基于"微观表征-化学分析-药效评价"三位一体探究土炒白术健脾止泻作用增强的炮制原理OA

Objective:To analyze the processing mechanism underlying the enhanced effect of invigorating spleen and stopping diarrhea of soil-fried Atractylodis Macrocephalae Rhizoma(AMR)by analyzing the changes of microstructure,chemical composition and anti-ulcerative colitis(UC)activity before and after soil stir-frying.Methods:The microstructure and elemental composition of AMR before and after soil stir-frying were analyzed by scanning electron microscopy-energy dispersive spectroscopy(SEM-EDS),to investigate the differences in microstructure and the underlying causes.Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS)coupled with UNIFI 1.9.2 natural product analysis platform were used to analyze and identify the chemical constituents in raw and soil-fried products,and multivariate statistical methods including principal component analysis(PC A)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were used to explore the differences and sources of chemical constituents between them.A dextran sulfate sodium(DSS)-induced UC mouse model was established.The method of disease activity index(DAI)was used to evaluate the severity of intestinal inflammation.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of colon tissue,enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors,Real-time quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to analyze the expressions of key genes and proteins involved in the intestinal mucosal barrier.The 16S rRNA sequencing was used to evaluate the diversity of intestinal flora,headspace gas chromatography-mass spectrometry(HS-GC-MS)was used to explore the levels of short-chain fatty acids(SCFAs)in feces.Base on the above findings,this paper investigated the effects of raw and soil-fried AMR on the biological,chemical,mechanical and immune barriers of model animals,and the differences in pharmacological effects and underlying mechanisms from the perspective of regulating the intestinal mucosal barrier in UC mice.Results:SEM observation revealed numerous hearth soil particles on the surface of soil-fried AMR,accompanied by bubble-like bulges.At the same time,there were many cracks and folds on the surface of the hearth soil.EDS analysis revealed that the contents of Si,Al,Mg and Ca in soil-fried AMR were significantly higher than those of raw products,and these elements constituted the primary components of hearth soil.UPLC-Q-TOF-MS combined with database comparison was used to identify the chemical constituents of raw and soil-fried AMR.In positive ion mode,a total of 132 components were identified,primarily comprising three categories of terpenoids,polyphenols and amino acids.In negative ion mode,a total of 40 components were identified,primarily polyphenolic and glycoside compounds.Among them,the contents of sesquiterpenes and polyphenolic acids were changed significantly before and after processing.Soil-fried AMR could reduce the DAI score of UC mice,alleviate the shortening of colon length,reduce the levels of pro-inflammatory factors such as interleukin(IL)-17,IL-18,γ-interferon(IFN-γ)and tumor necrosis factor(TNF)-α in serum,increase the levels of anti-inflammatory factors such as secretory immunoglobulin A(sIgA),IL-10,IL-4 and transforming growth factor-β(TGF-β)in serum,increase the expressions of key genes and proteins of intestinal mucosal barrier such as tight junction protein-1(ZO-1),Occludin,Claudin-1 and mucin 2(MUC2)in colonic mucosa,and improve the disorders of intestinal flora diversity and the levels of SCFAs(P＜0.05,P＜0.01).The raw and stir-fried products of AMR also exhibited the aforementioned effects,but they were weaker than the soil-fried products.Additionally,the auxiliary material hearth soil also had a certain pharmacodynamic effect.Conclusion:Soil-fried AMR can enhance the protective effect on intestinal mucosal barrier in UC mice.These changes or heating-induced alterations in the microscopic structure and chemical composition of AMR may be attributed to the dual effects of adsorption of hearth soil.