"Q-markers targeted screening" strategy for comprehensive qualitative and quantitative analysis in fingerprints of Angelica dahurica with chemometric methods.

Angelica dahurica is a famous functional food and herb. To guarantee quality of A. dahurica, a strategy "Q-markers targeted screening" was successfully developed by sufficient extraction of compounds and the targeted screening of qualitative and quantitative markers calculated through chemometric methods based fingerprints. Accelerated solvent extraction was selected due to its prominent advantages exhibiting the maximum extraction yields and varieties of compounds and especially excellent reproducibility (RSD < 1). After extraction, the fingerprints of A. dahuricae samples were established. For the preliminary herb authenticity, the targeted screening of 23 quantitative markers were performed by similarity analysis and hierarchical cluster analysis based on the fingerprints, which were identified by liquid chromatography tandem mass spectrometry (LC-MS). Subsequently, for further quality control, the targeted screening of nine quantitative markers were done by similarity analysis & linear discriminant analysis, which were determined by LC. Lastly, the strategy was successfully applied to quality assessment of A. dahurica samples.

Design, Synthesis, Antibacterial, Antifungal and Anticancer Evaluations of Novel ß-Pinene Quaternary Ammonium Salts.

ß-pinene is a monoterpene isolated from turpentine oil and numerous other plants' essential oils, which has a broad spectrum of biological activities. In the current work, six novel ß-pinene quaternary ammonium (ß-PQA) salts were synthesized and evaluated in vitro for their antifungal, antibacterial and anticancer activities. The in vitro assay results revealed that compounds 4a and 4b presented remarkable antimicrobial activity against the tested fungi and bacteria. In particular, compound 4a showed excellent activities against F. oxysporum f.sp. niveum, P. nicotianae var.nicotianae, R. solani, D. pinea and Fusicoccumaesculi, with EC50 values of 4.50, 10.92, 9.45, 10.82 and 6.34 µg/mL, respectively. Moreover, compound 4a showed the best antibacterial action against E. coli, P. aeruginosa, S. aureus and B. subtilis, with MIC at 2.5, 0.625, 1.25 and 1.25 µg/mL, respectively. The anticancer activity results demonstrated that compounds 4a, 4b, 4c and 4f exhibited remarkable activity against HCT-116 and MCF-7 cell lines, with IC50 values ranged from 1.10 to 25.54 µM. Notably, the compound 4c displayed the strongest cytotoxicity against HCT-116 and MCF-7 cell lines, with the IC50 values of 1.10 and 2.46 µM, respectively. Furthermore, preliminary antimicrobial mechanistic studies revealed that compound 4a might cause mycelium abnormalities of microbial, cell membrane permeability changes and inhibition of the activity of ATP. Altogether, these findings open interesting perspectives to the application of ß-PQA salts as a novel leading structure for the development of effective antimicrobial and anticancer agents.

A simple liquid chromatography tandem mass spectrometric method for fast detection of hydrogen sulfide based on thiolysis of 7-nitro-2, 1, 3-benzoxadiazole ether.

The long identified toxic gas, hydrogen sulfide (H2S), which has also been confirmed as the third gaseous signaling molecule following NO and CO, plays important roles in various physiological and pathological process. The current most established quantification method for H2S is HPLC method coupled with fluorescence detection after derivatization with a costly fluorescent reagent, Monobromobimane (MBB). However, The MBB method is characterized by strict reaction condition, long reaction time, tedious operation, and inconsistent reported results. In this study, based on the thiolysis reaction of 7-nitro-2, 1, 3-benzoxadiazole (NBD) ether, the commonly used chromatographic modifier 4-chloro-7-nitro-2,1,3- benzoxadiazole (NBDCl) and four probes (NBDOMe, NBDOEt, NBDOTFE and NBDOCMR) synthesized from NBDCl were tested as alternatives for fast quantification of H2S by LC-MS/MS. The reaction product between NBD ethers/NBDCl and H2S showed special pink color visible to the naked eye and was easy to synthesize and separate in lab; it also showed good retention on common chromatographic columns and high instrument response; therefore it is a good determinand. After establishment of LC-MS/MS methods for all the related compounds, the reaction conditions were optimized for all the probes with H2S. Then the stability, selectivity, reaction rate, sensitivity and quantitative linear relationship between the reaction product and H2S concentration were studied for each probe. Finally, NBDOEt was selected for LC-MS/MS detection of H2S. In comparision with the MBB method, the established NBDOEt method showed matched sensitivity and linearity, better selectivity, and higher repeatability; and had the advantages of easy operation, simple reaction condition, and cheap raw materials. The method was successfully validated and applied to determination of Na2S content in Na2S∙9H2O bulk drug and injection. In conclusion, NBDOEt is a promising option for quantification of H2S in abiotic matrix.

