Exploring the mechanism of the antithrombotic effects of Pueraria lobata and Pueraria lobata var. thomsonii based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. thomsonii (Benth.) Maesen are nutritious medicine food homology plants that are widely used in the food and health products industry and are excellent natural materials for the development of new health foods, with great potential for domestic and foreign markets. Clinically, P. lobata and P. thomsonii are used to treat coronary heart disease, atherosclerosis, cerebral infarction and other cardiovascular diseases, and antithrombotic actions may be their core effect in the treatment of thrombotic diseases. However, the underlying mechanisms of the antithrombotic properties of P. lobata and P. thomsonii have not been clarified. METHODS: First, P. lobata and P. thomsonii were identified by high-performance liquid chromatography (HPLC). An arteriovenous bypass thrombosis rat model was established. Thrombus dry‒wet weight, platelet accumulation rate and the four coagulation indices, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB), were detected in plasma to manifest the P. lobata and P. thomsonii antithrombotic function. Network pharmacology and molecular docking methods were used to obtain key targets and verify reliability. David 6.8 was used for GO and KEGG analyses to explore pathways and potential targets for P. lobata and P. thomsonii antithrombotic functions. Prostaglandin I2 (PGI2), thromboxane A2 (TXA2), cyclooxygenase 2 (COX-2), myeloperoxidase (MPO) and endothelial nitric oxide synthase (eNOS) were tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results indicated that P. lobata and P. thomsonii can reduce thrombus dry‒wet weight and platelet accumulation in rats and inhibit TT, APTT, FIB, and PT. A comprehensive network pharmacology approach successfully identified 9 active ingredients in P. lobata and P. thomsonii. The main active ingredients include polyphenols, amino acids and flavonoids. A total of 15 antithrombotic function targets were obtained, including 3 key targets (PTGS2, NOS3, MPO). Pathway analysis showed 10 significant related pathways and 29 biological processes. P. lobata and P. thomsonii inhibited platelet aggregation by upregulating PGI2 and downregulating TXA2, inhibited PTGS2 to reduce inflammation, and increased the level of eNOS to promote vasodilation. In addition, P. lobata and P. thomsonii alleviated oxidative stress by increasing SOD levels and significantly decreasing MDA contents. CONCLUSION: The results of the study further clarify the antithrombotic mechanism of action of P. lobata and P. thomsonii, which provides a scientific basis for the development of new drugs for thrombogenic diseases and lays the foundation for the development of P. lobata and P. thomsonii herbal resources and P. lobata and P. thomsonii health products.

The development and validation of a sensitive HPLC-MS/MS method for the quantitative and pharmacokinetic study of the seven components of Buddleja lindleyana Fort.

