透析-高效液相色谱法在当归补血汤药效物质基础研究中的应用

将脂质体作为模拟生物膜,采用平衡透析与液相色谱联用技术,建立了一种研究中药成分与模拟生物具有相互作用的新方法。应用该方法对当归补血汤进行了分析,同时考察了模拟生物膜的浓度、pH值、缓冲系统和胆固醇的加入等因素对当归补血汤与模拟生物膜相互作用的影响。结果表明:当归补血汤中有7个组分与模拟生物膜相互作用明显;模拟生物膜的浓度影响最大,pH值对酸性组分阿魏酸的作用影响较大,其它因素的影响较小。该方法可用于预测药物在体内的吸收情况,进而研究中草药及复方的药效物质基础。     

竹红菌乙素对模拟生物膜-脂质体环境变化的荧光响应

脂质体不但可以作为药物传输体系, 而且还作为一种模拟生物膜的简单模型. 近期报道竹红菌素的荧光性质决定于其分子内质子转移, 而分子内质子转移环境敏感特性决定其分子的环境敏感荧光性质. 文中以脂质体为模拟生物膜研究竹红菌乙素(HB)在膜上微环境敏感的荧光性质, 包括生理范围pH值变动(6.0~8.0)、胆固醇浓度和离子强度的变化对竹红菌素荧光的影响. 发现HB的荧光强度对微环境敏感且可被调控. 保持磷酸缓冲液(PBS)中胆固醇和离子强度的浓度不变, 发现脂质体中HB的荧光强度在pH7.4时有一极大值而在缓冲溶液中pH7.0时HB的荧光强度有一极小值. 保持pH值7.2不变, HB脂质体的荧光强度分别在离子强度为0.12 mol/kg和胆固醇浓度为6 × 10^-4mol/L达到峰值. 对于HB的PBS溶液来说, 它的荧光强度与离子强度成反比. 这种环境敏感的荧光特性可能用于探测生物组织或生物大分子微环境特征.     

卵磷脂涂敷生物膜色谱固定相的制备及其稳定性的考察

 将卵磷脂涂敷的硅胶作为生物膜固定相 ,并以此研究了药物与生物膜的相互作用。实验发现 ,卵磷脂涂敷的硅胶固定相的适宜使用温度为 2 0℃～ 30℃ ,而且卵磷脂中合适的胆固醇含量会使制备的生物膜固定相的稳定性有所增加。对药物在这种生物膜色谱固定相上的保留行为的研究表明 ,胆固醇含量、缓冲试剂组成、流动相中盐浓度及pH值等都会影响色谱柱对药物的选择性。卵磷脂涂敷生物膜色谱固定相的制备方法简便 ,通过固定相及流动相的改变可以很方便地模拟人体的生理环境 ,因而可以用于研究药物在体内的吸收和分布状况以及药物的初步筛选。     

毛细管区带电泳法测定油田水中的脂肪酸

采用二硝基本甲酸一十六烷基三甲基溴化脓缓冲体系对油田水中短链脂肪酸进行了分析。应用间接紫外法,使脂肪酸在数分钟之内得以分离检测。研究表明,当缓冲溶液pH值为9.0、电解质浓度为5.0mmol／L、表面活性剂浓度为0.5mmol／L和甲醇含量为5％时可达到最佳的分高效果。     

甲基紫探针吸收光谱法测定阿魏酸钠

在pH 4.2的乙酸-乙酸钠缓冲溶液中,甲基紫与阿魏酸反应生成具有负吸收峰的紫色络合物。其最大负吸收波长位于565nm,阿魏酸的质量浓度在0.1~3.9mg·L-1范围内与吸光度呈线性关系,服从比耳定律,表观摩尔吸光率(ε)为4.36×104L·mol-1·cm-1。方法用于市售药物中阿魏酸钠含量的测定,回收率在99.5%~103%之间,测定值的相对标准偏差(n=6)在1.3%~1.5%之间。     

