Liquid chromatography-mass spectrometry-based strategy for systematic profiling of chemical components and associated quantitative analysis of quality markers in Qi-Wei-Tong-Bi oral liquid.

Qi-Wei-Tong-Bi oral liquid (QWTB), a famous Chinese medicine preparation composed of seven crude drugs has a good therapeutic effect on rheumatoid arthritis and is widely used in China. However, its chemical composition and quality control have not been comprehensively and systematically investigated. In this study, high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was employed for its chemical profiling. As a result, 100 components were chemically characterized. Additionally, high-performance liquid chromatography coupled with a quadrupole linear ion trap mass spectrometry method was developed to simultaneously quantify nine bioactive components (hyperoside, ononin, quercetin, sinomenine, magnoflorine, gallic acid, protocatechuic acid, monotropein, and cyclo-(Pro-Tyr)) in multiple-reaction monitoring mode. After successful validation in terms of linearity, precision, repeatability, and recovery, the assay method was applied for the determination of 10 batches of QWTB. The results showed that QWTB was enriched in sinomenine and magnoflorine with the highest amount up to hundreds or even thousands of µg/mL, while quercetin, ononin, cyclo-(Pro-Tyr), and hyperoside were much lower with the lowest content below 10 µg/mL. This study work would help to reveal the chemical profiling and provide a valuable and reliable approach for quality evaluation and even pharmacodynamic material basis studies of QWTB.

Impaired bone morphogenetic protein signaling pathways disrupt decidualization in endometriosis.

It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in individuals with endometriosis. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from individuals with or without endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified differences in key pathways between the two groups, including defective bone morphogenetic protein (BMP)/SMAD4 signaling (ID2, ID3, FST), oxidate stress response (NFE2L2, ALOX15, SLC40A1), and retinoic acid signaling pathways (RARRES, RARB, ALDH1B1). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 and H3K27Ac in the decidualized stromal cells from individuals without endometriosis relative to those with endometriosis, with target genes enriched in pathways related to signaling by transforming growth factor ß (TGFß), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation in endometriosis patient-derived assembloids elevated the expression of decidualization markers. In summary, transcriptomic and genome-wide binding analyses of patient-derived endometrial cells and assembloids identified that a functional BMP/SMAD1/5/4 signaling program is crucial for engaging decidualization.

Chemical profile and miscarriage prevention evaluation of Jiao-Ai Decoction, a classical traditional Chinese formula.

Jiao-Ai Decoction (JAD), a classical traditional Chinese formula composed of seven Chinese herbs, has been widely used in clinical practice for the treatment of abortion for a long time. However, the material basis and pharmacological mechanism remain unclear. An integrative method combining ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) analysis and therapeutic effect evaluation based on the hypothalamus-pituitary-ovarian axis (HPOA) was employed to elaborate these problems. Firstly, the chemical profile of JAD was identified by UPLC-Q-TOF-MS. Secondly, the main target ingredients from JAD were determined by UPLC-T-Q-MS. Finally, the miscarriage prevention of JAD on threatened abortion pregnant rats induced by mifepristone was investigated. Threatened abortion model in rats were replicated, uterine bleeding quantity (UBQ) and histopathological sections were measured, the contents of luteinizing hormone (LH), follicular stimulating hormone (FSH), estradiol (E2) and progesterone (P) were determined by ELISA, related genes and protein expression levels were detected by RT-PCR and western blotting. As a result, a total of 101 compounds were identified and 27 ingredients were determined to evaluate the quality of JAD. In the model rats, JAD could effectively regulate the HPOA to achieve miscarriage prevention, and the mechanism might be related to the regulation of gene and protein expression on the HPOA. This work could provide a novel and valuable approach for the quality evaluation of JAD and were expected to provide ideas and methods for the basic research on the scientific application of similar traditional Chinese medicine prescriptions.

The toxicity mechanism of toxic compounds from Euphorbiae pekinensis Radix on zebrafish embryos.

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research.

Metabolomics analysis of the therapeutic effects of Qiwei Tongbi oral liquid on rheumatoid arthritis in rats.

