Study On Extraction And Purification Of Total Flavonoids From Hairy Ligularia And Anti-inflammatory Mechanism

To optimize the extraction and purification conditions of total flavonoids from hairy Ligularia,at the same time,to explore its anti-inflammatory activity and related mechaorous.In this study,total flavonoids were extracted by ultrasonic method in hairy Ligularia as raw materia,ultrasonic power,extraction time,ethanol concentration and liquid/solid ratio,extraction times were evaluated basing on single factor experiments,the Design-Expert8.05 b software was used to get the best extraction conditions;total flavonoids from hairy Ligularia were purified by AB-8 macroplumeadsorption resin,the purification effecton 6 factors of sample concentration,sample volume,sample flow rate,sample pH,ethanolconcentration,eluent volume were investigated,the orthogonal experiment analysis was used to elect the best purification conditions;the cycle of inhibiting bacterium as index,e.coli was selected as supply bacteria to detect the bacteriostasis with total flavonoids by disk fussion method;lipopolyssacharide(LPS)-activated macrophages have been used to establish vitro models ofinflammation,the effects of total flavonoids on LPS-induced inflammatory cytokines TNF-? and IL-6 secretion level were detected by ELISA,the effects of total flavonoids on LPS-induced inflammatory cytokines TNF-? and IL-6 mRNA were detected by RT-PCR,the effects of total flavonoids on I?B-? expression was detected by Western blotting to research the inhibition and the effect of NF-?B signal transduction pathway.Test results are as follows:1.Through the Box-Behnken Design-Expert8.05 b software in the analysis of ultrasonic power(A),extraction time(B),ethanol volume fraction(C)and the ratio of material to liquid(D)optimum conditions influence sequence for B>C>D>A.The results show that the optimal extraction ratio of total flavonoids was 1.48% with 670 W and 77% for 27 min in liquid/solid ratio of 34:1(mL:g).2.By orthogonal test analysis,sample concentration(A),Sample pH(B),sample volume(C)optimum conditions influence sequence for C>A>B.The results showed that the optimal conditions of AB-8 macroporous adsorption resin for the purification of total flavonoids from hairy Ligularia were: sample concentration of 0.8 mg/mL,sample volume of 30 mL,sample flow rate of 1 mL/min,sample pH 3.0,ethanol concentration of 90%,eluent volume of 40 mL,the purity of total flavonoids from hairy Ligularia was 67.3% under this condition.3.Antimicrobial test showed that hairy ligularia flavonoids concentration in 0.3125~5 mg/mL has significant inhibition to e.coli.When the concentration of total flavonoids in the range of 0.3125~1.25 mg/mL was low antibacterial;in the concentration range from 1.25 to 2.5 mg/mL was moderately inhibitory;in the concentration range from 2.5 to 5 mg/mL was highly bacteriostatic.4.The results of RT-PCR and ELISA showed that total flavonoids significantly inhibited TNF-a,IL-6 mRNA expression and TNF-a,IL-6 secretion in a dose-dependent manner.At concentration of 25,50,100 ?g/mL of total flavonoids caused 37.4%,57.5%,79.1% decrease of TNF-? level and 57.7%,77.1%,88.3% inhibition of IL-6 release.Hairy Ligularia flavonoids has significant anti-inflammatory effect.5.The results of Western blot showed that total flavonoids significantly inhibited phosphorylation of I?B-? protein expression.At concentration of 25,50,100 ?g/mL of total flavonoids caused 10.7%,42.5%,48.8% decrease.The activation of NF-?B signaling pathway induced by LPS has been inhibited by flavonoids from hairy Ligularia.