海洋硫酸鼠李寡糖的制备研究

目的 探究海洋硫酸鼠李寡糖的制备方法并对所得寡糖的特征进行了研究。方法 采用三氟乙酸(TFA)和盐酸(HCl)对海洋硫酸鼠李多糖进行降解,通过薄层色谱法和凝胶渗透色谱法对降解条件进行分析,确定寡糖的制备方法;制备得到一系列硫酸鼠李寡糖,采用质谱法对其特征进行研究。结果 0.1 mol.L-1 TFA 80℃及0.1 mol.L-1 HCl 80℃水解条件对多糖的降解作用较好,且通过Bio-Gel P4凝胶色谱柱对寡糖能进行有效分离;最终选取0.1 mol.L-1 HCl 80℃10 h的水解条件对硫酸鼠李多糖进行降解,采用Bio-Gel P4凝胶色谱柱对寡糖混合物进行分离纯化,负离子模式下的电喷雾质谱分析表明,这些寡糖为聚合度为1–11的硫酸化鼠李寡糖。结论 首次建立了海洋新型硫酸鼠李寡糖的制备方法,这些硫酸鼠李寡糖为新型海洋特征寡糖。     

海参硫酸多糖的高温高压降解工艺及其降解机制

目的 分析海参硫酸多糖的高温高压降解工艺和降解产物的结构,探究硫酸多糖的高温高压降解的机理。方法 采用聚丙烯酰胺凝胶电泳法、分子排阻凝胶色谱法、薄层层析法、离子高效液相色谱法和PMP柱前衍生-高效液相色谱法分析多糖的分子量、硫酸根含量和单糖组成的变化,并利用液相色谱-质谱联用技术分析降解产物中硫酸寡糖的结构特征。结果当pH>7时,海参硫酸多糖在高温加压条件下未见明显降解。添加醋酸使溶液的pH降至6以下,海参硫酸多糖在100～121 ℃加热后,pH越低、温度越高、加热时间越长,分子量降低越明显。海参硫酸多糖的硫酸根和单糖组成在降解后变化很小,降解产物中存在一系列奇数和偶数的硫酸岩藻寡糖和硫酸软骨素寡糖,说明高温高压降解为无规断裂型和解聚断裂混合型。相同条件下,岩藻聚糖硫酸酯比硫酸软骨素更易被降解。 结论 本研究建立了高温高压法高效降解海参硫酸多糖的技术,该方法是一种回收率高、易控制、绿色环保的低分子量海参硫酸多糖的制备方法。     

果胶多糖可控性降解及果胶寡糖的制备

目的建立一套果胶多糖可控性降解和果胶寡糖分离分析的完整方法。方法采用酸水解和生物酶法等不同降解方法对柑橘果胶多糖进行可控性降解,以所得果胶寡糖的聚合度范围及相对含量为指标,确定最优降解条件,对降解所得的果胶寡糖进行分离分析。结果以果胶酸为底物,用果胶酶40℃反应1h,可得聚合度范围1~5的寡聚半乳糖醛酸混合物;经强阴离子交换树脂Dowex-1×4分离、薄层色谱和电喷雾电离质谱检测,得到聚合度范围1~5的单一聚合度的半乳糖醛酸寡糖。结论建立的果胶多糖可控性降解和果胶寡糖分离的分析方法为果胶多糖的系统研究提供了参考。     

麦冬多糖单糖组成的分析方法研究

目的:建立一种麦冬多糖中单糖组成的高效液相色谱(HPLC)-蒸发光散射(ELSD)测定方法。方法:以三氟乙酸(TFA)水解麦冬多糖,采用Waters XBridgeTMAmide色谱柱,乙腈-水-三乙胺(75∶25∶0.2)为流动相,ELSD检测器监测麦冬多糖的酸水解产物,优化麦冬多糖的全水解条件,并对麦冬多糖的单糖组成进行分析。结果:麦冬多糖由果糖和葡萄糖组成,其全水解条件为以0.02 mol·L-1TFA为水解试剂,在110℃下水解2 h。HPLC-ELSD方法验证结果表明,该单糖组成测定方法的精密度和重复性良好,果糖和葡萄糖的加样回收率分别为95.9%～104.4%和91.3%～104.1%(RSD<5.9%)。麦冬多糖中果糖和葡萄糖的摩尔比约为15∶1。结论:本研究为麦冬多糖中单糖组成的准确测定提供了方法。     

