An efficient in vitro and in vivo HPLC method for hydnocarpin in nanomicelles formulation

For quantitative and other related bioactive studies of hydnocarpin, there is a need to establish an efficient, specific and sensitive analytical method (in vitro and in vivo). In this paper, an efficient HPLC method has been established and validated to analyze hydnocarpin in a nanomicelle formulation for the first time. Various chromatographic conditions for in vitro and in vivo determinations were investigated, with the application examined by pharmacokinetics and tissue distribution studies. The analysis was carried out using an HPLC system with a Waters symmetry C₁₈ column (4.6 × 150 mm, 5 µm) at 25°C with a detecting wavelength of 342 nm. Eluting at a rate of 1.0 mL/min, a 65% methanol and 35% acetic acid solution (0.1%) served as the mobile phase for the in vitro determinations while a 62% methanol and 38% acetic acid solution (0.1%) was used for in vivo analysis with isoliquiritigenin as internal standard. The established in vitro linearity range for hydnocarpin was 0.2–20 µg/mL (R² = 0.9996), with the biological (in vivo) samples following the same trend. The accuracy of the method was >99% (in vitro) and 92.4–105.3% (in vivo). Also, the precision met the acceptance criterion. These results indicate that the established method exhibited high specificity, accuracy, linearity and precision. Additionally, this efficient HPLC method was applied to pharmacokinetics and tissue distribution studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil triacylglycerols for their stereospecific analysis

An innovative procedure to separate the 3 isomeric sn-monoacylglycerols (MAG) classes (sn-1-, sn-2-, sn-3-MAG) is described. MAGs, obtained by chemical deacylation of triacylglycerols (TAGs), have been derivatized with (S)-(+)-1-(1-naphtyl)ethyl-isocyanate, and the resulting urethane derivatives have been separated by normal-phase high-performance liquid chromatography. This procedure allows resolution as diasteroisomers of the 2 enantiomeric classes (sn-1-MAG and sn-3-MAG), without the need of a chiral column, and to separate also the isomeric sn-2-MAG class; moreover, by introducing a chromophoric moiety, this reagent makes possible the ultraviolet detection of the analyte molecules. This procedure has been used to obtain the stereospecific analysis of the TAG fraction of extra virgin olive oil samples. The use of a nondestructive detector permitted the collection of the individual urethane classes; the fatty acid composition of each was determined by high-resolution gas chromatography, obtaining directly from the data the fatty acid distribution within each sn- position of TAGs. To validate this new method, the results have been compared with those obtained by 2 other procedures for TAG stereospecific analysis, and the obtained results were satisfactory since the proposed method gave data very similar to the other procedures.     

Effects of Ganoderma,Rhodiola and grape seed extracts on the glucuronidation and oral bioavailability of resveratrol in rats

Herb extracts were shown to inhibit the activity of UDP-glucuronosyltransferases (UGTs) in vitro. However, the actual in vivo effect of the inhibitory ability on oral bioavailability is yet verified. In this study, resveratrol (RES) was used as a model drug to study the effect of three Chinese herb extracts, Ganoderma, Rhodiola and grape seed, on the in vitro and in vivo inhibition of glucuronidation and the in vivo bioavailability of RES. Overall, although herb extracts might show inhibition on glucuronidation of RES in vitro and in vivo, the inhibition of glucuronidation did not necessarily mean to improve the in vivo bioavailability of RES.     