用分布系数表示的多元弱酸体系的缓冲容量公式

多元弱酸,如磷酸、柠檬酸、邻苯二甲酸等及其盐,常用于配制缓冲溶液。其中柠檬酸由于各级离解常数相差较小,是一个较好的应用于广泛pH范围的缓冲溶液。对于任一缓冲体系,如能了解它在不同pH值时的缓冲容量,则对于掌握其缓冲性质具有很大的作     

毛细管区带电泳法测定卷烟中的生物碱

在磷酸缓冲体系中采用毛细管区带电泳法测定卷烟中的生物碱时,检测灵敏度低,分离度差。考察了卷烟中生物碱的 提取条件,分离缓冲溶液的类型、pH值和浓度,卷烟中生物碱测定方法的线性范围、检出限、重现性和回收率。结果发 现,当采用410 mmol/L的酒石酸溶液（pH 2.8）为缓冲体系时,卷烟中生物碱的检测灵敏度和分离度均有明显改善,烟碱 的线性范围为0.06~0.80 mg/L（其他生物碱为0.006~0.10 mg/L）,检出限为0.002~0.01 mg/L,相对标准偏差为2.2%~10%,回收率为87.6%~102%。     

线性扫描伏安法研究大豆甙元与牛血清白蛋白的相互作用

采用线性扫描伏安法研究了大豆甙元与牛血清白蛋白(BSA)之间的相互作用;探讨了缓冲体系、pH、反应时间、反应温度、扫描速率等对相互作用的影响.结果表明,在pH为7.4的B-R缓冲溶液中,大豆甙元与BSA在相互作用20min后形成1∶1型的单一的非电活性超分子化合物,两者的结合常数β=8.29×105 L·mol-1,转移系数α=0.367 9,表观电子传递速率常数ks为1.39s-1.     

脂筏微区超分子聚集体结构的稳定性

生物膜中脂筏微区结构的动态特征与稳定性决定着生物膜的功能。通过从动物细胞提取脂筏,实验不但观测到质膜微囊烧瓶状凹陷结构,而且还观测到大量的球状和椭球状结构.通过模拟脂筏微区结构,重点对二元体系和三元体系的超分子聚集体结构的多形性进行了研究和探索。研究发现随着表面压力的增加,鞘磷脂和胆固醇双层膜出现了紧密聚集不规则的微区结构,在 SM/Chol/DOPC双层膜中,SM/Chol形成的微区结构漂浮在液态DOPC小颗粒上部。当 DOPE加入到SM/Chol中,三种成份形成不稳定的双层膜结构.Ceramide促进了SM/Chol结构发生重排,微区形状从原来的不规则向着紧密聚集的圆形结构演变;混合单层膜的分子面积与表面吉布斯自由能决定了分子间的相互作用, 当过量分子面积与过量吉布斯自由能为负值时,分子间相互作用表现为吸引力, 出现凝聚现象; 为正值时,分子间相互作用表现为排斥力, 促使单层膜出现相分离现象. 过量吉布斯自由能值越小, 单层膜的热稳定性越高.通过动物细胞提取脂筏与体外模拟脂筏相结合的方法,从超分子水平阐述了脂筏微区结构与功能的生物学意义,为生物膜的研究提供了理论依据和实验支持。     

