Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti‐influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well‐known traditional Uighur medicine for the treatment of colds. In the present study, high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

A volatolomic approach using cerumen as biofluid to diagnose bovine intoxication by Stryphnodendron rotundifolium

An innovative volatolomic approach employs the detection of biomarkers present in cerumen (earwax) to identify cattle intoxication by Stryphnodendron rotundifolium Mart., Fabaceae (popularly known as barbatimão). S. rotundifolium is a poisonous plant with the toxic compound undefined and widely distributed throughout the Brazilian territory. Cerumen samples from cattle of two local Brazilian breeds (‘Curraleiro Pé-Duro’ and ‘Pantaneiro’) were collected during an experimental intoxication protocol and analyzed using headspace (HS)/GC–MS followed by multivariate analysis (genetic algorithm for a partial least squares, cluster analysis, and classification and regression trees). A total of 106 volatile organic metabolites were identified in the cerumen samples of bovines. The intoxication by S. rotundifolium influenced the cerumen volatolomic profile of the bovines throughout the intoxication protocol. In this way, it was possible to detect biomarkers for cattle intoxication. Among the biomarkers, 2-octyldecanol and 9-tetradecen-1-ol were able to discriminate all samples between intoxicated and nonintoxicated bovines. The cattle intoxication diagnosis by S. rotundifolium was accomplished by applying the cerumen analysis using HS/GC–MS, in an easy, accurate, and noninvasive way. Thus, the proposed bioanalytical chromatography protocol is a useful tool in veterinary applications to determine this kind of intoxication.     

Metabolic profiles of dioscin in rats revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Dioscin (DIS), one of the most abundant bioactive steroidal saponins in Dioscorea sp., is used as a complementary medicine to treat coronary disease and angina pectoris in China. Although the pharmacological activities and pharmacokinetics of DIS have been well demonstrated, information regarding the final metabolic fates is very limited. This study investigated the in vivo metabolic profiles of DIS after oral administration by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry method. The structures of the metabolites were identified and tentatively characterized by means of comparing the molecular mass, retention time and fragmentation pattern of the analytes with those of the parent compound. A total of eight metabolites, including seven phase I and one phase II metabolites, were detected and tentatively identified for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. In addition, a possible metabolic pathway on the biotransformation of DIS in vivo was proposed. This study provides valuable and new information on the metabolism of DIS, which will be helpful for further understanding its mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of major components of Huangqi–Honghua extract in rat plasma using LC–MS/MS and application to a pharmacokinetic study

A sensitive and reliable LC–MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi–Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin‐7‐O‐β‐d ‐glucoside (CAG), calycosin, calycosin‐3′‐O‐glucuronide (C‐3′‐G) and calycosin‐3′‐O‐sulfate (C‐3′‐S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C₁₈ column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra‐ and inter‐assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from ?8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S in rat after an oral administration of Huangqi–Honghua extract.     

Diastereoselective Cyclobutenol Synthesis: A Heterogeneous Palladium-Catalyzed Oxidative Carbocyclization-Borylation of Enallenols

A highly selective and efficient oxidative carbocyclization/borylation of enallenols catalyzed by palladium immobilized on amino-functionalized siliceous mesocellular foam (Pd-AmP-MCF) was developed for diastereoselective cyclobutenol synthesis. The heterogeneous palladium catalyst can be recovered and recycled without any observed loss of activity or selectivity. The high diastereoselectivity of the reaction is proposed to originate from a directing effect of the enallenol hydroxyl group. Optically pure cyclobutenol synthesis was achieved by the heterogeneous strategy by using chiral enallenol obtained from kinetic resolution.     

Metabolites of A Novel Antibiotic Bitespiramycin in Rat Urine and Bile

A sensitive analytical method to identify active metabolites of bitespiramycin in rat urine and bile was developed by liquid chromatography-electrospray ionization tandem mass spectrometry(LC/ESI-MS^n).Bitespiramycin and its major active metabolites in rat urine and bile were isolated and identified as M1 serial(spiramycin Ⅰ，Ⅱ，Ⅲ),M2 serial(platenomycin A1,josamycin and leucomycin A1) and M3 serial(deisovalerylplatenomycin A1,deisovaleryljosamycin,deisovalerylleucomycin A1).     

