Pharmacokinetics of four phenolic acids from Danshen-Chuanxiong drug pair in plasma and heart tissue of rats by UPLC-MS/MS / 中国中药杂志

This study is to investigate the compatibility mechanism of Danshen-Chuanxiong drug pair on the pharmacokinetics of four phenolic acids. A UPLC-MS/MS method for quantitative determination of salvianolic acid B( Sal B),rosmarinic acid( RA),lithospermic acid( LA) and ferulic acid( FA) in plasma and heart tissue of rats was established. After single salvianolic acids and Chuanxiong-extract or combined intravenous infusion was given to rats,plasma samples and heart tissues in different time were collected. The chromatographic separation was performed on a BEH C18 column using 0. 15% formic acid-acetonitrile as mobile phase for gradient elution. A triple-quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating on multiple-reaction monitoring( MRM) scanning in negative ionization mode. Full validation of UPLC method including calibration curves,accuracy,precision,repeatability and matrix effect was investigated to comply with quantitative analysis requirements for biological samples. There were significant differences in the major pharmacokinetic parameters of Sal B,FA and RA for intravenous infusion of salvianolic acids and Chuanxiong-extract or combined in rat plasma. The AUC of Sal B and FA were increased above 40% and100%,respectively. Their Vd and CL were dropped evidently. t1/2 and Vd of RA increased above 130%. The concentration of four phenolic acids were all increased obviously in heart tissue comparing with single infusion. These results demonstrated that the compatibility mechanism of Danshen-Chuanxiong drug pair showed synergistic effect.

Pharmacokinetic research strategies of compatibilities and synergistic effects of classical Danshen herb pairs based on pharmacokinetics of "Danshen-Bingpian" and "Danshen-Honghua" / 中国中药杂志

Herb pairs are usual clinical compatibility forms and one of compound prescription sources in Chinese medicine. Pharmacokinetic research in vivo is one of the important items in elucidating the mechanism for synergistic and attenuated mechanisms of herb pairs. The paper comprehensively summarized and systemized the pharmacokinetic researches of marker-ingredients about Danshen-Honghua and Danshen-Bingpian in order to elucidate the rationality and scientificity of herb pairs and provide some feasible suggestions on the pharmacokinetics of drugs in the future. In view of complicated system of Traditional Chinese medicines and a chemical system that is not separated from its natural state, comparative pharmacokinetic researches on marker-ingredients from the herb pairs are reasonable to elucidate the synergistic and attenuated mechanisms of monarch-subjects compatible herbs and monarch-guide compatible herbs. Such pharmacokinetic research can better explain the mechanism of drug compatibility, while the pharmacokinetic researches based on the monomer chemical compositions and marker-ingredients that have been separated from complex chemical environment of traditional Chinese Medicine are still unreasonable and should be discussed deeply.

Multi-index integrated evaluation method optimizes water-extraction and ethanol precipitation of Xuanbi'antong formula (XBF) / 中国中药杂志

To determine the process parameters of optimal water-extraction and ethanol precipitation method for Xuanbi'antong (XBF) extract, which is a clinically experience formula for coronary disease. Orthogonal test L9(34) was conducted for the study of XBF water-extraction and ethanol precipitation process. Extractum, salvianolic acid B, rhizoma coptidis alkaloid, paeoniflorin, puerarin, ginsenoside Rb₁, ginsenosides and echinacoside were selected as marker components and multi-index comprehensive weighted score was used to select and verify optimal water-extraction and ethanol precipitation process. The optimal extraction process was as follows： XBF was added with 10 times distilled water, decocted for half an hour for 3 times. The best ethanol-precipitation process was established where the ethanol was added up to 70% and precipitated for 24 hours in 1.12 extract density (20 ℃). The optimized water-extraction and ethanol precipitation method is stable and reliable, and can provide reference for further development and utilization of the formula.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry / 中国天然药物

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Study on compatibility of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma based on pharmacokinetics of effective components salvianolic acid B and ferulic acid in rat plasma / 中国中药杂志