D-青霉胺酰化产物的水解工艺及其动力学研究

目的:研究将D-青霉胺酰化产物直接进行水解以得到L-青霉胺的工艺。方法:将D-青霉胺酰化产物、盐酸加入三口烧瓶中,在N2保护的条件下进行水解,筛选得到D,L-青霉胺消旋体的最佳水解条件并对水解过程的动力学进行分析。结果:最佳水解条件为1mol D-青霉胺酰化物在100mL、3mol.L-1的盐酸中水解10h,回流温度102℃;水解过程的动力学方程为ln(Ct/C0)=0.051 71+0.062 96t。结论:该工艺反应条件缓和,反应时间短,为L-青霉胺的制备提供了直接的拆分原料D,L-青霉胺。     

酶法制备低分子量肝素及其活性研究

用肝素酶Ⅰ(heparinaseⅠ,EC 4.2.2.7)对肝素进行控制性降解,分别在反应16,32,48 h终止反应,超滤离心粗分级后,用Bio-Gel P6低压柱色谱分离纯化,得到3组不同分子量范围的肝素寡糖混合物。用生色底物法和羊血浆法测定抗Ⅹa活性和抗Ⅱa活性。不同反应时间得到的肝素寡糖的抗Ⅹa和抗Ⅱa活性分别为82.4/40.3、21.5/11.8和5.6/4.5 IU/mg,其抗Ⅹa/抗Ⅱa活性值分别为2.04、1.82和1.24。结果表明降解16 h所得寡糖可供制备符合小分子肝素药物质控要求的产品。     

绿藻硫酸多糖UCS2的结构及抗凝血活性研究

目的 对绿藻多糖UCS2的结构特征和抗凝血活性进行研究。方法 通过冷水提取、强阴离子交换色谱和凝胶渗透色谱,从绿藻花石莼中提取分离得到硫酸多糖UCS2;采用高效凝胶渗透色谱（HPGPC）、高效液相色谱、红外光谱和气质联用色谱对多糖UCS2的结构进行表征;通过活化部分凝血活酶时间（APTT）、凝血酶时间、凝血酶原时间对多糖UCS2体外抗凝血活性进行研究。结果 绿藻多糖UCS2分子量为39.55kDa,硫酸根和糖醛酸含量分别为19.74%和18.66%;UCS2主要由鼠李糖组成,含有少量葡糖醛酸和木糖。糖链中含有→3,4)-α-L-Rhap-(1→, →4)-α-L-Rhap-(1→, →4)-β-D-Xylp-(1→和→4)-β-D-GlcAp-(1→,硫酸基位于→4)-α-L-Rhap-(1→的C-3位。多糖UCS2对APTT有显著延长作用,具有较高的抗凝活性。结论 绿藻多糖UCS2是1种抗凝活性的葡萄糖醛酸-木糖-鼠李糖型硫酸多糖。     

绿藻硫酸多糖SHW及其低分子量片段的结构及抗凝活性研究*

目的 对绿藻多糖SHW及其低分子量片段的结构及抗凝活性进行研究。方法 通过热水提取、强阴离子交换色谱和凝胶渗透色谱,从绿藻硬毛藻中提取分离得到硫酸多糖SHW;通过可控酸解方法制备SHW低分子量片段;采用高效凝胶渗透色谱、高效液相色谱、红外光谱及甲基化方法对多糖结构进行表征;通过活化部分凝血活酶时间、凝血酶时间、凝血酶原时间研究多糖SHW及其低分子量片段体外抗凝血活性。结果 多糖SHW及其低分子量片段主要由阿拉伯糖组成,含有少量半乳糖和氨基葡萄糖;糖链中阿拉伯糖主要以→4)-Arap-(1→和→3,4)Arap-(1→的形式存在,半乳糖以→4)-Galp-(1→存在形式,硫酸根主要位于→4)-Arap-(1→的C-3位。SHW及其低分子量片段A1～A6的分子量分别为492 kDa、200 kDa、170kDa、100kDa、26kDa、14kDa 和10 kDa。SHW 具有较高的抗凝血活性,且其抗凝活性随着其分子量的减少而降低。结论 海藻多糖SHW是一种具有抗凝活性的新颖硫酸多糖,其抗凝活性与分子量密切相关。     

