| Background: Short-chain fatty acids(SCFAs)refer to organic fatty acids with less than six carbon atoms in the carbon chain,which are produced by intestinal flora metabolism.They mainly contain seven kinds,namely acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,pentanoic acid and hexanoic acid.The main SCFAs in organisms are acetic acid,propionic acid and butyric acid,accounting for 90% to 95% of the total SCFAs.Studies have shown that SCFAs can regulate intestinal flora balance,improve intestinal function,as well as anti-tumor,anti-inflammatory and regulate gene expression.Detecting the changes of SCFAs in vivo can reveal the mechanism of drug action and assist the diagnosis and treatment of clinical diseases.Due to the volatility,polarity and low content of SCFAs in biological samples,the current SCFAs analysis methods have the disadvantages of complex pretreatment and poor detection stability.Therefore,it is particularly important to establish a more accurate and efficient SCFAs analysis method.Objective: In order to apply the trend analysis of SCFAs content in different samples,the qualitative semi-quantitative analysis method of SCFAs in different biological samples was established,and to explore the regulatory mechanism by polysaccharide of Polygonatum kingianum and water extract of Polygonatum kingianum on lipid metabolism disorder and non-alcoholic fatty liver in rats.Methods: Qualitative analysis of SCFAs in biological samples: feces,hepatic portal vein blood,tissues(liver,pancreas,heart,spleen,kidney,lung)of SPF male SD rats were collected.SCFAs in feces were extracted by adjusting p H after protein precipitation with methanol.SCFAs in hepatic portal vein blood and tissues were enriched by solid phase extraction column with anion exchange resin(PAX).The samples were detected by gas chromatography/mass spectrometry(GC/MS).The information of the compounds was identified by computer searching standard mass spectrometry library.The composition of SCFAs in the samples was identified by comparing with the mixed reference substances.A semi-quantitative analysis method for SCFAs in biological samples was established.Feces,hepatic portal vein blood and tissues liver of SPF male SD rats were collected.Fecal samples were purified by adjusting p H after protein precipitation with methanol.High-throughput purification of hepatic portal vein blood and tissue samples(96 samples could be purified simultaneously at one time)by solid phase extraction column with PAX as filler was carried out and analyzed by GC/MS.The linearity and range,precision,repeatability and stability of the method are investigated.Semi-quantitative analysis and application of SCFAs in biological samples: The established semi-quantitative analysis method was used to determine SCFAs in rats with lipid metabolism disorder and non-alcoholic fatty liver(feces,hepatic portal vein blood,liver).Pearson correlation analysis was used to analyze the correlation between SCFAs content and intestinal flora,lipid parameters,and to explore the regulatory effect of Polygonatum kingianum polysaccharide on SCFAs in rats with lipid metabolism disorder and the regulatory effect of water extract of Polygonatum kingianum on SCFAs in rats with non-alcoholic fatty liver disease.Results: Seven kinds of SCFAs were identified from feces and hepatic portal vein blood,including acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid.Five kinds of SCFAs were identified from liver,spleen,kidney and lung,including acetic acid,propionic acid,isobutyric acid,butyric acid and valeric acid.Six kinds of SCFAs were identified from some pancreatic and heart samples,including acetic acid,propionic acid,isobutyric acid and butyric acid,pentanoic acid,hexanoic acid.The semi-quantitative analysis method was established by methodological investigation.The linear relationship of SCFAs in different biological samples(feces,hepatic portal vein blood and tissues)was good in the range of 0.005-0.1 mol/L(r > 0.999).The instrument precision RSD was less than 2.23%,the sample stability RSD was less than 5.07%,and the method repeatability RSD was less than 3.05%.All of the methods met the requirements of quantitative analysis of biological samples.The content of butyric acid in feces,hepatic portal vein blood and liver of rats with lipid metabolism disorder was increased after administration of Polygonatum kingianum polysaccharide.The correlation analysis showed that the abundances of Butyrivibrio in feces were negatively correlated with propionic acid,while those of Peptococcus,Allobaculum and Blautia were negatively correlated with acetic acid,among which Allobaculum and Blautia were negatively correlated with propionic acid.Clostridium abundance was positively correlated with isobutyric acid and butyric acid.At the same time,the abundance of Roseburia was positively correlated with butyric acid.There was a significant positive correlation between the abundance of Bacteroides and valeric acid.LDL-C content in hepatic portal vein blood was positively correlated with acetic acid,propionic acid,isobutyric acid,butyric acid and pentanoic acid.There was a significant positive correlation between the content of TG and butyric acid in liver.It is presumed that this pathway is a new pathway for Polygonatum kingianum to improve lipid metabolism disorder.The content of butyric acid in hepatic portal vein blood and liver of rats with non-alcoholic fatty liver was increased by the water extract of Polygonatum kingianum.The correlation analysis showed that TC and ALT were negatively correlated with butyric acid.Butyric acid may increase liver lipid oxidation,increase glycogen storage and decrease liver fatty acid synthase activity through AMPK acetyl Co A carboxylase pathway to interfere with non-alcoholic fatty liver.Conclusion: SCFAs are found in feces,hepatic portal vein blood and tissues(liver,heart,spleen,lung and kidney),and the types of SCFAs in different samples are different.The semi-quantitative analysis method based on high throughput solid phase extraction coupled with gas chromatography-mass spectrometry can be used for rapid and efficient analysis of SCFAs in biological samples(feces,hepatic portal vein blood,liver and spleen).Polysaccharide and water extract of Polygonatum kingianum can significantly regulate lipid metabolism disorder and SCFAs in non-alcoholic fatty liver rats,which are important mechanisms of drug efficacy. |
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