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ObjectiveTo investigate the role and mechanism of hyodeoxycholic acid （HDCA） in the progression of metabolic associated fatty liver disease （MAFLD）， and to provide a new theoretical basis for further clarifying the pathogenesis of MAFLD. MethodsL02 hepatocytes were used as experimental cells， and palmitic acid was used to induce steatosis in L02 cells. The farnesoid X receptor （FXR） siRNA interference chain technique was used to construct a hepatocyte cell line with low FXR expression. CCK8 assay was used to observe the effect of HDCA on L02 steatosis hepatocytes at different concentrations （0， 100， 200， 300， and 400 μmol/L） and time points （12， 24， 36， and 48 hours）. The method of qRT-PCR was used to measure the mRNA expression levels of FXR， proliferating cell nuclear antigen （PCNA）， Cyclin D1， phosphatidylinositol 3-kinase （PI3K）， and protein kinase-B （AKT）， and Western blot was used to measure the protein expression levels of FXR， Cyclin D1， PCNA， PI3K， phosphorylated PI3K （p-PI3K）， AKT， and phosphorylated （p-AKT）. A one-way analysis of variance was used for comparison of normally distributed continuous data with homogeneity of variance between multiple groups， and the Tukey HSD test was used for further comparison between two groups； the Welch analysis of variance was used for comparison of normally distributed continuous data with heterogeneity of variance between multiple groups， and the Games-Howell test was used for further comparison between two groups. The independent-samples t test was used for comparison between two groups. ResultsCCK8 assay showed a significant reduction in the viability of L02 cells and steatosis hepatocytes treated by 300 μmol/L HDCA （P<0.05）， and qRT-PCR showed a significant increase in the mRNA expression level of FXR and significant reductions in the mRNA expression levels of PCNA， Cyclin D1， PI3K， and AKT （all P<0.05）. Western blot showed a significant increase in the protein expression level of FRX （P<0.05）， and after interference of FXR expression in L02 cells， there were significant increases in the protein expression levels of PCNA， PI3K， p-PI3K， AKT， and p-AKT （all P<0.05）. ConclusionHDCA inhibits the PI3K/AKT signaling pathway by upregulating FXR expression， thereby inducing a reduction in the viability of steatosis hepatocytes.
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OBJECTIVE To establish the method for determination of bile acid active constituents （cholic acid， hyodeoxycholic acid and sodium taurocholate） in Babao jingfeng powder， and to compare the differences of 3 constituents in Babao jingfeng powder from different manufacturers. METHODS UPLC-MS method was adopted to determine the contents of cholic acid， hyodeoxycholic acid and sodium taurocholate in 9 batches of Babao jingfeng powder from 3 manufacturers. The separation was performed on a Phenomenex Luna® C18 column with mobile phase consisted of acetonitrile-water （gradient elution） at a flow rate of 0.5 mL/min. The column temperature was 40 ℃ and the sample size was 2 μL. MS system was operated in a negative ion single quadrupole mode， and the m/z of cholic acid， hyodeoxycholic acid and sodium taurocholate were 407.2， 392.2 and 514.2， respectively. With the contents of three components as the index， cluster analysis and principle component analysis were performed on 9 batches of samples. RESULTS The linear ranges of cholic acid， hyodeoxycholic acid and sodium taurocholate were 5.48- 548.40， 5.38-538.40， 4.74-474.05 ng/mL， respectively （r≥ 0.999 3）. RSDs of precision， repeatability and stability tests（24 h） were all lower than or equal to 3.7% （n＝6）， and the average recoveries were 103.3%， 103.3% and 101.6% with RSDs of 3.3%， 3.4%， 4.2% （n＝6）， respectively. The contentsof cholic acid ranged 0.702-1.711 mg/g， those of deoxycholic acid ranged 0.599-2.049 mg/g， and those of sodium taurocholate ranged 0.664-1.752 mg/g in 9 batches of samples. The average content of 3 components in samples from manufacturer A was high， and the difference between batches was the smallest； the average content of 3 components from manufacturer C was the lowest， and the difference between batches was large. Cluster analysis could basically distinguish samples from different manufacturers； cluster analysis was conducted by using the comprehensive score of principal components as an indicator， the results of which were basically consistent with those of cluster analysis for content. CONCLUSIONS The method is established successfully for the content determination of the 3 bile acid active constituents of artificial bezoar in Babao jingfeng powder. The contents of 3 components in Babao jingfeng powder from 3 manufacturers are significantly different.