岩藻聚糖硫酸酯及其模拟物的制备研究进展

岩藻聚糖硫酸酯（fucoidan）是1种富含α-L-岩藻糖的硫酸化多糖，通常存在于海洋褐藻和棘皮动物中。天然fucoidan多糖主要从海洋生物中提取获得，并经物理、化学或生物酶法可控降解制备其低聚糖与寡糖，其多样的生物活性已被广泛报道；化学合成是获得结构清晰的fucoidan寡糖的高效途经之一，有助于进行深入的构效关系研究；利用结构清晰的fucoidan寡糖，开展多价fucoidan模拟物的合成研究，也是有效评价fucoidan的结构与功能关系的重要手段。本文全面总结了近年来fucoidan及其模拟物的制备策略和方法，以期为该类糖药物和功能制品的研发提供重要参考。     

抗HSV1单克隆抗体-阿糖胞苷偶联物制备条件的分步优化

目的:建立高效液相凝胶过滤色谱法测定偶联物的偶联率、中间产物的生成量;通过制备条件分步优化实验来提高偶联率.方法:制备条件从投料比、pH、温度、反应时间等方面来逐项优化反应条件以期能提高偶联率;检测方法采用高效液相凝胶过滤色谱法,以0.02 mol·L-1 pH 6.8磷酸盐缓冲溶液为流动相,流速为0.5 mL·min-1,检测波长为280 nm.结果:通过建立高效液相凝胶过滤色谱法来指导优化实验,摸索得到新的投料比和pH,使偶联率从10.19%提高到13.48%.结论:本实验建立的高效液相凝胶过滤色谱法简便、快速、灵敏、准确,对优化实验有指导意义,使偶联率得到了提高.     

Sulfation of resveratrol in human liver: Evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites (M1–3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4′-O-sulfate, and trans-resveratrol-3-O-4′-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K_i of 21.3?±?8.73?µM and a V_max/K_m of 1.63?±?0.41?µL?min^?1mg^?1 protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V_max/K_m values for M3 than for M2 (2.23?±?0.14 and 0.04?±?0.01?µL?min^?1?mg^?1). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.     

抗癌药细胞色素P450 1B1抑制剂的研究进展

介绍了有关人细胞色素P450 1B1(CYP1B1)抑制剂的研究概况。CYP1B1是一类在导致癌变的雌激素代谢和激活芳香烃化合物致癌性中起重要作用的酶，在正常组织中通常不表达而在许多肿瘤组织中表达活跃，同时对许多抗癌药物的代谢具有重要影响。这表明CYP1B1抑制剂有望成为又一类肿瘤治疗药物。     

Real-time PCR-analysis of the cytochrome P450 1B1 codon 432-polymorphism

The aim of the present study was to develop and validate a rapid assay for genotyping of CYP1B1 codon 432-polymorphism. The described method is a single tube assay and combines both rapid-cycle polymerase chain reaction (PCR) with real-time monitoring by amplification and generation of the melting profiles of an allele-specific fluorescent probe. With this method 300 samples were analysed from healthy, unrelated Germans. Genotype frequency determined for the mutated allele CYP1B1*2 was 0.40. The results show that genotyping of CYP1B1 codon 432-polymorphism with a real-time fluorescence PCR method is a rapid and reliable assay for the analysis of large numbers of samples. Received: 1 July 1999 / Accepted: 18 August 1999     

膀胱癌患者CYP1A1基因MSPI多态性的相关分析

目的探讨细胞色素CYP1A1基因MSPI多态性与膀胱癌遗传易感性的关系。方法应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析技术,检测44例膀胱癌患者(病例组)和85例同期住院非膀胱癌患者(对照组),检测CYP1A1基因MSPI多态性位点的3种基因型及等位基因的分布频率。结果在病例组CYP1A1基因MSPI位点基因型分布频率为:TT(54·5%)、TC(36·4%)、CC(9·1%),等位基因分布频率为T(72·7%)、C(27·3%);在对照组CYP1A1基因MSPI位点基因型的分布频率为TT(61·2%)、TC(31·2%)、CC(7·1%),等位基因分布频率为T(77·1%)、C(22·9%)。各个基因型在两组中所占的比例差异无统计学意义(P>0·05);T、C等位基因频率两组比较差异亦无统计学意义(P>0·05)。结论CYP1A1基因MSPI位点多态性的单独存在可能与本地区膀胱癌易感性无关。     

