Qualitative and Quantitative Analysis of Chemical Components in Yinhua Pinggan Granule with High-Performance Liquid Chromatography Coupled with Q-Exactive Mass Spectrometry.

Yinhua Pinggan Granule (YPG) is an approved compounded traditional Chinese medicine (TCM) prescription for the treatment of cold, cough, viral pneumonia, and related diseases. Due to its complicated chemical composition, the material basis of YPG has not been systematically investigated. In this study, an analytical method based on high-performance liquid chromatography (HPLC) coupled with Q-Exactive mass spectrometry was established. Together with the help of a self-built compound database and Compound Discoverer software 3.1, the chemical components in YPG were tentatively identified. Subsequently, six main components in YPG were quantitatively characterized with a high-performance liquid chromatography-diode array detector (HPLC-DAD) method. As a result, 380 components were annotated, including 19 alkaloids, 8 organic acids, 36 phenolic acids, 27 other phenols, 114 flavonoids, 75 flavonoid glycoside, 72 terpenes, 11 anthraquinones, and 18 other compounds. Six main components, namely, chlorogenic acid, puerarin, 3'-methoxypuerarin, polydatin, glycyrrhizic acid, and emodin, were quantified simultaneously. The calibration curves of all six analytes showed good linearity (R2 > 0.9990) within the test ranges. The precision, repeatability, stability, and recovery values were all in acceptable ranges. In addition, the total phenol content and DPPH scavenging activity of YPG were also determined. The systematic elucidation of the chemical components in YPG in this study may provide clear chemical information for the quality control and pharmacological research of YPG and related TCM compounded prescriptions.

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.

Role of the neurovascular unit in the process of cerebral ischemic injury.

Cerebral ischemic injury exhibits both high morbidity and mortality worldwide. Traditional research of the pathogenesis of cerebral ischemic injury has focused on separate analyses of the involved cell types. In recent years, the neurovascular unit (NVU) mechanism of cerebral ischemic injury has been proposed in modern medicine. Hence, more effective strategies for the treatment of cerebral ischemic injury may be provided through comprehensive analysis of brain cells and the extracellular matrix. However, recent studies that have investigated the function of the NVU in cerebral ischemic injury have been insufficient. In addition, the metabolism and energy conversion of the NVU depend on interactions among multiple cell types, which make it difficult to identify the unique contribution of each cell type. Therefore, in the present review, we comprehensively summarize the regulatory effects and recovery mechanisms of four major cell types (i.e., astrocytes, microglia, brain-microvascular endothelial cells, and neurons) in the NVU under cerebral ischemic injury, as well as discuss the interactions among these cell types in the NVU. Furthermore, we discuss the common signaling pathways and signaling factors that mediate cerebral ischemic injury in the NVU, which may help to provide a theoretical basis for the comprehensive elucidation of cerebral ischemic injury.

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.

Amygdalin prevents multidrug-resistant Staphylococcus aureus-induced lung epithelial cell injury by regulating inflammation and oxidative stress.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that can cause severe bacterial pneumonia. Amygdalin is the main active pharmaceutical ingredient of bitter almond, which has broad-spectrum antibacterial, anti-inflammatory, anti-oxidation and immunomodulatory effects. It is also the main ingredient of Yinhua Pinggan granule, which is commonly used to moisten the lung and relieve cough. However, little is known about the effects of amygdalin on MRSA. In this study, we found that amygdalin exhibited good antimicrobial activity in vitro against MRSA. Amygdalin has a protective effect on MRSA infected cells, and the effect is better when combined with levofloxacin. It also can reduce the adhesion and invasion of MRSA to cells. Amygdalin has anti-inflammatory and antioxidant effects, which can significantly reduce the increase of inflammatory factors and the production of ROS caused by infection. The protective mechanism of amygdalin on cells may be related to inhibiting the expression of NLRP3, ASC and IL-1ß pyroptosis pathways. Taken together, our study suggests that amygdalin exerts antibacterial effects by affecting biofilm formation, the expression of virulence factors, and drug resistance genes. Amygdalin combined with levofloxacin has a protective effect on A549 cells infected with MRSA, and the mechanism may be related to the inhibition of inflammatory response, oxidative damage and pyroptosis.

