Rapid identification, quantitation, and antioxidant activity evaluation of the components in Guanxin Shutong capsule with liquid chromatography and mass spectrometry.

Guanxin Shutong capsule (GSC) is a traditional Chinese medicinal prescription used in the treatment of coronary heart disease (CHD) and angina pectoris in clinic. However, the chemical profile of GSC is still uncovered, which hindered the progress of pharmacological study and clinical application. Herein, high performance liquid chromatography (HPLC) together with high resolution mass spectrometry (HR-MS) techniques were employed to analyze the quality consistency and to identify chemical components in GSC. As a result, a total of 111 compounds were tentatively annotated. Quantitative analysis based on HPLC-ultraviolet detection (UV) was performed for 6 main components and fingerprints of 10 different batches of GSC were established. The developed method was validated for linearity, precision, repeatability, stability, and recovery. The quality evaluation and similarity analysis of the 10 batches were also performed. Furthermore, in vitro antioxidant activity assays demonstrated that GSC exhibited potential DPPH and hydroxyl radical scavenging capacities. Especially, salvianolic acids showed the strongest free radical scavenging capacities, which might be the main component for quality control of GSC.

Rapid identification and quantitative determination of chemical compositions in Buyang Huanwu decoction based on HPLC-Q-Exactive mass spectrometry.

OBJECTIVE: To establish an analytical method for rapid identification of chemical compositions and quantitative determination of major compositions in Buyang Huanwu decoction (BYHWD) based on high performance liquid chromatography-quadrupole orbitrap mass spectrometry (HPLC-Q-Exactive MS) and high performance liquid chromatography-ultraviolet detection (HPLC-UV). METHODS: The mass spectrometry information was collected in Full MS/dd-MS 2 negative ion mode with HPLC-Q-Exactive MS system; the chemical compositions of BYHWD were subsequently annotated with Compound Discoverer 3.0 software and a self-built in-house compound library. Eight major compositions (paeoniflorin, gallic acid, hydroxysafflor yellow A, ferulic acid, calycosin-7-glucoside, ononin, calycosin, formononetin) were picked out and their contents were quantitatively determined with HPLC-UV analysis. RESULTS: A total of 178 compounds in BYHWD were tentatively identified. The results of HPLC-UV quantitative analysis showed that 8 compositions had a good linear relationship in their respective concentration range ( R 2≥0.9990), the relative standard deviations (RSD) of precision and stability were all less than 15%, and the recovery rate RSD was between 1.6% and 2.4%. CONCLUSIONS: The method established in this study can realize the rapid identification and accurate quantification of the major compositions in BYHWD. Paeoniflorin, hydroxysafflor yellow A and gallic acid may be used as quality control markers.

The combination of danhong injection plus tissue plasminogen activator ameliorates mouse tail thrombosis-induced by κ-carrageenan.

BACKGROUND: After thrombosis, t-PA thrombolysis is the first choice, but the use of t-PA can easily lead to hemorrhagic injury and neurotoxicity. The combination of Danhong injection (DHI) and tissue plasminogen activator (t-PA) therapy may be a new strategy to find high-efficiency anti-thrombosis and low bleeding risk. However, nothing is about the effect of DHI plus t-PA on platelet activation. PURPOSE: The present research was to explore the optimal dose of DHI and t-PA in vivo and mechanisms involved with the treatment of combining DHI and t-PA for thrombotic disease and determined whether DHI plus t-PA affects thrombotic processes related to platelet activation. METHODS: Mice were induced by administering κ-carrageenan intraperitoneally, the ratio of different doses of DHI and t-PA in vivo, and the optimal dose effects on platelet aggregation, platelet adhesion, thrombosis formation, and platelet activation were determined. The effects of the αIIbß3 signaling pathway were analyzed in mice. RESULTS: In vitro, DHI (62% v/v), t-PA (1 mg/ml), and DHI + t-PA (62% v/v + 1 mg/ml) decreased rat platelet aggregation and adhesion, with a stronger effect from the combination as compared to t-PA monotherapy. In vivo, injections of κ-carrageenan were used to induce BALB/c mice. The optimal dose of DHI, t-PA, and DHI + t-PA is 12 ml/kg, 10 mg/kg, and 12 ml/kg + 7.5 mg/kg. The administration of DHI (12 ml/kg), t-PA (10 mg/kg), and DHI + t-PA (12 ml/kg + 7.5 mg/kg) decreased thrombi in mouse tissue vessels. Furthermore, the reduction of thrombosis formation by DHI, t-PA, and DHI + t-PA was related to lower collagen deposition, and lowered expressions of collagen I, matrix metalloproteinase 2 (MMP-2), and metalloproteinase 9 (MMP-9) in mouse tails, with increased efficacy in combination as compared to t-PA alone. The anti-thrombosis actions of DHI, t-PA, and their combination regulated the expression of CD41, purinergic receptor (P2Y12), guanine nucleotide-binding protein G (q) subunit alpha (GNAQ), phosphatidylinositol phospholipase c beta (PLCß), Ras-related protein 1 (Rap1), RIAM, talin1, fibrinogen alpha chain (FG), kindlin-3, and RAS guany1-releasing protein 1 (RasGRP1). CONCLUSIONS: Based on expression, the mechanism responsible for thrombosis may be attributed to platelet activation via the αIIbß3 signaling pathway. Combination therapy with DHI and t-PA exerted potent thrombolytic effects. Thus, our data can be used as a foundation for further clinical studies examining the efficacy of traditional Chinese medicines for the treatment of thrombosis.