选择性鞘胺醇-1-磷酸受体1激动剂研究进展

鞘胺醇-1-磷酸 (S1P) 是调节细胞内外多种生物学功能的重要信号分子。S1P可通过结合5种G蛋白偶联受体亚型 (S1PR₁₋₅) 介导多重下游信号通路产生相关的细胞生理功能, 其中激动S1PR₁可以达到调节免疫的作用。新型S1PR₁激动剂芬戈莫德 (Fingolimod, FTY-720) 已完成临床研究成为自身免疫性疾病——多发性硬化症的治疗药物。选择性S1PR₁激动剂的研究已成为发现免疫性疾病治疗药物的热点。本文总结了近年来该领域的研究进展, 着重介绍S1PR₁激动剂的结构类型和构效关系研究, 并探讨了此类激动剂的发展方向。     

Effect of SLCO1B1 genetic polymorphism on the pharmacokinetics of nateglinide

AIMS: Nateglinide is a meglitinide analogue with antidiabetic action. A recent study showed that SLCO1B1 (which codes the OATP1B1 gene, also known as OATP-C, OATP2) is a major determinant which markedly affects the pharmacokinetics of repaglinide. Our objective was to assess the association between single nucleotide polymorphisms (SNPs) of SLCO1B1 and the pharmacokinetics of nateglinide. METHODS: Seventeen healthy volunteers with different SLCO1B1 genotypes (11 with 521TT, four with 521TC and two with 521CC) were enrolled in this study. Each was given a single oral dose of 90 mg nateglinide. Plasma concentrations of nateglinide were measured up to 8 h by HPLC. RESULTS: The C(max) and AUC(0,infinity) of nateglinide were 83% (P = 0.002) and 82% (P = 0.001) higher in the SLCO1B1521TC subjects (n = 4), and 76% (P = 0.016) and 108% (P = 0.001) higher in the SLCO1B1521CC subjects (n = 2) than in the SLCO1B1521TT subjects (n = 11), respectively. The t(1/2) of nateglinide in SLCO1B1521CC subjects was 78% longer than that in 521TT subjects (P = 0.036). The difference in t(max) values among the three genotypic groups was not statistically significant. CONCLUSIONS: Our results suggest that OATP1B1-mediated hepatic uptake of nateglinide may be the prior step for its metabolism and elimination. SLCO1B1521T > C SNP might play an important role in the pharmacokinetics of nateglinide.     

白细胞介素-1、基质金属蛋白酶-1在膝关节骨性关节炎发病中的作用探讨

目的探讨白细胞介素-1（IL-1）、基质金属蛋白酶-1（MMP-1）在膝关节骨性关节炎（OA）发病中的作用。方法提取23例OA病变轻微患者（OA第一组）、28例OA病变严重患者（OA第二组）、6例对照者（盘状半月板组）的滑膜组织及关节液，采用免疫组织化学方法检测膝关节滑膜组织中IL-1、MMP-1的阳性细胞率及通过夹心ELISA法测定膝关节滑液中IL-1、MMP-1含量。结果本组57份膝关节滑膜组织及关节滑液标本中，无论是对照组还是OA组中，均能测出IL-1、MMP-1。OA病变轻微组膝关节滑膜组织及关节滑液标本中IL-1、MMP-1含量与对照组比较无显著性差异（P〉0．05），OA病变严重组膝关节滑膜组织及关节滑液标本中IL-1、MMP-1含量显著高于病变轻微组和对照组（P〈0．05）。结论IL-1和MMP-1在骨性关节炎的发病中起重要作用。OA患者滑膜细胞高水平自分泌性分泌IL-1和MMP-1可能与其炎性发生与发展密切相关，可能是关节滑液中IL-1和MMP-1的主要来源。     

No significant effect of SLCO1B1 polymorphism on the pharmacokinetics of rosiglitazone and pioglitazone

AIMS

To examine possible effects of polymorphism in the SLCO1B1 gene, encoding the hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1, on the pharmacokinetics of rosiglitazone and pioglitazone in a prospective genotype panel study.