Overlapping and unique roles played by ROCK1 and 2 in the modulation of coding and long noncoding RNA expression.

BACKGROUND: Our previous study described the crucial role of Rho-associated coiled-coil containing-kinases (ROCK) in hepatocellular carcinoma (HCC). However, the potential significance of long noncoding RNA downstream of ROCK is largely unknown. Here, a comprehensive comparative bioinformatics analysis of a microarray of an MHCC-97H cell line overexpressing ROCK1 or ROCK2 was performed. RESULTS: Numerous lncRNAs and mRNAs were deregulated by Rho-associated coiled-coil containing kinases 1 and 2. These results were consistent with the qRT-PCR results. Compared with MHCC-97H-Con, which was transfected with a null vector, the GO analysis revealed differentially expressed mRNAs (DEmRNAs) in MHCC-97H-ROCK1 (ROCK1 was overexpressed) enriched in apoptotic cell clearance, the cyclooxygenase pathway and bone trabecula morphogenesis; the DEmRNAs in MHCC-97H-ROCK2 (ROCK2 was overexpressed) were enriched in VEGF production, chemokine-associated signaling pathways, acute inflammatory response and vasoconstriction. Compared with MHCC-97H-ROCK2, the DEmRNAs in MHCC-97H-ROCK1 were involved in the JAK-STAT cascade, the Akt signaling pathway and the activity of several different peptidases. The pathway analysis of ROCK1 and ROCK2 revealed an overlap in the VEGF signaling pathway, ECM-receptor interaction, and adhesion and differences in the PPAR signaling pathway and mismatch repair. The predicted targets of the differentially expressed lncRNA (DElncRNAs) were enriched in the p53 signaling pathway, Jak-STAT signaling pathway, etc. Several hub DElncRNAs were identified. CONCLUSIONS: ROCK1 and 2 modulate the expression of numerous mRNAs and lncRNAs and may participate in several signaling pathways in HCC. Several hub molecules were identified in the lncRNA-mRNA networks. Our results provide baseline data for ROCK1 and 2 regulation in HCC that might have implications for further research.

[The study on the fluorescence spectrometry for a novel photosensitizer of chlorine e6-C15-monomethyl ester and its application in biosamples analysis].

This paper studied the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester in different solvents to develop a fluorescence spectrometry for determining the concentration of chlorin e6-C15-monomethyl ester in plasma. By comparing the fluorescence spectral characteristics of chlorin e6-C15-monomethyl ester in six different solvents including methanol, ethanol, acetone, acetonitrile, phosphate buffered saline and water, the influence of different solvents on the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester has been examined. The results indicated that methanol and acetonitrile are the ideal solvent system, and the effect of phosphate buffered saline is better than water. The effect of organic solvent content and solution pH value on emission wavelength and fluorescence intensity was further evaluated. It was found that the fluorescence intensity of chlorin e6-C15-monomethyl ester was strong and stable in pH 7.2 phosphate buffered saline -acetonitrile (3 : 7) solution and can be detected at the excitation wavelength of 498.00 nm and the emission wavelength of 664.05 nm. Based on this, we developed the fluorescence spectrometry for determining chlorin e6-C15-monomethyl ester. It is specific and sensitive with good linearity over the range of 0.5-50 microg x mL(-1). The intra-batch and inter-batch precisions (RSD) were less than 10%, the extraction recoveries were all over 90%. The established method in this paper was simple, fast, effective and could be successfully applied to study the pharmacokinetics of chlorine e6-C15-monomethyl ester in SD rats.

