Comparative Analysis of Compatibility Influence on Invigorating Blood Circulation for Combined Use of Panax Notoginseng Saponins and Aspirin Using Metabolomics Approach.

The combined use of Panax notoginseng saponins (PNS)-based drugs and aspirin (ASA) to combat vascular diseases has achieved good clinical results. In this study, the superior efficacy was observed via the combined use of PNS and ASA on acute blood stasis rats, and untargeted metabolomics was performed to holistically investigate the therapeutic effects of coupling application and its regulatory mechanisms. The combined use of PNS and ASA exhibited better improvement effects when reducing the evaluated hemorheological indicators (whole blood viscosity, plasma viscosity, platelet aggregation, and fibrinogen content) in the blood stasis rats vs. single use of PNS or ASA at the same dose. The combined use of both drugs was the most effective application method, as shown by the relative distance in partial least-squares discriminant analysis score plots. Twelve metabolites associated with blood stasis were screened as potential biomarkers and were mainly involved in amino acid metabolism, lipid metabolism, and energy metabolism. After coherently treated with PNS and ASA, the altered metabolites could be partially adjusted to be closer to normal levels than single use. The collective results revealed that PNS could cooperate with ASA to treat blood stasis and provided a scientific explanation for the superior efficacy of their combined use.

Chicory ameliorates hyperuricemia via modulating gut microbiota and alleviating LPS/TLR4 axis in quail.

BACKGROUND: High-purine diet can cause gut microbiota disorder, which is closely related to the occurrence of hyperuricemia (HUA). At the same time, the development of HUA is often accompanied by renal impairment. Chicory, a natural medicine, has a significant effect on lowering uric acid. However, whether its concrete mechanism is associated with the regulation of gut microbiota and renal damage is still unclear. METHODS: Hyperuricemic quails induced by high-purine diet were used, and quails were divided into control (CON), model (MOD), and model plus high, middle, low doses of chicory. The uricosuric effect was evaluated by detecting the uric acid levels in serum and feces. Meanwhile, the morphology of intestine and kidney were observed by hematoxylin and eosin (HE) staining, and the expression of intestinal barrier junction proteins Occludin, Claudin-1 were detected by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Furthermore, the latent mechanism was clarified by analyzing 16S rRNA amplicon of gut microbiota and measuring the changes of LPS/TLR4 axis inflammatory response of the kidney by western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that serum uric acid levels were significantly decreased, and the feces uric acid levels were noticeably increased after the intervention of chicory. In addition, chicory could repair the damage of intestinal mucosa and improve the permeability of intestinal barrier. Moreover, the 16S rRNA sequencing analysis uncovered that chicory restored gut microbiota by increasing the probiotics flora (Bifidobacterium, Erysipelotrichaceae) and reducing the pathogenic bacteria group (Helicobacteraceae). Furthermore, it was found that chicory reduced the LPS/TLR4 axis inflammatory response by down regulating the serum LPS and TLR4/NF-κB inflammatory pathway in kidney, thus promoting the excretion of uric acid in kidney. CONCLUSION: Chicory intervention ameliorated HUA via modulating the imbalance of gut microbiota and suppressing LPS/TLR4 axis inflammatory reaction in quail model, which may be a promising candidate for hyperuricemia-relieving properties.

Uncovering the pharmacological mechanism of motherwort (Leonurus japonicus Houtt.) for treating menstrual disorders: A systems pharmacology approach.

Leonurus japonicus (motherwort) is a traditional Chinese medicine that is widely used to treat menstrual disorders (MDs). However, the pharmacological mechanisms that underlie its clinical application remain unclear. In this study, a network pharmacology-based approach was used that integrated drug-likeness evaluation, oral bioavailability prediction, target exploration, network construction, bioinformatic annotation and molecular docking to investigate the mechanisms that underlie motherwort treatment for MDs. In total, 29 bioactive compounds were collected from 51 compounds in motherwort, which shared 17 common MDs-related targets. Network analysis indicated that motherwort played a therapeutic role in MDs treatment through multiple components that acted on multiple targets. Pathway enrichment analysis showed that the putative targets of motherwort were primarily involved in various pathways associated with the endocrine system, cancers, vascular system, and anti-inflammation process. Notably, five targets (i.e., AKT1, PTGS2, ESR1, AR and PPARG) were screened as hub genes based on a degree algorithm. Moreover, most of the bioactive components in motherwort had good binding ability with these genes, implying that motherwort could regulate their biological function. Collectively, this study elucidated the molecular mechanisms that underlay the efficiency of motherwort against MDs and demonstrated the potential of network pharmacology as an approach to uncover the action mechanism of herbal medicines.