Simultaneous HPLC determination of ergosterol and 22,23‐dihydroergosterol in Flammulina velutipes sterol‐loaded microemulsion

An efficient HPLC method was developed and validated for the simultaneous determination of ergosterol and 22,23‐dihydroergosterol in Flammulina velutipes sterol‐loaded microemulsions (FVSMs). The different chromatographic conditions for in vitro and in vivo determinations were investigated, with the application examined by tissue distribution. Chromatographic separation was achieved on an Inertsil ODS‐SP (250 × 4.6 mm, 5 µm) analytical column using a mobile phase of 98% methanol (in vitro), and 93% methanol for stomach samples and 96% methanol for other samples (in vivo) at 1.0 mL/min. The sterol content was detected at 282 nm. The established in vitro linearity ranges for ergosterol and 22,23‐dihydroergosterol were 0.58–72.77 µg/mL (r1 = 0.9999) and 0.59–73.25 µg/mL (r2 = 0.9999), respectively, with the biological (in vivo) samples following the same trend. The accuracy of the method was >99% (in vitro) and between 93%–108% (in vivo). The LOQ was 2.15 µg/L for ergosterol and 2.41 µg/L for 22,23‐dihydroergosterol in the in vitro studies. Also, the precisions met the acceptance criterion. These results indicate that the established HPLC method was specific, linear, accurate, precise and sensitive for the separation and simultaneous determination of ergosterol and 22,23‐dihydroergosterol. Copyright © 2013 John Wiley & Sons, Ltd.     

The influence of triphenylantimony(V) catecholate and its spiroendoperoxide on lipid peroxidation

The effects of triphenylantimony(V) catecholate Ph₃Sb(Cat) (1) and its spiroendoperoxide Ph₃Sb(L‐O₂) (2) (Cat = 3,6‐di‐tert‐butyl‐4,5‐dimethoxycatecholate) on lipid peroxidation (LP) in vitro and in vivo were examined in BALB/c line mice. A comparative study of the impact of compounds 1 and 2 on LP under similar conditions was made by measuring the formation of thiobarbituric acid reactive substances (TBARS). The anti‐ or pro‐oxidant action of complexes 1 and 2 may be caused by the different redox level of the ligand acting as radical scavenger and/or by the bound molecular oxygen promoting the oxidation process. Biological experiments (in vitro and in vivo) were performed using mouse tissue homogenates. Decreasing TBARS concentration was observed in all examined tissues and blood serum (in vitro as well as in vivo) for catecholate 1. These results indicate inhibition of LP in the presence of complex 1. In contrast to 1, spiroendoperoxide 2 increases the level of TBARS in tissue homogenates. Minor fluctuations of TBARS concentration in erythrocytes and in blood serum indicate the absence of an obvious anti/pro‐oxidant influence of 2 on the LP process in vivo. The role of catecholate fragment was found to be essential in explaining antioxidant properties. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies on Antifungal Agents. Part 25. 1-[(3,5-Bisaryl-2-methylisoxazolidin-3-yl)methyl]-1H-1,2,4-triazoles

The synthesis and antifungal activity of a novel series of 1-[(3,5-bisaryl-2-methylisoxazolidin-3-yl)methyl]-1H-1,2,4-triazoles 6 and 7 (i.e. 8 – 19 ) are discussed. The preparation of 8 – 19 was straightforward and highlighted by a regiospecific 1,3-dipolar cycloaddition of α-substituted (E)-ketonitrones 4 with appropriate atyrene derivatives 5 that led to a cis/trans-diastereoisomeric mixture of the corresponding triazoles (Scheme). The title compounds were evaluated for in vitro antifungal activity in solid agar cultures against a broad array of yeast and systemic mycoses and dermatophytes. The in vivo activity was determined in an immune-compromised mouse model of systemic candidiasis. While the in vitro activity was evident throughout the series, it was moderate in potency. However, some of the triazole derivatives demonstrated a potent in vivo activity comparable to that of the standard drug ketoconazole. Analogue 12 (PR 988-399) emerged as the best overall compound demonstrating potent antifungal activity in both in vitro and in vivo assays.     

Triacylglycerols of the seeds and fruit flesh ofMandragora turcomanica

The triacylglycerols (TAGs) of the seeds and fruit flesh ofManagora turcomanica have been investigated for the first time using the methods of mass spectrometry and enzymolysis. It has been found that in the TAGs of the seeds the 182 acids undergo specific esterification in the sn-2 position, while in the TAGs of the flesh this position is acylated by 181 and 182 acids. For both parts of the fruit the main TAG species is sn-1(3)-oleoyldilinoleate, and the main types are represented by triunsaturated and monounsaturated-diunsaturated glycerides.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 322–326, May–June, 1999.     