Qiwei Tongbi oral liquid (QWTB), a classical traditional Chinese medicine (TCM) formula, has a good therapeutic effect on rheumatoid arthritis (RA) and is widely used in China. To comprehensively elucidate the therapeutic mechanism of QWTB in the treatment of RA, the effects of QWTB on biomarkers and metabolic pathways in a rat model of kidney deficiency arthritis were investigated in this study. The effects of QWTB on pharmacodynamic indicators, including paw swelling, arthritis score; interleukin-1ß, interleukin-6, interleukin-17 F, tumor necrosis factor-α, tartrate-resistant acid phosphatase 5b, bone alkaline phosphatase, bone-specific alkaline phosphatase, bone glaprotein, urea, and creatinine levels; and histopathology, suggested that QWTB significantly improved renal function, inhibited the inflammatory response, and reduced bone loss. In total, 39 differential metabolites were screened by comparing the endogenous components between blank and model rat plasma, among which 16 metabolites were altered by QWTB. The metabolism pathway analysis revealed that α-linolenic acid metabolism, phenylalanine metabolism, sphingolipid metabolism, histidine metabolism and glycerophospholipid metabolism were greatly disturbed. Thus, the biomarkers investigated included (1) α-linolenic acid, (2) hippuric acid, (3) phosphatidylethanolamine (15:0/22:2(13Z,16Z)), (4) phenylpyruvic acid, (5) sphinganine, and (6) urocanic acid. QWTB affected three abnormal biomarkers: (3), (4), and (6). Phenylphruvic acid, sphinganine and urocanic acid were significantly associated with pharmacodynamic indicators, as shown by Pearson correlation analysis. These results indicated that RA-related biomarkers had certain reliability and biological significance. In summary, QWTB regulated the metabolic disorders in rats with RA. Its therapeutic mechanism may involve the regulation of phenylalanine metabolism, histidine metabolism, and glycerophospholipid metabolism. The results of this study are useful for understanding the therapeutic mechanisms of TCM.

Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish.

Rubiae Radix et Rhizoma (called "Qiancao", QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called "Qiancaotan", QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC-MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile-water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.

Serum and urine metabolomics based on UPLC-Q-TOF/MS reveals the antipyretic mechanism of Reduning injection in a rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Reduning injection (RDN), a patented traditional Chinese medicine, has the obvious antipyretic effect and has been widely used in China. Although some previous studies proved its antipyretic effect by animal efficacy experiment or clinical observation, its holistic mechanism in vivo was still unclear. AIM OF THE STUDY: To comprehensively elucidate the antipyretic mechanism of RDN, the investigation of fever-related potential biomarkers and metabolic pathways in the rat fever model is described in this paper. MATERIALS AND METHODS: Rat fever model was established by dry yeast. A large number of endogenous metabolites in serum and urine were detected by UPLC-Q-TOF/MS, and fever-related potential biomarkers were screened and identified by multivariate analysis and metabolite databases. The reliability and biological significance of the largely disturbed biomarkers was verified by the metabolic network and the correlation with pharmacodynamic indicators, which contained IL-1ß, IL-6, TNF-α, PGE2 and cAMP. RESULTS: The established UPLC-Q-TOF/MS analytical method afforded satisfactory results in terms of precision, repeatability and stability, which met the requirements of biological sample determination. A total of 32 potential biomarkers associated with fever were screened and identified, among which 22 species could be adjusted by RDN. The metabolism pathway analysis revealed that valine, leucine and isoleucine biosynthesis, and sphingolipid metabolism were greatly disturbed. Their biomarkers involved L-leucine, L-valine, sphinganine and phytosphingosine, all of which showed a callback trend after RDN was given. These 4 biomarkers had a certain correlation with some known fever-related small molecules and pharmacodynamic indicators, which indicated that the selected fever-related biomarkers had certain reliability and biological significance. CONCLUSIONS: RDN has a good regulation of the metabolic disorder of endogenous components in dry yeast-induced fever rats. Its antipyretic mechanism is mainly related to the regulation of amino acid, lipid and energy metabolism. The study is useful to better understand and analyze the pharmacodynamic mechanism of complex systems, such as traditional Chinese medicine.

Systematically investigating the pharmacological mechanism of Dazhu Hongjingtian in the prevention and treatment of acute mountain sickness by integrating UPLC/Q-TOF-MS/MS analysis and network pharmacology.

