size,polydispersity index(PDI)and zeta-potential of EGCG as indicators,an EGCG liposome containing bile salt,called EGCG bile salt liposome,was prepared.The optimal conditions were as follows:cholesterol:Tween40 of1:4(w/w),sodium cholate:Tween40 of 1:8(w/w),and the addition amount of EGCG of 50 mg.Under these conditions,the encapsulation efficiency of EGCG was 85.93±1.43%,the particle size was 189.2±12.7 nm,the PDI was 0.234±0.012,and the Zeta potential was-39.2±1.8 m V.The storage stability and simulated gastrointestinal stability of the EGCG encapsulation systems were determined using the retention rate,particle size and zeta potential of EGCG as indicators.The retention of EGCG in niosomes and bilosomes was 1.19 and 1.20 times that of free EGCG,respectively,when stored at p H 6 until week 8.When stored in Na Cl solution(0,20,50 and 100 mmol/L)until week 8,the retention rates of EGCG in liposomes were 1.08,1.08,1.09 and 1.21 times that of free EGCG respectively,while the retention rates of free EGCG in bile salt liposomes were 1.07,1.05,1.10 and 1.22 times that of free EGCG respectively.The retention rates of EGCG in the liposomes were 1.08 and 4.94 times that of free EGCG,while the retention rates of EGCG in bile salt liposomes were 1.07 and 4.33 times that of free EGCG,respectively,when stored at 25?and 37?until week 8.These results indicated that the niosomes and bilosomes could improve the stability of EGCG at p H 6,different concentrations of Na Cl solution(0–100 mmol/L)and different temperatures(25?and 37?).The results of simulated digestion experiments showed that the retention rates of free EGCG,EGCG liposomes,niosomes and bilosomes after gastrointestinal simulated digestion were 3.09±0.41%,24.02±3.93%,55.74±6.85%and 71.67±4.05%,respectively,indicating that the gastrointestinal stability of EGCG bilosomes was the highest.The EGCG bilosomes with the highest gastrointestinal stability was selected for pharmacokinetic study in rats.The results showed that encapsulation of bilosomes could delay the release of EGCG in vivo and increase the area under the pharmacokinetic curve of EGCG by about 1.98 times.The mice colitis model was established using dextran sodium sulfate(DSS)and the effects of EGCG and EGCG bilosomes on colitis were explored.At the dose of 20 mg/kg/d,compared with EGCG,EGCG bilosomes significantly reduced mice body weight loss,disease activity index(DAI)and tissue damage score,and restored colon length.The research explored the mechanism of EGCG in alleviating colitis through the measurement of oxidative stress and inflammatory factor indexes,and the results showed that EGCG could reduce the myeloperoxidase(MPO),in the colon by recovering glutathione(GSH)and superoxide dismutase(SOD)in the colon and serum of mice,down-regulating the levels of tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)in mice colon tissue and serum,and up-regulating the level of interleukin-10(IL-10)in mice colon tissue to improve mice colon inflammation,compared with EGCG,EGCG bilosomes were significantly superior to EGCG in increasing the level of SOD in mice serum,reducing the content of malondialdehyde(MDA)in mice serum and MPO activity in mice colon,and down-regulating the levels of TNF-?and IL-6 in mice serum,thus better alleviating the symptoms of mice colitis. |
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