Buddleja lindleyana Fort., a traditional Chinese medicine, has demonstrated anti-inflammatory, immunomodulatory, antidementia, neuroprotective, antibacterial, and antioxidant effects. Its flowers, leaves, and roots have been used as traditional Chinese medicines. A simple and rapid high-performance liquid chromatography method coupled with mass spectrometry (HPLC-MS/MS) was applied in the multicomponent determination of Buddleja lindleyana Fort., and the discrepancies in the contents from ten different habitats were analyzed. The present study simultaneously determined the concentrations of seven chemical compounds of Buddleja lindleyana Fort. extract in rat plasma via HPLC-MS/MS, which was applied in the pharmacokinetic (PK) study of Buddleja lindleyana Fort. A C18 column was used for chromatographic separation, and ion acquisition was achieved by multiple-reaction monitoring (MRM) in negative ionization mode. The optimized mass transition ion-pairs (m/z) for quantization were 591.5/282.8 for linarin, 609.4/300.2 for rutin, 284.9/133.0 for luteolin, 300.6/151.0 for quercetin, 268.8/116.9 for apigenin, 283.0/267.9 for acacetin, 623.3/160.7 for acteoside, and 252.2/155.8 for sulfamethoxazole (IS). A double peak appeared in the drug-time curve of apigenin, which was associated with entero-hepatic recirculation. There were discrepancies in the contents of seven chemical compounds from 10 batches of Buddleja lindleyana Fort., which were associated with the growth environments. Herein, the pharmacokinetic parameters of seven analytes in Buddleja lindleyana Fort. extract are summarized. The maximum plasma concentration (C max) of linarin, rutin, luteolin, quercetin, apigenin, acacetin and acteoside were 894.12 ± 9.34 ng mL-1, 130.76 ± 18.33 ng mL-1, 77.37 ± 25.72 ng mL-1, 20.15 ± 24.85 ng mL-1, 146.42 ± 14.88 ng mL-1, 31.92 ± 17.58 ng mL-1, and 649.78 ± 16.42 ng mL-1, respectively. The time to reach C max for linarin, rutin, luteolin, quercetin, apigenin, acacetin, and acteoside were 10, 5, 5, 5, 180, 10 and 10 min, respectively. This is the first report on the simultaneous determination of seven active components for 10 different growing environments and the pharmacokinetic studies of seven active components in rat plasma after the oral administration of Buddleja lindleyana Fort. extract. This study lays the foundation for a better understanding of the absorption mechanism of Buddleja lindleyana Fort., and the evaluation of its clinical application.

[Determination of 14 components in Bazibushen capsule by UPLC-ESI-MS/MS].

The study developed a method for the determination of 14 components in Bazibushen capsule by UPLC-ESI-MS/MS. Waters ACQUITY BEH C(18) column (50 mm × 2.1 mm,1.7 µm) was used and the column temperature was 40 ℃.A linear gradient elution of eluents A (acetonitrile) and B（0.1% acetic acid） was used for the separation. The source temperature was set at 150 ℃.The capillary voltage was set at 2.0 k V. The source offset voltage was kept at 50 V. The desolvation temperature was set at 500 ℃.The desolvation flow was 800 L·h(-1).The cone flow was 150 L·h(-1). The nebuliser pressure was 7.0 Bar . Multiple reaction monitoring mode (MRM) is adopted. All of the 14 components showed good linearity (r2 > 0.999 1) in the test ranges. The LOQs for the compounds ranged from 0.11-4.52 ng·m L(-1), respectively.The RSDs were 0.8%-2.1%.The overall recoveries were between 97.89% and 101.9% for all compounds. The method is simple, rapid, accurate and highly reproducible, and may be used in the determination of 14 components in Bazibushen capsule.

[Simultaneous determination of 4 diterpenoids in Rabdosia japonica var.glaucocalyx by HPLC-ESI-MS/MS and cluster analysis].

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of four major diterpenoids in Rabdosia japonica var.glaucocalyx, including glaucocalyxin A, oridonin, hebeirubesensin and enmenol. Analysis was performed on an Agilent ZORBAX SB-C18(4.6 mm×250 mm, 5 µm ) column eluted in a gradient program with methanol and water. The flow rate was 0.8 mL•min⁻¹. Multiple reaction monitoring (MRM) scanning mode was performed in negative ion switching mode to apply for the quantitative determination. The calibration curves for the above four compounds were linear in corresponding injection amount. The average recoveries of the compounds ranged from 92.40% to 105.9%, with RSDs of 1.7%-6.5%. The method is simple, rapid, accurate with good repeatability, which can provide a reference for overcalling evaluation the quality of R. japonica var.glaucocalyx. The result of cluster analysis- showed that the quality of R. japonica glaucocalyx var. greatly varied between areas and parts.

Qualitative and quantitative determination of 15 main active constituents in Fructus Sophorae pill by liquid chromatography tandem mass spectrometry.

BACKGROUND: Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill. MATERIALS AND METHODS: Chromatographic separation was performed on a C18 column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes. RESULTS: The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents. CONCLUSION: The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill.