Members of the genus Rhodiola L. have been widely used in Tibetan medicines for preventing and treating acute mountain sickness (AMS) for a long time. However, the pharmacological mechanisms of these medicines in treating AMS remain unclear. To address this problem, an integrative method combining ultra-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS)analysis and network pharmacology was employed. First, the chemical profiles of Dazhu Hongjingtian (DZ, a Chinese medicine preparation composed of R. kirilowii (Regel) Maxim) were identified or tentatively characterized. Second, the targets of DZ were predicted using the SwissTargetPrediction and STITCH databases; the targets of AMS were also collected from the Drugbank and TTD databases. Then, networks between targets and compounds or diseases were constructed by Cytoscape 3.6.1. Third, GO and pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). As a result, 40 ingredients of 53 compounds in DZ might be biologically active. These activities were related to the regulatory effects of the ingredients on 68 significant signaling pathways, such as the inflammation pathway, apoptosis pathway, HIF-1 signaling pathway, and others, by targeting 33 proteins, including PTGS2 and PTGS1, ALOX5 and ALOX15, BCL2 and BCL2L1, the protein kinase C (PKC) family and HIF1A, among others.

Cellular pharmacokinetics and pharmacodynamics mechanisms of ginkgo diterpene lactone and its modulation of P-glycoprotein expression in human SH-SY5Y cells.

Ginkgo diterpene lactone (GDL) is the raw material for ginkgo diterpene lactone meglumine injection, which is used for treating cerebral ischemia. The aims of this study were to explore the cellular pharmacokinetics of GDL in whole cells and subcellular fractions, and detect cellular pharmacodynamics on the human SH-SY5Y cells induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Firstly, a simple, sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for assessing the amount of ginkgolide A (GA), B (GB) and K (GK) in cellular/subcellular samples. Then, phosphatidylserine and mitochondria membrane potential were assayed to evaluate the extent of apoptosis effect. The study showed that the cellular/subcellular accumulation of GA and GB were increased in a concentration-dependent manner; the levels of GA and GB in cytosol were the highest among these subcellular organelles. Meanwhile, GDL also attenuated the OGD/R-induced increases in the percentage of apoptotic and mitochondria membrane potential. In addition, verapamil increased the rate and amount of GA and GB entering cellular/subcellular compartments through inhibition of P-glycoprotein activity, and promoted the protective effect of GDL. The present study reports the cellular pharmacokinetics profiles of GA and GB in normal and OGD/R-induced SH-SY5Y cells in vitro for the first time, which provided valuable information for clinical safety application.

[Rapid identification of chemical components in compound Nanxing acesodyne plaster by UPLC-Q-TOF-MS/MS].

The study aims to qualitatively analyze the chemical composition of compound Nanxing acesodyne plaster by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS). The analysis was performed on Agilent Zorbax SB-C_(18)( 4. 6 mm×250 mm,5 µm) column. The mobile phase consisted of methanol and 0. 2% formic acid-water was used as gradient elution. The flow rate was 1 mL·min~(-1) and column temperature was 30 ℃. The Mass spectrometry was acquired in both positive and negative ion modes using ESI. The components were identified by the precise mass-to-charge ratio,secondary fragmentation and other information combined with reference substance and literature data. As a result,58 compounds were identified and predicted,including alkaloids,flavonoids,coumarins,organic acids and lactone compounds,of which 12 compounds were verified by the reference substances. The results provide reference for the quality control of compound Nanxing acesodyne plaster,and lay the foundation for elucidating the active components mechanism.

