Encapsulation of methyl and ethyl salicylates by beta-cyclodextrin HPLC, UV-vis and molecular modeling studies

The complexation of methyl salicylate (MS) and ethyl salicylate (ES), non-steroidal analgesic, anti-inflammatory and antirrheumatic drugs with beta-cyclodextrin (betaCD) has been studied from thermodynamic and structural points of view. The complexation with betaCD has been investigated using reversed-phase liquid chromatography. Retention behavior has been analyzed on a reverse-phase column Luna 18(2) 5 microm. The mobile-phase was methanol:water in different ratios (55:45 to 70:30) in which betaCD (1-9 mM) was incorporated as a mobile-phase additive. The decrease in retention times with increasing concentrations of betaCD enables the determination of the apparent stability constant of the complexes. Values at 30 degrees C with 55% methanol were K(MS:betaCD): 15.84 M(-1) and K(ES:betaCD): 12.73 M(-1) for MS and ES, respectively. The apparent stability constants decrease as the polarity of the solvent decreases. The low solubility of MS and ES in aqueous solution has been improved by complexation with betaCD (1-9 mM). The stability constants of the complexes obtained from the phase-solubility diagrams using a UV-vis spectrophotometric method were K(MS:betaCD): 229 M(-1) and K(ES:betaCD): 166 M(-1). In addition, semi-empirical quantum mechanics calculations using AM1 and PM3 methods in vacuum were performed. The energetically favorable inclusion structures were identified and the most favorable orientation for the inclusion process was found to be the head-down orientation for both complexes. Enthalpy for encapsulation processes was found to be favorable (DeltaH degrees <0), while entropy (DeltaS degrees <0) and Gibbs free energy were unfavorable (DeltaG degrees >0). By means of HPLC and UV-vis measurements and quantum mechanics calculations, it was found that MS and ES form a 1:1 inclusion complex with betaCD. The theoretical results are in agreement with the experimental parameters associated with the encapsulation process.     

鸵鸟心脏细胞色素C的制备及性质

目的从鸵鸟心脏中分离提纯细胞色素C(cyt.c)并对其理化性质、活性进行考察。方法在酸性条件下从鸵鸟心脏中提取细胞色素C粗制品后 ,在离子交换色谱柱上进行纯化 ,用HPLC法、IEF法及SDS PAGE法检测所得细胞色素C纯度 ,用SDS PAGE法测定其分子质量 ,用IEF法测定其等电点。用药典方法进行含量测定、活力测定。结果经纯化后所得样品具有等电点均一性 ,达到了高效液相纯及SDS PAGE凝胶电泳纯。测得分子质量为 1 1 6kDa ,等电点为 1 0 1 0。单位重量鸵鸟心的细胞色素C产量为 92～ 1 47mg/kg,鸵鸟细胞色素C的平均活力为 81 5 0 %。结论鸵鸟心的细胞色素C含量丰富 ,用同样的工艺过程 ,单位重量原料的产量高于猪心 ,且有着比猪心细胞色素C更高的活性。     

In silico predication of nuclear hormone receptors for organic pollutants by homology modeling and molecular docking

Homology modeling and molecular docking were used to in silico predict the rat nuclear hormone receptors of different organic pollutants. Rat aryl hydrocarbon receptor (rAhR), constitutive androstane receptor (rCAR) and pregnane X receptor (rPXR) were chosen as the target nuclear receptors. 3D models of ligand binding domains of rAhR, rCAR and rPXR were constructed by MODELLER 9V6 and assessed by the Procheck and Prosa 2003. Surflex-Dock program was applied to bind the different organic pollutants into the three receptors to predict their affinities. The results of docking experiments demonstrated that three polybrominated dibenzofurans (PBDFs, including TretaBDF, PentaBDF and HexaBDF) and 3,3′,4,4′,5′-pentachlorobiphenyl (PCB126) would be better categorized by rAhR-dependent mechanism, but four polybrominated diphenyl ethers (PBDEs, including BDE47, BDE80, BDE99 and BDE153) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) by rCAR and rPXR-dependent mechanism. For benzo(a)pyrene and pyrene, they have high affinities with the three target receptors, which suggests that “crosstalk” among the receptors might occur during the receptor induction. The results of this study are consistent with those of animal experiments reported by previous literatures, which suggest that homology modeling and molecular docking would have the potential to predict the nuclear hormone receptors of environmental pollutants.     

