不同胆汁制胆南星中胆酸类成分及其解热作用比较

目的比较猪、牛、羊胆汁制胆南星中胆酸类成分及其对发热小鼠体温的影响,为规范胆南星发酵工艺提供依据。方法分别采用猪、牛、羊胆汁与天南星生品发酵制备胆南星。采用HPLC-CAD法检测3种胆南星中胆酸类成分胆酸、去氧胆酸、猪去氧胆酸、鹅去氧胆酸。以干酵母制备小鼠发热模型,观察猪、牛、羊胆汁制胆南星对发热小鼠体温的影响。结果不同胆汁制胆南星中胆酸类成分存在差异,牛胆汁制胆南星中胆酸类成分质量分数最高,胆酸、猪去氧胆酸、鹅去氧胆酸、去氧胆酸的质量分数分别为0.035 9%、0.843 4%、2.260 2%、0.037 2%。猪胆汁制胆南星中鹅去氧胆酸和去氧胆酸升高,胆酸和猪去氧胆酸降低;羊胆汁制胆南星中胆酸类成分除去氧胆酸外均降低。不同胆汁制胆南星对发热小鼠均有一定的清热作用,猪胆汁制胆南星组小鼠体温先升高后降低,且与模型组比较差异有显著性,牛胆汁制胆南星亦表现出清热作用,但与模型组比较无显著性差异。羊胆汁制胆南星清热作用较弱。结论综合比较猪、牛、羊胆汁制胆南星中胆酸类成分质量分数和清热作用,作为制备胆南星的辅料,牛胆汁、猪胆汁优于羊胆汁。     

猪去氧胆酸合成熊去氧胆酸的研究进展

临床上用于治疗肝胆疾病的熊去氧胆酸最初是从熊的胆汁中提取得到,受熊胆汁来源的限制目前主要是以胆酸为原料合成而得。之后随着鹅去氧胆酸提取工艺和鹅去氧胆酸合成熊去氧胆酸工艺的成熟,鹅去氧胆酸逐渐代替胆酸成为生产熊去氧胆酸的主要原料。由于猪去氧胆酸来源丰富、价格低廉,近几年由猪去氧胆酸合成熊去氧胆酸的研究成为甾类药物合成研究的热点之一。本文对这一领域的研究进展进行综述,以期为相关研究提供有价值的参考。     

猪胆粉中猪去氧胆酸含量测定方法的探讨

建立猪胆粉中猪去氧胆酸提取方法的改进。猪胆粉中猪去氧胆酸的含量测定，中国药典2000年版一部收载，是用醋酸乙酯为溶媒进行提取。该法易产生乳化现象，杂质多，使含量测定结果偏低。我们采用氯仿为溶媒提取猪去氧胆酸，在高效硅胶G薄层板上以正乙烷．醋酸乙酯-醋酸-甲醇(20：25：2：3)的上层溶液展开，以薄层扫描(双波长，反射法，锯齿扫描)测得猪去氧胆酸的含量。结果平均回收率为98．9％，RSD=1．83％。该方法准确，分离效果好，杂质少，结果重现性好。     

小活络丸中胆南星的薄层色谱鉴别方法

目的：建立小活络丸中胆酸、猪去氧胆酸和鹅去氧胆酸的薄层色谱鉴别方法,用以帮助提高胆南星的鉴别手段。方法：采用薄层色谱方法,在异辛烷-乙醚．冰醋酸-正丁醇－水（10：5：5：3：1）上层溶液的展开条件下对所抽取45批样品进行实验。结果：经过对13个被抽样单位共45批样品的薄层色谱结果进行汇总统计分析,有73．33％的样品捡出胆酸,86．67％的样品检出猪去氧胆酸,93．33％的样品检出鹅去氧胆酸。结论：建立的胆酸、猪去氧胆酸和鹅去氧胆酸的薄层色谱鉴别方法,可作为小活络丸中胆南星鉴别的辅助手段。     