糖和盐类物质对生物膜超分子结构稳定性影响的研究

用原子力显微镜(AFM)和小角X射线(SAXS)技术, 研究了NaCl、KCl、胆固醇、葡萄糖和蔗糖等与膜脂的相互作用. 研究发现它们能引起脂质膜超分子体系液晶态结构的变化. 葡萄糖和蔗糖对脂双层膜结构有稳定作用. 在NaCl溶液中制成的脂质膜, 随着NaCl浓度的增加, 它们的双层膜更稳定. 在KCl溶液中结果恰好相反. AFM研究发现液晶态脂双层膜结构与双亲性分子的结构、浓度以及介质的组分和pH等因素有关. 在1,2-反十八碳-3-磷脂酰乙醇胺(DEPE)液晶态中, 钠盐诱导形成Q²²⁹(Im3m)立方相. 油酸的含量对DEPE-PVP(聚乙烯吡咯烷酮)超分子结构也有一定的影响, 当油酸含量达到某一临界值时, 则发生从Im3m(Q²²⁹)到Pn3m(Q²²⁴)的转变. 胆固醇能促使形成Pn3m(Q²²⁴)和六角相H_II共存相. 研究结果表明, 生物膜超分子聚集体的氢键、分子van der Waals力、离子的静电力等这些弱相互作用的协同性、方向性和选择性, 可能决定着生物膜的结构和功能.     

PH effects on drug interactions with lipid bilayers by liposome electrokinetic chromatography

Liposome electrokinetic chromatography (LEKC) provides convenient and rapid methods for studying drug interactions with lipid bilayers using liposomes as a pseudostationary phase. LEKC was used to determine the effects of pH on the partitioning of basic drugs into liposomes composed of zwitterionic phosphatidylcholine (PC), anionic phosphatidylglycerol (PG), and cholesterol, which mimic the composition of natural cell membranes. An increase in pH results in a smaller degree of ionization of the basic drugs and consequently leads to a lower degree of interaction with the negatively charged membranes. From the LEKC retention data, the fractions of drugs distributed in the bulk aqueous and the liposome phase were determined at various pH values. Finally, lipid mediated shifts in the ionization constants of drugs were examined.     

Interactions between local anesthetics and lipid dispersions studied with liposome electrokinetic capillary chromatography

In the case of local anesthetic intoxication, intravenous administration of lipid-based Intralipid dispersion (Fresenius Kabi) can be used for the entrapment of hydrophobic drugs. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances. In this study liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between local anesthetics and Intralipid or liposome dispersions. Intralipid dispersion and extruded liposomes with different concentrations of 1-palmitoyl-2-oleyl-sn-glycerophosphatidylcholine (POPC), phosphatidylglycerol, cardiolipin, cholesterol, oleic acid, and linoleic acid were used as a pseudostationary phase in LEKC and their interactions with lidocaine, prilocaine, and bupivacaine were studied. POPC liposomes containing 1 mol% of palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine as a fluorescent marker were used for the first time in LEKC connected with laser-induced fluorescent detection in order to calculate the retention factor for anesthetics.     

Liposome electrokinetic capillary chromatography in the study of analyte-phospholipid membrane interactions. Application to pesticides and related compounds

Interactions between low-molar mass analytes and phospholipid membranes were studied by liposome electrokinetic capillary chromatography (LEKC). The analytes were pesticides, some degradation products, and compounds associated with the manufacture of pesticides. Negatively charged liposome dispersions with different zwitterionic lipids (PC) were applied to the determination of retention factors (k) of 15 charged and uncharged compounds. The liposome dispersions consisted of 80:20 mol% of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/POPS, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS. Retention factors were calculated from the effective electrophoretic mobilities of the analytes under LEKC and CZE conditions and from the effective electrophoretic mobilities of the liposomes, determined by CZE with a polyacrylamide-coated capillary. Determining the liposome mobilities in this way proved to be a good alternative to the conventional method employing a liposome marker compound. The log k values of the analytes for the different liposome dispersed phases were correlated with one another. In addition, correlation curves were determined between log k and calculated octanol-water partition coefficients. The results showed that the zwitterionic phospholipid in the liposome has a major impact on the interactions between the tested compounds and the lipid membranes.     