[Simultaneous determination of 3 phenolic acids in Usnea by HPLC-ESI-MS/MS].

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of three chemical compositions in Usnea, including usnic acid, diffractaic acid, and ramalic acid. The separation was performed on a chromatographic column of Agilent ZORBAX SB-C, (4.6 mm x 250 mm, 5 µm), and the mobile phase was methanol (0.05% formic acid)-0.05% formic acid solution (4 mmol ammonium acetate), with an isocratic elution at a flow rate of 0.8 ml · min⁻¹. Multiple reaction monitoring scanning mode (MRM) was performed combined with the ion switching technology in positive and negative ion switching mode to apply for the quantitative determination. The calibration curves for the above three compounds were linear in corresponding injection amount. Their average recoveries were 95.0%-105.1%, with RSDs of 1.1%-5.2%. The method was simple, rapid, accurate with high repeatability, which could provide a reference for overcalling evaluation the quality of Usnea.

Simultaneous determination of 3 phenolic acids in Usnea by HPLC-ESI-MS/MS / 中国中药杂志

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of three chemical compositions in Usnea, including usnic acid, diffractaic acid, and ramalic acid. The separation was performed on a chromatographic column of Agilent ZORBAX SB-C, (4.6 mm x 250 mm, 5 µm), and the mobile phase was methanol (0.05% formic acid)-0.05% formic acid solution (4 mmol ammonium acetate), with an isocratic elution at a flow rate of 0.8 ml · min⁻¹. Multiple reaction monitoring scanning mode (MRM) was performed combined with the ion switching technology in positive and negative ion switching mode to apply for the quantitative determination. The calibration curves for the above three compounds were linear in corresponding injection amount. Their average recoveries were 95.0%-105.1%, with RSDs of 1.1%-5.2%. The method was simple, rapid, accurate with high repeatability, which could provide a reference for overcalling evaluation the quality of Usnea.

A comparative study on the pharmacokinetics of a traditional Chinese herbal preparation with the single herb extracts in rats by LC-MS/MS method.

The Er-Mu preparation (EMP) is a well-known traditional Chinese prescription that has been clinically employed for the treatment of asthma and bronchial inflammation for hundreds of years. Neomangiferin, mangiferin, peimine, peiminine, timosaponin BII and timosaponin AIII are the major active ingredients of EMP for their anti-inflammatory or anti-asthmatic effects. The aim of this study was to investigate the pharmacokinetics of the target compounds from the recipe of EMP and the single herb extracts of Anemarrhenae asphodeloides Bge. (ARR) and Fritillariae cirrhosae D.Don (FCB), and the influence of compatibility on the pharmacokinetics of the main active ingredients. The rats were randomly assigned to three groups and orally administered with the recipe of EMP and the single herb extracts of ARR and FCB, respectively. The concentrations of the target compounds in rat plasma were determined by an optimal liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and multiple reaction monitoring (MRM) with a multi-switching monitoring mode coupled with simple protein precipitation method, and the main pharmacokinetic parameters were estimated. Significant differences (p<0.05) were found in the pharmacokinetic parameters of neomangiferin, mangiferin, peimine and peiminine between the single ARR or FCB extract and the combination treatment (p<0.05). The developed HPLC-ESI-MS method by switching positive and negative ESI sources in a single run was successfully applied to study the pharmacokinetics of six compounds in SD rat, which was powerful in terms of sensitivity, selectivity, time savings and solvent consumption in the quantitative analysis of complex herbal medicines. It was surmised that formula compatibility could significantly influence the pharmacokinetics of EMP and our study has preliminarily elucidated the priority in the compatible administration of EMP based on pharmacokinetic studies.

[Studies on fingerprints of Bupleurum chinense from Hebei Province with HPLC-UV].