Panax notoginseng saponins suppress lipopolysaccharide-induced barrier disruption and monocyte adhesion on bEnd.3 cells via the opposite modulation of Nrf2 antioxidant and NF-κB inflammatory pathways.

Dysfunction of the blood-brain barrier (BBB) is a prerequisite for the pathogenesis of many cerebral diseases. Oxidative stress and inflammation are well-known factors accounting for BBB injury. Panax notoginseng saponins (PNS), a clinical commonly used drug against cerebrovascular disease, possess efficient antioxidant and anti-inflammatory activity. In the present study, the protective effects of PNS on lipopolysaccharide (LPS)-insulted cerebral microvascular endothelial cells (bEnd.3) were assessed and the underlying mechanisms were investigated. The results showed that PNS mitigated the decrease of Trans-Endothelial Electrical Resistance, increase of paracellular permeability, and loss of tight junction proteins in bEnd.3 BBB model. Meanwhile, PNS suppressed the THP-1 monocytes adhesion on bEnd.3 monolayer. Moreover, PNS prevented the pro-inflammatory cytokines secretion and reactive oxygen species generation in bEnd.3 cells stimulated with LPS. Mechanism investigations suggested that PNS promoted the Akt phosphorylation, activated Nrf2 antioxidant signaling, and inhibited the NF-κB activation. All the effects of PNS could be abolished by PI3K inhibition at different levels. Taken together, these observations suggest that PNS may act as an extrinsic regulator that activates Nrf2 antioxidant defense system depending on PI3K/Akt and inhibits NF-κB inflammatory signaling to attenuate LPS-induced BBB disruption and monocytes adhesion on cerebral endothelial cells in vitro.

Transcriptomic-proteomics-anticoagulant bioactivity integrated study of Pheretima guillemi.

ETHNOPHARMACOLOGICAL RELEVANCE: Earthworms, a type of animal drugs from traditional Chinese medicine, have been used to treat coagulation for many years with less adverse effects and similar anticoagulant effects compared to the commonly used anticoagulants. There are four species of earthworms recorded in Chinese Pharmacopoeia, while few of them were studied and deficient information were involved in the NCBI and UniProt earthworm protein database. We have adopted a transcriptomic-proteomics-anticoagulant bioactivity integrated approach to investigate a seldom-studied Chinese Pharmacopoeia recorded species, Pheretima guillelmi. AIM OF THE STUDY: In the present study, we aimed to reveal the anticoagulant bioactivity of Pheretima guillelmi, and identify its functional proteins via LC-MS/MS-transcriptome cross identification. METHODS AND RESULTS: With the aid of fibrinogen-thrombin time assay, Pheretima guillelmi was found to possess strong anticoagulant activity, and the bioactivity was quite stable under 30-50 °C and near-neutral conditions. A comprehensive non-reference transcriptome assembly of P. guillelmi was first established to supplement the currently inadequate earthworm protein database and to illustrate the active proteins. Illumina RNA sequencing generated 25,931,175 of clean reads with over 97% high-quality clean reads (Q20) and assembled an average of 133,228 of transcript and 106,717 of unigenes. A total of 11,259 coding sequences were predicted via ESTScan (3.0.3). The P. guillelmi unigenes were searched and annotated against public database. The bioactive proteins in P. guillelmi were with broad distribution of molecular weight. With bottom-up proteomics analysis, ten proteins were identified against UniProt and NCBI earthworm database; and 31 proteins with high-confidence were matched against transcriptomic established P. guillelmi database. CONCLUSION: This study illuminated the therapeutic potency of P. guillelmi for antithrombus and provide a new strategy to investigate animal drugs of Chinese materia medica.