Action spectra for modification of Escherichia coli B/r menaquinone by near-ultraviolet and visible radiations (313-578 nm)

–Menaquinone-8 (MQ-8) was irradiated in vivo in Escherichia coli B/r and in vitro after extraction from E. coli B/r, using monochromatic radiation in the range 313–578 nm. Within experimental error, the action spectra for loss of chromatographic mobility after irradiation in vivo and in vitro agree with each other and with the absorption spectrum of pure MQ-8. The MQ-8 is extremely sensitive to near-UV light (300–380 nm), showing an F₃₇in vivo at 334 nm of 1.3 kj/m², a value 15 times lower than that required for growth delay, and 150 times lower than that for killing, of E. coli B/r. The quantum yield for this reaction in vivo at 334 nm has the very high value of 0.26. The high sensitivity of MQ-8 suggests involvement in near-UV-induced effects on the plasma membrane.     

Hollow fibre cell fishing and hollow fibre liquid phase microextraction research on the anticancer coumarins of Radix Angelicae dahuricae in vitro and in vivo

Hollow fiber cell fishing (HFCF) and hollow fiber liquid phase microextraction (HF-LPME) with high performance liquid chromatography were utilized to research the anti-hepatoma HepG-2 or anti-renal tubular ACHN cells coumarins from Radix Angelicae dahuricae (RAD) in vitro and vivo. Before application, the coumarins of psoralen, bergapten, oxypeucedanin, imperatorin and isoimperatorin were selected as model compounds, the experiment conditions and methodology parameters of both HFCF and HF-LPME were investigated. Under the optimized conditions, cell growth state and survival rate, cell specific binding, repeatability for HFCF, and linear ranges, limits of quantification (1.3–21.0?ng/mL), precisions (<15.2%), recoveries (80.4%–109.3%) and stability for HF-LPME were achieved. Results showed that the five model compounds and xanthotoxol, xanthotoxin were screened in RAD extraction; xanthotoxin and xanthotoxol were fished by HepG-2 cell from both plasma and liver, xanthotoxol, xanthotoxin, bergapten and imperatorin were fished by ACHN cell from plasma and kidney; and these coumarins maintained a certain concentration in vivo. The study results provide experimental basis for researching the active anticancer components and expound the medicinal material base in RAD. The combination of HFCF and HF-LPME is simple and effective in studying bioactive anticancer compounds in traditional Chinese medicine in vitro and in vivo.     

Microbiological control and antibacterial action of a propolis-containing mouthwash and control of dental plaque in humans

Propolis is a bee product with several biological properties. This study aimed at investigating a propolis-containing mouthwash, its organoleptic properties, microbial contamination and its antibacterial action in vitro. This mouthwash was assessed in vivo to control dental plaque in humans. The presence of microorganisms was analyzed and the minimum inhibitory concentration against Streptococcus mutans was determined. A comparative study was done in vivo using propolis, chlorhexidine, and propolis plus chlorhexidine in lower concentrations for 14 days. Dental plaque was analyzed by the Patient Hygiene Performance (PHP) index. The odontological product was yellow, cloudy, free of microbial contamination, and exerted an inhibitory action in vitro. Individuals who used a propolis-containing mouthwash for 14 consecutive days in combination or not to chlorhexidine showed a similar PHP index to chlorhexidine alone. The product exerted an antibacterial action in vitro and in vivo, exhibiting a positive action in the control of dental plaque.     

