对乙酰氨基酚中对氯乙酰苯胺的限量分析

以Ｎｏｖａ－ＰａｃｋＣ１８为色谱柱，０．０１ｍｏｌ／Ｌ庚烷磺酸钠－甲醇－甲酸（８５１５０．５）为流动相，检测波长２５４ｎｍ，采用ＨＰＬＣ法测定对乙酰氨基酚中对氯乙酰苯胺的限量     

小儿氨酚那敏颗粒中对乙酰氨基酚有关物质的测定

目的用HPLC法测定对乙酰氨基酚中的有关物质,以控制小儿氨酚那敏的质量。方法用HPLC法测定对乙酰氨基酚中的有关物质。结果对乙酰氨基酚的线性范围为6．558-15．30μg／mL（r=0．9998,n=5）;平均回收率为98．7％。结论该方法准确可靠,可用于小儿氨酚那敏中对乙酰基酚有关物质的测定。     

HPLC法测定对乙酰氨基酚软胶囊的有关物质

目的建立对乙酰氨基酚软胶囊有关物质的高效液相色谱测定方法。方法采用Agilent XDB-C18(150 mm×4.6 mm,5μm)色谱柱,甲醇-水-磷酸(20∶80∶0.1)为流动相,流速1.0 mL.min-1,检测波长为272 nm。结果对氨基酚最低检出限为1.25 ng.mL-1,对乙酰氨基酚与对氨基酚及其他有关物质达到基线分离。结论本方法简便、准确、专属性高,可用于本制剂的有关物质控制。     

HPLC法测定对乙酰氨基酚缓释干混悬剂的有关物质

目的建立高效液相色谱法测定对乙酰氨基酚缓释干混悬剂的对氨基酚及有关物质的方法。方法采用Diamonsil C18（4.6mm×150mm,5μm）色谱柱;流动相：甲醇-水（20∶80）;流速：1.0mL·min-1;检测波长为275nm;进样量10μL。结果方法的最低检测限浓度为0.03μg.mL-1,对氨基酚及有关物质与主峰达到基线分离。结论本方法简便、准确、专属性强,可用于对乙酰氨基酚缓释干混悬剂的对氨基酚及有关物质的测定。     

对乙酰氨基酚栓与双氯酚酸钠栓治疗小儿急性发热的对比研究

目的对比对乙酰氨基酚栓与双氯酚酸钠栓治疗小儿急性发热的疗效与安全性,探讨后者临床应用优势。方法将我院收治的145例急性发热小儿依据退热给药方式差异予以分组,其中72例给予对乙酰氨基酚栓治疗设为对照组,其余73例采用双氯酚酸钠栓治疗设为观察组,对比两组用药后疗效、再次用药例数与不良反应情况。结果对照组患儿退热效果明显不及观察组(P<0.05);再次用药情况观察组例数明显较少(P<0.05);两组不良反应差异不具显著性(P>0.05)。结论双氯酚酸钠栓应用于急性发热患儿退热,具见效快、维持时间长、用药方便且无明显不良反应等优点,深具临床推广价值。     

复方氯唑沙宗片中有关物质的GC测定

建立了GC法测定复方氯唑沙宗片中的有关物质.采用DB-624毛细管柱,程序升温.4种已知杂质对氯苯酚、2-氨基-4-氯苯酚、对氨基酚和对氯苯乙酰胺分别在1.62～324、1.25～250、1.78～357和1.60～321 μg/ml浓度范围内线性关系良好,平均回收率为94.5%～107.3%,RSD为0.8%～3.9%.     

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.     

Differential effects of the organochlorine pesticide DDT and its metabolite p,p'-DDE on p-glycoprotein activity and expression

1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) is an organochlorine pesticide. Its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethene (p,p'-DDE) is a persistent environmental contaminant and both compounds accumulate in animals. Because multidrug resistance transporters, such as p-glycoprotein, function as a defense against xenobiotic exposure, we analyzed the ability of DDT and p,p'-DDE to act as efflux modulators. Using a competitive intact cell assay based on the efflux of the fluorescent dye rhodamine 123, we found that DDT, but not p,p'-DDE, stimulated dye retention. Subsequent studies using verapamil as competitor suggested that DDT is a weak p-glycoprotein inhibitor. Further studies addressed the ability of DDT and p,p'-DDE to induce MDR1, the gene encoding p-glycoprotein. In HepG2 cells, we found that both compounds induced MDR1 by twofold to threefold. Similar results were observed in mouse liver after a single dose of p,p'-DDE, although some gender-specific induction differences were noted. By contrast, p,p'-DDE failed to induce MDR1 in HeLa cells, indicating some cell-specific effects for induction. Further expression studies demonstrated increased levels of the endoplasmic reticulum molecular chaperone, Bip, in response to DDT, but not p,p'-DDE. These results suggest that DDT, but not p,p'-DDE, induces an endoplasmic reticulum stress response.     

Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl-L-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl-L-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.     