Panax notoginseng Saponins Protect Cerebral Microvascular Endothelial Cells against Oxygen-Glucose Deprivation/Reperfusion-Induced Barrier Dysfunction via Activation of PI3K/Akt/Nrf2 Antioxidant Signaling Pathway.

Oxidative stress plays a critical role in cerebral ischemia/reperfusion (I/R)-induced blood-brain barrier (BBB) disruption. Panax notoginseng saponins (PNS) possess efficient antioxidant activity and have been used in the treatment of cerebral ischemic stroke in China. In this study, we determined the protective effects of PNS on BBB integrity and investigated the underlying mechanism in cerebral microvascular endothelial cells (bEnd.3) exposed to oxygen-glucose deprivation/reperfusion (OGD/R). MTT and LDH release assays revealed that PNS mitigated the OGD/R-induced cell injury in a dose-dependent manner. TEER and paracellular permeability assays demonstrated that PNS alleviated the OGD/R-caused disruption of BBB integrity. Fluorescence probe DCFH-DA showed that PNS suppressed ROS generation in OGD/R-treated cells. Immunofluorescence and western blot analysis indicated that PNS inhibited the degradation of tight junction proteins triggered by OGD/R. Moreover, mechanism investigations suggested that PNS increased the phosphorylation of Akt, the activity of nuclear Nrf2, and the expression of downstream antioxidant enzyme HO-1. All the effects of PNS could be reversed by co-treatment with PI3K inhibitor LY294002. Taken together, these observations suggest that PNS may act as an extrinsic regulator that activates Nrf2 antioxidant signaling depending on PI3K/Akt pathway and protects against OGD/R-induced BBB disruption in vitro.

Effects of Panax Notoginseng Saponins on Esterases Responsible for Aspirin Hydrolysis In Vitro.

Herb⁻drug interactions strongly challenge the clinical combined application of herbs and drugs. Herbal products consist of complex pharmacological-active ingredients and perturb the activity of drug-metabolizing enzymes. Panax notoginseng saponins (PNS)-based drugs are often combined with aspirin in vascular disease treatment in China. PNS was found to exhibit inhibitory effects on aspirin hydrolysis using Caco-2 cell monolayers. In the present study, a total of 22 components of PNS were separated and identified by UPLC-MS/MS. Using highly selective probe substrate analysis, PNS exerted robust inhibitory potency on human carboxylesterase 2 (hCE2), while had a minor influence on hCE1, butyrylcholinesterase (BChE) and paraoxonase (PON). These effects were also verified through molecular docking analysis. PNS showed a concentration-dependent inhibitory effect on hydrolytic activity of aspirin in HepaRG cells. The protein level of hCE2 in HepaRG cells was suppressed after PNS treatment, while the level of BChE or PON1 in the extracellular matrix were elevated after PNS treatment. Insignificant effect was observed on the mRNA expression of the esterases. These findings are important to understand the underlying efficacy and safety of co-administration of PNS and aspirin in clinical practice.

Network pharmacology-based identification of protective mechanism of Panax Notoginseng Saponins on aspirin induced gastrointestinal injury.

BACKGROUND & AIMS: Aspirin is the first line therapy for cardiovascular and cerebrovascular diseases and is widely used. However aspirin-induced gastrointestinal injury is one of its most common side effect which limits long-term use. Panax Notoginseng Saponins(PNS) which is also used to prevent thrombus may alleviate this side effect according to previous clinical evidences. Owing to the complexity of drug combination, the protective mechanism of PNS on aspirin-induced gastrointestinal injury remains unclear. Therefore, a network pharmacology-based strategy was proposed in this study to address this problem. METHODS: A network pharmacology approach comprising multiple components, candidate targets of each component, known therapeutic targets, network analysis has been used in this study. Also, we establish aspirin-induced gastrointestinal injury model by the oral administration of aspirin (0.5 g/kg body weight) to verify the predicted targets from network pharmacology. All rats was randomly allocated to control groups (n = 6),aspirin groups (n = 6)and aspirin + PNS groups (n = 6) and conducted H&E staining and ELISA for VEGFA. RESULTS: The comprehensive systematic approach was successfully to identify 5 compounds and 154 candidate targets in PNS and 479 candidate targets in aspirin. After network establishment and analysis, 27 potential targets hit by PNS, aspirin and 6 kind of gastrointestinal diseases were found. The experiments results indicated that aspirin group has visible inflammation and lesions while aspirin + PNS group have not. The higher expression of VEGFA in aspirin + PNS group verified the predicted potential protective targets of PNS. CONCLUSIONS: PNS may have protective function for aspirin-induced gastrointestinal injury through increasing VEGFA expression. Network pharmacology strategy may provide a forceful tool for exploring the mechanism of herb medicine and discovering novel bioactive ingredients.

