| Acanthopanax senticosus belongs to perennial deciduous shrubs and is a bulk herb for clinical animal medicine.It contains active ingredients such as polysaccharides,saponins,flavonoids and triterpenoids.Clinical studies have confirmed the ability of Acanthopanax senticosus to inhibit inflammation,and flavonoids are an important material basis for the anti-inflammatory function of Acanthopanax senticosus.Using modern extraction techniques and pharmacological studies to improve the utilization efficiency of Acanthopanax senticosus and to investigate the pharmacological mechanism of its active ingredients is an important way to modernize and develop Acanthopanax senticosus.The present study focuses on the purification and in vitro and in vivo anti-inflammatory pharmacological mechanisms of the total flavonoids of Acanthopanax senticosus to investigate the molecular level mechanisms of the anti-inflammatory effects of the total flavonoids of Acanthopanax senticosus.The main studies are as follows.1.Separation and purification of total flavonoids of Acanthopanax senticosusThe best resin for the purification of total flavonoids of Acanthopanax senticosus was selected from six different types of macroporous resins（CAD-40,DM301,D-101,HPD-600,S-8,AB-8）,and the adsorption mechanism was investigated.The changes of total flavonoids composition of Acanthopanax senticosus before and after purification were compared using ultra performance liquid chromatography tandem mass spectrometry（UHLPC-MS/MS）,and the antioxidant activity was compared.The results revealed that the optimal macroporous resin was HPD-600,and the optimal conditions were determined as the p H of the loading solution was 3,the flow rate and volume of the loading solution were 3 BV·h-1and 2.5 BV,the desorption ethanol concentration was 60%,and the desorption flow rate and volume were 3 BV·h-1and 4 BV,and the purity of total flavonoids increased from 28.79%to 50.57%.The results on the adsorption mechanism showed that the adsorption process of total flavonoids by HPD-600 resin was in accordance with the quasi-second-order kinetic model and Freundlich isotherm model,and the thermodynamic parameters indicated that this adsorption process was spontaneous and heat-absorbing.The results of UHLPC-MS/MS showed that the purified total flavonoids（ASTF）had an increased content of seven flavonoids compared with the crude extract（EAS）of total flavonoids of Acanthopanax senticosus.In addition,the antioxidant capacity of ASTF was higher but lower than that of L-ascorbic acid.2.Effect of ASTF on LPS-induced inflammation model in RAW264.7 cellsA LPS-induced inflammation model of RAW264.7 cells was established to investigate the effect of ASTF on inflammation of RAW264.7 cells.The changes of NO content were detected by Griess,and the contents of IL-6,IL-1β,TNF-α,PGE2 were detected by ELISA.The m RNA expression of IL-6,IL-1β,TNF-α,i NOS,COX-2 was detected by RT-PCR.The results showed that the ASTF group could inhibit the secretion of NO,IL-1β,IL-6,PGE2,TNF-αin a dose-dependent manner and down-regulate the expression levels of i NOS,COX-2,IL-1β,IL-6,TNF-αm RNA.The intestinal inflammation model was established in LPS-induced mice,and the DEX and ASTF low,medium and high dose groups（200,400 and 800 mg-Kg-1）were administered by gavage to investigate the mechanism of the effect of ASTF on intestinal inflammation in mice.3.Study on the mechanism of action of ASTF on the inflammation model of LPS-induced miceThe intestinal health of small intestinal segments of mice in each group was compared by HE staining and PAS staining,the contents of IL-6,IL-1β,TNF-αand PGE2 in mice serum were detected by ELISA,the expression of IL-6,IL-1β,TNF-αand COX-2 m RNA in mice small intestinal tissues were detected by RT-PCR,and the TLR4,My D88,NF-κB p65,NF-κB p-p65 protein expression levels were detected by Western blot,and analyzed the changes of mouse intestinal microbial community by bacterial taxa 16S r RNA amplification sequences.The results showed that the intestinal tracts of ileum,jejunum and duodenum of LPS-induced mice showed broken conditions such as thinning of intestinal wall,disappearance of crypt,shortening of villi and decreased number of cupped cells secretion,and the intervention of ASTF at various concentrations could improve the intestinal morphology,length of villi and increase the number of cupped cells in ileum,jejunum and duodenum;compared with LPS mice,the mice in ASTF group had more IL-1β,IL-6,PGE2,and TNF-αlevels were significantly lower in the ASTF group compared with LPS mice,and the expression levels of related m RNAs,TLR4,My D88,and p-p65/p-65 proteins were also significantly lower;in addition,16S r RNA sequencing results showed that LPS disrupts the structural composition of mouse intestinal microorganisms,and ASTF could increase the diversity of mouse intestinal flora,increase the proportion of the thick-walled phylum,and In addition,the 16S r RNA sequencing results showed that LPS disturbed the structural composition of mouse intestinal microflora,and ASTF could improve the diversity of mouse intestinal flora,increase the proportion of thick-walled phylum and decrease the proportion of mimic phylum,and normalize the disrupted microbiota to some extent.In this experiment,a macroporous resin purification process of ASTF was successfully obtained,and ASTF had stronger antioxidant effect in vitro.The cellular assay study confirmed that ASTF can alleviate the LPS-induced inflammation in RAW264.7cells.The mouse intestinal inflammation assay showed that ASTF can reduce inflammation by regulating NF-κB pathway-related protein expression and m RNA expression to inhibit inflammatory factor secretion,and can regulate intestinal flora. |
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