Detection of an anti-angina therapeutic module in the effective population treated by a multi-target drug Danhong injection: a randomized trial.

It's a challenge for detecting the therapeutic targets of a polypharmacological drug from variations in the responsed networks in the differentiated populations with complex diseases, as stable coronary heart disease. Here, in an adaptive, 31-center, randomized, double-blind trial involving 920 patients with moderate symptomatic stable angina treated by 14-day Danhong injection(DHI), a kind of polypharmacological drug with high quality control, or placebo (0.9% saline), with 76-day following-up, we firstly confirmed that DHI could increase the proportion of patients with clinically significant changes on angina-frequency assessed by Seattle Angina Questionnaire (ΔSAQ-AF ≥ 20) (12.78% at Day 30, 95% confidence interval [CI] 5.86-19.71%, P = 0.0003, 13.82% at Day 60, 95% CI 6.82-20.82%, P = 0.0001 and 8.95% at Day 90, 95% CI 2.06-15.85%, P = 0.01). We also found that there were no significant differences in new-onset major vascular events (P = 0.8502) and serious adverse events (P = 0.9105) between DHI and placebo. After performing the RNA sequencing in 62 selected patients, we developed a systemic modular approach to identify differentially expressed modules (DEMs) of DHI with the Zsummary value less than 0 compared with the control group, calculated by weighted gene co-expression network analysis (WGCNA), and sketched out the basic framework on a modular map with 25 functional modules targeted by DHI. Furthermore, the effective therapeutic module (ETM), defined as the highest correlation value with the phenotype alteration (ΔSAQ-AF, the change in SAQ-AF at Day 30 from baseline) calculated by WGCNA, was identified in the population with the best effect (ΔSAQ-AF ≥ 40), which is related to anticoagulation and regulation of cholesterol metabolism. We assessed the modular flexibility of this ETM using the global topological D value based on Euclidean distance, which is correlated with phenotype alteration (r2: 0.8204, P = 0.019) by linear regression. Our study identified the anti-angina therapeutic module in the effective population treated by the multi-target drug. Modular methods facilitate the discovery of network pharmacological mechanisms and the advancement of precision medicine. (ClinicalTrials.gov identifier: NCT01681316).

[Pharmacokinetics of four phenolic acids from Danshen-Chuanxiong drug pair in plasma and heart tissue of rats by UPLC-MS/MS].

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"].

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.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry.

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.

[Multi-index integrated evaluation method optimizes water-extraction and ethanol precipitation of Xuanbi'antong formula (XBF)].

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.

[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].

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.

Comparative analysis of two species of Asari Radix et Rhizoma by electronic nose, headspace GC-MS and chemometrics.

Traditional Chinese medicines (TCM) can be identified by experts according to their odors. However, the identification of these medicines is subjective and requires long-term experience. In this paper, electronic nose, headspace gas chromatography-mass spectrometry (GC-MS) and chemometrics methods were applied to differentiate two species of Asari Radix et Rhizoma by their odors. The samples used were the dried roots and rhizomes of Asarum heterotropoides var. mandshuricum (AH) and Asarum sieboldii (AS). The electronic nose was used to determine the odors of the samples and enabled rapid differentiation of AH and AS when coupled with principal component analysis. Headspace GC-MS was utilized to reveal the differences between the volatile constituents of AH and AS. In all, 54 volatile constituents were identified, and 9 major constituents (eucalyptol, eucarvone, 3,5-dimethoxytoluene, 3,4,5-trimethoxytoluene, methyleugenol, 2,3,5-trimethoxytoluene, croweacin, pentadecane and asaricin) could be used as chemical markers to distinguish these two species. AH contained higher relative contents of eucarvone (1.79-16.76%), 3,5-dimethoxytoluene (6.64-26.52%), 3,4,5-trimethoxytoluene/methyleugenol (6.43-31.67%) and 2,3,5-trimethoxytoluene (1.64-6.66%), whereas AS had higher relative contents of eucalyptol (14.06-24.95%), croweacin (5.64-13.55%), pentadecane (8.44-20.82%) and asaricin (7.03-13.45%). Moreover, AH and AS could be distinguished according to the contents of either all 54 identified volatile constituents or only the 9 major constituents by employing cluster analysis. The proposed method is rapid, simple, eco-friendly and can successfully differentiate these two species of Asari Radix et Rhizoma by their odors.

[Species differentiation and quality assessment of Ziziphus jujuba var. spinosa based on the HPLC fingerprint and quantitative analysis].

