Polypharmacology-based approach for screening TCM against coinfection of Mycoplasma gallisepticum and Escherichia coli.

Natural products and their unique polypharmacology offer significant advantages for finding novel therapeutics particularly for the treatment of complex diseases. Meanwhile, Traditional Chinese Medicine exerts overall clinical benefits through a multi-component and multi-target approach. In this study, we used the previously established co-infection model of Mycoplasma gallisepticum and Escherichia coli as a representative of complex diseases. A new combination consisting of 6 herbs were obtained by using network pharmacology combined with transcriptomic analysis to reverse screen TCMs from the Chinese medicine database, containing Isatdis Radix, Forsythia Fructus, Ginkgo Folium, Mori Cortex, Licorice, and Radix Salviae. The results of therapeutic trials showed that the Chinese herbal compounds screened by the target network played a good therapeutic effect in the case of co-infection. In summary, these data suggested a new method to validate target combinations of natural products that can be used to optimize their multiple structure-activity relationships to obtain drug-like natural product derivatives.

Supramolecular self-assembly of amantadine hydrochloride with ferulic acid via dual optimization strategy establishes a precedent of synergistic antiviral drug-phenolic acid nutraceutical cocrystal.

To display the capability of the phenolic acid nutraceutical ferulic acid (FLA) in optimizing the in vitro/in vivo properties of the antiviral drug amantadine hydrochloride (AMH) and achieve synergistically enhanced antiviral effects, thereby gaining some new insights into pharmaceutical cocrystals of antiviral drugs with phenolic acid nutraceuticals, a cocrystallization strategy of dual optimization was created. Based on this strategy, the first drug-phenolic acid nutraceutical cocrystal of AMH with FLA, namely AMH-FLA-H2O, was successfully assembled and completely characterized by employing single-crystal X-ray diffraction and other analytical techniques. The cocrystal was revealed to be composed of AMH, FLA, and water molecules in the ratio of 3 : 1 : 1.5, and charge-assisted hydrogen bonds containing chloride ions crucially maintained the crystal lattice together with water molecules. The in vitro/in vivo properties of the cocrystal were systematically evaluated via both theoretical and experimental methods, and the results indicate that the dissolubility of AMH is down-regulated by two-thirds in the cocrystal, resulting in its potential for sustained pharmacokinetic release and the elimination of the adverse effects of AMH. More importantly, the enhanced antiviral effects of the current cocrystal were proven against four viral strains, and the pharmaceutical synergy between AMH and FLA was realized with a combination index (CI) of less than 1. Thus, the present work provides a novel crystalline product with bright commercial prospect for the classical antiviral drug AMH and also establishes an avenue for the synergetic antiviral application of nutraceutical phenolic acids via the cocrystallization strategy of dual optimization.

Supramolecular self-assembly and perfected in vitro/vivo property of 5-fluorouracil and ferulic acid on the strength of double optimized strategy: the first 5-fluorouracial-phenolic acid nutraceutical cocrystal with synergistic antitumor efficacy.

For highlighting the predominance of phenolic acid nutraceutical ferulic acid (FR) in regulating the in vivo/vitro performances of anticancer drug 5-fluorouracil (Flu) and strengthening their cooperativity in antitumor effect, thus achieving a major breakthrough in the development of drug-nutraceutical cocrystal with synergistic antitumor action, a cocrystallization strategy of dual optimization is created, in which both the in vivo and vitro natures of Flu are improved by exploiting the FR's excellent physicochemical property. Moreover, Flu's anticancer effects were promoted by exerting the assistant antitumor peculiarity of FR. Such dual optimization of FR for Flu in physicochemical properties and anticancer activities is beneficial for realizing synergistic augmentation effect by taking the benefit of the cooperativeness of Flu and FR in the anticancer ability. Based on this idea, a novel cocrystal of Flu and FR, namely, Flu-FR-H2O, is successfully assembled as the first 5-fluorouracil-nutraceutical cocrystal with synergistic antitumor effect and its explicit structure is resolved. The single-crystal X-ray diffraction demonstrates that Flu and FR have a ratio of 1 : 1 with one equivalent of solvent water in the cocrystal, where one-dimensional hydrogen-bonding helices and FR-Flu hydrogen-bonding pairs, together construct a three-dimensional supramolecular network. By combining experimental evaluation with theoretical analysis, in vitro/vivo pharmaceutical properties are scientifically investigated. Results show that the permeability and aqueous solubility of Flu are respectively elevated by 5.08 and 1.64 folds, which has brought about ameliorated pharmacokinetics, thus providing prolonged retention time and increased oral bioavailability. More interestingly, the cocrystal shows synergistic inhibition ability of Flu and FR against tested tumor cell strains, hence laying the groundwork for reducing the dosage and even the toxic side effects of Flu. As a result of this, the present research not only provides a new strategy for Flu to optimize its physicochemical properties and antitumor activities simultaneously but also offers some opinions for the development of synergistic antitumor pharmaceutical cocrystals.