Enantioselective separation of mirtazapine and its metabolites by capillary electrophoresis with acetonitrile field-amplified sample stacking and its application.

A simple, rapid and sensitive chiral capillary zone electrophoresis coupled with acetonitrile-field-amplified sample stacking method was developed that allows the simultaneous enantioselective separation of the mirtazapine, N-demethylmirtazapine, 8-hydroxymirtazapine and mirtazapine-N-oxide. The separation was achieved on an uncoated 40.2 cm×75 µM fused silica capillary with an applied voltage of 16 kV. The electrophoretic analyses were carried out in 6.25 mM borate-25 mM phosphate solution at pH 2.8 containing 5.5 mg/mL carboxymethyl-ß-cyclodextrin. The detection wavelength was 200 nm. Under these optimized conditions, satisfactory chiral separations of four pair enantiomers were achieved in less than 7 min in vitro. After one step clean-up liquid-liquid extraction using 96-well format, sample was introduced capillary zone electrophoresis with acetonitrile-field-amplified sample stacking to enhance the sensitivity of enantiomers. The method was validated with respect to specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and stability. The lower limit of quantification was 0.5 ng/mL with linear response over the 0.5-50 ng/mL concentration range for each mirtazapine, N-demethylmirtazapine and 8-hydroxymirtazapine enantiomer. The developed and validated method has been successfully applied to the enantioselective pharmacokinetic studies in 12 healthy volunteers after oral administration of rac- mirtazapine.

[Near-infrared spectroscopy technology for online monitoring of the column separation and purification process of active components of Centella asiatica L. Urban].

The present paper is to study and develop a method for online monitoring of the column separation and purification process of active components that are madecassoside and asiaticoside of Centella asiatica L. Urban using near-infrared (NIR) spectroscopy technology. After collecting 50%-ethanol eluant, we detected their NIR spectra and developed the high performance liquid chromatography (HPLC) assay method of active components. Then, partial least square (PLS) was used to develop linear correlation between their NIR spectra and contents. During modeling, correlation coefficient (R2) and root mean square errors of cross-validation (RMSECV) were regarded as the indexes to select optimal wavenumbers and preprocessing methods. The optimal wavenumbers of madecassoside and asiaticoside were in the range of 12 000.8-7 499.8 cm(-1) and 12 000.8-9 750.3 cm(-1), respectively; R2 were 96.44 and 96.07, respectively, and RMSECV were 0.084 80 and 0.000 99, respectively. The above developed model was used for online monitoring of the contents of madecassoside and asiaticoside during the column separation and purification process of Centella asiatica L. Urban. The predicted results were satisfactory. This method was proved to be fast, convenient and precise. It can be used in online monitoring and quality control of the manufacturing of madecassoside and asiaticoside.

[Establishment of the model for online monitoring of the column separation and purification process by near-infrared spectroscopy and determination of total ginsenosides in Folium Ginseng].

A method was developed for online monitoring of the constituents of ginsenoside of Folium Ginseng in the column separation and purification process using near-infrared (NIR) spectroscopy technology. Determination method of ginsenoside Rg1, Re and Rb1 was developed by high performance liquid chromatography (HPLC). After collecting 40%-ethanol eluant, their NIR spectra were detected and the contents of Rg1, Re and Rb1 were determined by the above HPLC method. The quantitative analysis models of the above three compounds and the total ginsenosides were established using partial least squares (PLS). During modeling, coefficient of determination (R2) and root mean square errors of cross-validation (RMSECV) were regarded as the indexes to select optimal wave numbers and preprocessing methods. The optimal wave numbers of ginsenoside Rg1, Re, Rb1 and total ginsenosides were all in the range of 12 000. 8 approximately 7499.8 cm-1; R2 were 0.9887, 0. 9603, 0.9905 and 0.9701, respectively; RMSECV were 0.0597, 0.0722, 0.00488 and 0.0755, respectively. A lot of samples, collected during the column separation and purification process of Folium Ginseng extract, were used to validate the predicttion effect of quantitative analysis model of total ginsenosides. As a result, the correlation coefficient of NIR predicted value and HPLC value of total ginsenosides was 0.9928 and the mean prediction recovery was 100.52%, which indicated that the prediction effect of the developed model was satisfactory. This method was proved to be fast, convenient and precise. It can be used for assaying and quality control of total ginsenosides in manufacture.