Lipid regulation of protocatechualdehyde and hydroxysafflor yellow A via AMPK/SREBP2/PCSK9/LDLR signaling pathway in hyperlipidemic zebrafish.

The consumption of a high-cholesterol diet is known to cause hyperlipidemia, which is one of the main risk factors for cardiovascular disease. Protocatechualdehyde (PCA) and hydroxysafflor yellow A (HSYA) are the active components of Salvia miltiorrhiza and safflower, respectively. However, their exact mechanism is still unclear. The aim of this study is to investigate its effects on lipid deposition and liver damage in hyperlipidemic zebrafish and its mechanism of anti-hyperlipidemia. The results showed that the use of PCA and HSYA alone and in combination can improve lipid deposition, slow behavior, abnormal blood flow and liver tissue damage, and the combined use is more effective. Further RT-qPCR results showed that PCA + HSYA can regulate the mRNA levels of PPAR-γ, SREBP2, SREBP1, HMGCR, PCSK9, mTOR, C/EBPα, LDLR, AMPK, HNF-1α and FoxO3a. The PCA + HSYA significantly improves lipid deposition and abnormal liver function in hyperlipidemic zebrafish larvae, which may be related to the AMPK/SREBP2/PCSK9/LDLR signaling pathway.

Naoxintong capsule remodels gut microbiota and ameliorates early-stage atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Naoxintong capsule (NXT) is a compound traditional Chinese medicine prescription with demonstrated effect for the treatment of cardiovascular and cerebrovascular diseases including atherosclerosis (AS). However, the pharmacological mechanisms of NXT in ameliorating early-stage AS are still unclear, especially regarding the role of gut microbiota. PURPOSE: This study is aiming to evaluate the therapeutic effect of NXT against early-stage AS, and further illustrate the potential correlations among AS, gut microbiota, and NXT. METHODS: Thirty-two male ApoE knockout mice (C57BL/6 background) were fed with a high cholesterol diet (HCD) for 4 weeks to establish an early-stage AS model. NXT in two different dosages and simvastatin (Simv) were than administrated for another 8 weeks. Lipid metabolism indicators and inflammation levels were measured with corresponding assay kits. Changes in blood vessels, liver lesions, and intestinal barrier proteins were evaluated with different staining methods. Furthermore, the gut microbiota structure was analyzed using 16S rRNA sequencing technology, while GC-MS was utilized to determine the fecal contents of short-chain fatty acids (SCFAs). RESULTS: Administration of NXT significantly ameliorated obesity, hyperlipidemia, systemic inflammation, vasculopathy, liver injury, and intestinal barrier disorder in AS mice. Administration of NXT also significantly regulated the gut microbiota disturbance and increased the total contents of fecal SCFAs in AS mice. Furthermore, acetic acid content and the relative abundance of Faecalibacterium in feces were proposed as potential therapeutic biomarkers of NXT for AS treatment as indicated via the correlation analysis. CONCLUSION: This study demonstrated that NXT could effectively treat early-stage AS induced by HCD in mice. NXT regulated the gut microbiota and metabolites, maintained intestinal homeostasis, and improved the systemic inflammatory response. These findings may provide robust experimental support for the clinical use of NXT for AS treatment.

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.

Amygdalin Reverses Macrophage PANoptosis Induced by Drug-Resistant Escherichia coli.

Infectious diseases caused by drug-resistant Escherichia coli (E. coli) pose a critical concern for medical institutions as they can lead to high morbidity and mortality rates. In this study, amygdalin exhibited anti-inflammatory and antioxidant activities, as well as other potentials. However, whether it could influence the drug-resistant E. coli-infected cells remained unanswered. Amygdalin was therefore tested in a cellular model in which human macrophages were exposed to resistant E. coli. Apoptosis was measured by flow cytometry and the lactate dehydrogenase (LDH) assay. Western immunoblotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to quantify interleukin-18 (IL-18), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). The production of reactive oxygen species (ROS) in macrophages was detected by ROS kit. The expression of panapoptotic proteins in macrophages was measured by qRT-PCR and Western immunoblotting. Drug-Resistant E. coli inhibited cell viability and enhanced apoptosis in the cellular model. In cells treated with amygdalin, this compound can inhibit cell apoptosis and reduce the expression of pro - inflammatory cytokines such as IL-1ß, IL-18 and IL-6. Additionally, it decreases the production of PANoptosis proteins, Furthermore, amygdalin lowered the levels of reactive oxygen species induced by drug-resistant E. coli, in cells, demonstrating its antioxidant effects. Amygdalin, a drug with a protective role, alleviated cell damage caused by drug-resistant E. coli in human macrophages by inhibiting the PANoptosis signaling pathway.

