Glucuronidation of Entacapone, Nitecapone, Tolcapone, and Some Other Nitrocatechols by Rat Liver Microsomes

Purpose. Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. Methods. The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltrans-ferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. Results. Apparent K_m values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold V_max values compared with 4-nitrophenol (68.6 nmol min^–1 mg^–1), while all disubstituted catechols exhibited much lower glucuronidation rate. V_max/K_m values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K_m nitecapone >> entacapone > tolcapone; V_max nitecapone > entacapone > tolcapone; V_max/K_m tolcapone > nitecapone > entacapone. Conclusions. Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.     

银杏聚戊烯醇同系物的提取及其HPTLC法的初步分析

目的 :确定银杏聚戊烯醇各同系物的链长。方法 :从银杏叶的石油醚提取物中分离获得了纯度较高的聚戊烯醇同系物的混合物。进而利用反相高效薄层色谱 ,以丙酮为展开剂 ,碘蒸气显色后呈显五个斑点。结果 :经与标准品对照 ,可确定为聚戊烯醇 16～聚戊烯醇 2 0五个单体。结论 :用HPTLC能快速、简便地确定各同系物链长 ,较HPLC方法费用低廉。     

结肠腺癌与正常结肠神经节苷脂的比较研究

我们对成人结肠腺癌和同一个体的远端正常结肠,分别分离、纯化出细胞膜神经节苷脂(GLS),经GLS定量分析,结果表明:癌组织中GLS的含量较正常结肠显著增加,结肠腺癌平均每克湿重组织含GLS39.7nmol,正常结肠平均为16.5nmol。高效薄层层析图谱显示,癌组织中,在含有单唾液酸GLS区段,存在二条肿瘤“特异”的条带,位于通常的GM2和GM1条带之后,它们分别占总神经节苷脂的3.9—6.0％、3.9—7.5％;在含有二唾液酸GLS区段,发现一条异常的GLS条带,位于GDla与GDlb之间,占总GLS5.1—6.7％,而5例正常对照中,有一例存在痕量,一例占总GLS的1.4％。比较结肠腺癌和正常结肠的不同GLS的百分含量可知:癌组织中GM3、GM2增加;GD3、GDla减少,同时GDlb和含三唾液酸以上的GLS呈减少的趋势。     

加压溶剂提取-高效薄层扫描法测定三七中皂苷类成分

目的：建立加压溶剂提取-高效薄层扫描法测定三七中皂苷类成分含量的方法。方法：采用加压溶剂提取三七中皂苷类成分，高效薄层扫描进行含量测定，使用高效薄层板、半自动点样、自动展开，10％硫酸乙醇液显色，光密度扫描，测定波长534am，参比波长700nm。结果：人参皂苷Rb1、Rd、Rg1和三七皂苷R1的线性范围为：0．402～2．010,μg(r=0．9995)；0．154～1．275,μg(r=0．9965)；0．198～1．980,μg(r=0．9998)和0．156～1．400,μg(r=0．9978)，回收率在95．3～99．3％之间。结论：高效薄层扫描法可同时测定三七中的人参皂苷Rb1、Rd、Rg1和三七皂苷R1。     

HPTLC evaluation of oleanolic acid and ursolic acid from the methanol extract of Wattakaka volubilis

ObjectiveTo find out the secondary metabolites present in the methanol extract of Wattakaka volubilis (W. volubilis).MethodsHigh performance thin layer chromatography method for the quantification of triterpenoids in soxhlet methanol extract of W. volubilis is described by densitometric scanning. The linear regression data from the calibration curve was plotted over the range of 10–25 μg/mL, r² = 0.992 46, 0.950 42 respectively. A mixture of toluene: methanol (9:1) was used as mobile phase for oleanolic acid were petroleum ether: chloroform: ethyl acetate: methanol (4:1:0.1:0.1) were used for ursolic acid.ResultsThe results showed that the presence of oleanolic acid and ursolic acid in methanol extract. The content found to be 218.30 ng and 509.99 ng/10 mg of extract.ConclusionFor conclusion, above study scientifically validated as a useful traditional medicine with the identification of bioactive secondary metabolites.     

羌活薄层色谱和HPLC指纹图谱结合多成分含量测定的质量评价方法研究

目的?建立薄层色谱、HPLC指纹图谱、多成分含量测定分析方法,评价18批不同产地羌活市售药材,为提高羌活质量标准提供技术支持。方法?采用高效薄层色谱法对羌活进行鉴别;采用HPLC-DAD法建立羌活指纹图谱,运用相似度软件对18批药材进行质量评价,建立HPLC法测定羌活中多个有效成分（阿魏酸苯乙醇酯、羌活醇、异欧前胡素、镰叶芹二醇）的含量。结果?建立了高效便捷的羌活薄层鉴别方法;所建立的指纹图谱出峰时间适中、方法稳定,共标定了14个共有峰,并通过对照品比对指认了6个色谱峰。18批市售药材指纹图谱相似度按共有峰计算在0.887~0.992;建立羌活多类型有效成分含量测定方法,阿魏酸苯乙醇酯、羌活醇、异欧前胡素、镰叶芹二醇在该色谱条件下分离良好,线性关系良好（r>0.999?8）,平均回收率分别为95.3%、99.5%、98.5%、98.5%。结论?建立的羌活薄层鉴别方法和HPLC指纹图谱稳定可靠,能有效区别羌活、宽叶羌活及其易混淆品;同时建立了羌活中多个有效成分的HPLC含量测定方法,为全面提高羌活质量控制方法提供指导。      

