Ya-Jun Yang

A 15-day oral dose toxicity study of aspirin eugenol ester in Wistar rats.

The subchronic toxicity of aspirin eugenol ester (AEE) was evaluated after 15-day intragastrically administration in rats at daily doses of 50, 1000, and 2000 mg/kg. AEE at low-dose showed no toxicity to the tested rats. Following repeated exposure to medium- or high-dose of AEE, apparent changes were observed in the levels of blood glucose, AST, ALP, ALT and TB in both male and female rats, and appeared to be dose-independent. There were no significant gender differences in most indexes of subchronic toxicity throughout the experimental period with the exception of food consumption and body weight. The no-observed-adverse-effect level (NOAEL) of AEE was considered to be 50 mg/kg/day under the present study conditions.

Synthesis of aspirin eugenol ester and its biological activity

Aspirin, as starting precursor, was reacted with SOCl2 to generate acyl chloride compound via esterifying eugenol to aspirin eugenol ester (AEE) with a yield of 65%. Tests of AEE for acute toxicity, anti-inflammatory, analgesic, and antipyretic effects were carried out in mice and rats. The results showed that toxicity of AEE was significantly reduced, about 50 and 3.7 times lower than aspirin and eugenol, respectively. Its strength of anti-inflammation, analgesia, and antipyresis was similar as aspirin and eugenol, but the effects lasted longer.

In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with fewer side effects than its precursor, aspirin. Investigation into its metabolic process in target animal species will help to illustrate its mechanism of action and to establish its residual mark compound to formulate its dosage. Six beagle dogs were orally given a dose of 20 mg kg(-1) of AEE and one dog was used to prepare blank liver microsomes. Their liver microsomes were prepared for in vitro study and their plasma and urine were collected for in vivo metabolic analysis using liquid chromatography tandem mass spectrometry. In this study we identified 10 metabolites, M1, M2, M3, M4, M5 in phase I and M6, M7, M8, M9, M10 in phase II. Based on the metabolites of AEE, the pathways of AEE metabolism in dog were demonstrated.

Genotoxic evaluation of aspirin eugenol ester using the Ames test and the mouse bone marrow micronucleus assay

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with less side effects and it is important to characterize its genotoxicity. In this study, the genotoxicity of AEE was assessed with two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and the mouse bone marrow micronucleus assay. In the Ames test, Salmonella strains TA97, TA98, TA100, TA102 and TA1535 were treated with or without the metabolic activation with a S9 fraction from Acroclor-induced rat liver. The doses of AEE were 5 mg/plate, 2.5 mg/plate, 1.25 mg/plate, 0.625 mg/plate and 0.3125 mg/plate, respectively. In the above tested strains, mutagenicity with or without the S-9 mixture was not detected. In the mammalian erythrocyte micronucleus assay, fifty mice were divided into five groups evenly and the AEE dose at 5000 mg/kg, 2500 mg/kg and 1250 mg/kg and the cyclophosphamide dose at 40 mg/kg as a positive control, the 0.5% of CMC-Na as negative control were administered. The results showed that AEE did not induce any significant increase in micronucleated erythrocytes after 24 h (p<0.01). Our results suggested that AEE was non-genotoxic in vivo or in vitro.

A non-biological method for screening active components against influenza virus from traditional Chinese medicine by coupling a LC column with oseltamivir molecularly imprinted polymers.

To develop a non-biological method for screening active components against influenza virus from traditional Chinese medicine (TCM) extraction, a liquid chromatography (LC) column prepared with oseltamivir molecularly imprinted polymer (OSMIP) was employed with LC-mass spectrometry (LC-MS). From chloroform extracts of compound TCM liquid preparation, we observed an affinitive component m/z 249, which was identified to be matrine following analysis of phytochemical literatures, OSMIP-LC column on-line of control compounds and MS/MS off-line. The results showed that matrine had similar bioactivities with OS against avian influenza virus H9N2 in vitro for both alleviating cytopathic effect and hemagglutination inhibition and that the stereostructures of these two compounds are similar while their two-dimensional structures were different. In addition, our results suggested that the bioactivities of those affinitive compounds were correlated with their chromatographic behaviors, in which less difference of the chromatographic behaviors might have more similar bioactivities. This indicates that matrine is a potential candidate drug to prevent or cure influenza for human or animal. In conclusion, the present study showed that molecularly imprinted polymers can be used as a non-biological method for screening active components against influenza virus from TCM.

