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。     

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。     

HPLC-ELSD法同时测定生物转化样品中牛磺熊去氧胆酸等5种成分的含量

目的:建立同时测定生物转化样品中牛磺熊去氧胆酸、牛磺鹅去氧胆酸、熊去氧胆酸、鹅去氧胆酸、牛磺7-酮石胆酸5种成分含量的方法。方法:采用高效液相色谱法,检测器为蒸发光散射检测器。色谱柱为Agilent XBD-C18,流动相为0.05%三氟乙酸-乙腈,梯度洗脱,流速为1.0 ml/min;漂移管温度为80℃,N2流量为3.0 ml/min,柱温为40℃。结果:牛磺熊去氧胆酸、牛磺鹅去氧胆酸、熊去氧胆酸、鹅去氧胆酸、牛磺7-酮石胆酸检测质量浓度线性范围分别为10.00²00.08、10.22200.08、10.22²04.32、10.00204.32、10.00¹99.92、9.85199.92、9.85¹97.08、10.25197.08、10.25²04.92μg/ml(r=0.999 5204.92μg/ml(r=0.999 5⁰.999 8);精密度、稳定性、重复性试验的RSD均不大于2.0%。5种成分的平均回收率分别为97.9%、98.4%、97.9%、98.1%、98.3%,RSD分别为0.49%、0.63%、0.81%、0.72%、0.78%(n=3)。结论:该方法快速、准确、重复性好,可用于生物转化样品中牛磺熊去氧胆酸等5种成分的定量测定。     

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。     

薄层扫描法测定熊胆引流物中胆汁酸含量

熊胆向以贵重药材闻名,被称之为稀有药品,为开发熊胆资源,解决熊胆奇缺问题,我校解剖教研室已成功地完成了人工引流熊胆汁技术,可随时进行人工引流获取熊胆汁。为了确定胆汁的质量指标,了解其主要成分,我们进行了引流胆汁与天然熊胆的分析。文献报道,熊胆中主要含熊去氧胆酸(ursodesoxycholic acid,UDCA)、鹅去氧胆酸(cheno desoxycholic acid,CDCA)、胆酸(cholic acid,CA)、去氧胆酸(deoxycholic,acid DCA)等。     

猪胆汁中三种主要胆汁酸的提取分离

目的从提取胆红素下脚料中同时提取三种胆汁酸。方法混合猪胆汁酸粗品在硫酸催化下形成甲酯,利用猪去氧胆酸甲酯与苯形成的加合物不溶于苯的性质将其分离出来;用乙酸酐将剩余胆汁酸甲酯中的所有羟基转化为乙酸酯,利用猪胆酸甲酯三乙酸酯在正己烷中溶解度低的性质,使其分离,最后用无水乙醇结晶分离出鹅去氧胆酸甲酯二乙酸酯。所得各种猪胆汁酸酯经乙醇-NaOH溶液水解得到纯猪去氧胆酸、猪胆酸和鹅去氧胆酸,并对产物结构进行了表征,纯度经HPLC测定均大于98%。结果通过一次工艺流程分离出三种胆酸,含量均大于98%。结论该提取工艺提高了原料的利用率,适合工业化生产。     

从猪胆膏中分离纯化鹅去氧胆酸的新工艺

鹅去氧胆酸(chenodeoxycholic acid,CDCA),又称3α,7α-二羟基-5β-胆烷酸,是动物体胆汁中的一种生化物质,临床上不仅用作溶解胆固醇类结石和纠正饱和胆汁的药物[1],还具有显著的平喘、抗炎、镇咳和祛痰作用[2,3].鹅去氧胆酸可以作为昂贵的熊去氧胆酸合成原料,它们都是治疗胆道疾病的有效药物[4].     

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。     

双波长薄层扫描法测定清开灵注射液中牛胆酸及猪去氧胆酸的含量

本实验应用双波长薄层扫描法,同时测定了清开灵注射液中牛胆酸及猪去氧胆酸的含量,方法简便、快速、可靠。牛胆酸及猪去氧胆酸的回收率分别为100.45%,98.45%。     

荧光薄层扫描法测定藿胆丸中猪去氧胆酸(HDCA)和鹅去氧胆酸(CDCA)的含量

应用荧光薄层扫描法测定藿胆丸中猪去氧胆酸和鹅去氧胆酸的含量.对实验条件进行下比较,排除干扰,使含量测定获满意的效果.     