人工引流熊胆粉中结合胆汁酸成分的研究

目的研究人工引流熊胆粉中的结合型胆汁酸成分。方法采用反相树脂柱层析法对人工引流熊胆粉进行分离,然后通过IR1、H-NMR和13C-NMR等波谱数据进行结构鉴定。结果从人工引流熊胆粉中分离得到3个化合物,鉴定为:牛磺熊去氧胆酸(Ⅰ)、牛磺鹅去氧胆酸(Ⅱ)和牛磺熊去氧酮胆酸(Ⅲ)。结论直接得到结合型胆汁酸Ⅰ和Ⅱ,化合物Ⅲ为首次从熊胆中分离。     

从猪去氧胆酸合成具有生物活性的甾体化合物

综述了用猪去氧胆酸合成具有生物活性的甾体化合物如6α-甲基黄体酮、熊去氧胆酸、鹅去氧胆酸、油菜甾醇内酯及其类似物和维生素 D_3活性代谢物等的概况。     

RP-HPLC法同时测定猪胆粉药材中3种牛磺结合型胆酸的含量

目的:建立同时测定猪胆粉药材中牛磺猪胆酸、牛磺猪去氧胆酸和牛磺鹅去氧胆酸含量的RP-HPLC方法。方法:采用Welchrom C18色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇-磷酸二氢钠溶液(0.03 mol·L-1)(70∶30),用磷酸调节pH为4.4,流速为1.0 mL·min-1,检测波长200 nm,柱温25℃。结果:牛磺猪胆酸、牛磺猪去氧胆酸和牛磺鹅去氧胆酸进样量分别在1.65～16.5μg(r=0.9998),2.03～20.3μg(r=0.9998),1.09～10.9μg(r=0.9996)范围内线性关系良好;检测限分别为26.4,7.6,38.2 ng;平均加样回收率(n=9)分别为101.2%(RSD=1.9%),99.1%(RSD=1.7%),100.6%(RSD=2.1%)。结论:该方法简便、准确,重复性好,在同一色谱条件下实现多指标成分的同时测定,为猪胆粉的全面质量评价提供参考。     

鹅去氧胆酸制备新工艺

用氯化钙取代老工艺中所用的氧化钡,利用鹅去氧胆酸钙盐与杂质钙盐在水中溶解度的不同,分离纯化了鹅去氧胆酸,收率3.2％。     

猪去氧胆酸的纯化工艺

目的研究猪去氧胆酸中的杂质成分和纯化方法。方法运用重结晶和柱层析法进行分离和纯化。结果与结论从猪去氧胆酸中分离出一种主要杂质,建立了纯化猪去氧胆酸的方法。     

藿胆丸含量测定方法的改进

目的：研究测定藿胆丸中猪去氧胆酸和鹅去氧胆酸含量的方法。方法：采用荧光薄层扫描法对其有效成分猪去氧胆酸和鹅去氧胆酸的含量进行测定。结果：猪去氧胆酸在0.1～1．0μg范围内呈良好的线性关系,回归方程为：Y=201.14X 34．41,r=0.9992;平均回收率为100．78％(RSD1．91％),重复性RSD为1．80％;鹅去氧胆酸在0．1～1.2μg范围内呈良好的线性关系,回归方程为Y=370．38X 24．22,r=0.9998,平均回收率为101．18％(RSD为2．13％),重复性RSD为1．42％。结论：本法可靠、准确度高、重现性好,可有效控制藿胆丸的质量。     