VALIDATION OF A HIGH SENSITIVE IMMUNOENZYMATIC ASSAY TO ESTABLISH THE ORIGIN OF IMMUNOGLOBULINS IN FEMALE GENITAL SECRETIONS

ABSTRACT

Several studies were carried out to characterize the humoral immune response on mucosal genital surfaces. However, the results obtained so far were particularly conflicting due to the absence of validation methods. The aim of this study was to develop and validate a quantitative ELISA method, which is sensitive and reproducible, to measure immunoglobulin and secretory immunoglobulin concentrations in various biological fluids. This quantitative, sensitive (detection limit = 1 µg/L) and reproducible (coefficient of variation <15%) method could be of interest to study the effects of viral infections on mucosal non-specific immune response in genital tract. To explore the humoral response, serum, saliva, vaginal secretions, and cervicovaginal secretions from 18 women, 20–45 years old, were evaluated for total-IgA, secretory IgA, IgM, and IgG. Albumin level was also evaluated by immuno-nephelometry. The secretion rates of immunoglobulins were measured by calculating their relative coefficients of excretion by reference to albumin. Despite large individual variations, median immunoglobulin levels were higher in the endocervical secretions than in the cervicovaginal secretions. When we compared the rates of immunoglobulins in genital fluids, IgG prevalence was higher (80%) in cervicovaginal and endocervical secretions than IgA prevalence (12%). In contrast, digestive mucosal secretions, such as saliva, contained mostly IgA (80%). In cervicovaginal and endocervical secretions, IgG and IgM originated mainly from serum, whereas a local synthesis provided total-IgA and secretory IgA. These results allowed us to raise a possible hypothesis for the origin of immunoglobulins in the genital tract. They illustrated the peculiar feature of the female reproductive tract and the difficulty for this tissue to contribute in the mucosal associated lymphoid tissue. The low secretory-IgA and total-IgA levels could explain the particular sensitivity of the vagina and the cervix to infections.     

Optimization of the extraction condition for benzyl isothiocyanate contents in Salvadora persica roots “Siwak”

Benzyl isothocyanate is the major active antibacterial metabolite in Salvadora persica roots “Siwak” beside two minor isothiocyanate derivatives namely; 3-methoxy benzyl isothiocyanate and 3-hydroxy benzyl isothiocyanate. The extraction condition effect on the amount of benzyl isothiocyanate was explored in detailed study. Both cold and hot extraction with different solvents was applied. The amount of benzyl isothiocyanate was estimated using HPLC and HPTLC. The results indicated that cold extraction of the fresh samples with chloroform offers the maximum amount of benzyl isothiocyanate. Drying process leads to great loss of the active component of Siwak.     

黄精炮制品多糖多元指纹图谱的建立及化学模式识别

目的 构建黄精炮制品多糖的酶水解产物指纹图谱,结合化学计量学方法评价不同蒸晒次数黄精炮制品多糖的差异。方法 采用糖苷酶水解,结合高效薄层色谱（high thin-layer chromatography,HPTLC）、亲水作用色谱-高效液相色谱-蒸发光检测器（hydrophilic interaction liquid chromatography-high performance liquid chromatography-evaporative light scattering detector,HILIC-HPLC-ELSD）和超高效液相色谱-四级杆飞行时间串联质谱（ultra high pressure liquid chromatography-quadrupole time of flight tandem mass spectrometry,UHPLC-QTOF/MS）对相应的酶解产物进行高效分离和检测。由此产生的自下而上的指纹图谱反映了黄精炮制品多糖的变化,通过相似度和偏最小二乘-判别分析（partial least squares-discriminant analysis,PLS-DA）对指纹图谱进行综合评价。结果 HPTLC分析发现,果糖随着蒸晒次数的增加逐渐降低,结合色谱分析发现,六蒸六晒和十二蒸十二晒为黄精炮制过程中的转折点。同时根据酶水解中寡糖的变化,利用相似度和PLS-DA对不同蒸晒次数的黄精炮制品进行了清晰的聚类。结合UHPLC-QTOF/MS分析表明,不同蒸晒次数黄精炮制品中含有蔗糖、蔗果三糖、蔗果四糖、蔗果五糖、蔗果六糖、蔗果七糖及蔗果八糖。结论 基于酶解法分析不同蒸晒次数样品的质量动态变化,结合所建立的多元指纹图谱,进一步阐释了不同蒸晒次数黄精炮制品的科学内涵。