Blockade of Indoleamine 2, 3-dioxygenase 1 ameliorates hippocampal neurogenesis and BOLD-fMRI signals in chronic stress precipitated depression.

Indoleamine 2, 3-dioxygenase 1 (IDO1) has been implicated in the pathogenesis of depression, though its molecular mechanism is still poorly understood. We investigated the molecular mechanism of IDO1 in depression by using the chronic unpredictable mild stress (CUMS) model in Ido1-/- mice and WT mice. The brain blood oxygen level dependent (BOLD) signals in mice were collected by functional magnetic resonance imaging (fMRI) technology. IDO1 inhibitor INCB024360 was intervened in dorsal raphe nucleus (DRN) through stereotactic injection. We found an elevation of serum IDO1 activity and decreased 5-HT in CUMS mice, and the serum IDO1 activity was negatively correlated with 5-HT level. Consistently, IDO1 was increased in hippocampus and DRN regions, accompanied by a reduction of hippocampal BDNF levels in mice with CUMS. Specifically, pharmacological inhibition of IDO1 activity in the DRN alleviated depressive-like behaviour with improving hippocampal BDNF expression and neurogenesis in CUMS mice. Furthermore, ablation of Ido1 exerted stress resistance and decreased the sensitivity of depression in CUMS mice with the stable BOLD signals, BDNF expression and neurogenesis in hippocampus. Thus, IDO1 hyperactivity played crucial roles in modulating 5-HT metabolism and BDNF function thereby impacting outcomes of hippocampal neurogenesis and BOLD signals in depressive disorder.

A field amplification enhanced paper-based analytical device with a robust chemiluminescence detection module.

A sensitive detection method combined with an effective on-line concentration may improve the analytical performance of a paper-based analytical device (PAD), and its merits of low cost and portability in POCT are fully demonstrated. Here, a sensitive PAD system with chemiluminescence (CL) detection and electrokinetic preconcentration was introduced, and the performance was demonstrated by the detection of hemin. A commercially available low cost and miniaturized optical detection module was used for the CL detection. Firstly, hemin was stacked on a simple paper fluidic channel based on field amplified stacking (FAS), and then a CL reagent (luminol-H2O2) was loaded on the stacked band to initiate the CL reaction. The photons were directly detected using the detection module. With optimization of the background electrolyte (BGE), voltage and CL reagent, a limit of detection (LOD) of 0.58 nM for hemin was obtained with a linear range of 1-1000 nM (R2 = 0.995). With FAS, the signal intensity was about 13-fold enhanced. This PAD also exhibited satisfactory selectivity over possible interfering components at a 104 times higher concentration. The applicability of the PAD was demonstrated by the detection of hemin from iron supplements and human serum samples. With total manual operation, recovery rates of 84.8-115.6% were obtained with an RSD of less than 14.3%. With the introduction of the optical detection model, and together with FAS, both the LOD and dynamic range of this PAD were effectively improved.

Polydatin alleviated alcoholic liver injury in zebrafish larvae through ameliorating lipid metabolism and oxidative stress.