OBJECTIVE: To establish a HPLC fingerprint analysis method for identification of Bupleurum chinense of Hebei Province and compare the fingerprints of Radix Bupleuri collected from different habitats so as to establish a sensitive and specific method for controlling the quality of Radix Bupleuri. METHODS: The HPLC-UV fingerprints of Radix Bupleuri from different habitats were obtained from Waters 1525 instruments. The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and water at a flow rate of 1.0 ml/min with UV detector at 203 nm. The temperature of column was 30 degrees C. RESULTS: The mutual mode of HPLC-UV fingerprints was set up, and the similar degrees to the crude drugs of different habitats were compared. CONCLUSION: It is simple and quick to differ Bupleurum chinense from different habitats with the method that can be used as a quality control item for Radix Bupleuri.

[Study on the metabolite of stilbene glucoside in mice by liquid chromatography tandem-mass spectrometry].

AIM: To study the metabolite of stilbene glucoside in the Chinese traditional medicine Polygonum multiflornm in mice and elucidate its chemical structure by liquid chromatography tandem-mass spectrometry. METHODS: The stilbene glucoside was injected into the tail vein of mice. Blood samples were collected from artery in the eyepit. The methanol-protein-precipitated plasma sample was introduced into the liquid chromatography-tandem mass spectrometer directly. The analytical column was C18 column (250 mm x 4.6 mm ID, 5 microns). The mobile phase consisted of acetonitrile-methanol-water-formic acid (15:18:66:1) for ES+, acetonitrile-methanol-water (15:18:67) for ES-. The UV detection wavelength was set at 320 nm. The mass ion source type was ESI. HV capillary was 3 kV. The dry gas was nitrogen gas and the flow rate was set at 318 L.h-1. The ion source temperature was 150 degrees C. RESULTS: The stilbene glucoside and its metabolite were separated completely under the chromatography condition. The ions at m/z 600 and m/z 605 were detected under positive ion polarity while the ions at m/z 581 and m/z 402 were detected under negative ion polarity. CONCLUSION: It was proposed that the metabolite of stilbene glucoside injected in vein was its glucuronide conjugate.

[Studies on pharmacokinetics of loganin and morroniside in Cornus officinalis injection in mice].

OBJECTIVE: To establish a revered-phase HPLC method for the study of pharmacokinetics of loganin and morroniside in Cornus officinalis injectionin mice after a single oral and intravenous administrations. METHOD: The Diamonsil C18 column (4.6 mm x 250 mm, 5 microns) was used as the analytical column and the mobile phase consisted of acetonitrile-methanol-0.2% formic acid (12:8:80) with the flow rate at 1.0 mL.min-1. The UV detection was set at 237 nm. RESULT: The calibration curves of loganin and morroniside were linear in the range from 0.38 to 68.25 mg.L-1(r = 0.9999), and from 0.66 to 117.22 mg.L-1(r = 0.9999), respectively. The lowest determination concentrations of loganin and morroniside were 0.10 and 0.16 mg.L-1, respectively. The recoveries and relative standard deviations of loganin were 99.6% (2.0%), 102.0% (1.0%), 87.9% (7.2%), and those of morroniside were 99.2% (2.5%), 104.1% (1.2%), 92.7% (4.2%), respectively. The relative standard deviations of within-day and between-day precision for the method were all less than 6.8%. After a single intravenous administration of Cornus officinalis injection to mice, the mean plasma concentration-time courses were found to fit a two-compartment open model, the main pharmacokinetic parameters of loganin were as follows: T1/2(alpha), T1/2(beta), K21, K12, K10, V(c), AUC, CL were 3.2 min, 25.1 min, 5.997 h-1, 4.981 h-1, 3.564 h-1, 0.551 L.kg-1, 13.59 mg.L-1.h, 1.965 L.kg-1.h-1, respectively and those of morroniside were 3.6 min, 21.5 min, 5.926 h-1, 3.833 h-1, 3.797 h-1, 0.647 L.kg-1, 27.15 mg.L-1.h, 2.457 L.kg-1.h-1, respectively. CONCLUSION: It is the first time to establish the revered-phase HPLC method to determine concentrations of loganin and morroniside in plasma and to obtain their pharmacokinetic parameters and characteristics.