Inhibitory Influence of Panax notoginseng Saponins on Aspirin Hydrolysis in Human Intestinal Caco-2 Cells.

Herb-drug interactions are important safety concerns in clinical practice. The interactions occur firstly in the intestinal absorption for orally administered drugs. Aspirin and Panax notoginseng saponins (PNS)-based drugs are often combined in China to prevent larger-artery atherosclerosis. Here, we aimed to characterize the aspirin transport across Caco-2 cell monolayers, a model of the intestinal absorption, and further to evaluate the influence of PNS on aspirin hydrolysis and the relating mechanisms. Transcellular transport of aspirin and the influence of PNS were explored using Caco-2 cell monolayers. The protein expression of human carboxylesterase 1 (hCE1) and hCE2 in Caco-2 cells after PNS treatment was analyzed by ELISA, and the mRNA level were determined by qRT-PCR. In the study, Caco-2 cells showed high level of hydrolase activity, and most aspirin was hydrolyzed inside the cells during the transport process. Interestingly, PNS were demonstrated to inhibit the esterase activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of hCE1 and hCE2, whereas exhibited minor effect on the mRNA expression. These results indicated that oral administration of PNS-based drugs might inhibit the hydrolysis of aspirin during intestinal absorption thus promoting its bioavailability.

[Characteristic Chromatograms Analysis and Simultaneous Determination of Six Marker Components in Xinnaojian Capsules].

OBJECTIVE: To establish an HPLC method for characteristic chromatograms analysis and simultaneous determination of six marker components in Xinnaojian Capsules from different manufactories. METHODS: Using HPLC, an Agilent C18 column (100 mm x 4.6 mm, 2.7 µm) was adopted with acetonitrile-0.05% phosphoric acid as the mobile phase in a gradient elution mode, the flow ratewas 0.4 mL/min, the detection wavelength was 280 nm, and the column temperature was 40 degrees C. RESULTS: Totally eleven common peaks were recognized with epigallocatechin-3-gallate as the reference peak. There were good similarities between the standard characteristic chromatogram and each characteristic chromatogram of the 26 samples with their similarities over 0.99. The six marker components (gallic acid, gallocatechin, caffeine, epigallocatechin, epigallocatechin-3-gallate and epicatechin gallate) were separated well. Good correlation coefficients were found (r > 0.9990) and the average recovery rates ranged from 95.29% to 102.3%. CONCLUSION: The established method has high sensitivity and specificity, and can be used for the quality control of Xinnaojian Capsules.

[Study on purification of total organic acid and chlorogenic acid in Herba Artemisiae scopariae with macroporous adsorption resin].

OBJECTIVE: To optimize the technological parameters of the purification process for total organic acid and chlorogenic acid in Herba Artemisiae scopariae with macroporous adsorption resin. METHODS: Static and dynamic adsorption-desorption methods were adopted to choose the optimal type of resin. Then orthogonal design L9 (3(4)) and single factor experiment were used to select the optimum purification process conditions. RESULTS: The optimal purification process for total organic acid and chlorogenic acid with HPD200A macroporous adsorption resin were as follows: the diameter height ratio of the resin was 1:6, the sample concentration was 1 g/mL, the absorption velocity was 1 BV/h (1 BV represented one column volume), the ratio of sample to HPD200A macroporous adsorption resin was 1.5:1 (W/W), 3 BV of water was used as purificant and 2 BV of 90% ethanol was used as eluant. Under these conditions, the purity of total organic acid and chlorogenic acid was 588.74% and 567.89%, respectively. CONCLUSION: The established purification process for total organic acid and chlorogenic acid in Herba Artemisiae scopariae with HPD200A macroporous adsorption resin is simple and stable and can be used in industrial production.