Bile acids in porcine fetal bile

A study of the biliary bile acid composition in porcine fetus compared with that of the adult pig is described. Biles, collected during gestation (weeks 4, 15 to 17 and at birth), aged six months and two years old, were analyzed by gas-liquid chromatography and capillary GC-MS. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, piperidinohydroxypropyl Sephadex LH-20. All and one fourth of the total bile acids in the bile of weeks 4 and 15 of gestation, respectively, were present as unconjugated form, however, only a trace of unconjugated bile acids was present in bile of late gestation, the young and the adult pigs. The ratio of glycine/taurine (G/T) conjugates in the conjugated fraction of the fetal bile at 15 weeks gestation was less than 1, which markedly contrasted with the conjugation pattern for adult bile where the ratio of G/T conjugates was approximately more than 9. The predominant acids identified in porcine fetal bile of the 4 weeks gestation were cholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-chola n-24-oic acid) and chenodeoxycholic acid (3alpha,7alpha -dihydroxy-5beta-cholan-24-oic acid). However, cholic acid in late gestation, young, and adult bile was the smallest component, whereas chenodeoxycholic acid was still the major constituent of these biles. The presence of small but valuable amounts of allocholic acid (3alpha,7alpha,12alpha-trihydroxy-5alpha-chol an-24-oic acid) and cholic acid in early gestation suggested the presence of 12alpha-hydroxylase activity of steroid nucleus in fetal liver. Considerable amounts of glycine-conjugated hyodeoxycholic acid were found in the bile of the gestation periods, suggesting the placental transfer of this bile acid from maternal circulation.     

Cattle bile but not bear bile or pig bile induces lipid profile changes and fatty liver injury in mice: mediation by cholic acid

Three types of animal bile preparation, bear bile (BB), cattle bile (CB) and pig bile (PB) differ in bile acid composition and are supposed to exert different pharmacotoxicological actions. Dietary supplementation with CB at 1% (w/w) for 4 weeks decreased triacylglycerol (TAG) level but increased total cholesterol (CHO) level in serum, which were associated with fatty liver injury in mice. The increased levels of cholesterol esters (CE) and monounsaturated fatty acids (MUFA) in the serum and liver were observed in the mice fed the CB-supplemented diet. Lipid abnormalities and fatty liver injury observed in the mice fed the CB diet were not induced by the supplementation with BB and PB. The supplementation with cholic acid (CA), the most abundant bile acid in CB, could induce lipid abnormalities and fatty liver injury, which were indistinguishable from those induced by CB supplementation. CB and CA supplementation induced similar changes in the expression levels of mRNAs in the liver. Thus, CB induced lipid abnormalities and fatty liver injury, which can be attributed to the actions of CA contained in CB. The inabilities of BB and PB to induce lipid abnormalities and fatty liver injury are supposed to be due to their limited contents of CA.     

Isolation and determination of bile acids.

In this article, the methods of isolation and determination of bile acids are reviewed. Methods for separation of bile acids from cattle and pig bile are given in detail. Isolation of a mixture of cholic acid and deoxycholic acids from cattle bile and their subsequent purification are described. The isolation and purification of hyodeoxycholic acid and other components of pig bile are also included. Methods for the determination of bile acids in various biological samples are reviewed, including enzyme assays, radioimmunoassay, enzyme immunoassay and chromatographic methods. Among chromatographic methods, separation and determination of bile acids by thin-layer chromatography, gas chromatography and high performance liquid chromatography are reviewed. Particular attention is given to the use of high performance liquid chromatography since this has recently been the most commonly applied method for the separation and determination of bile acids.     

Chromatographic determination of bile acids in biological fluids with sensitive and selective detection