Studies on pharmacokinetics of loganin and morroniside in Cornus officinalis injection in mice / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a revered-phase HPLC method for the study of pharmacokinetics of loganin and morroniside in Cornus officinalis injectionin mice after a single oral and intravenous administrations.</p><p><b>METHOD</b>The Diamonsil C18 column (4.6 mm x 250 mm, 5 microns) was used as the analytical column and the mobile phase consisted of acetonitrile-methanol-0.2% formic acid (12:8:80) with the flow rate at 1.0 mL.min-1. The UV detection was set at 237 nm.</p><p><b>RESULT</b>The calibration curves of loganin and morroniside were linear in the range from 0.38 to 68.25 mg.L-1(r = 0.9999), and from 0.66 to 117.22 mg.L-1(r = 0.9999), respectively. The lowest determination concentrations of loganin and morroniside were 0.10 and 0.16 mg.L-1, respectively. The recoveries and relative standard deviations of loganin were 99.6% (2.0%), 102.0% (1.0%), 87.9% (7.2%), and those of morroniside were 99.2% (2.5%), 104.1% (1.2%), 92.7% (4.2%), respectively. The relative standard deviations of within-day and between-day precision for the method were all less than 6.8%. After a single intravenous administration of Cornus officinalis injection to mice, the mean plasma concentration-time courses were found to fit a two-compartment open model, the main pharmacokinetic parameters of loganin were as follows: T1/2(alpha), T1/2(beta), K21, K12, K10, V(c), AUC, CL were 3.2 min, 25.1 min, 5.997 h-1, 4.981 h-1, 3.564 h-1, 0.551 L.kg-1, 13.59 mg.L-1.h, 1.965 L.kg-1.h-1, respectively and those of morroniside were 3.6 min, 21.5 min, 5.926 h-1, 3.833 h-1, 3.797 h-1, 0.647 L.kg-1, 27.15 mg.L-1.h, 2.457 L.kg-1.h-1, respectively.</p><p><b>CONCLUSION</b>It is the first time to establish the revered-phase HPLC method to determine concentrations of loganin and morroniside in plasma and to obtain their pharmacokinetic parameters and characteristics.</p>

Study on the metabolite of stilbene glucoside in mice by liquid chromatography tandem-mass spectrometry / 药学学报

<p><b>AIM</b>To study the metabolite of stilbene glucoside in the Chinese traditional medicine Polygonum multiflornm in mice and elucidate its chemical structure by liquid chromatography tandem-mass spectrometry.</p><p><b>METHODS</b>The stilbene glucoside was injected into the tail vein of mice. Blood samples were collected from artery in the eyepit. The methanol-protein-precipitated plasma sample was introduced into the liquid chromatography-tandem mass spectrometer directly. The analytical column was C18 column (250 mm x 4.6 mm ID, 5 microns). The mobile phase consisted of acetonitrile-methanol-water-formic acid (15:18:66:1) for ES+, acetonitrile-methanol-water (15:18:67) for ES-. The UV detection wavelength was set at 320 nm. The mass ion source type was ESI. HV capillary was 3 kV. The dry gas was nitrogen gas and the flow rate was set at 318 L.h-1. The ion source temperature was 150 degrees C.</p><p><b>RESULTS</b>The stilbene glucoside and its metabolite were separated completely under the chromatography condition. The ions at m/z 600 and m/z 605 were detected under positive ion polarity while the ions at m/z 581 and m/z 402 were detected under negative ion polarity.</p><p><b>CONCLUSION</b>It was proposed that the metabolite of stilbene glucoside injected in vein was its glucuronide conjugate.</p>