A study was made on the pharmacokinetic regularity of effective components salvianolic acid B and ferulic acid in Salviae Miltiorrhizae Radix et Rhizoma (SMRR) and Chuanxiong Rhizoma(CR) in rats, so as to discuss the compatibility mechanism of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma. Rats were randomly divided into three groups and intravenously injected with 50 mg x kg(-1) salvianolic acid B for the single SMRR extracts group, 0.5 mg x kg(-1) ferulic acid for the single CR extracts group and 50 mg x kg(-1) salvianolic acid B + 0.5 mg x kg(-1) ferulic acid for the SMRR and CR combination group. The blood samples were collected at different time points and purified by liquid-liquid extraction with ethyl acetate. With chloramphenicol as internal standard (IS), UPLC was adopted to determine concentrations of salvianolic acid B and ferulic acid. The pharmacokinetic parameters of salvianolic acid B and ferulic acid were calculated with WinNonlin 6.2 software and analyzed by SPSS 19.0 statistical software. The UPLC analysis method was adopted to determine salvianolic acid B and ferulic acid in rat plasma, including linear equation, stability, repeatability, precision and recovery. The established sample processing and analysis methods were stable and reliable, with significant differences in major pharmacokinetic parameters, e.g., area under the curve (AUC), mean residence time (MRT) and terminal half-life (t(1/2)). According to the experimental results, the combined application of SMRR and CR can significantly impact the pharmacokinetic process of their effective components in rats and promote the wide distribution, shorten the action time and prolong the in vivo action time of salvianolic acid B and increase the blood drug concentration and accelerate the clearance of ferulic acid in vivo.

Effect of accutase or trypsin dissociation on the apoptosis of human striatum-derived neural stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the apoptosis of neural stem cells (NSCs) at differential time points after the dissociation of neurospheres by Accutase or trypsin.</p><p><b>METHODS</b>The NSCs were isolated from striatum of human fetals that suffered abortion at 12-16 weeks of pregnancy. The 3(rd)-5(th) passages of NSCs were digested by Accutase or trypsin. Only vortexing was applied, and the triturating by Pasteur pipette was avoided to attenuate the injury to the cells during the dissociation. The single cells were then stained by Annexin V/propidium iodide and Hoechst 33342. The apoptosis rates 2 and 24 hours after passaging were evaluated.</p><p><b>RESULTS</b>The trypan blue staining confirmed that immediately after the dissociation,the viability of cells digested by trypsin was (83.10 ± 6.76)%, which was significantly lower than that digested by Accutase,which was (91.65 ± 4.43)% (P<0.05). The apoptosis of the NSCs digested by Accutase was higher than that digested by trypsin at both 2 and 24 hours after passaging (P<0.01). Four days after the passaging, both the new clone formation rate and diameter of new spheres after trypsin digestion were significantly higher than those after Accutase digestion (P<0.01).</p><p><b>CONCLUSIONS</b>Although the viability of NSCs immediately after the disassociation by trypsin is lower than that digested by Accutase, the apoptosis of NSCs subsequently caused by trypsin is lower than that caused by Accutase. Trypan blue test immediately after the disassociation can not be used as an indicator in estimating the apoptosis of NSCs during the expanding.</p>

The cardioprotective effects of ischemic postconditioning on myocardial interstitium following ischemic/reperfusion in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of ischemic postconditioning (IPTC) on the changes of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) protein and mRNA levels in rat heart subjected to ischemia/reperfusion, and explore the mechanism by which IPTC protects myocardial interstitium following ischemic/reperfusion (I/R).</p><p><b>METHODS</b>Twenty four healthy male SD rats were randomly divided into 3 groups (n = 8): sham control (SC) group, I/R group and IPTC group. The parameters of left ventricular function including left ventricular systolic pressure (LVSP) and its derivate (±dp/dt) were measured; the amount of myocardial collagen contents was determined by hydroxyproline quantification; the plasma activity of creatine kinase (CK) and lactate dehydrogenase (LDH) was detected; the protien levels of MMP-2 and TIMP-2 was measured by Western blot and the mRNA levels of MMP-2 and TIMP-2 was detected by real-time PCR.</p><p><b>RESULTS</b>The myocardial collagen contents, left ventricular function and the protein and mRNA levels of TIMP-2 were significantly decreased in I/R group compared with those of SC group, wherease the activities of CK and LDH in the plasma and the protein and mRNA levels of MMP-2 were significantly enhanced in I/R group when compared to SC group. Compared with I/R group, the myocardial collagen contents, left ventricular function and the protein and mRNA levels of TIMP-2 were increased in IPTC group, the activities of CK and LDH in the plasma and the protein and mRNA level of MMP-2 were decreased in IPTC group.</p><p><b>CONCLUSION</b>These findings indicate that IPTC has protective effects on myocardial interstitial after the myocardial ischemia/reperfusion injury, and IPTC may exert its cardioprotectve effect via inhibiting MMP-2 and enhancing TIMP-2 expression in cardiac muscle.</p>