Comparison of the choleretic properties of bile acids

The choleretic properties of cholic, chenodeoxycholic, and deoxycholic acid and their taurine and glycine conjugates were compared to their ability to form micelles. It has previously been concluded that deoxycholate has the lowest critical micellar concentration; chenodeoxycholate is slightly higher and cholic is much higher. Conjugation with glycine and taurine has little or no effect on the critical micelle concentration. Since the choleretic properties of bile salts are thought to be directly proportional to their osmotic activities, one might suspect that deoxycholic acid would be the least choleretic, chenodeoxycholic slightly more choleretic and cholic much more choleretic, with little difference between the conjugated and unconjugated forms. However, in the present study, cholic, chenodeoxycholic and taurocholic acid produced similar increases in bile flow (450–700 μl/kg) after an equimolar dose (55 μM/kg). Except for the conjugation of deoxycholic acid with taurine, conjugation of these bile acids with glycine or taurine always decreased the choleretic properties of the bile acids. Therefore, it has been concluded that there is not a good correlation between the in vitro osmotic properties of bile acids and their ability to increase bile flow.     

Altered bile acid metabolism in alloxan diabetic rats

Changes of cholesterol, phospholipid, triglyceride or bile acid levels in serum liver, bile and feces after the treatment with alloxan were examined in Wistar strain male rats. Serum cholesterol, phospholipid and triglyceride levels and liver cholesterol level markedly increased but liver phospholipid and triglyceride levels remained unchanged. The lipid levels in serum very low density and low density lipoproteins were elevated but those in high density lipoprotein were not. Bile flow was not changed but biliary secretion of cholesterol, phospholipid and bile acids markedly increased. Among the biliary bile acid components, cholic acid markedly increased but the amount of chenodeoxycholic acid was similar to that of normal rats. Fecal excretion of deoxycholic acid increased but that of lithocholic and hyodeoxycholic acids decreased, and alpha, beta- and omega-muricholic acids did not change, thus, the total amount of fecal bile acids remained unchanged. Hepatic cholesterol synthesis was markedly depressed, while cholesterol 7 alpha-hydroxylase activity did not change and cytochrome P-450 content was elevated by about 40%. From such evidence, it was apparent that synthesis of cholic acid increased while that of chenodeoxycholic acid decreased and the total amount of bile acids synthesized did not change in the diabetic rats. Furthermore, marked increase of the pool size of cholic acid and hepatic secretion of cholic acid stimulated the absorption of lipids and produced a hyperlipidemia in the diabetic rats.     

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.     

反相高效液相色谱-蒸发光散射检测法同时测定人工牛黄中多组分含量

目的 :建立人工牛黄中多组分色谱分离条件 ,并对除无机盐外各成分同时测定进行方法学考察。方法 :用反相高效液相色谱 蒸发光散射检测法 ,蒸发光散射检测器参数 :漂移管温度 10 5℃ ,雾化气体 (N2 )流速 :2 0 5SLPM。结果 :在选定色谱条件下 ,胆固醇、各种胆汁酸及无机盐在室温下可达很好分离 ,胆酸与猪去氧胆酸的非衍生分离尚属首次。除无机盐外 ,各物质色谱峰面积与浓度呈良好线性关系 (γ >0 998)。 3个浓度水平的回收率测定值为 98 3 %～ 10 2 4 %。进样 2 0 μL组分最低检出量为 0 0 5～ 0 10 6μg。 结论 :蒸发光散射检测器与高效液相色谱法联用 ,可使不含生色团物质的分离、分析更为准确、灵敏。可为人工牛黄的质量控制提供更为科学的依据     

Mesophase formation during cholesterol gallstone dissolution in human bile: effect of bile acid composition

Duodenal bile obtained from patients with gallstones who were acutely infused with chenodeoxycholic acid, ursodeoxycholic acid, or cholic acid were examined for the propensity toward the formation of a liquid crystalline mesomorphic phase when cholesterol gallstones were incubated in these bile acids. Bile taken from patients infused with ursodeoxycholic acid was found to be enriched in ursodeoxycholic acid; mesophase formation was detected in these samples but not in bile from patients infused with chenodeoxycholic acid or cholic acid.     