Hepatic steatosis is the early stage of alcoholic liver disease (ALD), may progress to steatohepatitis, fibrosis even cirrhosis. Polydatin, the primary active component of Polygonum cuspidatum Sieb. et Zucc, has been recognized to possess hepatoprotective and anti-inflammatory properties. To investigate whether polydatin alleviates ethanol induced liver injury and to elucidate the underlying molecular mechanisms, zebrafish larvae at 4 days post-fertilization (dpf) were exposed to 350 mmol/L of ethanol for 32 h, then treated with polydatin for 48 h. Oil red O, Nile Red and H&E staining were used to analyze the pathological changes in liver. The mRNA levels were measured by quantitative PCR and the antioxidant capacity was detected using H2O2-specific fluorescent probe. Here, polydatin strongly alleviated hepatic steatosis and decreased the expression levels of alcohol and lipid metabolism-related genes, including CYP2Y3, CYP3A65, HMGCRa, HMGCRb and FASN. Additionally, polydatin inhibited oxidative stress in the liver according to fluorescent probe. Moreover, significantly up-regulated expression of DNA damage-related genes (CHOP, GADD45αa) revealed that polydatin attenuated hepatic apoptosis in larvae. In conclusion, polydatin may improve the liver function of zebrafish with acute alcoholic liver injury through attenuating hepatic fat accumulation, ameliorating lipid and ethanol metabolism and reducing oxidative stress and DNA damage.

Optimization of baicalin water extraction process from Scutellaria baicalensis (a traditional Chinese medicine) by using orthogonal test and HPLC

ABSTRACT In this study, we optimized the baicalin water extraction process from Scutellaria baicalensis Georgi, Lamiaceae (a traditional Chinese medicine). Orthogonal test design L9(3)4 was used to analyze the optimization of water extraction process of baicalin from S. baicalensis. The effect of solid-liquid ratio, extraction time and soaking time on the yield of baicalin were investigated and optimized by orthogonal test. High-performance liquid chromatography was employed for the determination of extraction yield of baicalin. Analysis of variance was carried out to study the effects of the above three factors. The results showed that solid-liquid ratio plays a significant role in attaining maximum extraction yields of baicalin. However, the other two factors had some effect (not statistically significant) on the extraction yield of baicalin. Conclusively, the optimum experimental conditions such as the solid-liquid ratio (1:12), extraction time (30 min) and soaking time (1 h) for the water extraction of baicalin were proposed which can provide the maximum extraction yield of baicalin. In addition, the score based on the content of baicalin and total solid residues yield were used as evaluation indexes for baicalin uterus suppositories evaluation.

Plasma Riboflavin Level is Associated with Risk, Relapse, and Survival of Esophageal Squamous Cell Carcinoma.

Riboflavin is an essential micronutrient for normal cellular activity, and deficiency may result in disease, such as cancer. We performed a case-control study to explore the association of riboflavin levels with risk and prognosis of esophageal squamous cell carcinoma (ESCC). Plasma riboflavin levels, as measured by enzyme-linked immunosorbent assay (ELISA), in ESCC patients were significantly lower than in those of healthy controls (7.04 ± 6.34 ng/ml vs. 9.32 ± 12.40 ng/ml; P < 0.05). Moreover, there was an inverse relationship between riboflavin level and risk of ESCC (odds ratio (OR) = 0.97, 95% confidence interval (CI) = 0.95-0.99, P =  0.02). The 5-year relapse-free and overall survival rates were significantly lower when riboflavin levels were ≤0.8 ng/ml than >0.8 ng/ml (relapse-free survival rate: 29.4% vs. 54.8%; overall survival rate: 28.6% vs. 55.6%). Plasma riboflavin level was an independent protective factor for both relapse-free (hazard ratio (HR) = 0.325, 95% CI = 0.161-0.657, P = 0.002) and overall survival of ESCC patients (HR = 0.382, 95% CI = 0.190-0.768, P = 0.007). In conclusion, plasma riboflavin levels are significantly related to risk and prognosis of ESCC patients, suggesting that moderate supplementation of riboflavin will decrease risk and prevent recurrence of ESCC and also improve prognosis of ESCC patients.