Effect of ischemic postconditioning on the expression of myocardium matrix metalloproteinase-2 induced by ischemia/reperfusion in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of ischemic postconditioning on the expression of rat myocardium matris metalloproteinase-2 (MMP-2) induced by ischemia/reperfusion (I/R) and relationship between its expression and interstitium and the effect on left ventricular function.</p><p><b>METHODS</b>Twenty-four rats were randomly divided into 3 groups (n = 8): sham control (SC) group, ischemic/reperfusion (I/R) group and ischemic postconditioning (IPTC) group. The left ventricular peak systolic pressure and its derivate (+/- dp/dt) were calculated; The amount of myocardium collagenous were determined; The vitality of superoxide dismutase (SOD) and content of malondialdehyde (MDA) of plasma were detected; The activity of myocardium MMP-2 was measured by Western blot and RT-PCR.</p><p><b>RESULTS</b>As compared with I/R group, IPTC could lower the expression of MMP-2, ameliorate left ventricular function and increase the content of myocardium collagenous. In the meantime, the vitality of superoxide dismutase (SOD) of plasma were greatly enhanced and the content of malondialdehyde (MDA) of plasma were reduced in IFC group.</p><p><b>CONCLUSION</b>Protective effect of IPIC on myocardium may be due to reduce free radical, lower expression of MMP-2 and protect myocardial interstitium. MMPs plays an important role in the myocardial protection provided by IPTC.</p>

HPLC-UV-ELSD characteristic figure and chemical pattern recognition of Panacis Quinquefolii Radix / 药学学报

The paper is to report the establishment of a method of characteristic figure analysis for the quality control of Panacis Quinquefolii Radix. Application of HPLC-UV-ELSD techniques was connected in series and applied. The separation was carried out on the Agilent Extend-C18 (250 mm x 4.6 mm, 5 microm) column. The mobile phase consisted of water and acetonitrile with gradient elution. The flow rate was 1.0 mL x min(-1) and the wavelength of measurement was 203 nm. The temperature of drift tube was maintained at 106.5 degrees C and the flow rate of air was set at 2.9 L x min(-1). Twenty batches of the Panacis Quinquefolii Radix were determined. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were applied to study on the HPLC characteristic figure and chemical pattern recognition. The HPLC-UV and HPLC-ELSD characteristic figure of Panacis Quinquefolii Radix was developed, the ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd and the pseudoginsenoside F11 were identified. This method is accurate and reliable, and it can be used to control the quality of the Panacis Quinquefolii Radix.

UPLC fingerprint for quality assessment of ginsenosides of ginseng radix et rhizoma / 药学学报

This paper is aimed to establish the method of fingerprint analysis of chemical constituents by reversed-phase ultra-performance liquid chromatography (UPLC) for the quality control of the roots and rhizomes of Panax ginseng (Ginseng Radix et Rhizoma). The method was performed on a ACQUITY UPLC BEH C18 (50 mm x 2.1 mm ID, 1.7 microm) with a mixed mobile phase of water and acetonitrile in a gradient mode. The flow rate was 0.3 mL x min(-1) and the wavelength of measurement was 203 nm. Eleven batches of the Ginseng Radix et Rhizoma were determined. The UPLC chromatographic fingerprints of chemical constituents were established from the eleven batches of the Ginseng Radix et Rhizoma and showed fifteen characteristic common peaks, among which fifteen peaks were recognized and nine compounds (ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) were determined by comparison with chromatographic behaviors and UV spectra of the authentic compounds. The eleven batches of samples were classified as two clusters by hierarchical clustering analysis (HCA) and principle component analysis (PCA), and six samples were confirmed to establish the mutual model. The quality was assessed by similarity evaluation system for chromatographic fingerprint of TCM (2004B Version). The convenient and high specific method can be used to identify and evaluate the quality of the Ginseng Radix et Rhizoma.

Affection of different preparations on the content of toxic constituents in traditional Chinese medicines, examplified with Caulis Aristolochiae Manshuriensis / 中国中药杂志

<p><b>OBJECTIVE</b>Taking Caulis Aristolochiae Manshuriensis (Guanmutong in Chinese, derived from the stem of Aristolochia manshuriensis) as an example, to study the affection of different preparations on the content of toxic constituents in traditional Chinese medicines.</p><p><b>METHOD</b>The separation was performed on a zorbax SB-C18 column with mobile phase of acetonitrile-3.7 mmol x L(-1) phosphoric acid buffer, detected at 260 nm.</p><p><b>RESULT</b>The extraction percentage of aristolochic acids I, II and IV a in water extraction (1 h x 2) of Guanmutong were 53.4%, 75.5% and 61.9%, respectively; the remaining quantity of aristolochic acids I, II and IVa in the dregs of the decoction were 22.3%, 15.7% and 30.3%, respectively; Aristolochic acid I was still main substance among these aristolohic acids in the decoction of Guanmutong.</p><p><b>CONCLUSION</b>The content of toxic constituents of the traditional Chinese medicines varies evidently with different preparations of Guanmutong. So the preparation methods of traditional Chinese medicines should be suitably selected according to characteristics of the toxic constituents so as to lessen the body damages of human.</p>

