| Bile acids(BA) is the main organic acid in bile, which is a type of cholanic acid synthesized from cholesterol in the liver. BA has many very important physiological functions because of its special structure and metabolism diversity: the amphiphilic structure of BA can promotes the digestion and absorption of lipid; BA can combine lecithin to be micelle, which can transport cholesterol to the small intestine, so BA can help to maintain the balance of body cholesterol and prevent the formation of biliary calculi; BA can regulate the metabolism of lipid, glucose, energy and itself through three signaling mechanism(1) activate mitogen-active protein kinase(MAPK)(2) as the natural ligand of G-protein-coupled receptors(TGR5)(3) activate Farnesoid X receptor(FXR), which is a nuclear hormone receptor. The metabolic processes of BA are in close contact with Gastrointestinal and hepatobiliary system. Diseases such as hepatitis, cirrhosis, liver cancer and cholestasis can influence the synthesis and metabolism of BA, affecting the concentration of bile acids in serum, so serum BA is an indicator with high specifity and sensitivity in the diagnosis of hepatobiliary system diseases, and also a sensitive indicator reflecting the substantial damage and pathological stage of liver and gall. Intestinal canal(IC) is one of the main metabolic locus of BA, part of the primary BA that have been secreted into IC transfers into secondary BA and tertiry BA under the action of bacteria in jejunum, ileum and upper sectiong of colon. BA is reabsorbed into portal blood mainly in terminal ileum, forming the enterohepatic circulation(EC) of BA. So IC is in close contaction with BA, so lesion in IC such as enteritis, diarrhea, adenocarcinoma of small intestine and Colorectal Cancer may lead to BA metabolism disorder and affecting the EC. So it has great significance to study the BA metabolic spectrum for early diagnosis, course monitoring, efficacy diagnosis and prognosis of these diseases. The detection methods of BA in biologic sample at present are mainly: enzymatic analysis, immunoassay, nuclear magnetic resonance method, chromatography and chromatography tandem mass spectrometry, the first three methods have large deficiency in detection sensitive, specificity and applications. The common chromatography can hardly separate all the components perfectly because of the complexity of biological sample, and the common detector has low sensitive, all these limit its application in the detection of biological sample. GC-MS has a strict requirement to samples, which need the hydrolysis and derivative according to BA types, only the unconjugated BA can be detected and unable to quntified all the conjugated BA, all these limit the application of GS-MS. LC-MS is a better method to detect BA in high throughput complex biological samples at present, because of its higher separating ability, detection sensitivity and specificity.Our study established the sensitive, rapid and stable serum BA detection method based on 43 mixed BA standards, human serum and rats serum by UPLC/QTOFMS, which can simultaneously determine 28 BA in human serum and 35 BA in rats serum. Methodological evaluation showed that most BA have good linear relationship and low limit of detection and limit of quantitation. BA in the three samples showed good precision and stability, also the recovery of most BA are 80-120%. So the detection method of BA spectrum by UPLC/QTOFMS meet the requirement in qualitative and quantitative detection of biologic samples, and it’s appropriate for the BA detection of high throughput biological samples and serum BA analysis in diagnosis of related diseases. Based on the above results,we established obesity rats model through high fat diet and determined the serum BA spectrum of obesity and normal rats by the established method, then compared BA of the two group by one-dimensional and multi-dimensional statistical analysis, the results showed that there are significant difference of BA spectrum between them, and 17 differences BA produced by metabolism of bacteria in IC, which present downward trend in obesity rats. So we deduct that obesity induced by high-fat diet may has direct relationship with the structure of the intestinal microflora, which affect BA and its metabolism. The study give the BA spectrum of obesity animal serum for the first time, broaden the observation window of the study on obesity and the related metabolic diseases, provide cues for investigating the formation of obesity.Besides, we studied on the chemical constituents of Thunbergia. Thunbergia belongs to the Acanthaceae and Thunbergia family. It has been reported to contain chemical constituents such as flavonoid glycosides, iridoids and phenols in this genus plant. Thunbergia has many pharmacological activities, such as hypoglycemic acitivity, heat-clearing and detoxifying, antibiosis and hepatoprotective. Based on our previously research, the ethyl acetate extract of Thunbergia ethanol total extract has a significant inhibition effect on aldose reductase, and showed significant dose-dependence manner. From this active extract, 9 chemical compounds were obtained. Future separation of the ethyl acetate extract, 8 compounds were obtained and their structures were identified as Thunbergin C(1), Thunbergin D(2), Thunbergin E(3), Thunbergin F(4), Thunbergin A(5), Thunbergin B(6), 4,3-Dihydro-8,5,4- trihydroxy-2-(3-methyl-2-butenyl)naphtha[2,3-b]oxiren-1(2H)-one(7) and 8-(β-Glucopyranosyloxy)-4,3-dihydro-5,4-dihydroxy-2-(3-methyl-2-butenyl) naphtha [2,3-b]oxirene-1(2H)-one(8) by the analysis the spectra of IR, EI-MS, 1H-NMR, 13C-NMR and 2D-NMR.. Among these compounds, Thunbergin C~F are new compounds. These compounds have similar basic molecular structure, providing basis for further screening hypoglycemic active ingredients in this species. |
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