Syntheses and crystal structures of tetracopper(II) complexes bridged by asymmetric N,N'-bis(substituted)oxamides: Molecular docking, DNA-binding and in vitro anticancer activity.

Two new tetranuclear copper(II) complexes of the formulae [Cu4(oxbm)2(phen)2](NO3)2⋅6H2O (1) and [Cu4(oxbpa)2(phen)2](ClO4)2·4H2O (2), where H3oxbm and H3oxbpa stand for N-(2-aminopropyl)-N'- (2-carboxylatophenyl)oxamide and N-hydroxypropyl-N'-(2-carboxylatophenyl)oxamide, respectively, and phen is 1,10-phenanthroline, have been synthesized and characterized by elemental analyses, molar conductivity measurements, IR and electronic spectrum studies, and X-ray single crystal diffraction. In the two tetracopper(II) complexes, the presence of the circular tetracopper(II) cations is assembled by a pair of cis-oxamido-bridged dicopper(II) units through carboxyl bridges, in which Cu1 is located in a distorted square-planar environment, while Cu2 is in a distorted square-pyramidal geometry. Numerous hydrogen bonds link complex 1 or 2 into a 2-D infinite network. The interactions of the two tetracopper(II) complexes with DNA are investigated both theoretically and experimentally, revealing that these tetracopper(II) complexes can interact with HS-DNA in the mode of intercalation, and complex 1 possesses stronger intercalating ability. The molecular docking of the two tetranuclear copper(II) complexes with the self-complementary DNA duplex of sequence d(ACCGACGTCGGT)2 facilitates the binding events. Cytotoxicity experiments indicate that the two tetracopper(II) complexes exhibit cytotoxic effects against human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549. Interestingly, the cytotoxic activities of the two tetracopper(II) complexes are consistent with their DNA-binding abilities, following the order of 1>2. The main results suggest that different bridging ligands in tetracopper(II) complexes may play an important role in the DNA-binding properties and cytotoxic activities.

[Effects of rosuvastatin on monocrotaline-induced pulmonary artery hypertension in rats].

OBJECTIVE: To investigate the effects of rosuvastatin on monocrotaline (MCT)-induced pulmonary artery hypertension in rats. METHODS: Pulmonary arterial hypertension was induced by a single subcutaneous injection of monocrotaline (50 mg/kg) in rats. In the prevention protocol, 32 male Sprague-Dawley rats were randomly divided into four groups (n = 8 each): low-dose rosuvastatin prevention group (2 mg×kg(-1)×d(-1)), high-dose rosuvastatin prevention group (10 mg×kg(-1)×d(-1)), pulmonary arterial hypertension group, normal control group. Beginning on the MCT injection day, rats were treated with rosuvastatin by daily gavage for 4 weeks. Normal control group and pulmonary arterial hypertension group received vehicle by gavage. In the treatment protocol, 52 male Sprague-Dawley rats were randomly divided into four groups (n = 13 each): low-dose rosuvastatin treatment group (2 mg×kg(-1)×d(-1)), high-dose rosuvastatin treatment group (10 mg×kg(-1)×d(-1)), pulmonary arterial hypertension group, normal control group. Four weeks after MCT injection, rats were treated with rosuvastatin by daily gavage for 4 weeks. Normal control group and pulmonary arterial hypertension group received vehicle by gavage. At the end of study, survival rates, mean pulmonary arterial pressure (mPAP), wall thickness of small pulmonary artery and right ventricular hypertrophy among groups were compared. The expression levels of proliferating cell nuclear antigen (PCNA) and endothelial nitricoxide synthase (eNOS) protein in small pulmonary artery, the expression levels of Rho kinase 1(ROCK-1) and eNOS mRNA in lung tissue were also detected. RESULTS: All rats in the prevention protocol survived. Rosuvastatin treatment improved survival in the treatment protocol (58%, 75% vs.30%, P < 0.05). Rosuvastatin therapy in both preventive or treatment protocols significantly lowered mPAP [prevention protocol: (27.53 ± 3.43), (25.72 ± 1.76) vs. (36.05 ± 2.45) mm Hg (1 mm Hg = 0.133 kPa), P < 0.01; treatment protocol: (30.39 ± 3.17), (27.59 ± 1.99) vs. (40.68 ± 1.39) mm Hg, P < 0.01], reduced thickening of small pulmonary artery wall (P < 0.01) and right ventricular hypertrophy (P < 0.01). Rosuvastatin also inhibited PCNA expression of SMC (P < 0.01), restored eNOS expression of EC (P < 0.05) and inhibited ROCK-1 mRNA expressions in lung tissue (P < 0.05). CONCLUSIONS: Rosuvastatin therapy reduced mPAP in monocrotaline-induced pulmonary arterial hypertension rat model and this effect is linked with inhibition of ROCK-1 expression, inhibition of smooth muscle cell proliferation and restoration of endothelial cell functions.

