| Epigallocatechin-3-gallate(EGCG), the principal active component of tea polyphenols,has been proved to possess plenty of benefits, such as anti-inflammation, antimicrobial,mutant prevention, anticancer, antiviral and antioxidation as well as protecting neuron.Aiming to develop a green, safe and industrialized process technics, a method of preparativepurifying EGCG monomer from tea polyphenols contained fifty percents EGCG viaadsorption column chromatography was developed in this paper. Meanwhile, the inhibitingeffect of EGCG prepared towards Pseudomonas aeruginosa(P. a) biofilm was also studied toprovide some reference to EGCG being applied in medical field.Firstly, polar adsorption packing LX-8and semipolar adsorption packing LSA-10werescreened to distinguishingly separate catechins by investigating distinguishing adsorptionability of packing applied towards catechins. Adsorption behavior of catechins on LSA-10was studied to make some contribution to dynamic column chromatography, and the resultrevealed that adsorption process, a kind of intermediate speed adsorption type, reached theprocess terminal in120minutes. The adsorption process can be simulated bypseudo-second-order better than pseudo-first-order kinetic model, and rate constant of totalcatechins was about0.0019g/mg/min. The adsorption behavior can be well interpreted byFreundlich model, and a lower temperature promoted the adsorption reaction of catechins onLSA-10better, a higher catechins concentration promoted the selected adsorption betweenester-catechins and non-ester-catechins better. kFvalue of EGCG was higher than43.0473mg(n-1)/n/g/mL-1/n, and it meant that LSA-10would absorb catechin monomersdistinguishingly.Secondly, technics of preparative purifying EGCG monomer via adsorption columnchromatography were optimized and confirmed as follows: column packed with LX-8washired to separate ester-catechins from non-ester-catechins, bed volume of the column was600mL. Tea polyphenols with a300mg/mL EGCG was used as material at an1.5BV/h flow rate,and the sample volume was60mL. The column was sequentially eluted with distilled water,10%ethanol,25%ethanol and80%ethanol with a volume of4BV,6BV,6BV and4BVrespectively at an1.5BV/h flow rate. Product with (73.13±0.64)%EGCG purity and(61.21±0.48)%recovery can be obtained after the fraction of25%ethanol being concentratedand dried. Then column packed with LSA-10was employed to separate EGCG fron ECG, andBV of the column was210mL. Fraction of25%ethanol from LX-8column was loaded in thecolumn with an1.5BV/h flow rate,20mg/mL loaded concentration and1.5BV loaded volume. After that, the column was sequentially eluted with5%ethanol,15%ethanol and80%ethanol with a volume of3BV,5BV and3BV respectively at an1.5BV/h flow rate. Productwith (93.16±1.04)%EGCG purity and (89.82±0.82)%recovery was achieved after thefraction of15%ethanol being concentrated and dehydrated. And product with95.37%EGCGpurity and49.58%total recovery can be gotten via crystallization. In a large scale, productwith95%EGCG purity can also be obtained, and it meant that the process technics was ableto be applied in large scale and operated in an industrial production.Lastly, inhibiting effect of EGCG towards P. a biofilm was also studied and the resultsindicated that minimum inhibitory concentration(MIC) value of EGCG towards P. a was0.125mg/mL and the minimum biofilm inhibitory concentration(MBIC) value of EGCGtowards P. a was0.0625mg/mL. After treated with EGCG with an MBIC concentration,inhibition rates of P. a virulence factors elastase activity, rhamnolipids and pyocyaninproduction were (37.73±1.25)%,(41.70±1.92)%and (35.98±1.34)%respectively, andinhibition rates of P. a quorum sensing signal molecules C4-HSL and3-oxo-C12-HSL were(31.98±1.26)%and (15.91±0.82)%respectively. EGCG can inhibit the formation of biofilmand release of virulence factors via the inhibiting effect on cell to cell quorum sensing signalmolecules. |
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