基于UPLC/Q-TOF-MS分析附子半夏配伍相反的物质基础

利用超高效液相色谱-飞行时间质谱联用技术(UPLC/Q-TOF-MS)分析附子半夏药对配伍相反的物质基础,从化学成分层次阐释其配伍相反机制.基于UPLC/Q-TOF-MS建立附子半夏药对配伍后生物碱类成分的化学指纹图谱,通过主成分分析法和正交偏最小二乘判别法分析药对配伍在合煎过程中的生物碱类成分的含量变化,找出差异变化显著的化学成分.结果表明正离子模式时附子半夏药对合煎液中次乌头碱,中乌头碱,乌头碱,去氧乌头碱,10-OH-中乌头碱,10-OH-乌头碱等的含量明显增高,而中乌头原碱,去乙酸中乌头原碱,去乙酸次乌头原碱,苯甲酰乌头原碱,苯甲酰次乌头原碱,10-OH-苯甲酰中乌头原碱等含量降低.附子半夏药对配伍应用时双酯型二萜生物碱的含量明显增高,而单酯型二萜生物碱的含量明显降低,这可能是附子半夏药对配伍相反作用的物质基础.     

复方中药四逆汤中乌头碱类二萜生物碱的电喷雾串联质谱研究

利用电喷雾串联质谱方法不经柱分离而直接分析复方中药四逆汤中二萜生物碱的组成,乌头碱类二萜生物碱在质谱条件下形成质子化分子,一种生物碱对应一个分子离子峰.检测到了苯甲酰单酯型、双酯型和脂类生物碱等18种二萜生物碱,其中苯甲酰中乌头原碱等单酯型生物碱是四逆汤中的主要生物碱成分.     

大孔树脂对麻黄细辛附子汤生物碱成分静态吸附及解吸附性能研究

采用高效液相色谱法(HPLC)测定麻黄细辛附子(MXF)汤复方水提液中盐酸麻黄碱、盐酸伪麻黄碱、苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱含量,考察了4种大孔树脂(HPD–100,HPD–722,HPD–400,HPD–600型)对5种生物碱的静态吸附、解吸附性能,筛选出分离纯化麻黄细辛附子汤中生物碱的最佳树脂。结果显示,HPD–722型树脂对5种生物碱的吸附过程稳定且吸附量大,具有较高的解吸附率(87.26%),优于其它3种树脂。HPD–722型大孔树脂适用于富集麻黄细辛附子汤中的生物碱类化合物。     

人血清白蛋白生物色谱柱的性能研究及应用于铁棒锤中活性成分的筛选

以大孔硅胶为基质,采用羰基咪唑法合成了人血清白蛋白(HSA)生物色谱填料。详细评价了该填料对药物的分离性能,温度和pH对药物保留的影响。结果表明,该种生物色谱填料的分离性能优良,可以在短时间内基线分离4种药物分子。用HSA色谱柱对铁棒锤中有效成分进行了分离,通过LC-MS分析结果显示,该色谱柱可以分离出其中4种活性成分(脱乙酰去氧乌头碱、苯甲酰脱氧乌头碱、乌头原碱、16-O-去甲基乌头次碱)。     

近红外光谱法快速测定附子中6个生物碱的含量

应用近红外光谱(NIRS)技术结合偏最小二乘(PLS)和最小二乘支持向量机(LS-SVM)建立了附子中多指标成分的快速无损检测方法。选取38批样品建立了同时测定附子样品中6种成分含量的高效液相色谱(HPLC)方法;通过采集附子样品的NIRS图,分别采用PLS和LS-SVM建立了各个成分HPLC测定值与NIRS图的定量校正模型。所建立的苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱、单酯型生物碱总量和双酯型生物碱总量LS-SVM模型的相对预测偏差(RPD)分别为3.3、3.2、4.1、7.7、8.8、7.6、4.0和8.6;验证集相关系数(rpre)分别为0.9486、0.9475、0.9668、0.9909、0.9946、0.9969、0.9669和0.9927,且LS-SVM模型优于PLS模型,说明NIRS模型验证集与HPLC测定值具有良好的非线性关系,模型预测效果良好。采用NIRS技术结合LS-SVM模型可以快速对附子中的上述6个生物碱含量以及单酯型生物碱总量和双酯型生物碱总量进行检测,方法操作简便,对控制附子中的生物碱含量具有一定的指导作用。     

乌头碱水解产物的研究

乌头碱为C_(19)二萜类双酯型生物碱,是乌头类中药的主要毒性成分和活性成分,乌头碱具有很强的抗炎和镇痛活性~([1]),在临床上被用于抗肿瘤药物,由于其治疗剂量与中毒剂量接近,必须经炮制后使用.乌头碱水解产物已有报道,但主要集中在苯甲酰乌头原碱和乌头原碱上,焦乌头碱很少提及,本研究利用硅胶柱色谱法对乌头碱的水解产物进行了分离和纯化,利用质谱和核磁共振谱确定了3种主要产物的结构,为研究乌头碱类生物碱的水解产物提供了新的数据.     