Comparative analysis of sixteen active compounds and antioxidant and anti-influenza properties of Gardenia jasminoides fruits at different times and application to the determination of the appropriate harvest period with hierarchical cluster analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gardenia jasminoides fruit (GJF) is used as a well-known traditional folk medicine, a food and a natural colorant in Asia with a long history. The herbal medicine has usually been harvested in the autumn from September to November. However, this time span is too long and might result in the quality instability of GJF. AIM OF STUDY: We aimed to conduct the comprehensive quality evaluation of GJF including the quantitative analysis of the bioactive components and the main bioactivities, and further to determine the most appropriate harvest time of this phytomedicine. MATERIALS AND METHODS: In this study, an UFLC-Q-TRAP-MS/MS method was established to quantify 7 iridoid glycosides (geniposide, geniposidic acid, secoxyloganin, gardenoside, genipin 1-gentiobioside, scandoside methyl ester, and shanzhiside), 7 phenylpropanoid acids (chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid) and 2 carotenoids (crocin-1 and crocin-2) in GJF. With this method, nine samples of GJF harvested at different times were analyzed and compared. These samples were also investigated and compared in terms of their antioxidant activity (DPPH free radical scavenging, ABTS free radical scavenging, ferric-reducing antioxidation) and anti-influenza activity (neuraminidase inhibition), which are closely related to the GJF efficacies. Then, hierarchical cluster analysis (HCA) was separately performed for the quantitative analysis and bioactivity evaluation in vitro. RESULTS: The HCA results demonstrated that three GJF samples (S5, S6, and S7) were clustered into one group for both quantitative analysis and bioactivity evaluation in vitro; these three samples were found to have the highest standardized scores in both the former (12.775, 12.106, 10.817) and the latter (3.406, 3.374, 3.440). Based on the comprehensive results, the optimum harvest period was confirmed to extend from mid-October to early-November. CONCLUSIONS: This study firstly validated the use of UFLC-Q-TRAP-MS/MS method for the determination of 16 bioactive components in GJF. It was also the first time that a quantitative analysis and a bioactivity assay in vitro were integrated for the determination of the most appropriate harvest period of GJF. We hope this paper may provide some reference to studies of appropriate harvest periods and even the quality control of TCMs.

[Rapid identification of chemical components in Xingbei Zhike Keli by UPLC-Q-TOF-MS/MS].

To analyze and identify the chemical components in Xingbei Zhike Keli by using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS/MS). The analysis was performed on Agilent Zorbax SB-C18(4.6 mm×250 mm, 5 µm) column, with methanol-0.08% formic acid solution (including 0.1% ammonium formate) as the mobile phase for gradient elution. The flow rate was 1 mL·min⁻¹ and column temperature was 30 °C. The MS spectrum was acquired in both negative and positive ion modes by using electron spray ionization (ESI). These components were further analyzed based on accurate m/z, secondary fragmentation and other information combined with reference substance and literature data. As a result, 87 compounds were successfully identified and predicted, including alkaloids, flavonoids, coumarins and saponins, of which 23 compounds were verified by comparing with reference substances. These results provide reference for the quality control of Xingbei Zhike Keli, and lay the foundation for elucidating their effective components and mechanism of action.

[Research progress of platelet activating factor(PAF) receptor antagonist].

Platelet activating factor(PAF), an endogenous synthesized phospholipid transmitter, has widely biological activities. It has "signal transmission" effect in various life processes, but abnormality of concentration in vivo will promote or aggravate the diseases, such as, cerebral ischemia, myocardial injury, multi-organ failure, asthma, injury of liver and kidney, severely affecting the normal life activities of body. In recent years, with the development of medical science and technology, more and more attention has been paid to the research of platelet activating factor receptor antagonist. Components of animals, plants, microbial fermentation, and synthetic composition all can reflect such activity. Ginkgolide B and cytopone are the most representative herbal compositions at present. This paper referred to the research status of platelet activating factor receptor antagonist in recent years, made a summary of the researches on biological effect of platelet activating factor and platelet receptor antagonist of different sources, so as to provide a reference for the exploration of effective and safe platelet activating factor receptor antagonists.

[¹H-NMR quantitative analysis and fingerprints of ginkgo diterpene lactone raw material].

Ginkgo diterpene lactone raw material, as a raw material for ginkgo diterpene lactone meglumine injection, is extracted and purified from ginkgo leaf. ¹H-NMR content determination method and fingerprint analysis method were respectively established for ginkgo diterpene lactone raw material. Content determination was conducted in 3 batches of samples by using ¹H-qNMR, and then the results were basically consistent with the results in HPLC method. Twenty-four proton peaks were identified as common fingerprint peaks, and the fingerprint peaks were identified by using the control product and NMR information. Furthermore, 10 batches of samples were analyzed by ¹H-NMR fingerprint. The similarities were all higher than 0.99 and the common peaks were identified with the reference standards. This method is easy, fast, with good precision, stability and repeatability and could provide basis and new ideas for quality evaluation of ginkgo diterpene lactone raw material and its preparations.

[Research development of ginkgo terpene lactones].