Inhibition of rat hepatic CYP2E1 by quinacrine: molecular modeling investigation and effects on 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutagenicity

Increased activity of CYP2E1 has been associated with increased risk of chemically-mediated cancers, through enhanced activation of a variety of procarcinogens. In this context, inhibition of CYP2E1 is potentially of significance in xenobiotic toxicity. The aim of the present study was to test the hypothesis that quinacrine inhibits hepatic CYP2E1. For this purpose, disulfiram (75 mg/kg i.p) as an inhibitor and isoniazid (100 mg/kg i.p) as an inducer of CYP2E1, as well as quinacrine (50 mg/kg i.p) were administered to Wistar rats and the hepatic activity of CYP2E1 was measured. The expression of CYP2E1 was further assessed by Western blot analysis. As expected, disulfiram inhibited, while isoniazid induced the activity and expression of the enzyme. Interestingly, treatment with quinacrine resulted in a significant decrease of CYP2E1 activity and expression. To investigate any similarities in the inhibition of CYP2E1 by quinacrine and disulfiram, molecular modeling techniques were adopted and revealed that quinacrine molecule anchors inside the same binding pocket of the protein where disulfiram is also attached. Finally, as assessed by the sister chromatid exchanges (SCE) assay, quinacrine was demonstrated to reduce the mutagenic effects of the tobacco-specific N-nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is known to be converted to active mutagen in the liver principally through CYP2E1. We suggest that these antimutagenic effects of quinacrine could be possibly attributed, at least in part, to its ability to block the bioactivation of NNK, mainly by the inhibition of CYP2E1. Our results, even preliminary, indicate that quinacrine as an inhibitor of CYP2E1 might be protective against chemically-induced toxicities such as NNK-induced mutagenicity.     

Retinoic acid binding properties of the lipocalin member beta-lactoglobulin studied by circular dichroism,electronic absorption spectroscopy and molecular modeling methods

Interaction between the Vitamin A derivative all-trans retinoic acid and the lipocalin member bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted pi-pi(*) absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD-pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the beta-barrel.     

槲皮素包合物微球的制备及其体外释放研究

目的:制备槲皮素β-环糊精包合物-壳聚糖微球(QT-CD-CM),并考察其理化性质和药物体外释放性能。方法:采用有机溶剂挥发法制备槲皮素β-环糊精包合物,再用乳化分散-离子交联法、以三聚磷酸钠为交联剂制备壳聚糖微球,并考察其形态、粒径、包封率、载药量和体外释放情况。结果:制备的QT-CD-CM形态规则、均质、无粘连,平均粒径(3.327±0.124)μm,包封率为32.4%,载药量为12.3%,在5%乙醇-磷酸盐缓冲液介质中72h可以达到完全释药,释药过程符合一级动力学模型。结论:QT-CD-CM理化性质及体外释药性能良好,制备工艺简单,有望成为理想的槲皮素给药系统。     

