Preparation Of Cryptochlorogenic Acid From Ageratina Adenophora And Its Anti-inflammatory Effect On RAW264.7 Cells

Ageratina adenophora is one of the most harmful invasive alien plants in China.Studies have shown that the extracts of A.adenophorahave many biological activities,among which the anti-inflammatory activity of 4-O-caffeoylquinic acid in A.adenophora is seldom studied.In this study,4-CQA extracted from A.adenophora was used to investigate the anti-inflammatory activity of 4-CQA in RAW264.7 cells induced by LPS.The results are as follows:1.Isolation,identification and extraction process optimization of 4-CQA from A.adenophorahaveUltra-high performance liquid chromatographic identification of 4-CQA in A.adenophorahave showed that the content of 4-CQA in 0.7 mg extract was 0.6 mg and the purity was 94.3%.The extraction solvent,macroporous resin type,material-liquid ratio,soaking time,soaking times and PH were selected by single factor screening,and orthogonal test.The optimum extraction process was determined as follows: extraction solvent was 90% ethanol,material-liquid ratio was 1:4,soaking time was 4 h,soaking time was 3 times,PH was 3,macroporous resin type was NKA II.2.Effects of 4-CQA from A.adenophora have on the growth status and cell viability of RAW264.7 cellsCell observation and CCK-8 assay showed that after LPS stimulation,RAW264.7cells in the blank control group grew smoothly and without pseudopods.Most of the pseudopods in the LPS group did not aggregate and grew,and the number of pseudopods in the LPS group decreased.There was no significant difference between the experimental group and the blank control group except 400 μg/m L.The cell viability of 400 μg/m L cells was significantly lower than that of the normal group,200-50 μg/m L group and LPS group.The concentration of 4-CQA at 200,100 and 50 μg/m L was chosen as the follow-up drug concentration..3.Effects of 4-CQA from A.adenophora on LPS-induced expression of inflammatory factors in RAW264.7 cellsOne-step NO test showed that 100 μg/m L 4-CQA had the best effect on inhibiting NO secretion in RAW264.7 stimulated by LPS;ELISA was used to investigate the effects of different doses of 4-CQA on the secretion of inflammatory cytokines TNF-α,IL-1β,IL-6,IL-8 and IL-10.The results showed that different concentrations of 4-CQA had different degrees of effects on the secretion of inflammatory cytokines such as TNF-α,IL-1β,IL-6and IL-8.The results showed that 4-CQA exerted anti-inflammatory effect by down-regulating pro-inflammatory factors TNF-α,IL-1β,IL-6,IL-8 and COX-2,and up-regulating anti-inflammatory factor IL-10.The best anti-inflammatory effect was 100μg/m L group.4.Effects of A.adenophora 4-CQA on LPS-induced expression of inflammatory factors and MAPKs pathway-related genes in RAW264.7 cellsRT-PCR results showed that the changes of TNF-α,IL-1β,IL-6,IL-8 and IL-10 after4-CQA treatment were consistent with ELISA.At the same time,the synthesis of related genes p38,JNK and ERK1/2 in the MAPKs pathway was increased and the MAPKs signaling pathway was activated.After 4-CQA treatment,the expression of p38,JNK and ERK1/2 genes in RAW264.7 cells was significantly down-regulated,and 4-CQA could transcribe water.Pingshang inhibited the activation of MAPKs pathway.These results suggest that 4-CQA can regulate the expression of inflammatory factors by inhibiting the transcription of genes in the MAPKs pathway.5.Effects of 4-CQA from A.adenophora on the expression of MAPKs pathwayrelated proteinsWestern Blot results showed that 4-CQA could inhibit the expression of p38,JNK1/2and ERK1/2 protein in RWA264.7 cells stimulated by LPS,inhibit the phosphorylation of JNK1/2 and ERK1/2 and inhibit the activation of MAPK pathway,thereby regulating the release and expression of inflammatory factors.These results suggest that 4-CQA can inhibit the activation of MAPKs pathway in RAW264.7 cells at the level of protein translation,and ultimately regulate the expression of inflammatory factors.In conclusion,94.3% 4-CQA can be obtained from the aqueous phase of the methanol extract of A.adenophora.When it acts on RAW264.7 cells induced by LPS,4-CQA can inhibit the activation of MAPKs pathway by inhibiting gene transcription and protein translation,and ultimately regulate the release and expression of pro-inflammatory and anti-inflammatory factors in cells to play an anti-inflammatory role.