[Study on purification of total flavones from Trollius ledebouri by macroporous resin].

OBJECTIVE: To study the optimal technologies for the purification of total flavones from Trollius ledebouri with macroporous resin. METHODS: Static and dynamic adsorption-desorption methods were adopted to choose the optimal type of resin. The adsorption function of D101 macroporous resin and the effects of sample concentration, pH, flow rate and eluant etc. were studied. Then the orthogonal design L9 (3(4)) was used to select the optimum purification process conditions. RESULTS: The appropriate technological conditions were as follows: the sample concentration was 0.5 g/mL, pH 6.0, the ratio of sample to D101 macroporous resin was 1:1 (W/W), 4 BV of water was used as purificant and 6 BV of 30% alcohol was used as eluant. Under these conditions, the average content of total flavonoids extract was over 60%.

Perivascular adipose tissue-derived visfatin is a vascular smooth muscle cell growth factor: role of nicotinamide mononucleotide.

AIMS: Perivascular adipose tissue (PVAT) inhibits vascular smooth muscle cell (VSMC) contraction and stimulates VSMC proliferation by releasing protein factors. The present study was to determine whether visfatin is involved in these paracrine actions of PVAT, and if so, to explore the underlying mechanisms. METHODS AND RESULTS: Visfatin was preferentially expressed in Sprague-Dawley rat and monkey aortic PVAT, compared with subcutaneous and visceral adipose tissues. The PVAT-derived visfatin was found to be a VSMC growth factor rather than a VSMC relaxing factor, which was proved by visfatin-specific antibody/inhibitor and direct observation of recombinant visfatin. Exogenous visfatin stimulated VSMC proliferation in a dose- and time-dependent manner via extracellular signal-regulated kinase (ERK 1/2) and p38 signalling pathways. This proliferative effect was further confirmed by enhancement of DNA synthesis and upregulation of proliferative marker Ki-67. Visfatin had no anti-apoptotic effect on normal cultured VSMCs, and it exerted an anti-apoptotic effect only during cell apoptosis induced by H2O2, excluding a role of anti-apoptosis in the visfatin-induced VSMC proliferation. Insulin receptor knockdown did not show any action on the visfatin effect. However, visfatin acted as a nicotinamide phosphoribosyltransferase to biosynthesize nicotinamide mononucleotide (NMN), which mediated proliferative signalling pathways and cell proliferation similar to the visfatin effect. CONCLUSION: Visfatin stimulates VSMC proliferation via NMN-mediated ERK1/2 and p38 signalling. The present study provides a molecular link of visfatin to the paracrine action of PVAT, demonstrates a novel function of visfatin in promoting VSMC proliferation, and reveals NMN as a novel signalling molecule that triggers the proliferative process.

[Studies on interaction of sinafloxacin with bovine serum albumin and effect of the coexistent metal ions on the reaction].