Separation and determination of bile acids in biological fluids by high-performance liquid chromatography are reviewed. The capacity ratios of bile acids on an ODS column were affected by the number, position and configuration of the hydroxyl group on the steroid nucleus, and the chromatographic behavior was markedly influenced by the pH of a mobile phase according to the conjugated form at C-24. A new pre-column derivatization reagent, 1-anthroyl nitrile, was developed and applied to the analysis of bile acids in biological fluids. Bile acids were derivatized through the 3 alpha-hydroxyl group into the corresponding esters, separated on an ODS column, and monitored by a fluorescence detector with detection limit of 20 fmol. The sensitive method for the determination of bile acids in biological materials by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is also described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to positive ion current. A characteristic carboxylate anion [M-181]- was produced as the most abundant ion by the loss of the PFB group in NICI. PFB esters of bile acids were further derivatized into the dimethylethylsilyl ethers and then separated by GC. The detection limit was 2 fg when the characteristic anion was monitored in the NICI mode. The preparation of 18O-labelled bile acids, as the internal standard for the trace analysis or the tracer for the metabolic study, was developed. Finally, the clean-up procedure for bile acids in biological fluids was investigated. The combined use of solid-phase extraction with a Sep-pak C18 or Bond Elut cartridge and group separation on a lipophilic ion-exchange gel, piperidinohydroxypropyl Sephadex LH-20, was found most effective for this purpose.     

Species-specific and age-dependent bile acid composition: aspects on CYP8B and CYP4A subfamilies in bile acid biosynthesis

The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12alpha or 6alpha position, respectively, has a profound effect on the hydrophilic-hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12alpha-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6alpha-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6alpha-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.     

Chemical and metabolic transformations of selected bile acids.

This article surveys chemical transformations of selected bile acids. Chemical transformations were initially carried out with the aim of determining the structure of bile acids. More recently they have been concerned with bile acid interconversions as well as with the synthesis of steroid hormones, vitamins and therapeutc agents. Studies of similarities and differences in the biosynthesis of bile acids from cholesterol have occupied many researches. However, this article reviews only papers dealing with the synthesis of potential intermediates in the biosynthesis of bile acids. Steroid hormones such as pregnenolone, progesterone and testosterone are synthesized from methyl thiodeoxycholate whereas cortisone is synthesized from methyl deoxycholiate. Numerous papers and patents devoted to the synthesis of ursodeoxycholic acid from cholic or chenodeoxycholic acid testify to its effectiveness in the treatment of cholelithiasis. Chenodeoxycholic acid appears to be an excellent precursor in the synthesis of steroid plant growth regulators, as well as in the synthesis of metabolites and vitamin D analogues. Chirality of bile acids has been exploited in the synthesis of cyclic and acyclic receptors and solvents. Cholic and deoxycholic acids have been used to create new macrocyclic structures which show different capacities to bind and transport other compounds. Another important trend in the chemistry of bile acids is their application in combinatorial chemistry.     

Influence of drugs on bile acid excretion.

The hydroxylation of bile acids in rat liver microsomes in cyt P-450 dependent (Bj?rkhem et al., 1975). To find out possible interactions between drugs and bile acid hydroxylation and/or active transport mechanisms we investigated the influence of the microsomal inhibitor metyrapon, the microsomal inducer phenobarbital and the intrahepatic cholestasis producing agents chlorpromazine, phenylbutazone and progesteron on bile flow and bile acid excretion. The excretion in monohydroxy (MBA), dihydroxy (DBA) and trihydroxy (TBA) bile acids were estimated in bile-fistula rats in three one hour periods. MBA, DBA and TBA were separated with thinlayer-chromatography and estimated fluorimetrically. Bile flow, bile acid excretion and relation TBA/DBA were influenced by acute and subchronic administration of the above mentioned drugs in different ways.     

Interaction of fusidates with bile acid uptake by isolated rat hepatocytes

Summary The interaction of fusidic acid and two of its conjugates with carrier-mediated uptake of bile acids was investigated in isolated rat hepatocytes. All three fusidates inhibited the uptake of both cholate and taurocholate competitively suggesting a direct interaction of fusidates with bile acid carrier. The inhibition constants for all three fusidates for the inhibition of cholate uptake were significantly different from the respective inhibition constants for the inhibition of taurocholate uptake. This would indicate that both cholate and taurocholate are transported by more than one carrier into hepatocytes. The results may also indicate that taurine conjugated bile acids may be transported preferentially by one transport system while unconjugated bile acids may be preferentially transported by another transport system.Part of this publication was presented in the 18. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (1977)