Polydatin prevent lung epithelial cell from Carbapenem-resistant Klebsiella pneumoniae injury by inhibiting biofilm formation and oxidative stress.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes severe inflammation in various infectious diseases, such as bloodstream infections, respiratory and urinary tract infections, which leads to high mortality. Polydatin (PD), an active ingredient of Yinhuapinggan granule, has attracted worldwide attention for its powerful antioxidant, anti-inflammatory, antitumor, and antibacterial capacity. However, very little is known about the effect of PD on CRKP. In this research, we evaluated the inhibitory effects of PD on both the bacterial level and the bacterial-cell co-culture level on anti-biofilm and efflux pumps and the other was the inhibitory effect on apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) after CRKP induction. Additionally, we validated the mechanism of action by qRT-PCR and western blot in human lung epithelial cells. Firstly, PD was observed to have an inhibitory effect on the biofilm of CRKP and the efflux pump AcrAB-TolC. Mechanically, CRKP not only inhibited the activation of Nuclear Factor erythroid 2-Related Factor 2 (Nrf-2) but also increased the level of ROS in cells. These results showed that PD could inhibit ROS and activate Nrf-2 production. Together, our research demonstrated that PD inhibited bacterial biofilm formation and efflux pump AcrAB-TolC expression and inhibited CRKP-induced cell damage by regulating ROS and Nrf-2-regulated antioxidant pathways.

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.

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.

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.

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%.

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.

Neuroprotective Effect of Danhong Injection on Cerebral Ischemia-Reperfusion Injury in Rats by Activation of the PI3K-Akt Pathway.

Many traditional Chinese medicines, including Danhong injection (DHI), can be used to treat cerebral ischemia-reperfusion injury and have neuroprotective effects on the brain; however, few studies have explored the mechanism by which this effect is generated. In this study, we investigated the neuroprotective effect of DHI against cerebral ischemia-reperfusion injury mediated via the PI3K-Akt signaling pathway. After establishing the model of middle cerebral artery occlusion (MCAO), 60 male Sprague-Dawley rats were allocated to six groups as follows: sham, MCAO, DHI (MCAO + DHI), LY294002 (MCAO + LY294002 [PI3K-Akt pathway specific inhibitor]), DHI + LY294002 (MCAO + DHI + LY294002), and NMDP + LY294002 (MCAO + NMDP [nimodipine] + LY294002). Hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to evaluate the pathological changes of brain tissue and the degree of neuronal apoptosis. Real-time quantitative polymerase chain reaction (qRT-PCR), western blot analysis and enzyme-linked immunosorbent assays were used to measure the expression of Bad, Bax, Bcl-2, Bim, P53, MDM2, Akt, PI3K, p-Akt, p-PI3K, and Cyt-C. Compared with the MCAO group, brain tissue cell apoptosis was significantly reduced in the DHI group, and the brain function score was significantly improved. In addition, the expression of pro-apoptotic factors (Bad, Bax, and Bim) was significantly downregulated in the DHI group, while expression of the anti-apoptotic factor Bcl-2 was significantly upregulated, and expression of the apoptotic gene p53 was also significantly attenuated. Moreover, this neuroprotective effect was attenuated by the PI3K-Akt signaling pathway inhibitor (LY294002). Thus, our results confirmed the neuroprotective effects of DHI in rats with ischemia-reperfusion injury and indicate that these effects on the brain are partly generated by activation of the PI3K-Akt signaling pathway.