Ginkgo terpene lactones, as an important active ingredient from Ginkgo leaves, has high medicinal values and has been widely used in clinics. This article would review the researches both at home and abroad, including chemical composition, structure-activity relationship, analytical methods, pharmacological effects, pharmacokinetic characteristics, and so on, providing a reference for further development and utilization of ginkgo terpene lactones.

An integrated strategy for rapid discovery and identification of quality markers in Guanxin Kangtai preparation using UHPLC-TOF/MS and multivariate statistical analysis.

BACKGROUND: Guanxin Kangtai preparation (GXKT), consisting of Panax ginseng, Panax notoginseng and Ilex pubescens, is a new proprietary Chinese medicines under development for treating coronary heart disease. Like other Chinese medicines, the components of GXKT were complex and the bioactive compounds remained unclear. PURPOSE: To discover bioactive compounds as quality markers (Q-markers) for better quality control of GXKT. STUDY DESIGN: Chinese medicines was separated into fractions. The correlation between chemical information and bioactivity of these fractions were analyzed with multivariate statistical methods to discover bioactive compounds responsible for the actions of Chinese medicine. METHOD: GXKT was separated into fractions by using high-performance liquid chromatography (HPLC). Ultra HPLC coupled with time-of-flight mass spectrometer (UHPLC-TOF/MS) was applied to detect compound information from these fractions to form a chemical database. The bioactivity of these fractions in protecting cardiomyocytes from ischemia/reperfusion injury was examined in H9c2 cells that were exposed to hypoxia followed by reoxygenation (H/R). Then, partial least square model and orthogonal projections to latent structures discriminant analysis were employed to discover bioactive compounds from the chemical database that were positively correlated with the bioactivity of GXKT fractions. Finally, the bioactivity of these compounds was confirmed by bioassay in H9c2 cells. RESULTS: The chemical information of 120 fractions separated from GXKT was detected and extracted by UHPLC-TOF/MS, and a chemical database including 61 high abundance compounds were formed from all fractions. These fractions produced different extent of protective effect to H9c2 cell underwent H/R treatment with cell viability ranging from 33.43% to 74.91%, demonstrating the separation of bioactive compounds among different fractions. The multivariate analysis discovered 16 compounds from GXKT positively correlated with the bioactivity of GXKT. Of these compounds, 6 compounds, i.e.: ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were chemical identified and also confirmed for their responsibility to the action of GXKT by bioassay. CONCLUSION: Ginsenoside Rg1, Rb1, Rh1, Rc, ilexsaponin A1, and chikusetsusaponin IVa were bioactive compounds and qualified as Q-markers for quality control of GXKT. This research provided a useful reference for the quality research of Chinese medicines.

Pharmacokinetics of the prototype and hydrolyzed carboxylic forms of ginkgolides A, B, and K administered as a ginkgo diterpene lactones meglumine injection in beagle dogs.

Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg-1) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg-1).

Direct inhibition of Re Du Ning Injection and its active compounds on human liver cytochrome P450 enzymes by a cocktail method.

The aim of this study was to investigate the direct inhibitory effects of Re Du Ning Injection (RDN) and its active compounds on the major cytochrome P450 enzyme (CYP) isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomes by 'a cocktail method'. The activity of each CYP isform was represented as the formation rate of the specific metabolite from relevant substrate. Then a sensitive and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously analyze the seven metabolites. RDN (0.035-2.26 mg/mL) showed a strong inhibitiory effect on CYP2C8, followed by CYP2C9, CYP2B6, CYP2C19, CYP1A2 and CYP3A4. The IC50 value for each enzyme was 0.19, 0.66, 0.72, 1.27, 1.66 and 2.13 mg/mL, respectively. RDN competitively inhibited the activities of CYP1A2 (Ki = 1.22 mg/mL), CYP2B6 (Ki = 0.65 mg/mL) and CYP3A4 (Ki = 0.88 mg/mL); it also exhibited mixed inhibition of CYP2C8, CYP2C9 and CYP2C19 with a Ki value of 0.26, 0.64 and 0.82 mg/mL, respectively. However, the activity of CYP2D6 was not significantly inhibited even by 2.26 mg/mL RDN. Moreover, the data of nine active compounds on the CYPs showed that cryptochlorogenin acid, sochlorogenic acid B and sochlorogenic acid C were the major contributors to the inhibitory effect of RDN on CYP2C8, while the inhibitory effect of RDN on CYP2C9 might be caused by sochlorogenic acid A and sochlorogenic acid C. Moreover, neochlorogenic acid might be the major contributor to the inhibitory effect on CYP2B6. All of the findings suggested that drug-drug interactions may occur and great caution should be taken when RDN is combined with drugs metabolized by these CYPs.