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AIM To establish an HPLC method for the content determination of six constituents in Qingkailing Freeze-Dried Powder for Injection (cholic acid,hyodeoxycholic acid,Bubali Cornu,etc.).METHODS The content determination of adenosine,chlorogenic acid and gardenoside was performed on a 30 ℃ thermostatic XBridge C1s column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-water (containing 0.1％ formic acid) flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 254 nm.The content determination of baicalin,hyodeoxycholic acid and cholic acid was performed on a 35 ℃ thermostatic XBridge C1s column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of methanol-water (containing 0.1％ formic acid) flowing at 1.0 mL/min in a gradient elution manner.RESULTS Six constituents showed good linear relationships within the ranges of 2.244-56.108,2.658-66.445,4.347-108.682,122.01-1 016.75,131.94-1 099.50,152.22-1 268.50 μg/mL (r ＞ 0.999 0),whose average recoveries (RSDs) were 101.1％ (0.46％),98.0％ (1.74％),99.7％ (0.15％),100.9％ (1.31％),98.1％(0.18％),98.2％ (1.61％),respectively.CONCLUSION This stable and reproducible method can be used for the quality control of Qingkailing Freeze-Dried Powder for Injection.
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OBJECTIVE:To establish a method for the contents determination of cholic acid,hyodeoxycholic acid and deoxy-cholate acid in Naoxueshuan tablet. METHODS:HPLC-ELSD was performed on the column of SHIMADZU VP-ODS C18 with mo-bile phase of acetonitrile-0.2% formic acid(gradient elution)column temperature was 30 ℃,at flow rate of 1.0 ml/min,and the vol-ume injection was 10 μl. RESULTS:The linear range was 0.039 84-0.398 4 mg for cholic acid,0.032 064-0.320 64 mg for hyode-oxycholic acid and 0.0339 52-0.339 52 μg for deoxycholate acid(r≥0.999 0),and the logarithm value of volume injection and peak area showed good linear relationship;RSDs of precision,stability and reproducibility tests were lower than 2%;recoveries were 101.33%-104.05%(RSD=0.95%,n=6),95.56%-101.38%(RSD=2.54%,n=6)and 96.07%-97.12%(RSD=0.44%,n=6),re-spectively. CONCLUSIONS:The method is simple and accurate,and suitable for the contents determination of cholic acid,hyode-oxycholic acid and deoxycholate acid in Naoxueshuan tablet.
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Objective To establish a pre-column derivation HPLC-UV method for the content determination of hyodeoxycholic acid in artificial calculus bovis. Methods The hyodeoxycholic acid was derived by 2-bromo-2’-acetonaphthoneat using triethylamine as the catalyst in 60 ℃ water bath. After that, a HPLC-UV method was established to determine the content of hyodeoxycholic acid in artificial calculus bovis. Results When the derivatising time at 60 ℃ water bath was 50 min, the radio of the molar amount of derived reagents and hyodeoxycholic was over 20:1 and the radio of catalyst and hyodeoxycholic was over 15:1; the reaction was completed. The calibration curve was linear within the range of 0.1–2 μg for hyodeoxycholic acid (r=0.999 7), and the average recovery was 97.85% (RSD=1.6%). In this sample, the content of hyodeoxycholic is 4.12%. Conclusion The method is with high sensitivity, highly reproducible, reliable and accurate for the content determination of hyodeoxycholic acid in artificial calculus bovis.