Inhibition of Oxidative Stress: An Important Molecular Mechanism of Chinese Herbal Medicine (Astragalus membranaceus, Carthamus tinctorius L., Radix Salvia Miltiorrhizae, etc.) in the Treatment of Ischemic Stroke by Regulating the Antioxidant System.

Ischemic stroke is a severe cerebrovascular disease with high mortality and morbidity. Traditional Chinese medicine (TCM) has been utilized for thousands of years in China and is becoming increasingly popular all over the world, especially for the treatments of ischemic stroke. More and more evidences have implicated that oxidative stress has been closely related with ischemic stroke. This review will concentrate on the evidence of the action mechanism of Chinese herbal medicine and its active ingredient in preventing ischemic stroke by modulating redox signaling and oxidative stress pathways and providing references for clinical treatment and scientific research applications.

Naoxintong Capsule Alternates Gut Microbiota and Prevents Hyperlipidemia in High-Fat-Diet Fed Rats.

Background: Naoxintong Capsule (NXT) is a formulated Traditional Chinese Medicine (TCM) widely applied in the treatment of cardiovascular and metabolic diseases, most of which are closely related to hyperlipidemia as a major risk factor. Given the current limited understandings to the role of gut microbiota in the lipid-lowering effect of NXT and other TCM products, this study investigated the regulation of gut microbiota and lipid metabolism by NXT, and their potential relationship. Methods: The chemical components of NXT were firstly analyzed with HPLC-MS method. In high fat diet (HFD)-fed rat models, as well as normal rats as control, the histopathological and biochemical changes of serum and liver were examined, including total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C). In addition, the gut microbiota community was analyzed using 16S rRNA sequencing technique, the fecal levels of gut microbiota related metabolites, including bile acids (BAs) and short chain fatty acids (SCFAs) were determined with HPLC-MS. The correlations of the clinical indicators and gut microbiota related indicators were then investigated statistically. Results: The results showed that NXT exerted potential preventive effect on hyperlipidemia. Specifically, NXT significantly reduced the body weight, TC, TG and LDL-C in serum, increased HDL-C in serum, reduced the TC and TG in liver, as well as protected liver. The body weight, serum lipid levels and liver function were all significantly alleviated. The gut microbiota of the HFD-fed rats was reconstituted with supplementation of NXT. The fecal levels of gut microbiota related metabolites, including BAs and SCFAs were also altered. The correlation between the gut microbiota and clinical/metabolomic parameters was then studied. As the result, the amount of propionic aicd, Firmicutes/Bacteroidetes ratio (F/B) and the relative abundance of Collinsella in feces are the most possibly potential therapeutic biomarkers of NXT. Conclusion: NXT was effective in regulation of gut microbiota and prevention of hyperlipidemia in HFD fed rats. The present work might provide novel insights into the anti-hyperlipidemia effect of TCM and afford new scientific evidence for clinical application of TCM.