UPLC-Q-TOF/MS-based urine and plasma metabonomics study on the ameliorative effects of aspirin eugenol ester in hyperlipidemia rats

ABSTRACT The main objective of this study was to investigate the ameliorative effects of aspirin eugenol ester (AEE) in hyperlipidemic rat. After five‐week oral administration of AEE in high fat diet (HFD)‐induced hyperlipidemic rats, the impact of AEE on plasma and urine metabonomics was investigated to explore the underlying mechanism by UPLC‐Q‐TOF/MS analysis. Blood lipid levels and histopathological changes of liver, stomach and duodenum were also evaluated after AEE treatment. Without obvious gastrointestinal (GI) side effects, AEE significantly relieved fatty degeneration of liver and reduced triglyceride (TG), low density lipoprotein (LDL) and total cholesterol (TCH) (P < 0.01). Clear separations of metabolic profiles were observed among control, model and AEE groups by using principal component analysis (PCA) and orthogonal partial least‐squares‐discriminate analysis (OPLS‐DA). 16 endogenous metabolites in plasma and 18 endogenous metabolites in urine involved in glycerophospholipid metabolism, fatty acid metabolism, fatty acid beta‐oxidation, amino acid metabolism, TCA cycle, sphingolipid metabolism, gut microflora and pyrimidine metabolism were considered as potential biomarkers of hyperlipidemia and be regulated by AEE administration. It might be concluded that AEE was a promising drug candidate for hyperlipidemia treatment. These findings could contribute to the understanding of action mechanisms of AEE and provide evidence for further studies. Graphical abstract Figure. No Caption available. HighlightsAspirin eugenol eater (AEE) reduced blood lipid levels.AEE relieved fatty degeneration in liver in hyperlipidemic rat.36 biomarkers associated with hyperlipidemia were regulated by AEE.Potential mechanism of AEE on hyperlipidemia was revealed by pathway analysis.

Simultaneous determination of diaveridine, trimethoprim and ormetoprim in feed using high performance liquid chromatography tandem mass spectrometry.

This study developed and validated a simple and reliable method for detecting and quantifying DVD, TMP and OMP in feed using dichloromethane extraction followed by HPLC-MS/MS. A matrix effect evaluation was performed using the post-extraction spiking method, and levels were less than ±15% in all three feeds with their corresponding concentrations. LOD and LOQ, CCα and CCβ were 20μgkg(-1) and 40μgkg(-1), 8.68-15.55μgkg(-1) and 10.61-18.92μgkg(-1) for all analytes, respectively. Calibration curves were linear for DVD, TMP and OMP with R(2)⩾0.990 and r⩾0.995, respectively. Recoveries of low, medium and high concentrations using the proposed method ranged from 74.4 to 105.2%. Repeatability and within-laboratory reproducibility were <7.4% (RSD). The chosen seven factors had no a significant influence on robustness. The method showed good performance when it was applied to analyze other laboratory-prepared or actual feed samples.

Preventive Effect of Aspirin Eugenol Ester on Thrombosis in κ-Carrageenan-Induced Rat Tail Thrombosis Model

Based on the prodrug principle, aspirin eugenol ester (AEE) was synthesized, which can reduce the side effects of aspirin and eugenol. As a good candidate for new antithrombotic and anti-inflammatory medicine, it is essential to evaluate its preventive effect on thrombosis. Preventive effect of AEE was investigated in κ-carrageenan-induced rat tail thrombosis model. AEE suspension liquids were prepared in 0.5% sodium carboxymethyl cellulose (CMC-Na). AEE was administrated at the dosage of 18, 36 and 72 mg/kg. Aspirin (20 mg/kg), eugenol (18 mg/kg) and 0.5% CMC-Na (30 mg/kg) were used as control drug. In order to compare the effects between AEE and its precursor, integration of aspirin and eugenol group (molar ratio 1:1) was also designed in the experiment. After drugs were administrated intragastrically for seven days, each rat was injected intraperitoneally with 20 mg/kg BW κ-carrageen dissolved in physiological saline to induce thrombosis. The length of tail-thrombosis was measured at 24 and 48 hours. The blank group just was given physiological saline for seven days without κ-carrageenan administrated. The results indicated that AEE significantly not only reduced the average length of thrombus, PT values and FIB concentration, but also reduced the red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT) and platelet (PLT). The effects of AEE on platelet aggregation and anticoagulant in vitro showed that AEE could inhibit adenosine diphosphate (ADP)-induced platelet aggregation as dose-dependence but no notable effect on blood clotting. From these results, it was concluded that AEE possessed positive effect on thrombosis prevention in vivo through the reduction of FIB, PLT, inhibition of platelet aggregation and the change of TT and PT values.

Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography–tandem mass spectrometry

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with β-pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6×75mm, 3.5μm) using methanol: 5mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2-500ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC-MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration.

Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneous quantify artesunate and its metabolite in sheep plasma. The plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (250×4.6mm, 5μm) using methanol: water (60:40, v/v) (the water included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 1ng/mL to 400ng/mL (r(2)=0.9992 for artesunate, r(2)=0.9993 for its metabolite). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations for both artesunate and its metabolite. The recoveries ranged from 92% to 98% at the three concentrations for both. In summary, the LC-MS/MS metho described herein was fully successfully applied to pharmacokinetic studies of artesunate nanoemulsion after intramuscular delivery to sheep.