Quantification of triacylglycerols in butterfat by gas chromatography-electron impact mass spectrometry using molar correction factors for [M-RCOO]+ ions

A quantitative electron impact GC-MS method using molar correction factors (MCFs) for [M-RCOO]+ ions has been developed for determination of molecular species of triacylglycerols (TAGs). MCFs were determined by linear calibration for 226 ions of 104 TAG species with good reproducibility: on the average, coefficient of determination was 0.975 +/- 0.043 and 0.963 +/- 0.115 for saturated and unsaturated TAGs, respectively. The MCFs of the sn-1(3) regioisomers of short-chain TAGs were lower than those of sn-2 isomers indicating ca. 2-3-fold higher cleavage of butyroyl and caproyl groups from the primary positions than from the secondary position. The method enabled quantification of 139 and 135 individual TAG species of butterfat (BF) and interesterified butterfat, respectively, including several regioisomers of short-chain TAGs. The most abundant molecular species of the even-numbered TAGs in BF were butyroylpalmitoyloleoylglycerol (5.05 mol%), butyroyldipalmitoylglycerol (4.75 mol%), and palmitoyldioleoylglycerol (3.32 mol%). The method provides an alternative for elucidation of nutritional and technological properties of relatively saturated TAG mixtures.     

Identification of triacylglycerols containing two short-chain fatty acids at sn-2 and sn-3 positions from bovine udder by fast atom bombardment tandem mass spectrometry

Several triacylglycerol (TAG) molecular species, that contain two short-chain fatty acids (C4 to C8) at the sn-2 and sn-3 positions of the glycerol backbone, were isolated from bovine udder by using solvent extraction and silica gel column chromatography. Their structures were identified by fast atom bombardment (FAB) tandem mass spectrometry (MS/MS), based on the information obtained from collision-induced dissociation (CID) spectra of sodium-adducted molecules ([M + Na](+)) of model TAG compounds which had been synthesized from glycerol and appropriate fatty acids. For each species, the relative positions of the three fatty acids on the glycerol backbone, as well as fatty acid composition and double-bond position in the fatty acyl group, were determined. A majority of sodium-adducted molecules observed in the FAB mass spectrum were mixtures of at least two components that have different fatty acid composition but the same molecular mass. In addition, all the components present in mixtures of all the species contain a long-chain fatty acid (C12 to C18) at the sn-1 position, a short-chain fatty acid (C4 to C8) at the sn-2 position, and a butyric acid uniquely at the sn-3 position.     

Synthesis and Biological Evaluation of 14-Alkoxymorphinans. Part 10. 14-O-Methyl derivatives of 5-methylnaltrexone and 5-methylnaloxone

In several steps, 5, 14-O-dimethylnaltrexone ( 3 ) and 5, 14-O-dimethylnaloxone ( 4 ) were prepared starting from 5, 14-O-dimethyloxycodone ( 5 ). Compound 3 exhibited opioid agonism in vitro (guinea-pig ileum and mouse vas deferens preparations) and antagonism in vivo (AcOH-writhing test in mice), while compound 4 was found to be an agonist in vitro and in vivo.     

Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument

We describe features of tandem mass spectra of lithiated adducts of triacylglycerol (TAG) species obtained by electrospray ionization mass spectrometry (ms) with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument. The spectra distinguish isomeric triacylglycerol species and permit assignment of the mass of each fatty acid substituent and positions on the glycerol backbone to which substituents are esterified. Source CAD-MS2 experiments permit assignment of double bond locations in polyunsaturated fatty acid substituents. The ESI/MS/MS spectra contain [M + Li - (RnCO2H)]+, [M + Li - (RnCO2Li)]+, and RnCO+ ions, among others, that permit assignment of the masses of fatty acid substituents. Relative abundances of these ions reflect positions on the glycerol backbone to which substituents are esterified. The tandem spectra also contain ions reflecting combined elimination of two adjacent fatty acid residues, one of which is eliminated as a free fatty acid and the other as an alpha, beta-unsaturated fatty acid. Such combined losses always involve the sn-2 substituent, and this feature provides a robust means to identify that substituent. Fragment ions reflecting combined losses of both sn-1 and sn-3 substituents without loss of the sn-2 substituent are not observed. Schemes are proposed to rationalize formation of major fragment ions in tandem mass spectra of lithiated TAG that are supported by studies with deuterium-labeled TAG and by source CAD-MS2 experiments. These schemes involve initial elimination of a free fatty acid in concert with a hydrogen atom abstracted from the alpha-methylene group of an adjacent fatty acid, followed by formation of a cyclic intermediate that decomposes to yield other characteristic fragment ions. Determination of double bond location in polyunsaturated fatty acid substituents of TAG is achieved by source CAD experiments in which dilithiated adducts of fatty acid substituents are produced in the ion source and subjected to CAD in the collision cell. Product ions are analyzed in the final quadrupole to yield information on double bond location.     