Serum albumin is the most abundant protein in plasma. It can bind with many intrinsic and extrinsic materials. The study of the interaction between serum albumin and drugs is a very important task in life science and chemistry. Quinolone drug is a kind of antibacterial drugs which have been used widely in clinical medicine, but its pharmacology and toxicology still have to be studied further. Sinafloxacin is a new quinolone antibiotics. The interaction between sinafloxacin and bovine serum albumin (BSA) at different temperatures and pH was investigated by fluorescence and UV absorption spectroscopy. The experimental results demonstrate that the fluorescence quenching of BSA by sinafloxacin is a result of the formation of sinafloxacin-BSA complex and the quenching mechanism is mainly static quenching. The interaction association constants of BSA and sinafloxacin were determined from the double reciprocal Lineweaver-Burk plot. The binding distance(r = 3.64 Rnm) and energy transfer efficiency (E = 0.163) between donor (BSA) and acceptor (sinafloxacin) were obtained based on Förster's non-radiative energy transfer theory. There is a strong interaction between sinafloxacin and BSA. From thermodynamic coordination it can be judged that the binding force between sinafloxacin and BSA is mainly electro-static force. The effect of sinafloxacin on the conformation of BSA was analyzed by synchronous fluorescence spectrometry and three-dimensional fluorescence spectrometry. The emission maximum of tyrosine residues does not show a significant shift, while the small blue shift of tryptophan residues indicates that the hydrophobicity of microenvironment was increased. In addition, in the plasma, there are some metal ions, which can participate in many important vital actions and affect the reactions of the drugs with the serum albumins. So the effect of Cu(II), Fe(III), Zn( II) and Mg(II) on the binding constant of sinafloxacin to BSA was also discussed and it was shown that the interaction between BSA and sinafloxacin was decreased. In short, the results offer a reference for the studies on the biological effects and action mechanism of sinafloxacin with albumins in vivo.

Isolation of six isoflavones from Semen sojae praeparatum by preparative HPLC.

A method for the isolation of six isoflavones (genistein, genistin, daidzein, daidzin, glycitein and glycitin) with high purity from Semen sojae praeparatum, a famous traditional Chinese medicine, by preparative HPLC is described.

[Isolation and structure identification of chemical constituents from Patrinia villosa].

AIM: To study the chemical constituents of Patrinia villosa Juss. METHODS: Solvent extraction, silica gel column and preparative liquid chromatography were used to separate the chemical constituents, and the chemical structures were elucidated by physico-chemical properties and spectra data. RESULTS: Eight compounds were isolated and identified as bolusanthol B (1), (2S)-5, 7, 2', 6'-tetrahydroxy-6,8-di (gamma,gamma-dimethylallyl) flavanone (2), orotinin (3), (2S)-5, 7, 2', 6'-tetrahydroxy-6-lavandulylated flavanone (4), 3'-prenyl-apigenine (5), luteolin (6), quercetin (7) and apigenin (8). CONCLUSION: Compound 2 and 4 are new compounds, compounds 1, 3 and 5 were separated from Patrinia genius for the first time, compounds 6, 7 and 8 were isolated from Patrinia vollosa Juss for the first time.

[Studies on chemical constituents of Patrinia villosa].

OBJECTIVE: To investigate the chemical constituents of Patrinia villosa. METHOD: The chemical constituents were isolated by silica gel column chromatography and semi-preparative high-performance liquid chromatography, and identified by physicochemical properties and spectral analysis (MS, 1H-NMR and 13C-NMR). RESULT: Seven compounds were isolated from ethyl acetate and n-butanol extract and identified as: 5-hydroxyl-7, 3', 4'-trimethoxy flavone (I), 5-hydroxyl-7, 4'-dimethoxy flavone (II), luteolin (III), quercetin (IV), isoorientin (V), isovitexin (VI) and 8-C glucosylprunetin (VII). CONCLUSION: Compounds I , II, III, V, VI and VIII were obtained from the plant of genus Patrinia for the first time, compound IV was separated from P. villosa for the first time.

[Stereoselective pharmacokinetics of tetrahydropalmatine in rats].

AIM: To investigate the stereoselective pharmacokinetic process of tetrahydropalmatine (THP) in rats. METHODS: The concentrations of tetrahydropalmatine enantiomers in rat plasma were determined by coupled achiral and chiral HPLC method. The differences in plasma concentrations and pharmacokinetic parameters between the two enantiomers were compared by paired t-test. RESULTS: The plasma levels of l-THP were always higher than those of d-THP in eight rats. There was significant difference between the main pharmacokinetic parameters of the two enantiomers. CONCLUSION: Tetrahydropalmatine showed significant stereoselective pharmacokinetics in rats after an ig dose of the racemate.