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Objective: To synthesize and identify the hyodeoxycholic acid (HDCA) artificial antigen and to lay a foundation for the preparation of its specific monoclonal antibody, establishment of immunoassay method, and application in the pharmacological researches. Methods: The HDCA artificial immunogen (HDCA-BSA) and coating antigen (HDCA-OVA) were synthesized by mixed anhydride method and then examined by TLC method. BALB/c mice were immunized with the emulgator containing the prepared HDCA-BSA conjugate and Freund's adjuvant by the back sc multipoint injection for three times following with a booster immunization by the prepared HDCA-BSA conjugate alone. Then the blood of mice was obtained from tail vein 24 h after the booster, and the sera were isolated for the titer and specificity test by ELISA. Results: According to TLC, HDCA was successfully conjugated to BSA. The antibody against HDCA obtained from immunized-mice could bind to HDCA and the titer was over 1:8 000. As shown in the competitive inhibition curves, the antibodies in the serum were specifically bound to HDCA without cross-relativities with glycyrrhizic acid, baicalin, geniposide, etc, except cholic acid (90%). Conclusion: The HDCA-BSA is successfully synthesized, which could be used for the preparation of anti-HDCA monoclonal antibody and the research on pharmacological targets of HDCA.
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AIM: To establish a method for determining baicalin, jasminoidin, cholalic acid and hyodeoxycholic acid in Qingkailing Granules(chololic acid, hyodeoxycholic acid, baicalin, Fructus Gardeniae, Cornu Bubali, Flos Longicerae Japonicae, Radix lsatidis, Concha Margaritifera). METHODS: Part 1 (determination of baicalin and jasminoidin):The HPLC method was carried out on kromasil~(TM)C_(18) column(4.6 mm×150 mm,5 μm) with acetonitrile-water(10: 90)as mobile phase, gradient elution; the UV detection wavelength was at 238 nm. Part 2 (determination of cholalic acid and hyodeoxycholic acid):The HPLC method was carried out on kromasil~(TM)C_(18) column(4.6 mm×150 mm,5 μm) with acetonitrile-water-phosphonic acid(35:65:0.1) as mobile phase,gradient elution;the UV detection wavelength was at 192 nm. RESULTS: The average recoveries were 99.30% with RSD of 0.2% for baicalin; 99.50% with RSD of 0.4% for jasminoidin; 99.04% with RSD of 0.2% for hyodeoxycholic acid; 99.06% with RSD of 0.4% for cholalic acid; respectively. CONCLUSION: The assay demonstrats that the method is simple,it has the adequate accuracy and selectivity to quantify the four active components in Qingkailing Granules.
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Objective To establish a method for content determination of hyodeoxycholic acid in Qingkailing Injection. Methods An UltimateXB-C18 column (5 ?m, 250 mm?4.6 mm) was used with a mobile phase of acetonitril-water-phosphoric acid (35∶65∶0.1). The flow rate was 1 mL/min. The temperature of the column was 40 ℃. The detection wavelength was 192 nm. Results There was a good linear relationship between the concentration of hyodeoxycholic acid and absorption area value in range of 0.201 35～1.006 75 mg/mL, r=0.999 6. The average recovery was 98.25% with RSD=1.26%. Conclusion This method was accurate, credible and repeatable which can be used to control the quality of Qingkailing Injection.
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Objective To design and synthesize an antitubercular compound,24-ketoarguesterol.Methods The target compound was synthesized via 8 steps,including methyl esterification,grignard isopropylation,deportation,etc.,using hyodeoxycholic acid as the starting material.3?-tert-butyldimethylsilyoxyl-5?-hydroxyl-6?-di-me-thoxylmethyl-24-keto-B-norcholanate was the key intermediate and grignard isopropylation as the key step.Results The target compound was synthesized in a total yield of 42% and identified by mass spectrometry(MS),proton magnetic resonance spectroscopy(1HNMR),carbon magnetic resonance spectroscopy,(13CNMR)and infrared spectroscopy(IR).Conclusion The synthetic route is characterized by atomic economy and high efficiency and laid the foundation for development of novel antituberculous drugs.