Preparation of amino-modified active carbon cartridges and their use in the extraction of quercetin from Oldenlandia diffusa

Polyethyleneimine (PEI) and ethylenediamine (EDA) as modifiers were bonded on active carbon (AC) surface for specific selective extraction of quercetin from Oldenlandia diffusa. The characteristics of the modified AC materials that were obtained were investigated by field emission-scanning electron microscopy (FE-SEM) and Fourier transform infrared spectrometer (FT-IR). The interactions between quercetin and the AC materials were investigated by fitting the static adsorption data to four linear and nonlinear adsorption isotherm models. Of these four models, the Langmuir-Freundlich adsorption isotherm was proved the best for investigating quercetin on AC materials. Scatchard analysis was used to evaluate the binding properties of the AC materials for quercetin. Solvent extraction and solid-phase extraction (SPE) were optimized, and the effect of the mobile phase pH was investigated to improve the performance for the separation of quercetin on high performance liquid chromatography (HPLC). The results from the validation of the proposed analytical method demonstrated that the EDA-modified AC was the most suitable SPE cartridge for the purification of quercetin from O. diffusa.     

Quality Control Standards for the Roots of Three Plumbago Species

Physicochemical parameters of roots of three Plumbago species, Plumbago capensis, P. rosea and P. zeylanica belonging to Plumbaginaceae were analyzed. Microbial contamination, aflatoxins, pesticide residue and heavy metal content were also determined. Attempt has also been made to estimate the biologically active chemical plumbagin present in them and the data compared. The study ensures that the quality control parameters do help in the proper standardization of the crude drugs in drug development process for global acceptance.     

Dermal penetration and metabolism of p-aminophenol and p-phenylenediamine: Application of the EpiDerm™ human reconstructed epidermis model

To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm™ reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm™ tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm™ tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dye exposure), the majority (80–>90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5 h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-mono- and N,N′-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24 h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm™ tissue via N-acetylation. Characterising the metabolic capability of EpiDerm™ tissue is important for the evaluation of this model for use in genotoxicity testing.     

Exploring Protein Binding of Uremic Toxins in Patients with Different Stages of Chronic Kidney Disease and during Hemodialysis

As protein binding of uremic toxins is not well understood, neither in chronic kidney disease (CKD) progression, nor during a hemodialysis (HD) session, we studied protein binding in two cross-sectional studies. Ninety-five CKD 2 to 5 patients and ten stable hemodialysis patients were included. Blood samples were taken either during the routine ambulatory visit (CKD patients) or from blood inlet and outlet line during dialysis (HD patients). Total (C_T) and free concentrations were determined of p-cresylglucuronide (pCG), hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS) and p-cresylsulfate (pCS), and their percentage protein binding (%PB) was calculated. In CKD patients, %PB/C_T resulted in a positive correlation (all p < 0.001) with renal function for all five uremic toxins. In HD patients, %PB was increased after 120 min of dialysis for HA and at the dialysis end for the stronger (IAA) and the highly-bound (IS and pCS) solutes. During one passage through the dialyzer at 120 min, %PB was increased for HA (borderline), IAA, IS and pCS. These findings explain why protein-bound solutes are difficult to remove by dialysis: a combination of the fact that (i) only the free fraction can pass the filter and (ii) the equilibrium, as it was pre-dialysis, cannot be restored during the dialysis session, as it is continuously disturbed.     

Investigation of the immunogenicity of p-phenylenediamine and Bandrowski's base in the mouse

p-Phenylenediamine (PPD) exposure is associated with T-cell mediated contact dermatitis. T-cells from allergic patients proliferate following exposure to PPD and the oxido-conjugation product Bandrowski's base (BB). Both compounds are classified as sensitizers in the local lymph node assay; however, because of their instability the nature of the antigenic determinant remains ill-defined. The aim of this study was to explore the immunogenic potential of PPD and BB in mice. Spleen cell proliferation and cytokine secretion was measured ex vivo following antigen recall with soluble PPD or BB and either irradiated or glutaraldehyde fixed, antigen pulsed dendritic cells from syngeneic mice. Glutathione was added to certain incubations. LC–MS analysis and solvent extraction were used to monitor the fate of [¹⁴C]BB in culture and the extent of BB binding, respectively. Spleen cells from BB exposed, but not PPD- or vehicle-exposed, mice proliferated when stimulated with BB. Proliferating cells secreted high levels of IFN-γ, GM-CSF and IL-2. Stimulation with PPD instigated low levels of proliferation. Irradiated, but not fixed, dendritic cells pulsed with BB stimulated proliferation signifying a classical hapten mechanism involving irreversible BB binding to protein and processing. BB bound preferentially to serum protein when incubated together with cells and serum. Degradation of BB in the presence of glutathione was associated with a stronger stimulation of specific T-cells at higher BB concentrations. These data demonstrate that BB is a potent immunogen in the mouse.     

Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline

p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10 and 25 μM were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations ≥ 50 μM were cytotoxic. Cytostatic concentrations induced transient G₁ and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.