Ultrasound-assisted preparation of 'Ready-to-use' extracts from Radix Paeoniae Rubra with natural deep eutectic solvents and neuroprotectivity evaluation of the extracts against cerebral ischemic/ reperfusion injury.

Natural deep eutectic solvent (NaDES) is widely applied in the extraction of nutrients from natural resources as a greener alternative for fossil solvent. In the present work, 27 different NaDESs were screened for the extraction of paeoniflorin (PF) and galloyl paeoniflorin (GPF) from Radix Paeoniae Rubra (RPR). After screening and extraction parameter optimization, the extraction yields of PF and GPF reached up to 182.8 mg/g and 77.4 mg/g with the selected NaDES, ChCl-Sor. Furthermore, the antioxidant activity in vitro and neuroprotectivity in vivo of the 'ready-to-use' extracts were evaluated comprehensively. Especially in vivo, the cerebral ischemic/ reperfusion injury model was established in rats and the protective effects of the RPR extracts were determined. The results not only proved that NaDES is a valuable green extraction media, but also indicated the safety and potential pharmaceutical application of NaDES based 'ready-to-use' extracts from medical plants.

Spectrum-Effect Relationship between HPLC Fingerprints and Antioxidant Activity of Yangyin Tongnao Prescription.

Yangyin Tongnao (YYTN) prescription is used as a traditional Chinese herbal formula, and it has antioxidant activity that mainly contributes in the treatment of cardiovascular and cerebrovascular diseases. However, the compounds related to its antioxidant activity are still unknown. In the present study, the fingerprints of YYTN extracts under different extraction conditions were obtained by high performance liquid chromatography (HPLC) to identify the common peaks to all the samples processed. A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay and ferric reducing antioxidant power (FRAP) assay were carried out to evaluate the antioxidant activity of the extracts. Spectrum-effect relationship between HPLC fingerprints and antioxidant activity of YYTN was assessed by Pearson product-moment correlation coefficient (PPMCC) and multiple linear regression analysis (MLRA). The results showed that peaks 5, 6, 13, 15, and 24 of the fingerprints were closely connected to antioxidant activity. Five peaks were identified: vanillic acid (P5), puerarin (P7), ferulic acid (P13), daidzein (P21), and formononetin (P23). Our study successfully established the spectrum-effect relationship between HPLC fingerprints and antioxidant activity of YYTN, which provided a general method for establishing quality standards with a combination of chromatography and antioxidant activity.

Efficacy of Yangyin Yiqi Huoxue Granule () in Treatment of Ischemic Stroke Patients with Qi-Yin Deficiency and Blood Stasis Syndrome: A Randomized, Double-Blind, Multicenter, Phase-2 Clinical Trial.

OBJECTIVE: To evaluate the efficacy and safety of Yangyin Yiqi Huoxue Granule (, YYHG) in the treatment of ischemic stroke (IS) patients with qi-yin deficiency and blood stasis syndrome (QYDBSS), and to explore its effective dosage. METHODS: The total of 288 patients were randomly assigned to the YYHG high-dose, YYHG low-dose, positive control (administered Xiaoshuantong Granule, XSTG, ), or placebo control (administered inert granule) groups (72 cases per group) by software-drived competitive block randomization. The trial was conducted for a 28-day period, with a 180-day follow-up period. The primary outcome was the comprehensive curative evaluation, and secondary outcomes were the National Institute of Health Stroke Scale (NIHSS) score, Barthel activities of daily living (ADL) index score, the quality of life index (QLI) score, and the Chinese medicine syndrome (CMS) score. All analyses were done on an intention-to-treat basis. The clinical safety was also assessed. RESULTS: The total of 288 participants were recruited between June 1, 2008 and September 30, 2009, and 287 patients received intervention; the treatment groups were well balanced at baseline. The comprehensive cure rates of YYHG high-dose, low-dose, positive and placebo control groups were 63.38%, 31.94%, 36.11% and 6.14%, respectively; there was a statistical difference between the two groups (P<0.01), while the high-dose YYHG treatment group was significantly higher than the other 3 groups (P<0.01). The improvement of NIHSS, ADL, QLI and CMS scores of the YYHG high-dose and low-dose groups was significantly better than that of the positive control group and the placebo control group (P<0.05). In terms of improving the classification of the NIHSS scale and the assessment of the ADL scale, the YYHG high-dose group was significantly better than the other three groups (P<0.05), and the YYHG low-dose group was better than the placebo control group (P<0.01). At the same time, except for the QLI score, the high-dose group was better than the low-dose group (P<0.05). In terms of safety, adverse reactions after YYHG treatment were generally mild (3.78%), and no serious adverse reactions have been reported. CONCLUSION: YYHG is safe and effective in the treatment of IS patients with QYDBSS.