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution.

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl(3)-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl(3)-MeOH-0.2 M HCl (4:2:2, v/v) and CHCl(3)-MeOH-0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of (1)H-NMR, (13)C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

Role of PGI2 in the formation and maintenance of hyperdynamic circulatory state of portal hypertensive rats.

AIM: To investigate the role of prostacyclin (PGI(2)) and nitric oxide (NO) in the development and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats. METHODS: Ninety male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) group by injection of CCl(4), prehepatic portal hypertension (PHPH) group by partial stenosis of the portal vein and sham-operation control (SO) group. One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group (n = 23), Nomega-nitro-L-arginine (L-NNA) group (n = 21) group, indomethacin (INDO) group (n = 22) and high-dose heparin group (n = 24). The rats were administrated 1 mL of saline, L-NNA (3.3 mg/kg.d) and INDO (5 mg/kg.d) respectively through gastric tubes for one week, then heparin (200 IU/Kg/min) was given to rats by intravenous injection for an hour. Splanchnic and systemic hemodynamics were measured using radioactive microsphere techniques. The serum nitrate/nitrite (NO(2)(-)/NO(3)(-)) levels as a marker of production of NO were assessed by a colorimetric method, and concentration of 6-keto-PGF1alpha, a stable hydrolytic product of PGI(2), was determined by radioimmunoassay. RESULTS: The concentrations of plasma 6-keto-PGF1alpha (pg/mL) and serum NO(2)(-)/NO(3)(-) (micromol/L) in IHPH rats (1123.85+/-153.64, 73.34+/-4.31) and PHPH rats (891.88+/-83.11, 75.21+/-6.89) were significantly higher than those in SO rats (725.53+/-105.54, 58.79+/-8.47) (P<0.05). Compared with SO rats, total peripheral vascular resistance (TPR) and spanchnic vascular resistance (SVR) decreased but cardiac index (CI) and portal venous inflow (PVI) increased obviously in IHPH and PHPH rats (P<0.05). L-NNA and indomethacin could decrease the concentrations of plasma 6-keto-PGF1alpha and serum NO(2)(-)/NO(3)(-) in IHPH and PHPH rats (P<0.05). Meanwhile, CI, FPP and PVI lowered but MAP, TPR and SVR increased (P<0.05). After deduction of the action of NO, there was no significant correlation between plasma PGI(2) level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI(2), NO still correlated highly with the hemodynamic parameters, indicating that there was a close correlation between NO and the hemodynamic parameters. After administration of high-dose heparin, plasma 6-keto-PGF(1alpha) concentrations in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle (P<0.05). On the contrary, levels of serum NO(2)(-)/NO(3)(-) in IHPH, PHPH and SO rats were significantly lower than those in rats administrated Vehicle (P<0.05). Compared with those rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats, such as CI and PVI, declined significantly after administration of high-dose heparin (P<0.05), while TPR and SVR increased significantly (P<0.05). CONCLUSION: It is NO rather than PGI(2) that is a mediator in the formation and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.