Prediction of Human Drug Absorption Using Liposome Electrokinetic Chromatography

With the tremendously increasing numbers of novel drug candidates, there remains a compelling need for rapid screening methods for drug-like physiochemical and pharmacokinetic properties. Different technologies have emerged that enable rapid screening in vitro for sorting out new chemical entity (NCE) classes. It is invaluable for these technologies being developed early in the drug discovery process to avoid the loss of cost and time in late development due to poor absorption and/or bioavailability. In this study, liposome electrokinetic chromatography (LEKC) serves as a convenient, rapid and cost-effective tool to determine lipophilicity and to predict human oral absorption. Twenty-seven organic neutral molecules were evaluated by octanol/water system (log P _ow) and LEKC (log k), and linear solvation energy relationship (LSER) analysis was conducted to compare the retention mechanism between LEKC and octanol/water system. LEKC can provide a rapid indirect measurement of log P _ow for small organic neutral molecules. A clearly sigmoidal relationship could be seen by correlating log k with the fraction of 25 drugs absorbed in humans (Fa), and the outliers suggested the involvement of non-transcellular passive diffusion, e.g. active transport, paracellular route; on the contrary, it is not the case with the octanol/water system. Therefore, LEKC, in combination with other permeability prediction model, can provide a primary screen for a large number of drug candidates at early stage of the drug discovery process with high-throughput and at low-cost.     

Drug-liposome distribution phenomena studied by capillary electrophoresis-frontal analysis

The potential of using CE frontal analysis (CE-FA) for the study of low-molecular-weight drug-liposome interactions was assessed. The interaction of bupivacaine, brompheniramine, chlorpromazine, imipramine, and ropivacaine with net negatively charged 80/20 mol% 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine/egg yolk phosphatidic acid liposome suspensions in HEPES buffer at pH 7.4 was investigated. The fraction of free drug as a function of lipid concentration was measured and apparent liposome - buffer distribution coefficients were determined for the basic drug substances. The distribution coefficients increased in the order ropivacaine, bupivacaine, brompheniramine, imipramine, and chlorpromazine. The developed CE method was relatively fast allowing estimates of drug-liposome affinity to be obtained within 15 min. CE-FA may have the potential to become a valuable tool for the characterization of drug-liposome interactions in relation to estimation of drug lipophilicity and for the evaluation of drug distribution in liposomal drug delivery systems.     

Optimization of the conditions for extraction of serine protease from kesinai plant (Streblus asper) leaves using response surface methodology

Response surface methodology (RSM) using a central composite design (CCD) was employed to optimize the conditions for extraction of serine protease from kesinai (Streblus asper) leaves. The effect of independent variables, namely temperature (42.5,47.5, X?), mixing time (2-6 min, X?), buffer content (0-80 mL, X?) and buffer pH (4.5-10.5, X?) on specific activity, storage stability, temperature and oxidizing agent stability of serine protease from kesinai leaves was investigated. The study demonstrated that use of the optimum temperature, mixing time, buffer content and buffer pH conditions protected serine protease during extraction, as demonstrated by low activity loss. It was found that the interaction effect of mixing time and buffer content improved the serine protease stability, and the buffer pH had the most significant effect on the specific activity of the enzyme. The most desirable conditions of 2.5 °C temperature, 4 min mixing time, 40 mL buffer at pH 7.5 was established for serine protease extraction from kesinai leaves.     

脂质体电动色谱用于预测皮肤渗透性的定量保留活性关系的建立

脂质体电动色谱 （Liposome electrokinetic chromatography,LEKC）是一种简单快速的评价药物与生物膜相互作用的方法。本文建立了脂质体电动色谱作为高通量筛选皮肤渗透性的体外分析方法。将脂质体电动色谱中保留因子的对数值（log k）作为自变量建立了定量保留活性关系式。采用SPSS分析软件对于16种结构不同的化合物进行分析,结果表明log k与皮肤渗透性常数线性相关性良好( R²=0.886)。采用交互验证评价了该模型的预测能力。在定量保留活性关系中的一个变量和传统定量构效关系中的三个变量可解释的能力( R² =0.704)相似。文中建立的定量保留活性关系模型对于新化合物早期的筛选可提供一种有效快捷的方法。