Effect Of Polyphenols Extracted From Blueberries On Anti-inflammatory Factors In Macrophages

Blueberries have antioxidant, anti-cancer, anti-inflammatory and other effects, which arecaused by high levels of polyphenols. Polyphenols can be divided into extractable polyphenols(EPP) and non-extractable polyphenols (NEPP) according to the different link of polyphenols.In this paper, EPP and NEPP were extracted and purificated from blueberries; and the majorcompounds of EPP and NEPP were determinated by HPLC. Effect of EPP and NEPP on studyon the secrection and gene expression of cytokines (NO、iNOS、COX-2) in LPS-inducedmacrophage were further studied.EPP was extracted from freeze-dried powder of blueberries, the three major factors ofethanol concentration, solid/liquid ratio, ultrasonic time of polyphenol extraction were chosenand used in the response surface analysis on the basis of single-factor experiment. By theresponse surface, the optimized extraction conditions were as following: ultrasonic power400W, extraction times2h, ethanol concentration55%, solid/liquid ratio1:25, ultrasonic time1.5h, and the EPP yield of11.05mg/g was achieved.The extractable polyphenols in fresh blueberries were firstly removed, and the remainingresidues were hydrolyzed by acid solution to obtain NEPP. On the basis of single-factorexperiment, the three factors of temperature, time, solid/liquid ratio were chosen in the responsesurface analysis. By the response surface, the optimized extraction conditions were as following:extraction temperature74℃, time22.5h, solid/liquid ratio1:21. The NEPP yield of27.25mg/gwas achieved under optimized condition.According to static adsorption test, NAK-9, AB-8, XAD-7, HP-20and D101were used toselect the optimum adsorption resin of EPP. The results showed that HP-20is benefical topurify EPP. AB-8, DA-201, ADS-17, HPD-826, HD-20, HPD-750and HPD-100C were used toselect the optimum adsorption resin of NEPP. The results showed that HPD-100C is benefical topurify NEPP.The major compounds of EPP and NEPP were determinated by HPLC. The compounds ofcyanidins, delphinidins, petunidins, peonidins, malvidins and acylated anthocyandins weredetectd in EPP NEPP from blueberries.The different concentration EPP and NEPP were added to RAW264.7macrophages andcultured for the different time, the effect of EPP and NEPP on secretion and gene level ofcytokine (NO, iNOS, COX-2) were studied.The effects of EPP and NEPP from blueberries on the secretion of NO, iNOS and COX-2 in LPS-prestimulated RAW264.7cells were studied. The results showed that the secretion ofNO、iNOS、COX-2can be decreased by EPP and NEPP in the LPS-induced RAW264.7cell,and the inhibitory effect was enhanced with the increasing of concentration. At the sameconcentration, the inhibitory effect of NEPP was better than that of EPP and the positive control(TP). It is suggested that EPP and NEPP from blueberries have significant anti-inflammatoryeffect by inhibitting the secretion of NO、iNOS and COX-2in macrophages.The effects of different concentrations of EPP and NEPP from blueberries on iNOS andCOX-2mRNA in LPS-induced RAW264.7cells in the different time were studied in this paper.The results showed that EPP and NEPP inhibited the gene expression of iNOS and COX-2.Within12h incubation time, the inhibited effects of EPP and NEPP were significance, and theinhibited effect of EPP was significantly better than that of NEPP. It is suggested that EPP andNEPP have significant anti-inflammatory activity by inhibiting the expression of iNOS andCOX-2mRNA in macrophages.