Total synthesis of (±) maculalactone A, maculalactone B and maculalactone C and the determination of the absolute configuration of natural (+) maculalactone A by asymmetric synthesis

Maculalactones A, B and C from the marine cyanobacterium Kyrtuthrix maculans are amongst the only compounds based on the tribenzylbutyrolactone skeleton known in nature and (+) maculalactone A from the natural source possesses significant biological activity against various marine herbivores and marine settlers. We now report a concise synthesis of racemic maculalactone A in five steps from inexpensive starting materials. Maculalactones B and C were synthesized by a minor modification to this procedure, and the synthetic design also permitted an asymmetric synthesis of maculalactone A to be achieved in around 85% ee. The (+) and (−) enantiomers of maculalactone A were assigned, respectively, to the S and R configurations on the basis of the chiral selectivity expected for catecholborane reduction of an unsymmetrical ketone in the presence of Corey's oxazoborolidine catalyst. Surprisingly, it appeared that natural (+) maculalactone A was biosynthesized in K. maculans in a partially racemic form, comprising ca. 90-95% of the (S) enantiomer and 5-10% of its (R) enantiomer. Coincidentally therefore, the percentage enantiomeric excess of the product obtained from asymmetric synthesis almost exactly matched that found in nature.     

一种新颖的测定和分析羟自由基氧化损伤RNA及其分子机理的化学发光体系

近年来，羟自由基（^.OH)对DNA氧化损伤已受到广泛关注，但是很少研究^.OH对RNA的氧化损伤。其实，RNA与DNA一样，也是核酸的两大组分之一，也有许多重要功能。所以^.OH攻击RNA也会引起重后果，会造成细胞功能衰退甚至细胞死亡等。为此，我们建立了Vit.C-CuSO4-Phen-H2O2-PNA这一产生和测定^.OH氧化损伤RNA的化学发光体系，以便加强^.OH氧化损伤RNA的研究。通过对本体系测定条件的研究，得出了本体系最佳组方是：Vit.C,CuSO4,Phen,H2O2和RNA，浓度分别为350μmol/L,55μmol/L,350μmol/L,0.2mol/L和20μg/mL,体系pH为5．5，体系终体积为1mL。随后，利用本体系检测了槲皮素，咖啡酸，黄芩甙和芦丁抗^.OH氧化损伤RNA的作用，发现这四种抗氧化剂均能有效抑制^.OH氧化损伤RNA的分子机理，结果发现，^.OH清除剂硫脲几乎抑制全部发光，推测是因硫脲清除了引发剂^.OH所致；O^-.2清除剂SOD只能抑制小部分发光；^1O2清除剂叠氮化钠和苯甲酸都能抑制绝大部分发光。这些事实提示，^.OH是RNA氧化损伤的引发剂；O^-.2只是导致RNA氧化损伤的次要因素，^1O2才是导致RNA氧化损伤的最主要因素。     

Dibutyltin-3-hydroxyflavone titrates a dissociable component (cofactor) of mitochondrial ATP synthase: An energy-transfer component linked to the ubiquinone pool

Dibutyltin-3-hydroxyflavone, Bu₂Sn(of), is a new fluorescence probe inhibitor of F₁F₀-ATPase and oxidative phosphorylation which inhibits by titration of an unidentified component of F₀. Its site of action is closely related to that of the trialkyltins and of venturicidin. This F₀ component is part of a pool of this component which is present in the heart mitochondrial inner membrane at levels of 5–7 nmol (mg protein)^?1 [18 ± 3 Bu₂Sn(of) sites per mol F₁F₀-ATPase]. However, ATPase activity in submitochondrial particles is near maximally inhibited by titration of approx. three Bu₂Sn(of) sites per mol F₁F₀-ATPase. Over 60% (60–80%) of the Bu₂Sn(of) interaction sites can be lost during the purification of F₁F₀-ATPase from submitochondrial particles. The number of Bu₂Sn(of) interaction sites in various F₁F₀-ATPase preparations is variable. The high numbers of Bu₂Sn(of) sites per mol F₁F₀-ATPase for heart mitochondria (18–21) and submitochondrial particles (15–19.5) decline in ATP synthase (11–15) to the low values obtained in Complex V (7–10.5) and the minimal values observed in highly purified F₁F₀?ATPase (3.5–5.6), thus indicating a variable dissociable component or cofactor of ATP synthase. The Bu₂Sn(of) interaction site, a component of ATP synthase, is responsive to the redox status of the respiratory chain and the interaction with Bu₂Sn(of) is with the reduced form of this component. Fluorescence titration studies show that this component is in redox equilibrium with the ubiquinone pool of the respiratory chain. It is proposed that this redox component serves as an inhibitor titratable cofactor pool which cycles through an F₀ interaction site (or sites) via a system which serves as an energy-transfer link between the respiratory chain and ATP synthase.