UPLC-Q-Exactive/MS based analysis explore the correlation between components variations and anti-influenza virus effect of four quantified extracts of Chaihu Guizhi decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaihu Guizhi decoction (CGD) is a classic Traditional Chinese Medicine (TCM) prescription for the treatment of influenza and fever, composes of Bupleuri Radix (Chaihu), Cinnamomi Ramulus (Guizhi), Scutellariae Radix (Huangqin), Codonopsis Radix (Dangshen), Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle (Zhigancao), Pinelliae Rhizoma Praeparatum (Fabanxia), Zingiberis Rhizoma Recens (Shengjiang), Paeoniae Radix Alba (Baishao) and Jujubae Fructus (Dazao) in the ratio of 12:4.5:4.5:4.5:3:6:4.5:4.5:4. The efficacy of TCM, if there are differences, depends on the different extraction methods and extracted components. AIM OF THE STUDY: This study was to evaluate the anti-influenza virus effect of CGD extracts with different extraction methods, analyze the components and explore their correlation. MATERIALS AND METHODS: CGD were prepared with four extraction methods respectively, the traditional decoction (TD), two steps alcohol-water extraction (AWE), alcohol reflux extraction (AE) and water reflux extraction (WE). Based on the influenza mouse model, the efficacy of anti-influenza virus in vivo of the four CGD extracts were evaluated with the therapeutic index of body weight, rectal temperature, lung index, thymus index and lung viral load of mice. The chemical components in four CGD extracts, and compounds absorbed in rats blood with prototypes or metabolites were identified by UPLC-Q-Exactive/MS. The partial least squares (PLS) method was used to explore the correlation between the components variation in CGD extracts and the comprehensive efficacy index. The potential effective components were further accessed by molecular docking. RESULTS: Comparing with the other three extracts, AWE has the best anti-influenza effect. It could ameliorate the symptoms caused by influenza virus infection in mice, increase body weight and rectal temperature, reduce the lung index and virus load in lung tissue. 129, 144, 140 and 129 components were identified from TD, AWE, AE, and WE respectively. The identified components were mainly including flavonoids, terpenoids, organic acids, phenylpropanoids, amino acids, nucleosides, phenols, alkaloids, etc. 43 prototypes and 49 metabolites of CGD were detected in rat plasma after oral administration. Seven components, cinnamaldehyde, wogonoside, baicalin, baicalein, gallic acid, oroxylinA-7-O-glucuronide and coumarin, showed significant correlation with anti-influenza effects, all of which had good binding activity with NA, IL-6, STAT3, AKT1, EGFR and TNF. CONCLUSION: Two steps alcohol-water extraction was optimal for CGD preparation. Cinnamaldehyde, wogonoside, oroxylinA-7-O-glucuronide, coumarin, gallic acid, baicalein and baicalin play a certain essential role in anti-influenza effects and may be taken as a potential maker compounds for quality evaluation of CGD.

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking.

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-ß-D-glucoside, emodin-8-O-ß-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.

Mahuang Xixin Fuzi decoction ameliorates apoptosis via the mitochondrial-mediated signaling pathway in MCM cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mahuang Xixin Fuzi Decoction (MXF), as a classical prescription of traditional Chinese medicine (TCM), has been used to treat the symptoms of fever, nasal congestion and headache in elderly people for almost a thousand years. AIM OF THE STUDY: The purpose of this study was to evaluate the effects and possible mechanisms of MXF on thermal stimulation-induced mouse cardiac myocytes (MCM) cell apoptosis. MATERIALS AND METHODS: The apoptosis of the MCM cell model was induced by a PCR-calculated temperature control system with a gradual heating pattern at 43 °C for 1 h. The cytotoxic effects were determined using real-time cell analyzer (RTCA) technology. Annexin V-FITC/7-AAD staining, and JC-1 fluorescence were used to assess apoptosis. Specific substrates, enzyme-linked immunosorbent assays (ELISAs), and Western blotting were used to identify proteins in the mitochondrial-mediated pathway. The identification of chemical components in the mouse heart was performed by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry analysis. RESULTS: MXF inhibited apoptosis through the mitochondrial-mediated signaling pathway, including ameliorating ∆Ψm reduction, blocking mitochondrial Cyt C release, reducing Bax levels and increasing Bcl-2 levels, suppressing caspase-9 and caspase-3 activation in cytoplasmic fractions. Moreover, the components of MXF that act on the heart are mainly ephedra alkaloids and aconitine alkaloids. CONCLUSIONS: The findings demonstrated that MXF treatment markedly reduced MCM cell apoptosis induced by thermal stimulation, which may be ascribed to the mitochondrial-mediated signaling pathway.

