| Objective To optimize the flocculation process of stevia side production,to extract and purify chlorogenic acid components from flocculated waste plate mud,to establish the quality standard of stevia chlorogenic acid extract(SCAE),and to study the antibacterial,anti-inflammatory and antioxidant effects.Methods 1.With the comprehensive score of transfer rate of chlorogenic acid and isochlorogenic acid A and retention rate of rebaudioside A(RA)as evaluation index,the light transmittance was considered comprehensively,and three single factors of flocculant dosage,stirring time and standing time were selected to investigate the flocculation process.The optimal flocculation process of chlorogenic acid components was obtained and verified.2.Taking the comprehensive score of chlorogenic acid and isochlorogenic acid A content as the evaluation index,three single factors including ethanol concentration,pH and solidliquid ratio were selected to conduct the single factor investigation of the extraction process.Combined with the results of the single factor investigation,L9(34)orthogonal experiment was designed to obtain the optimal extraction process of chlorogenic acid components,and the best process was verified.3.With chlorogenic acid and isochlorogenic acid A as the index components,through the selection of resin,static adsorption behavior,pH of test solution,dynamic adsorption behavior,impurity removal conditions and elution conditions,the optimal purification process of chlorogenic acid components was obtained,and the optimal process was verified.4.The quality standard of SCAE was studied through the preparation method,properties,inspection,and content determination of chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,caffeic acid,isochlorogenic acid A,isochlorogenic acid B,isochlorogenic acid C.5.The inhibitory zone and minimum inhibitory concentration(MIC)of SCAE against common pathogens were determined by disk diffusion method and multiple dilution method.The inflammatory cell model was established by LPS induced RAW 264.7 cells,and the cytotoxicity test,nitric oxide(NO)release test,and the release of inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were measured.The acute model of ear swelling induced by xylene was established to measure the degree of ear swelling and the release of inflammatory factors TNF-α,IL-6 and IL-1β in ear tissue.The antioxidant capacity of SCAE was investigated by p-dibenzoyl hydrazine radical(DPPH)method,iron ion reduction/antioxidant capacity(FRAP)method,2,2-diazo-di(3-ethyl-benzothiazol-6-sulfonic acid)diamiammonium salt(ABTS)method.Results 1.The optimal flocculation process of chlorogenic acid components was determined by single factor investigation as the dosage of PAC(yellow)was 5%,stirring at room temperature for 3 min without standing;It has been proved that the optimized flocculation process is stable,reasonable and feasible.2.Through single factor investigation and orthogonal experiment,the optimal extraction process of chlorogenic acid components was determined as extraction of 60%ethanol at pH 1,8 times of the sample solution;It was proved that the optimized extraction process was stable and feasible.3.The optimal purification process is D101 macroporous resin,chlorogenic acid loading solution concentration 0.81 mg·mL-1,isochlorogenic acid A 0.91 mg·mL-1,loading volume 7 BV,flow rate 2 BV/h,2 BV ultra-pure water at the flow rate of 4BV/h,and then 5 BV,The chlorogenic acid was eluted with 80%ethanol and 3 BV/h flow rate.It has been proved that the optimized purification process is stable and feasible.4.Conduct quality standard research on SCAE,check the moisture content of SCAE,and the moisture content shall not exceed 6.5%;Combined with the content determination results,the content limits of chlorogenic acid,cryptochlorogenic acid,neochlorogenic acid,isochlorogenic acid A,isochlorogenic acid B,isochlorogenic acid C in SCAE were formulated,respectively,that the chlorogenic acid(C16H18O9)in SCAE should not be less than 9.9%,cryptochlorogenic acid(C16H18O9)should not be less than 1.32%.Neochlorogenic acid(C16H18O9)shall not be less than 1.73%,isochlorogenic acid A(C25H24O12)shall not be less than 8.43%,isochlorogenic acid B(C25H24O12)shall not be less than 0.96%,isochlorogenic acid C(C25H24O12)shall not be less than 3,8%;Based on the results of flocculation process,extraction process,purification process and quality standard,the draft quality standard of SCAE was preliminarily formulated.5.SCAE showed inhibitory effects on Staphylococcus aureus,Streptococcus pneumoniae,Escherichia coli,Proteus vulgaris and Pseudomonas aeruginosa,and the inhibitory effects increased with the increase of the concentration of SCAE.The MIC of Staphylococcus aureus was 0.0125 g·mL-1,Escherichia coli was 0.025 g·mL-1,Proteus was 0.025 g·mL-1,Pseudomonas aeruginosa was 0.2 g·mL-1,and Streptococcus pneumoniae was 0.2 g·mL-1.In vitro results showed that SCAE could significantly inhibit the release of NO(P<0.0001),and significantly reduce the release of TNF-α,IL-6 and IL-1β induced by LPS(P<0.001 or P<0.0001).In vivo,SCAE significantly inhibited xylene induced acute inflammation in mice(P<0.05,P<0.01 or P<0.0001),and the high-dose SCAE group significantly decreased the level of TNF-α(P<0.01).The levels of IL-6 in high-dose and medium-dose SCAE groups were significantly decreased(P<0.01 or P<0.001),and IL-1βlevels in high-dose and medium-dose SCAE groups were significantly decreased(P<0.05 or P<0.001),indicating that SCAE had better anti-inflammatory activity.SCAE has good antioxidant capacity,and its concentration is positively correlated with the antioxidant capacity.When the concentration reaches the critical point,the antioxidant capacity tends to be stable,and the antioxidant capacity of SCAE after purification is significantly improved compared with that before purification.Conclusion This study completed the optimization of the flocculation process of stevia water extract,extraction and purification of chlorogenic acid components in the flocculation plate mud.The process is simple,reasonable and feasible.The quality standard and preliminary pharmacodynamics of SCAE were studied,and a relatively standard quality standard draft was formulated.Through preliminary pharmacodynamic study,it was proved that SCAE had good antibacterial,anti-inflammatory and antioxidant activities,which provided a theoretical basis for further entering the market after secondary utilization of stevioside solid waste production. |
PDF Full Text Request