OBJECTIVE: To establish an HPLC method of a characteristical chemical fingerprint analysis in combination with simultaneous determination of four bioactive components for species differentiation and quality assessment of Ziziphus jujuba var. spinosa. METHODS: The chromatographic separation was performed on an Agilent TC-C18 BDS (250 mm x 4.6 mm, 5 microm) column. The mobile phase consisted of acetonitrile and water in a linear gradient elution procedure. The evaporator tube temperature of ELSD was set at 110.5 degrees C with the nebulizing gas flow rate of 3.1 mL/min and the flow rate of mobile phase was 1.0 mL/min. The column was maintained at 30 degrees C. The injection volume was 20 microL. RESULTS: HPLC methodology for both chemical fingerprint analysis and quantitative determination of four active ingredients were validated, respectively. According to the contents of the four ingredients and the chemical fingerprints of Ziziphus jujuba var. spinosa using principal component analysis, Ziziphus jujuba var. spinosa was different from the fake derived from the seeds of Ziziphus mauritiana. CONCLUSION: The developed HPLC method is exclusive and repetitive for the species identification and quality evaluation of Ziziphus jujuba var. spinosa.

[HPLC-UV-ELSD characteristic figure and chemical pattern recognition of Panacis Quinquefolii Radix].

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].

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.

[Constituents of essential oil from different processing drynesses of Radix Angelicae Dahuricae].

OBJECTIVE: To analyze the constituents of essential oil from different processing drynesses of Radix Angelicae Dahuricae. METHODS: Water steam distillation and GC-MS were used. Relative contents were determined by area. RESULTS: 37 compounds were identified. The constituents of essential oil the constituents from Radix Angelicae Dahuricae by insolation, drying and microwave dryness were similar, but one by dryness after sulfurizing and dryness after perspiring were different. CONCLUSION: Dryness after sulfurizing and dryness after perspiring are not fit for the dryness of Radix Angelicae Dahuricae.

[Study on HPLC fingerprint of processed Radix Angelicae Dahuricae].

OBJECTIVE: To establish HPLC fingerprint analysis for the quality control of processed Radix Angelicae Dahuricae. METHODS: HPLC fingerprint analysis of processed Radix Angelicae Dahuricae was developed Agilent C18 column (250 mm x 4.6 mm, 5 microm) was used, with mixture of acetonitrile and 5% phosphorous acid mobile phase in a gradient mode. The flow rate was 1.0 mL/min. The wavelength of measurement was 254 nm. Fifteen batches of processed Radix Angelicae Dahuricae were determined. RESULTS: The methodological evaluation showed that the method had a good repeatability. CONCLUSION: The method can be used to identify and evaluate the quality of Radix Angelicae Dahuricae conveniently.

Cytotoxicity of phenanthrenes extracted from Aristolochia contorta in human proximal tubular epithelial cell line.

BACKGROUND/AIMS: Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is predominantly a result of aristolochic acid I (AA-I) intoxication. However, other unidentified phytotoxins have indeed been postulated as the cause of this unique interstitial nephropathy. The purpose of this study was to investigate the cytotoxicity of other phenanthrene derivatives extracted from Aristolochia contorta in the human proximal tubular epithelial cell line HK-2. METHODS: After HK-2 cells were incubated with an indicated concentration of test compounds for 24 h, cell viability was assessed by lactate dehydrogenase (LDH) leakage assay (cell membrane damage) in combination with MTT assay (metabolic capability). Cellular morphologic assessments were performed with a phase-contrast inverted microscope and transmission electron microscope. RESULTS: In all test compounds at 5 microg/ml, AA-I, 7-methoxy-aristololactam IV and aristololactam IVa showed cytotoxic activity in HK-2 cells in both MTT assay and LDH leakage assay (p < 0.01). At high concentration (5-80 microg/ml), these three compounds caused a dose-dependent decrease in MTT reduction and a dose-dependent increase in LDH leakage compared to non-treated cells (p <0.01). In LDH leakage assay, 40 mug/ml 7-methoxy-aristololactam IV induced a 1.58-fold LDH leakage compared to AA-I at the same concentration (p < 0.01). Moreover, the IC50 of these three compounds were 16.675 microg/ml for AA-I, 4.535 microg/ml for 7-methoxy-aristololactam IV, and 30.244 microg/ml for aristololactam IVa in MTT assay. The cellular morphologic assessments suggest interactions with cell membrane and intracellular structures such as lysosome and mitochondria are likely to be involved in cell injury induced by these three compounds. CONCLUSION: The potency of cytotoxic activity of aristololactam IVa and 7-methoxy-aristololactam IV extracted from A. contorta is similar to or even stronger than that of AA-I.

[Affection of different preparations on the content of toxic constituents in traditional Chinese medicines, examplified with Caulis Aristolochiae Manshuriensis].

OBJECTIVE: 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. METHOD: 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. RESULT: 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. CONCLUSION: 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.

[Injury in renal proximal tubular epithelial cells induced by aristololactam I].

OBJECTIVE: To study whether aristololactam I (AL-I) induces injury in human renal proximal tubular epithelial cells. METHOD: 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. RESULT: 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. CONCLUSION: 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.