Urinary Metabolomics Study of the Intervention Effect of Hypoglycemic Decoction on Type 2 Diabetes Mellitus Rats Model.

The hypoglycemic decoction (HD) is a traditional Chinese medicine (TCM) preparation for the treatment of diabetes mellitus (DM), with a remarkable therapeutic effect. However, its mechanism of action is still unclear at the metabolic level. In this study, the biochemical markers from type 2 DM (T2DM) rats, induced by a high-sugar and high-fat diet combined with streptozotocin (STZ), were detected. The metabolomics-based analysis using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was conducted to evaluate urine samples from control, model, metformin, and HD groups. After oral administration of HD for 28 days, the general state, weight, fasting blood glucose (FBG), blood lipid level, oral glucose tolerance test (OGTT), fasting insulin (FINS), insulin sensitivity index (ISI), and homeostasis model assessment of insulin resistance (HOMA-IR) were significantly improved (P < 0.01). The western blotting showed that HD can enhance the protein expression of glucose transporter 4 (GLUT4) and adenosine monophosphate-activated protein kinase (AMPK). The metabolomics results revealed that after treatment with HD, the levels of L-carnitine, 1-methyladenosine, 1-methylhistamine, and 3-indoleacrylic acid were upregulated and the levels of riboflavin, phenylalanine, atrolactic acid, 2-oxoglutarate, citrate, isocitrate, cortisol, and glucose were downregulated. The main mechanism may be closely related to the regulation of the tricarboxylic acid (TCA) cycle, phenylalanine metabolism, glyoxylate metabolism, and dicarboxylate metabolism. Additionally, it was also found that HD can regulate the protein expression of GLUT4 and AMPK to interfere with TCA cycle and carbohydrate metabolism to treat T2DM.

[Action mechanism of Mahuang Xixin Fuzi decoction for mice with influenza based on metabolomics information].

This study aimed to analyze the endogenous metabolite changes in the serum of mice infected with H1N1 virus after intervention by Mahuang-Xixin-Fuzi decoction (MXF) based on metabolomics method, investigate potential biomarkers and related metabolic pathways, and explore the therapeutic mechanism of MXF through metabolomics technology. Thirty-six Kunming (KM) mice were randomly divided into three groups: normal group, model group and MXF group. Influenza virus H1N1 was used by nasal drip to establish influenza mice model. The mice in MXF group were orally administrated with MXF for 6 consecutive days after inoculation, and the other two groups were given with equal volume of saline solution in the same way. Body weight, rectal temperature, morbidity and mortality were recorded daily. Serum samples were collected 24 hours after the last administration for HPLC-TOF-MS analysis. The results showed that as compared with the normal group, the body weight and rectal temperature were decreased in model group, and their lung index and mortality rate were significantly increased (P<0.05); MXF had good therapeutic effects on the abnormity of body weight, rectal temperature, lung index and high mortality rate of mice infected with H1N1 virus. The original data collected from the serum samples were analyzed with R language, MPP, SIMCA-P and other software, and significant changes were found in 14 kinds of endogenous substances from mice serum (P<0.05). As compared with model group, the potential metabolic markers in MXF group recovered to normal levels to a certain degree after being intervened by MXF. Further analysis with MetPA data platform showed that, the pathways involved in 14 metabolites included glucose metabolism, arachidonic acid metabolism, glycerophospholipids and sphingolipids metabolism etc. The metabolomics study and pharmacological experiment showed that MXF might play a role of efficacy by improving glucose metabolism, regulating arachidonic acid metabolism, glycerophospholipid and sphingolipid metabolic pathways.

Qualitative and quantitative analysis of the chemical constituents in Mahuang-Fuzi-Xixin decoction based on high performance liquid chromatography combined with time-of-flight mass spectrometry and triple quadrupole mass spectrometers.

High-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF/MS) and high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang-Fuzi-Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC-QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC-QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter- and intra-day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang-Fuzi-Xixin decoction.