柱切换液相色谱法快速定量检测参附注射液中乌头碱类生物碱和人参皂苷

利用柱切换液相色谱,建立了参附注射液中苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱和乌头碱6种乌头碱类生物碱,以及Rg1、Re、Rf、Rb1、Rc、Ro、Rb2、Rb3、Rd 9种人参皂苷的分析方法。首先利用强阳离子交换的在线固相小柱选择性富集和净化样品中生物碱类成分,优化了色谱条件;并采用EC－C18柱作为人参皂苷的分析柱,通过优化实验条件,结合柱切换方式,去除了样品中辅料等大极性基质成分对色谱柱的污染,实现了生物碱分析和人参皂苷分析的自动切换。结果显示,样品中的生物碱和人参皂苷分离良好,线性相关系数（r2）均大于0.999,连续进样精密度的相对标准偏差（RSD） < 2.0%,重复性的RSD < 2.0%;其中6种生物碱的平均回收率为95.1%～98.6%,检出限为4.0～8.2 ng/mL;9种人参皂苷的平均回收率为91.7%～104%。所构建的基于柱切换液相色谱技术的在线固相萃取方法能够有效去除样品中的基质干扰,快速完成参附注射液中3种单酯型生物碱和9种人参皂苷的快速定量,同时也可对3种双酯型生物碱进行限量检测,可应用于药物的质量评价。     

RP-HPLC法测定黑顺片中6种酯型生物碱的含量

建立黑顺片中6种酯型生物碱含量的高效液相测定方法。采用岛津C18色谱柱(250 mm×4.6 mm,5μm),以乙腈–四氢呋喃(体积比为25∶15)为流动相A,以0.1 mol/L乙酸铵溶液(每1 000 m L加冰乙酸0.5 m L)为流动相B梯度洗脱,流量为1.0 ml/min,检测波长为235 nm,柱温为35℃。苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱进样量分别在0.222~4.430,0.060~1.119,0.144~2.878,0.015~0.297,0.025~0.496,0.011~0.229μg范围内与色谱峰面积线性呈良好的线性关系,相关系数r均大于0.999 5,检出限分别为2.10,2.70,2.20,2.60,1.60,3.10 ng,样品在12 h内稳定。苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱的加标回收率分别为98.7%~102.2%,98.6%~102.3%,99.0%~101.9%,102.0%~97.3%,102.1%~98.3%,95.7%~103.5%,测定结果的相对标准偏差分别为2.5%,2.5%,2.2%,2.3%,2.2%,1.7%(n=6)。该方法简便,测定结果准确、可靠,可为黑顺片的质量评价和质量控制提供参考。     

川乌与半夏配伍禁忌的化学研究

应用高效液相色谱和电喷雾质谱技术,通过分析生、制川乌单煎及其与生、法半夏共煎液中乌头碱类生物碱的含量和转变途径,发现生半夏在与川乌共煎时对乌头类剧毒生物碱的转化表现出明显的抑制作用,而法半夏却能很好的促进乌头碱类剧毒生物碱的转化,说明川乌与半夏是否可以共用与半夏的炮制与否和炮制方法存在着密切联系.     

离子液体均匀提取-高效液相色谱法测定参附注射液中4种单酯型乌头碱的含量

摘要 采用离子液体均匀提取-高效液相色谱法测定了参附注射液中苯甲酰乌头碱（BA）、苯甲酰新乌头碱（BM）、苯甲酰次乌头碱（BH）和苯甲酰脱氧乌头碱（BD）的含量,通过优化离子液体的用量,样品溶液的pH值,提取时间和盐浓度等参数,确定最佳提取条件为：pH为中性,盐浓度为5%,采用0.058g的[C6Mim][ BF4]和0.26g的 NH4PF6作为提取剂,提取1min,可有效地将目标物从5mL注射液中提取出来。在此实验条件下测得四种单酯型乌头碱的检测限分别为45.5（BM）,19.8(BA),26.3(BH)和12.2(BD)μg L-1,对参附注射液样品进行分析,加样回收率范围在93.74-107.91%,RSD值均小于4.34%。     

Determination of hydroxycamptothecin affinities to albumin and membranes by steady-state fluorescence anisotropy measurements

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.     

EVIDENCE FOR A MAJOR STRONG BINDING SITE FOR TETRACHLOROSALICYLANILIDE ON HUMAN SERUM ALBUMIN

Abstract— Solutions of human serum albumin(HSA) monomer were irradiated with UV light(360 nm) in the presence of [₁₄C]-3,3.4'S-tetrachlorosalicylanilide([₁₄C]-T₄CS).The [₁₄C]-T₄ CS-labeiled HSA was cleaved by cyanogen bromide and separated into two fractions. These fractions were reduced carboxymethylated and separated into their seven characteristic peptides and monitored for radioactivity. Tetrachlorosalicylanilide was found to bind mainly to one region of the sequence of HSA and this covalent binding site was located in residues 124 (Cys) to 298 (Met) of the molecule. The binding of 3,5-dichlorosalicylamido-4-(2,2,6.6-tetramethylpiperidine-l-oxyl (DCS-TEMPO),a spin-label analogue of T₄CS, to HSA was studied by electron spin resonance spectroscopy. In the absence of UV light. DCS-TEMPO bound non-covalently (k = 6.1 times 10₆M₁) to one major binding site on HSA. These results are evidence for the existence of a major strong binding site for the photochemical binding of T₄CS to HSA.     

Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates

This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions.     

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.     

Disposable Electrodes for Capacitive Immunosensor

Disposable electrodes were fabricated by coating chromium (5 nm) and gold (200 nm) on glass strips (5.0 mm×25.4 mm) and used in a label‐free immunosensor. Human serum albumin (HSA) and its antigen (anti‐HSA) were used as a model system. Electropolymerization of o‐phenylenediamine was used for the immobilization of anti‐HSA by covalent binding. A linear relationship was obtained in the range from 1.0×10^?14 to 1.0×10^?9 M with a limit of detection of 8.0×10^?15 M. Each modified electrode can be reused up to 30 times. The developed system was applied for human serum samples and compared to Albumin BCG method.     

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.     

Combinatorial dapoxyl dye library and its application to site selective probe for human serum albumin

The combinatorial fluorescent dapoxyl dye library was prepared by both solution- and solid-phase synthesis, generating 80 unique dapoxyl derivatives. A fluorescence-based screening toward human serum albumin (HSA) found one highly sensitive HSA binder ( A41-S) with over 55-fold intensity change. Displacement assay showed the selective binding of A41-S to the site I of HSA, addressing its potential to be a highly selective and sensitive HSA probe.     

Influence of positional isomers on the macroscale and nanoscale architectures of aggregates of racemic hydroxyoctadecanoic acids in their molecular gel, dispersion, and solid states

Inter/intramolecular hydrogen bonding of a series of hydroxystearic acids (HSAs) are investigated. Self-assembly of molecular gels obtained from these fatty acids with isomeric hydroxyl groups is influenced by the position of the secondary hydroxyl group. 2-Hydroxystearic acid (2HSA) does not form a molecular dimer, as indicated by FT-IR, and growth along the secondary axis is inhibited because the secondary hydroxyl group is unable to form intermolecular H-bonds. As well, the XRD long spacing is shorter than the dimer length of hydroxystearic acid. 3-Hydroxystearic acid (3HSA) forms an acyclic dimer, and the hydroxyl groups are unable to hydrogen bond, preventing the crystal structure from growing along the secondary axis. Finally, isomers 6HSA, 8HSA, 10HSA, 12HSA, and 14HSA have similar XRD and FT-IR patterns, suggesting that these molecules all self-assemble in a similar fashion. The monomers form a carboxylic cyclic dimer, and the secondary hydroxyl group promotes growth along the secondary axis.     

A Robust Analytical Approach for the Identification of Specific Protein Carbonylation Sites: Metal-Catalyzed Oxidations of Human Serum Albumin

The formation of protein carbonyls in the metal-catalyzed oxidation of human serum albumin (HSA) is characterized using a new analytical approach that involves tagging the modification site with multiple hydrazide reagents. Protein carbonyl formation at lysine and arginine residues was catalyzed with copper and iron ions, and the resulting oxidation patterns in HSA are contrasted. A total of 18 modification sites were identified with iron-ion catalysis and 14 with copper-ion catalysis. However, with the more stringent requirement of identification with at least two tagging reagents, the number of validated modification sites drops to 10 for iron and nine for copper. Of the 14 total validated sites, there were only five in common for the two metal ions. The results illustrate the value of using multiple tagging agents and highlight the selective and specific nature of metal-catalyzed protein oxidations.     

Binding of wogonin to human serum albumin: a common binding site of wogonin in subdomain IIA

The binding of wogonin to human serum albumin (HSA) has been studied by spectroscopic method including circular dichroism (CD), infrared spectra (IR) and fluorescence spectra. The fluorescence properties of HSA were examined in presence of wogonin and the fluorescence intensity of HSA was significantly decreased in the presence of wogonin. The binding parameters of wogonin were studied from the fluorescence decreasing of HSA by the fluoremetric titrations. The Stern-Volmer plots indicated that the binding of wogonin to HSA at 296, 303, 310 K is characterized by one binding site with the binding constant K(S-V) at 1.872 x 10(5), 1.561 x 10(5), 1.392 x 10(5), respectively, which are good agreement with the results from the Scatchard plots. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction, which were consistent with the result of molecule modelling study, and there are also a numbers of hydrogen bonds between wogonin and HSA. Furthermore, the displacement experiments indicate that wogonin can bind to the subdomain IIA, that is, the site I of HSA, which is also good agreement with the result of molecule modelling study.