Effects of bile acids on the mutagenicity and recombinogenicity of triethylene melamine in yeast strains MP1 and D61.M

When Saccharomyces cerevisia MP1 was treated with bile acids alone or in combination with triethylene melamine (TEM), cholic acid was found to be comutagenic and antirecombinogenic while lithocholic acid had the opposite effect. Other bile acids enhanced the mutagenic and recombinogenic effects of TEM. Chenodeoxycholic acid, deoxycholic acid and ursodeoxycholic acid had stronger comutagenic than corecombinogenic effects; hyodeoxycholic acid did not show this difference.Ox gall and a mixture of bile acids antagonized the genotoxic effect of TEM, and it therefore seems plausible that in normal composition bile acids neutralize each other, i.e., the comutagenic (corecombinogenic) effect of one substance is antagonized by the antimutagenic (antirecombinogenic) effect of another one. However, when the composition is altered, bile acids may become harmful.Experiments with yeast strain D61.M were performed in order to test for aneuploidy caused by bile acids; no effects were observed.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Cholic acid and ursodeoxycholic acid therapy in primary biliary cirrhosis

Summary We treated 6 patients with Stage II primary biliary cirrhosis with cholic acid (CA) 10 mg · kg^–1 per day for 3 months and then with the same dose of ursodeoxycholic acid (UDCA). A matching group of 6 patients was observed for 3 months without any therapy. Liver function tests and serum and stool bile acids were investigated before, during and at the end of CA and UDCA therapy.The results of liver function tests deteriorated after 6–8 weeks of CA therapy and the changes were correlated (r=0.92) with an increase in -dihydroxy-bile acids (chenodeoxycholic acid and deoxycholic acid) in the serum. The 24 h excretion of DCA in 24 h faeces was markedly increased.Ursodeoxycholic acid treatment improved liver function tests; after 4 weeks glutamate dehydrogenase (GLDH) had decreased. After 8–12 weeks of therapy ursodeoxycholic acid had increased to 50–60% of the total serum bile acids whereas the more apolar bile acids were significantly decreased. No changes in liver function tests or bile acid metabolism were found in the untreated group.Since CA and UDCA are non-toxic in man, this trial indicates that the apolar bile acids chenodeoxycholic acid and deoxycholic acid may be responsible for the deterioration of liver function in primary biliary cirrhosis. However, the therapeutic effect of UDCA cannot be explained merely by the decrease in -dihydroxy-bile acids in the serum, since the laboratory results had improved prior to the decrease in the serum apolar bile acids.     

Hepatic uptake and biliary secretion of bile acids in the perfused rat liver.

Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r = -0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.     

Relative inhibitory activity of bile acids against 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced inflammation,and chenodeoxycholic acid inhibition of tumour promotion in mouse skin two‐stage carcinogenesis

Objectives Bile acids are present in Bezoar Bovis and Fel Ursi, traditionally used as antipyretics and antispasmodics. However the anti‐inflammatory activity of individual bile acids and related compounds has not yet been investigated. In this paper, we report the structure–activity relationships influencing the anti‐inflammatory activity of a variety of structurally different bile acid derivatives and also the inhibitory activity of chenodeoxy‐cholic acid against tumour promotion. Methods Fifty derivatives of bile acids were examined for their inhibitory activity against the induction of oedema in mouse ear by application of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Also, the effect of chenodeoxycholic acid was studied in mouse skin in which tumours had been induced by topical application of 7,12‐dimethylbenz[a]anthracene (DMBA) and promoted by TPA. Key findings Many bile acid derivatives had an inhibitory effect against TPA‐induced ear oedema at a similar grade to that of indometacin. Chenodeoxycholic acid, methyl 3α,7α,15α‐trihydroxy‐5β‐cholan‐24‐oate and methyl 3α,7α,15β‐trihydroxy‐5β‐cholan‐24‐oate showed the most potent activity with an ID50 value of 71–110 nmol/ear, a level corresponding to that of hydrocortisone (69 nmol/ear). Furthermore, chenodeoxycholic acid markedly suppressed tumour‐promoting activity by TPA following initiation by DMBA in mouse skin. Conclusions This is the first report on the anti‐inflammatory activity of bile acids on TPA‐induced inflammatory ear oedema in mice. Chenodeoxycholic acid, methyl 3α,7α,15α‐trihydroxy‐5β‐cholan‐24‐oate and methyl 3α,7α,15β‐trihydroxy‐5β‐cholan‐24‐oate showed the most potent activity, at a level corresponding to that of hydrocortisone. Furthermore, chenodeoxycholic acid markedly inhibited tumour promotion in a two‐stage carcinogenesis model in mouse skin.