METHODS

Sixteen healthy volunteers with the homozygous SLCO1B1 c.521TT genotype (controls), 12 with the heterozygous c.521TC genotype and four with the homozygous c.521CC genotype ingested a single 4-mg dose of rosiglitazone and a single 15-mg dose of pioglitazone in a cross-over study with a wash-out period of at least 1 week.

RESULTS

SLCO1B1 polymorphism had no statistically significant effect on any of the pharmacokinetic variables of rosiglitazone, pioglitazone or their metabolites. The mean ± SD area under the plasma rosiglitazone concentration–time curve from time 0 to infinity (AUC_0–∞) was 2024 ± 561 ng ml⁻¹ h in the c.521TT subjects, 1763 ± 288 ng ml⁻¹ h in the c.521TC subjects (geometric mean ratio c.521TC/c.521TT 0.89; 95% confidence interval 0.72, 1.11) and 1729 ± 346 ng ml⁻¹ h in the c.521CC subjects (c.521CC/c.521TT 0.87; 0.63, 1.20). The AUC_0–∞ of pioglitazone averaged 6244 ± 1909 ng ml⁻¹ h in the c.521TT subjects, 5123 ± 1165 ng ml⁻¹ h in the c.521TC subjects (c.521TC/c.521TT 0.83; 0.65, 1.06) and 4851 ± 1123 ng ml⁻¹ h in the c.521CC subjects (c.521CC/c.521TT 0.79; 0.55, 1.14). There was a significant correlation between the AUC_0–∞ of rosiglitazone and pioglitazone (r = 0.717, P < 0.001).

CONCLUSIONS

The SLCO1B1 c.521T→C SNP does not affect the pharmacokinetics of rosiglitazone or pioglitazone, indicating that OATP1B1 plays no significant role in the disposition of these drugs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• A common single nucleotide polymorphism (SNP) (c.521T→C) of the SLCO1B1 gene, encoding the hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1, has been associated with marked changes in the pharmacokinetics of the antidiabetic drug repaglinide.
• Rosiglitazone and pioglitazone are competitive inhibitors of OATP1B1 and might thus be its substrates.
• Gemfibrozil, an inhibitor of OATP1B1 in vitro, considerably increases the plasma concentrations of rosiglitazone and pioglitazone in vivo in humans.

WHAT THIS STUDY ADDS

• The SLCO1B1 c.521T→C SNP was not associated with changes in rosiglitazone or pioglitazone pharmacokinetics in healthy volunteers.
• OATP1B1 is thus unlikely to play an important role in the disposition of rosiglitazone or pioglitazone.

     

结肠清颗粒对溃疡性结肠炎模型大鼠血清IL-1、ICAM-1含量的影响

目的：探讨结肠清颗粒对溃疡性结肠炎模型大鼠血清白细胞介素1（IL-1）、细胞黏附因子（ICAM-1）的影响。方法：用家兔结肠加弗氏佐剂复制大鼠溃疡性结肠炎模型,检测模型大鼠血清IL-1、ICAM—1含量。结果：结肠清颗粒能显著增加溃疡性结肠炎模型大鼠血清IL-1及ICAM-1的含量;显著降低溃疡性结肠炎模型大鼠结肠的病理改变,对模型大鼠结肠有显著的修复作用。结论：结肠清颗粒治疗溃疡性结肠炎的作用可能与其升高血清IL-1及ICAM-1的含量,从而改善免疫功能。减轻炎症损伤有关。     

1-磷酸鞘氨醇受体的研究进展

1-磷酸鞘氨醇S1P(Sphingosine 1-phosphate,S1P)是调节细胞内外多种生物学功能的重要信号分子之一,作用于S1P受体后,在很多生理和病理过程中发挥重要的调节作用。Sphk-S1P-S1PR信号通路及其在炎症、肿瘤、动脉粥样硬化、自体免疫系统疾病、神经系统疾病等多种疾病中的作用已成为目前研究的热点之一。越来越多的S1P受体调节剂被研发。