An integrative strategy for discovery of functional compound combination from Traditional Chinese Medicine: Danhong Injection as a model.

Traditional Chinese Medicine formulas, which are usually considered exerting their holistic clinical benefits via multi-component, multi-target manner, are unique resources for the discovery of multi-component drug combinations. In order to screen and optimize the functional compound combination (FCC) from TCM, we established a novel four-step 'GCIC' strategy, including 'Global profiling', 'Chemical structural classification', 'Intra-group screening' and 'Component-knockout optimization'. Following this strategy, an FCC consisted of four components from Danhong Injection (DHI) was identified, containing ferulic acid, cryptotanshinone, quercetin and anhydrosafflor yellow B. The holistic neuroprotective effects of the FCC were further investigated, indicating that the combination can both activate the antioxidative and anti-inflammatory responses in PC12 cells to protect them from oxidative stress. Major signaling pathways as Nrf2/ARE and Nrf2/AMPK/GSK3ß were involved in the protective process of FCC. The 'GCIC' strategy established in this study might provide an alternation to traditional strategies in discovering the bioactive components from herbal medicines, especially compounded TCM formulas.

Greener extraction process and enhanced in vivo bioavailability of bioactive components from Carthamus tinctorius L. by natural deep eutectic solvents.

Natural deep eutectic solvents (NaDESs) are promising green alternatives to conventional solvents widely applied in the extraction of natural products due to their physical and chemical superiorities. In present study, 22 NaDESs consisted from food grade ingredients were screened in ultrasonic assisted extraction (UAE) of bioactive compounds from safflower. The oral bioavailabilities of hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) in the extracts were then investigated in SD rats with the help of HPLC-MS technique. The results revealed that l-proline-acetamide (l-Pro-Am) was an effective solvent with the yields of HSYA and ASYB at 32.83 and 8.80 mg/g. Pharmacokinetic studies revealed that the blood level of HSYA and ASYB were significantly higher after oral administration of l-Pro-Am extract than that of aqueous extract. Especially, the relative bioavailabilities (to aqueous extract) of HSYA and ASYB were calculated 183.5% and 429.8%.

Transport properties of paeoniflorin and amygdalin across caco-2 cell monolayer model and their modulation of cytochrome p450 metabolism.

Paeoniflorin and amygdalin are two major active saponins constituents in some Chinese herbal formulas used for cardio-cerebrovascular diseases. However, their intestinal absorption property and metabolic characteristics have not been clarified. The aim of this work was to study the absorption property of Paeoniflorin and Amygdalin across Caco-2 cell monolayer and their metabolic characteristics on the activity of cytochrome P450 (CYP450) enzyme. The results showed that the transport amount of Paeoniflorin and Amygdalin was positively correlated with the time and concentrations, and the transport amount from AP side to BL side was higher than that from BL to AP. The absorptions of Paeoniflorin and Amygdalin were reduced by P-glycoprotein, which provided the pharmacokinetic basis for their clinical application. Furthermore, we demonstrated that Paeoniflorin and Amygdalin had obvious inhibiting effects on CYP2C9 and CYP2E1. The transports of Paeoniflorin and Amygdalin across Caco-2 cell monolayer model were deduced as the passive transport, which indicated that the present bioassay system was appropriate and reliable for the evaluation of the transport characteristics and metabolic characteristics of active ingredient groups in Bu-yang-huan-wu decoction. Moreover, this research method may also be suitable for the appropriate bioactivity and metabolic characteristics analysis of other plant extracts.