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Objective To synthesize 24-methylenecholestan-3?,5?,6?-triol,a compound same as a marine natural product,.Methods The target molecule was synthesized in 7 steps using hyodeoxycholic acid as the starting material,3?-(tetrahydropyran-2-yloxy)-cholest-5-ene-24-ketone as the key intermediate and isopropylation in Grignard reagents as the key step.Results The target molecule was synthesized in a total yield of 53% and confirmed by mass spectrometry(MS),proton magnetic resonance spectroscopy(1HNMR),carbon magnetic resonance spectroscopy,(13CNMR)and infrared spectroscopy(IR).Conclusion Our method to synthesize 24-methylene-cholestan-3?,5?,6?-triol is characterized by easily available and cheap starting material,short synthetic route,high yield and atom-economic.
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Objective To synthesize a marine natural product, 24-methylenecholesterol. Methods The target molecule was synthesized in 7 steps using hyodeoxycholic acid as the starting material, 3?-(tetrahydropyran-2-yloxy)-cholest-5-ene-24-ketone as the key intermediate and isopropylation as the key step. Results The target molecule was synthesized with a total yield of 66% and confirmed by MS, IR, 1HNMR and 13CNMR. Conclusion Our synthesis of 24-methylenecholesterol is characterized by easily acquired starting material, short synthetic route, high yield and atom-economic.
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OBJECTIVE:To determine the contents of cholalic acid and hyodeoxycholic acid in Qingkailing suppository by HPLC-ELSD(high performance liquid chromatography with evaporative light scattering detection). METHODS: The analytical column was Diamonsil C18(250 mm?4.6 mm, 5 ?m) and the mobile phase consisted of 0.1% formic acid-acetonitrile(gradient elution) with the temperature of the drift tube of the light scattering detector set at 110 ℃ and the flow rate of the nitrogen gas at 2.0 L?min-1. RESULTS: The linear ranges of cholalic acid and hyodeoxycholic acid were 0.59～7.4 ?g(r=0.999 6)and 0.38～4.8 ?g(r=0.999 7),respectively,with their average recoveries at 99.20%(RSD=1.4%,n=6)and 98.57%(RSD=1.8%,n=6), respectively. CONCLUSION: The method is simple, accurate and well-separated, and it is applicable for the quality control of Qingkailing suppository.
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AIM: To establish a method for determining baicalin,jasminoidin,cholalic acid and hyodeoxycho-lic acid in Qingkailing Granules(chololic acid,hyodeoxycholic acid,baicalin,Fructus Gardeniae,Cornu Bubali, Flos Longicerae Japonicae,Radix Isatidis,Concha Margaritifera). METHODS: Part 1(determination of baicalin and jasminoidin):The HPLC method was carried out on kromasilTMC_18 column(4.6 mm?150 mm,5 ?m) with acetonitrile-water(10∶90)as mobile phase,gradient elution;the UV detection wavelength was at 238 nm.Part 2(determination of cholalic acid and hyodeoxycholic acid):The HPLC method was carried out on kromasilTMC_18 column(4.6 mm?150 mm,5 ?m) with acetonitrile-water-phosphonic acid(35∶65∶0.1) as mobile phase,gradient elution;the UV detection wavelength was at 192 nm. RESULTS: The average recoveries were 99.30% with RSD of 0.2% for baicalin;99.50% with RSD of 0.4% for jasminoidin;99.04% with RSD of 0.2% for hyodeoxycholic acid;99.06% with RSD of 0.4% for cholalic acid;respectively.CONCLUSION: The assay demonstrats that the method is simple,it has the adequate accuracy and selectivity to quantify the four active components in Qingkailing Granules.