[Effects of Electroacupuncture on Hippocampal nNOS Expression in Rats with Anxiety-like Behavior].

OBJECTIVE: To compare the effects of electroacupuncture (EA) preconditioning and post-conditioning on hippocampal neuronal nitric oxide synthase (nNOS) expression in rats with anxiety-like behavior, so as to explore the reasonable EA intervention time. METHODS: Forty-two SD rats were randomly divided into 6 groups:normal 1, normal 2, anxiety model 1, an-xiety model 2, EA-pre-conditioning and EA-post-conditioning. The anxiety model was established by giving the rats with repeated foot shock stimulation (0.8 mA, 2-25 s/time, 10 times in 5 min) combined with isolation-raising. Before or after modeling, EA stimulation was applied to "Baihui" (GV 20) and "Yintang" (GV 29) for 20 min, once a day for 7 days. Elevated plus maze (EPM) tests were performed to determine the percentages of time spent in the open arms and the percentages of entries into the open arms in 5 min for evaluating the animals' anxiety-like behavioral activities. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect hippocampal nNOS mRNA expression and the immunohistochemical staining adopted to detect the expression of nNOS protein in the hippocampal CA 1 and CA 3 areas, respectively. RESULTS: Compared with their own normal control group 1 and 2, both the percentage of time spent in the open arms and the percentage of entries into the open arms were significantly decreased in model group 1 and 2 (P<0.05, P<0.01). After pre- and post-conditioning of EA, the decreased proportions of time spent and entries into the open arms were considerably increased (P<0.05), suggesting an improvement of anxiety-like behavior activities after EA intervention. The expression levels of hippocampal nNOS mRNA and nNOS protein in the hippocampal CA 3 region were significantly higher in the model group 1 and 2 than in their own normal control group 1 and 2 (P<0.01), but those of nNOS protein in the CA 1 area were markedly lower in the model group 1 and 2 than in their own normal control group 1 and 2 (P<0.01). Following pre- and post-conditioning of EA, the increased expression levels of nNOS mRNA and protein of CA 3 area and the decreased level of nNOS protein of CA 1 area were all notably reversed (P<0.05, P<0.01). CONCLUSIONS: Both pre- and post-conditioning of EA can improve anxiety-like behavior in anxiety rats, which may be associated with its effects in down-regulating hippocampal nNOS expression.

[Metabolism of six saponins by rat intestinal bacteria in vitro].

To investigate the metabolism of six saponins by rat intestinal bacteria in vitro.Six saponins, including notoginsenoside R1, ginsenoside Rg1, ginsenoside Rg2, ginsenoside Re, ginsenoside Rd and ginsenoside Rb1, were incubated for 8 and 24 h with rat intestinal bacteria under anaerobic environment, respectively. After the samples were precipitated by acetonitrile and extracted with ethyl acetate, LC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites. The potential metabolites in rat feces were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results showed that six saponins could be easily metabolized by rat intestinal bacteria. Notoginsenoside R1 was mainly metabolized into five metabolites, and it's metabolic pathway was notoginsenoside R1→ginsenoside Rg1→ginsenoside Rh1 and ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg1 was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rg1→ginsenoside Rh1 and ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg2 was mainly metabolized into two metabolites, and it's metabolic pathway was ginsenoside Rg2→ protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Re was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Re→ginsenoside Rg2→ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rd was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rd→ginsenoside Rg3 and ginsenoside F2→ginsenoside Rh2→protopanaxadiol. Ginsenoside Rb1 was mainly metabolized into five metabolites, and it's metabolic pathway was ginsenoside Rb1→ginsenoside Rd→ginsenoside Rg3 and ginsenoside F2→ginsenoside Rh2→protopanaxadiol. In summary, six saponins could be quickly metabolized by rat intestinal bacteria in vitro. Their major metabolic pathways were deglycosylation and dehydrogenation.