Research advances in the study of anticoagulant active molecules / 药学学报

The incidence of thrombosis-induced cardiovascular diseases is increasing worldwide and poses a serious threat to human health. Three factors, slow speed of blood flow, hypercoagulable blood and vascular damage, have been considered to be causes of thrombosis. Antithrombotic drugs have been classified into three categories based on the mechanism of thrombosis, including anticoagulants, platelet inhibitors and fibrinolytics. The coagulation and anticoagulation systems have drawn increasing attention because of the important role they play in the process of thrombosis. Novel compounds with anticoagulant activity are now emerging, alleviating to some extent some of the problems associated with the clinical use of early approved thrombotic drugs, such as high bleeding risk, slow onset of action and narrow therapeutic windows. In this review, we initially describe the mechanisms of coagulation as well as thrombosis. Meanwhile, a wide range of bioactive compounds and potential antithrombotic candidates reported in recent years have been summarized. In addition, the structure-activity relationship of certain compounds has been discussed, expecting to facilitate the development of molecules with anticoagulant biological activity for the treatment of thrombotic diseases.

Paeoniflorin regulates gut microbiota and bile acids metabolism in colitis mice / 药学学报

The current study was designed to evaluate the modulatory effects of paeoniflorin on the dysregulated gut microbiota as well as the disturbed fecal bile acids (BAs) in colitis mice. After approved by Xi'an Jiaotong University Ethics Committees (Approval No. XJTU2019-679), the animals were randomly distributed into the control (Con), colitis, low dose paeoniflorin (PF-L, 25 mg·kg-1·d-1), high dose paeoniflorin (PF-H, 50 mg·kg-1·d-1) and 5-aminosalicylic acid (5-ASA, 50 mg·kg-1·d-1) groups. Colitis was induced by administering 3% (w/v) DSS in drinking water for 7 days. Paeoniflorin and 5-ASA were dissolved in water and administered to the appropriate groups by oral gavage over the 7-day period. The mice were monitored daily, and the disease activity index (DAI) comprising of body weight loss, stool consistency and gross blood was measured. The pathological changes of colon were evaluated by HE staining; the levels of inflammatory cytokines in colonic tissue were determined by ELISA; the gut permeability was measured by FITC-dextran. Microbiota analysis based on 16S rDNA and targeted metabolomics for BAs were used to evaluate the composition of gut microbiota and fecal BAs pool. The results showed that administration of paeoniflorin markedly alleviated the inflammatory response and intestinal barrier dysfunction in DSS-induced colitis. Importantly, these ameliorative effects of paeoniflorin were accompanied by the improvements of disturbed composition of gut microbiota and the dysmetabolism of bile acids in feces. Finally, we performed Spearman's correlation analysis between the fecal BAs and gut microbiota genera, and found that Lactobacillus has a strong positive correlation with DCA and LCA which were reported to confer the beneficial effects of maintaining intestinal homeostasis. Taken together, paeoniflorin might improve the intestinal homeostasis in colitis mice via modulating gut microbiota and fecal BAs metabolism.

Establishment of a cocrystal characterization method for sacubitril valsartan sodium / 药学学报

Sacubitril valsartan sodium (LCZ696) is an ionic cocrystal drug. The purpose of this study was to explore the cocrystal features of LC696 by establishing a variety of characterization methods, and thus provide basic research data for effective quality control. The cocrystal characteristics of LCZ696 and its tablets were identified by applying analytical means including powder X-ray diffraction (PXRD), fourier transform infrared spectroscopy (FTIR), Raman spectra (RM), differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance spectroscopy (ssNMR). The crystalline water and hygroscopicity of LCZ696 were analyzed by thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), hygroscopicity test and Karl Fischer reaction method. The results show that PXRD, FTIR, DSC and ssNMR can effectively distinguish the features of LCZ696 cocrystal, sacubitril monomer, valsartan monomer, and sacubitril-valsartan (1∶1) mixture. RM can be used as a supplementary approach. Combined with the analysis by TGA, DVS, hygroscopicity test and Karl Fischer reaction method results, LCZ696 contains 2.5 crystalline water molecules and is very hygroscopic; we recommend that LCZ696 be stored in an environment with a relative humidity below 60%. By characterizing the crystal features we can establish quality control measure and evaluate the stability of the drug tablets. This study provides data in support for the establishment of the LCZ696 quality standard.

SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines, SKOV-3.</p><p><b>METHODS</b>hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' tetraacetic acid gadolinium (III) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).</p><p><b>RESULTS</b>The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r = 0.990, P < 0.01) and concentration-dependent manner (r = 0.964, P < 0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.</p><p><b>CONCLUSIONS</b>hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.</p>

Applications of HPLC/MS in the analysis of traditional Chinese medicines / 药物分析学报

In China, traditional Chinese medicines （TCMs） have been used in clinical applications for thousands of years. The successful hyphenation of high-performance liquid chromatography （HPLC） and mass spectrometry （MS） has been applied widely in TCMs and biological samples analysis. Undoubtedly, HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs. We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs. The present review focused on the applications of HPLC/MS in the component analysis, metabolites analysis, and pharmacokinetics of TCMs etc. 50% of the literature is related to the component analysis of TCMs, which show that this field is the most popular type of research. In the metabolites analysis, HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns. This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs. HPLC/MS in the fingerprint analysis is reviewed elsewhere.