Antiviral effects and mechanisms of Yinhuapinggan granule against H1N1 influenza virus infection in RAW264.7 cells.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250 µg/mL) significantly up-regulated levels of IFN-ß, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250 µg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

Protective effects of effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) compatibility after rat hippocampal neurons induced by hypoxia injury.

OBJECTIVE: To investigate the effective ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) on protective properties towards neonatal rat hippocampal neurons under hypoxia condition. METHODS: Primary culture of neonatal rat hippocampal neurons was used to model hypoxia damage on the hippocampus. Methyl thiazolyl tetrazolium (MTT) assay and preliminary experiments were conducted to identify the four effective ingredients of Danhong, along with the injection of positive drug onto hippocampal neurons at a non-toxic dosage level. The cultured cells were randomly divided into 12 groups: the normal group, model group, positive drug control group and nine compatibility groups of the four effective ingredients. Different test methods were applied to determine lactate dehydrogenasein (LDH), total superoxide dismutase (T-SOD), malondialdehyde (MDA), 6-keto prostaglandin Fla (6-keto-PGF1a), thromboxane B2 (TXB2), mitochondrial membrane potential (MMP), free calcium ions concentration ([Ca2+]i) and early onset cell apoptosis. RESULTS: Different compatibility groups could inhibit the content of LDH and intracellular calcium overload, increase activity in T-SOD, decrease level of MDA and TXB2, improve level of 6-keto-PGF1a and MMP, and prevent the early onset cell apoptosis. CONCLUSION: The compatibility of four effective ingredients of Danhong had protective effect toward hippocampi hypoxia. The mechanism might be related to inhibit oxidative stress damage and cell apoptosis, resist thrombosis, and reduce the intracellular calcium ion of overload.

Protective effect of Danhong Injection combined with Naoxintong Capsule on cerebral ischemia-reperfusion injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong Injection (DHI) and Naoxintong Capsule (NXT) are renowned traditional Chinese medicine in China. The drug combination of DHI and NXT is frequently applied for the treatment of cardiovascular and cerebrovascular diseases in clinic. However, there had been no pharmacological experiment studies of interaction between DHI and NXT. Due to the drug interactions, exploring their interaction profile is of great importance. MATERIAL AND METHODS: In this study, focal cerebral I/R injury in adult male Sprague-Dawley rats were induced by transient middle cerebral artery occlusion (tMCAO) for 1h followed by reperfusion. Rats were divided into 5 groups: sham group, ischemia reperfusion untreated group (IRU), DHI group (DHI 10mL/kg/d), NXT group (NXT 0.5g/kg/d), DHI plus NXT group (DHI-NXT, DHI 10mL/kg/d plus NXT 0.5g/kg/d). All drug-treated groups were respectively successive administrated for 7 days after ischemia/ reperfusion (I/R) injury. The effects on rat neurological function were estimated by neurological defect scores. Brain infarct volumes were determined based on 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining and transmission electron microscope (TEM). Levels of nitric oxide (NO), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) in serum were determined with enzyme-linked immunosorbent assay (ELISA). Immunohisto-chemistry and Western blot were used to detect the expressions of basic fibroblast growth factor (bFGF), von Willebrand factor-microvessel vascular density (vWF-MVD), vascular endothelial cell growth factor (VEGF), transforming growth factor-ß1 (TGF-ß1), angiogenin-1 (Ang-1), angiogenin-2 (Ang-2) and platelet derived growth factor (PDGF) at day 7 after ischemia/reperfusion (I/R) injury. RESULTS: Compared with IRU group and mono-therapy group (DHI group or NXT group), Danhong Injection combined with Naoxintong Capsule (DHI-NXT) group significantly ameliorated neurological deficits scores, infarct volume and pathological change, significantly decreased the overexpression of NO and the level of Ang-1, significantly increased the expressions of VEGF, Ang-2, G-CSF, GM-CSF, bFGF, PDGF, vWF, TGF-ß1. CONCLUSION: The protective benefits on rat brain against I/R injury were clearly produced when DHI and NXT were used in combination, which provided rational guidance for clinical combined application of DHI and NXT, and this protection maybe associated with the up-regulation expressions of the related chemokines and growth factors of angiogenesis.

Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells.

OBJECTIVE: To observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs). METHODS: VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. RESULTS: Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). CONCLUSIONS: Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.