糖尿病合并冠心病患者C反应蛋白、糖化血红蛋白水平与心力衰竭的相关性研究

目的：探讨糖尿病合并冠心病患者C反应蛋白（CRP）、糖化血红蛋白（ HbA1C）水平与心力衰竭的相关性。方法选取我院2013年3月-2014年3月收治的糖尿病患者137例,根据患者冠状动脉造影将其分为单纯糖尿病患者65例（对照1组）及冠心病合并糖尿病尿病及心力衰竭患者72例（观察组）,同时选取同期本院体检健康者50例作为对照2组。比较各组患者血浆HbA1C、CRP、血压（SBP、DBP）、左心室功能各指标（ESV、EDV、EF）、血脂指标（TC、TG、LDL-C、HDL-C）及血糖（FPG、2hPBG）水平。结果观察组及对照1组患者HbA1C、CRP、FPG、2hPBG、SBP、DBP、TC、TG、LDL-C、ESV、EDV水平明显高于对照2组,而HDL-C、EF明显低于对照2组,差异均有统计学意义（P＜0.05）;而观察组HbA1C、CRP、ESV、EDV水平高于对照1组,EF低于对照1组,差异均有统计学意义（P＜0.05）。经多因素Logsitic 回归分析可知, HbA1C、CRP是糖尿病合并冠心病并发心力衰竭的独立危险因素。结论 HbA1C、CRP可能是引起糖尿病合并冠心病患者发生心力衰竭的相关因素,通过监测HbA1C、CRP水平可以预测糖尿病合并冠心病患者心功能状况,预防心力衰竭的发生。     

HPLC法测定维尔康胶囊中维生素C和维生素B1的含量

目的：应用高效液相色谱法同时测定维尔康胶囊中维生素C和维生素B1的含量。方法：色谱条件：C18柱，0.03mol／L磷酸二氢钾缓冲液（用磷酸调节pH至3．0）-甲醇（99：1）为流动相，检测波长为246nm。结果：该方法的维生素C的回收率为99．7％（RSD=2．2％，n=9）；维生素B，的回收率为100．2％（RSD=0．5%，n=9）。结论：本法简便、灵敏、准确。     

三七总皂苷中代表性皂苷含量测定方法学的建立及稳定性研究

目的 建立同时测定三七总皂苷中三七皂苷R1、人参皂苷Rg1 和人参皂苷Rb1 三种成分的含量测定方法,并对其各种状态下的稳定性进行研究.方法：以乙腈-水系统作为流动相,采用高效液相-梯度洗脱法,同时测定三七总皂苷中三种成分的含量,并考察方法的专属性、线性、精密度和回收率;测定四种pH值溶液中三种皂苷成分的稳定性以及在高温、高湿和强光照射条件下,三七总皂苷粉末中三种皂苷成分的稳定性.结果：方法学验证结果显示,在选定的梯度洗脱条件下,方法专属性良好,三种皂苷可有效分离和定量;在各自的线性范围内线性良好;精密度RSD 值均小于2%;三种皂苷的平均回收率分别为（98.98 ± 0.60）%、（98.86 ± 0.34）%和（100.19 ± 0.64）%,方法准确性良好.稳定性研究结果显示,在溶液状态下,三种皂苷的稳定性趋势基本-致,稳定性与pH值密切相关,在pH 1.2 溶液中药物迅速降解,24 h 含量即降低了约80%;而在pH 4.5 溶液、pH 6.8 溶液以及水中,三种成分的稳定性均良好,24 h 内含量未发生明显变化;而在三七总皂苷粉末中,三种代表性皂苷对湿、热和光照均不敏感,10 d 内含量基本保持稳定;但是在高湿条件下,粉末有-定的吸湿性.结论：所建立的液相方法准确、可靠,溶液状态下三七皂苷R1、人参皂苷Rg1 和人参皂苷Rb1 的稳定性与pH密切相关,随pH的降低稳定性变差,而在固体状态下则稳定性良好.     

