| Taxifolin,also known as dihydroquercetin,is a flavonoid compound,which has many pharmacological activities.Its precursor,astilbin,has a good anti-inflammatory activity,but its stability is poor.When it is transformed into aglycone-taxifolin,its stability and activity may be increased.Taxifolin has attracted attention.There are few studies on the preparation,equilibrium solubility,apparent oil-water partition coefficient,absorption and transportation,and anti-inflammatory activity of Taxifolin.In this paper,the parameters of enzymolysis technology were investigated for the first time,and obtained a better enzymolysis process;measured the apparent oil-water partition coefficient;studied the absorption and transport mode by using Caco-2 cell model;explored the anti-inflammatory activity of Taxifolin by using HDMEC and RAW264.7 cell inflammation models.Objective: To study the preparation technology,the mechanism of absorption and transport,and the anti-inflammatory activity of Taxifolin.Method: 1.The enzymolysis method was used to prepare Taxifolin.Through the design of a single factor test,the screening of enzyme,enzymatic hydrolysis temperature,enzymatic hydrolysis p H,enzyme dosage and enzymatic hydrolysis time were investigated.The conversion rate of astilbin to Taxifolin was determined by the index to determine the enzymatic hydrolysis process,and its content was determined by high performance liquid chromatography(HPLC).2.The solubility and apparent oil-water partition coefficient of Taxifolin in0.1mol/L hydrochloric acid,p H2.0,p H5.8,p H6.8 and p H7.4 at 37℃ were determined by HPLC and saturated solubility method.3.CCK-8 study on the safe concentration of Taxifolin in Caco-2 cells,HDMEC cells and RAW264.7 cells.4.A single-layer model of Caco-2 cells was established and evaluated by transmembranne resistance.Based on the single-layer model of Caco-2 cells,the apparent permeability coefficient and transport mechanism of bilateral transmembrane transport of taxifolin.5.The inflammatory model was established by HDMEC cells induced by lipopolysaccharide,and the release of LDH was observed before and after the intervention of taxifolin;the inflammatory meodel of macrophage RAW264.7 was established by lipopolysaccharide,and the inflammatory model was intervened by taxifolin,and the effect of NO and IL-6 released by RAW264.7 cells was detected byELISA study on the anti-inflammatory activity of taxifolin.Result:1.Optimization of the technological parameters of enzymatic hyddrolysis of astilbin to obtain taxifolin.Finally,under the condition of glycosidase2,the p H of solution is 5.0,the ratio of enzyme to feed is 1:5,and the optimal enzymatic hydrolysisi time is 3 hours at 50℃.2.The lg P in the following solvents were 0.29(0.1mol/L hydrochloric acid),0.48(p H2.0),0.46(p H5.8),0.34(p H6.8),0.26(p H7.4),0.38(water),which indicated that the taxifolin is a hard-to-absorb drug.3.The mass concentration of taxifolin is not higher than 500μg/m L,which has no significant toxic effect on Caco-2 cells;the mass concentration of taxifolin is not higher than 300μg/m L,which has no significant toxic effect on HDMEC cells;the mass concentration of taxifolin is not higher than 500μg/m L,which has no significant toxic effect on RAW264.7 cells.4.study on the absorption and transport of taxifolin in Caco-2monolayer cell model: In this study,the Caco-2 cell model was established and cultured for 21 days to synthesize monolayer,and its transmembrane resistance value was more than 1000Ω/cm2.The results showed that the transport of different concentrations of taxifolin in Caco-2 monolayer cell model was time-dependent,and the cumulative infiltration increased with time;the was no significant difference in Papp of bilateral transport of different concentrations of taxifolin,and they were all less than 1×10-6 cm/s,suggusting that the transport model of taxifolin was passive diffusion and difficult to absorb.The lateral transport efflux rate(ER)of two-sided transport to different concentrations of taxifolin was less than 2,suggesting that there was no significant effux of taxifolin,and is not a P-gp substrate.5.Inflammation of HDMEC cells was induced by lipopolysaccharude,and inflammatory cells were intervened with different concentrations of taxifolin.Compared with the lipopolysaccharide(LPS)stimulation group,each medication group could significantly reduce the inflammation model cell lactate dehydrogenase(LDH)release(P<0.05).The inhibition of LDH release increased with the increase of the concentration.The inhibition of LDH activity tended to be stable in the range of 100-250μg/m L.It shows that the inhibitory effect of taxifolin on LPS induced HDMEC cell inflammation is concentration dependet.LPS-induced RAW264.7 cell model was used to induce inflammation,and different concentrations of taxifolin were used to intervene in inflammatory cells.The results showed thatcompared with the model group,different concentrations of taxifolin can inhibit inflammatory cells NO(P < 0.05).Release and reduce the expression of IL-6cytokines in inflammatory cells.Conclusion: Under the condition of glycosidase 2,the p H of the solution was5.0,the ratio of enzyme to feed was 1:5,and the optimal enzymatic hydrolysis time was 3 hours at 50℃.The mechanism of absorption and transport of taxifolin in the intestinal absorption cell model in vitro is clarified.Taxifolin is a difficult-to-absorb drug in the intestine,and its transmembrane transport mechanism is passive transport.It can inhibit the inflammatory injury of HDMEC cells induced by LPS,the release of NO(P<0.05)in RAW264.7 inflammatory cells induced by LPS,and the content of IL-6 cytokines in inflammatory cells.It is suggested that taxifolin has anti-inflammatory activity. |
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