A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg.</p><p><b>METHODS</b>The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database.</p><p><b>RESULTS</b>Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A.</p><p><b>CONCLUSIONS</b>PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.</p>

Metabolic kinetic of puerarin in beagle liver microsomal by HPLC-ESI-MS / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a HPLC-ESI-MS method for the determination of puerarin and its metabolite and study the metabolic kinetics in beagle dog liver microsomes.</p><p><b>METHOD</b>Beagle dog liver microsomes were prepared by using ultracentrifugation method. Chromatography was performed on a Shimadzu C18 column (2.0 mm x 150 mm, 5 microm). Amethanol-water gradient system was used. ESI interface was applied in the positive, and SIM m/z 417 was puerarin and m/z 531 was daidzein.</p><p><b>RESULT</b>The puerarin was metabolized by NADPH regenerating system in beagle dog microsomes. The Michaelis-Menten parameters Km and Vmax in beagle dog microsomes were initially estimated by analyzing Lineweave-Brurk plot. The Vmax Km of puerarin were (0.047 +/- 0.006) mg x min(-1) x g(-1), (1.22 +/- 0.53) mg x L(-1).</p><p><b>CONCLUSION</b>The puerarin and daidzein can be rapidly determined by HPLC-MS in beagle dog microsomes and the puerarin was metabolized to daidzein by CY P450. The study can give help for Baige capsule.</p>

Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent, diethylenetriamine pentaacetic acid gadolinium / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro.</p><p><b>METHODS</b>Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T(1)WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay (MTT).</p><p><b>RESULTS</b>The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW = 2163.34, Gd-DTPA-CPP MW = 2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T(1) weighted imaging (T(1)WI) signal, and that the T(1)WI signal intensity was increasing in a time-dependent manner (r = 0.972, P = 0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P = 0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F = 0.006, P = 1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group.</p><p><b>CONCLUSIONS</b>The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.</p>

Analysis of supercritical fluid extracts of Radix caulophylli with gas chromatography-mass spectrometry / 药学学报

To analyze the constituents in supercritical fluid CO2 extraction (SFE-CO2) of Radix caulophylli, the Radix caulophylli was extracted with SFE-CO2, and analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis with a DB-5MS capillary column (30 mm x 0.32 mm ID, 0.25 microm film thickness) was used. The inlet temperature was maintained at 280 degrees C. The column oven was held at 80 degrees C for 2 min, then programmed from 80 to 280 degrees C at 5 degrees C x min(-1) and, finally, held for 4 min. Helium at a constant flow rate of 2.0 mL x min(-1) was used as the carrier gas. The mass spectrometry conditions were as follows: ionization energy, 70 eV; ion source temperature, 200 degrees C. The mass selective detector was operated in the TIC mode (m/z was from 40 - 500). For the first time 49 peaks were separated and identified, the compounds were quantitatively determined by normalization method, and the identified compounds represent 97.44% of total GC peak areas. Viz, n-hexadecanoic acid (31.4%), (E, E) -9, 12-octadecadienoic acid (26.54%), (Z)-7-tetradecenal (9.4%), hexadecenoic acid (3.23%), 10-undecenal (3.22%), octadecanoic acid (2.25%), and caulophylline (1.76%) etc. The results will provide important foundation for understanding the constituents and further exploitation of Radix caulophylli.

Studies on differences of pharmacokinetic behavior and tissue distribution of nimodipine and its two enantiomers in rats using achiral and chiral liquid chromatography / 药学学报

<p><b>AIM</b>To investigate the differences of pharmacokinetic behavior and tissue distribution of nimodipine and its two enantiomers in rats.</p><p><b>METHODS</b>A high-performance liquid chromatographic method with an ODS column (150 mm x 4.6 mm ID) and a mobile phase of methanol-water (70:30) was used for racemic nimodipine assay. Another method with a Chiralcel OJ column (250 mm x 4.6 mm ID) and a mixture of n-haxane-ethanol (85:15) as mobile phase was used to determine its two enantiomers. Nimodipine was monitored at 236 nm wavelength.</p><p><b>RESULTS</b>The linearity, recoveries and the detection limits of the methods were found to be suitable for the determinations. The average results of within-day and between-day RSDs were 5.64% and 7.85% respectively, the mean recovery was 97.66% for the concentration ranges studied. The pharmacokinetic parameters Tmax, Cmax, AUC and CLs were: S-(-)-nimodipine (2.1 +/- 0.3) h, (197 +/- 5) microgram.L-1, (656 +/- 18) mL.min-1, (0.30 +/- 0.03) microgram.h.L-1, and R-(+)-nimodipine (1.7 +/- 0.5) h, (128 +/- 4) microgram.L-1, (381 +/- 4) mL.min-1, (0.53 +/- 0.03) microgram.h.L-1, respectively. The S-(-)-nimodipine concentration was 2.23 and 1.97 times as high as that of R-(+)-nimodipine in heart and in cerebrum respectively and there was almost only S-(-)-nimodipine in cerebellum. But R-(+)-nimodipine concentration was 1.57, 3.69 and 4.20 times as high as that of S-(-)-nimodipine in major excretion organs such as kidney, spleen and liver respectively.</p><p><b>CONCLUSION</b>The experimental results obtained by using the achiral and chiral liquid chromatography showed that the differences between enantiomers were apparent for the pharmacokinetics in rat plasma, and very significant for the distributions in major target tissues: heart, cerebrum and cerebellum, and main elimination tissues: kidney, spleen and liver.</p>