血清、尿Cystatin C检测在高血压肾病中的诊断价值

目的探讨血清、尿半胱氨酸蛋白酶抑制剂C（Cystatin C）在高血压肾病早期诊断中的价值。方法检测高血压患者尿常规、尿Cystatin C、尿α1微球蛋白（α1-MG）和尿白蛋白,以及血Cystatin C、血肌酐、尿素氮水平。通过分组,对各项指标进行比较分析。结果血Cystatin C和血肌酐水平、尿的Cystatin C和尿α1微球蛋白的水平、尿的Cystatin C和尿白蛋白的水平均呈正相关。蛋白尿阳性组的血、尿Cystatin C的水平均明显高于蛋白尿阴性组和健康对照组（P〈0.05）,蛋白尿阴性组的尿Cystatin C的水平高于健康对照组（P〈0.05）。血、尿Cystatin C对高血压肾损害的敏感性高于血肌酐、尿白蛋白和α1微球蛋白。结论血清、尿Cystatin C分别是判断高血压肾病患者肾小球功能和肾小管受损的早期敏感指标。     

Exploring the mechanism of interaction between 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one and human serum albumin: Spectroscopic, calorimetric and molecular modeling studies

In this paper, binding interaction of 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (EMMD) with human serum albumin (HSA) under physiological conditions was investigated by using spectroscopy, isothermal titration calorimetry (ITC) and molecular modeling techniques. The results of spectroscopic studies suggested that EMMD have a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. ITC investigations indicated that drug-protein complex was stabilized by hydrophobic forces and hydrogen bonds, which was consistent with the results of molecular modeling studies. Competitive experiments indicated the displacement of warfarin by EMMD, which revealed that the binding site of EMMD to HSA was located at subdomain IIA.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

蝉拟青霉中虫草素提取液的稳定性及酸碱溶解度探究试验

目的：研究虫草素提取液的稳定性及酸碱溶解度,为虫草素的进一步开发利用奠定基础。方法利用75％乙醇、45℃超声波浸提虫草素的提取液对虫草素的稳定性、在酸碱溶液中的溶解度等进行了研究。结果虫草素对光的稳定性较差,光照可导致其分解;对温度的稳定性不高;维生素中,VA 不利于虫草素的保存,VB、VC、VE均有利于虫草素的保存;随着溶液碱性的增强虫草素的溶解度略微降低,溶液酸性越强虫草素越不易溶解。结论通过对虫草素提取液稳定性的初步研究,以期为虫草素的进一步开发利用奠定基础。     

The physicochemical properties and the in vivo AChE inhibition of two potential anti-Alzheimer agents, bis(12)-hupyridone and bis(7)-tacrine

The lipophilicity and solubility profiles of bis(12)-hupyridone (B12H) and bis(7)-tacrine (B7T), two novel acetylcholinesterase inhibitors dimerized from huperzine A fragments and tacrine, respectively, were investigated over a broad pH range. Lipophilicity was assessed by both shake flask method with 1-octanol-water system and a reverse-phase HPLC system with methanol-water as mobile phase. The former method was used for determining the lipophilicities of the ionized forms (log D) of the dimers while the latter method was used for that of the neutral forms (log P). The log P values for B12H and B7T were found to be 5.4 and 8.2, respectively, indicating that the two dimers are highly lipophilic. The solubilities of both dimers were found to be affected by pH. The solubility of B12H was >1.41 mg/ml when the pH was <7, but <0.06 mg/ml when the pH was >8. The solubility of B7T was >0.26 mg/ml when the pH was <9, but <0.005 mg/ml when the pH was >12. The ionic strength of a solution could affect the solubilities considerably (11.16 mg/ml for B12H and 12.71 mg/ml for B7T in water; 2.07 mg/ml for B12H and 0.36 mg/ml for B7T in saline). The ionization constants (pK(a)) of the two dimers were determined by UV spectrophotometry. Both dimers were found to have two pK(a) values: 7.5+/-0.1 (pK(a1)) and 10.0+/-0.2 (pK(a2)) for B12H; and 8.7+/-0.1 (pK(a1)) and 10.7+/-0.4 (pK(a2)) for B7T. Furthermore, an in vivo pharmacological assay conducted in mice showed that a maximum AChE inhibition occurred 15 min after the single-dose and intraperitoneal administration of either dimer. This indicates that the two dimers may easily cross the blood-brain barrier. In summary, these physiochemical characteristics suggest that the two dimers may be promising candidates for the development of better drugs for Alzheimer's disease.     