Injury in renal proximal tubular epithelial cells induced by aristololactam I / 中国中药杂志

<p><b>OBJECTIVE</b>To study whether aristololactam I (AL-I) induces injury in human renal proximal tubular epithelial cells.</p><p><b>METHOD</b>Cultured human renal proximal tubular epithelial cell line HK-2 was used as the subject. Aristolochic Acid I (AA-I) was used as a positive control. Cell toxicity of AL-I was detected by LDH releasing rate. Cell apoptosis was evaluated by cellular morphology, DNA content and expression of cell membrane phosphatidylserine (PS). The secretion level of fibronectin (FN) and TGF-beta1 in HK-2 cells were assayed by ELISA.</p><p><b>RESULT</b>AL-I had a direct toxicity on HK-2 in a dose dependent manner from 2.5 mg x mL(-1) to 20 mg x mL(-1); In these range of concentration, AL-I could induce cell apoptosis which was detectable by measurements of morphology, DNA content and expression of PS. AL-I could stimulate the secretion of FN and TGF-beta1. The potency of AL-I cell toxicity was higher than AA-I at the same concentration. The effects of AL-I on apoptosis, secretion of FN and TGF-beta1 were all weaker than AA-I.</p><p><b>CONCLUSION</b>AL-I as one metabolite of AA-I in vivo induces direct injury in renal proximal tubular cells. Its effects are similar to those of AA-I. AL-I may be one of toxic metabolites in Chinese herbs containing AA which participate in renal damage and fibrosis.</p>

A case-control study on the vitamin D receptor gene polymorphisms and susceptibility to pulmonary tuberculosis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the association between the genetic polymorphisms of VDR gene and susceptibility to pulmonary tuberculosis.</p><p><b>METHODS</b>Case-control study was conducted. PCR-RFLP technique was used to detect the C/T polymorphism in VDR gene. Information on related factors of tuberculosis was collected using a pre-tested questionnaire. Univariate and multivariate logistic analyses were conducted with SPSS software package.</p><p><b>RESULTS</b>A sample of 76 cases and 171 controls was studied. The genotype frequencies of VDR-FF, VDR-Ff and VDR-ff were 38.2%, 44.7%, 17.1% and 52.6%, 40.9%, 6.4% respectively. VDR-ff was significantly overrepresented in case group, the OR (95% CI) was 3.668 (1.483 - 9.071) when comparing with FF genotype. The significant association remained after adjusting BCG immunization and smoking, the OR (95% CI) was 3.036 (1.117 - 8.253).</p><p><b>CONCLUSION</b>The VDR-ff genotype might be associated with the susceptibility to pulmonary tuberculosis in Chinese Han population.</p>

A case-control study on natural-resistance-associated macrophage protein 1 gene polymorphisms and susceptibility to pulmonary tuberculosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate association between the natural-resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms and susceptibility to pulmonary tuberculosis (TB) in Chinese Han population.</p><p><b>METHODS</b>Hospital-based case-control study design was adopted. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique were used to type three NRAMP1 polymorphisms (INT4, D543N and 3'UTR). Information on related factors of tuberculosis was collected using a pre-tested standard questionnaire. Univariate and multivariate unconditional logistic analyses were conducted using SPSS for window software package. Totally, 110 cases of TB were selected during April 2001 to June 2002, with an average age of (27.7 +/- 12.7) years. Also, 180 cases of healthy control were selected, aged (27.3 +/- 9.2) years in average. Locus of NRAMP1 polymorphism was analysed with univariate method.</p><p><b>RESULTS</b>Univariate analysis demonstrated that the D543N G/A and 3'UTR TGTG+/del genotype occurred more frequently in the cases than in the controls, with crude odds ratios (OR) (95% CI) of 2.22 (1.03 - 4.78) and 1.93 (1.14 - 3.26), respectively. No significant association was observed between TB and INT4 polymorphisms. In multivariate analysis, associations of TB and D543N G/A and 3'UTR TGTG+/del genotypes remained, adjusted for exposure history and bacille Camette-Guérin immunization. Adjusted OR (95% CI) was 3.04 (1.12 - 8.27) and 2.36 (1.20 - 4.64), respectively. Still, no significant association between INT4 polymorphisms and TB was found.</p><p><b>CONCLUSION</b>Polymorphisms of D543N and 3'UTR locus in NRAMP1 gene might affect their susceptibility to TB in Chinese Han population.</p>