聚合物囊泡的稳定性及H+透膜特性考察

目的 以嵌段聚合物制备聚合物囊泡并考察其稳定性，测定聚合物囊泡膜层的H⁺跨膜渗透特性，及1,4-二氧六环对膜渗透性能的影响，作为聚合物囊泡载药的基础。方法 以二嵌段共聚物PEG-PLGA在溶液中自组装制备聚合物囊泡，采用pH敏感荧光探针HPTS对囊泡的H⁺透膜特性进行考察，并与PBD-b-PEO、PS-b-PEO制备的囊泡及脂质体进行比较。考察不同浓度的1,4-二氧六环对聚合物囊泡膜渗透特性的作用。结果 HPTS的荧光激发光谱有pH依赖性，囊泡外水相中H⁺浓度与t^1/2呈线性相关，不同膜壁厚度的聚合物囊泡的膜渗透能力有显著区别。3种聚合物囊泡对比脂质体，H⁺透膜系数分别降低了2.39×10⁴ 、3.38×10⁴、5.48×10⁸倍。1,4-二氧六环对囊泡膜的渗透性具有调节作用，且存在浓度依赖关系。结论 聚合物囊泡的膜渗透显著低于脂质体，稳定性更好，1,4-二氧六环可调节囊泡膜的渗透性，从而调节药物的装载和释放。     

Utility of cyclodextrins in the formulation of genistein: Part 1. Preparation and physicochemical properties of genistein complexes with native cyclodextrins

Isoflavones are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs). The molecular encapsulation with CDs results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of isoflavones. Genistein, a key isoflavone constituent of Ononidis spinosae radix was found to form a supramolecular, non-covalent inclusion complex with both β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD), while it did not form a stable complex with α-CD. The guest genistein was found to spatially located in the less polar cavity of cyclodextrin. The isolated binary genistein/CD complexes appeared novel crystalline lattices. The in vitro dissolution of genistein entrapped into both β- and γ-CD, significantly surpassed that of the plain isoflavone.     

基于分子对接和体外大鼠肝微粒体抑制实验综合考察何首乌中潜在肝毒性成分研究

目的 基于UDP-葡萄糖醛酸转移酶1A1（UGT1A1）介导的胆红素代谢,构建UGT1A1酶蛋白模型,用于研究何首乌中潜在致肝毒性成分的毒性作用,并用体外大鼠肝微粒体抑制实验进行验证。方法 采用同源模建方法构建UGT1A1酶蛋白结构,将UGT1A1底物胆红素及何首乌中主要蒽醌类成分大黄素、大黄酚、大黄酸、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄素甲醚与UGT1A1蛋白进行分子对接,考察分子结合靶点及结合强弱。采用大鼠肝微粒体孵育体系（RLM）,加入系列浓度的底物胆红素对照品溶液及待测单体对照品溶液,检测表观抑制常数（K_i）,测定蒽醌类单体成分对UGT1A1酶的抑制作用。结果 分子对接结果显示,UGT1A1酶蛋白结构上共有9个活性口袋区,胆红素的结合口袋确定为位点F;6个单体主要集中在两个活性口袋区：大黄素、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄酚对接进入位点F;大黄素甲醚和大黄酸对接进入位点C。结合于UGT1A1酶蛋白位点C中的单体大黄酸和大黄素甲醚的结合自由能（IE）值较小;位点F区中,单体大黄素-8-O-β-D-葡萄糖苷、大黄素及羟基大黄素具有较高的IE值,结合能力强。体外抑制实验显示,大黄素-8-O-β-D-葡萄糖苷、大黄素表现为较强的竞争型抑制作用,羟基大黄素为较强的混合型抑制作用,与分子对接结果一致。结论 大黄素-8-O-β-D-葡萄糖苷、羟基大黄素、大黄素对UGT1A1酶介导的胆红素代谢产生较强的抑制作用,构建的UGT1A1酶蛋白模型可有效预测药物的潜在风险。