Estimation of244Cm intake by bioassay measurements following a contamination incident

An employee was contaminated with radioactive material consisting primarily of²⁴⁴Cm and²⁴⁶Cm as a consequence of handling a curium nitrate solution at a reprocessing facility.In vivo gamma analysis andin vitro (urine and fecal) analysis were initiated soon after the incident. Furtherin vivo measurements were performed regularly through hour 528, andin vitro bioassay measurements were obtained through day 74. A sample of the curium solution from the workplace was obtained to confirm that the nitrate was the chemical form in question and to identify the isotopes of curium present. The mass ratio of²⁴⁴Cm/²⁴⁶Cm was determined to be 91 to 7. Diethylenetriaminepentaacetate (DTPA) was administered on hours 33 and 71. Observed excretion rates were consistent with available information on curium. In this paper, the results of thein vivo andin vitro measurements are presented and intake estimates for the incident are developed using various excretion rate functions.     

Comparison of direct mass spectrometry methods for the on‐line analysis of volatile compounds in foods

For the on‐line monitoring of flavour compound release, atmospheric pressure chemical ionization (APCI) and proton transfer reaction (PTR) combined to mass spectrometry (MS) are the most often used ionization technologies. APCI‐MS was questioned for the quantification of volatiles in complex mixtures, but direct comparisons of APCI and PTR techniques applied on the same samples remain scarce. The aim of this work was to compare the potentialities of both techniques for the study of in vitro and in vivo flavour release. Aroma release from flavoured aqueous solutions (in vitro measurements in Teflon bags and glass vials) or flavoured candies (in vivo measurements on six panellists) was studied using APCI‐ and PTR‐MS. Very similar results were obtained with both techniques. Their sensitivities, expressed as limit of detection of 2,5‐dimethylpyrazine, were found equivalent at 12 ng/l air. Analyses of Teflon bag headspace revealed a poor repeatability and important ionization competitions with both APCI‐ and PTR‐MS, particularly between an ester and a secondary alcohol. These phenomena were attributed to dependency on moisture content, gas/liquid volume ratio, proton affinities and product ion distribution, together with inherent drawbacks of Teflon bags (adsorption, condensation of water and polar molecules). Concerning the analyses of vial headspace and in vivo analyses, similar results were obtained with both techniques, revealing no competition phenomena. This study highlighted the equivalent performances of APCI‐MS and PTR‐MS for in vitro and in vivo flavour release investigations and provided useful data on the problematic use of sample bags for headspace analyses. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro metabolism studies of desoxy‐methyltestosterone (DMT) and its five analogues,and in vivo metabolism of desoxy‐vinyltestosterone (DVT) in horses

The positive findings of norbolethone in 2002 and tetrahydrogestrinone in 2003 in human athlete samples confirmed that designer steroids were indeed being abused in human sports. In 2005, an addition to the family of designer steroids called ‘Madol’ [also known as desoxy‐methyltestosterone ( DMT )] was seized by government officials at the US–Canadian border. Two years later, a positive finding of DMT was reported in a mixed martial arts athlete's sample. It is not uncommon that doping agents used in human sports would likewise be abused in equine sports. Designer steroids would, therefore, pose a similar threat to the horseracing and equestrian communities. This paper describes the in vitro metabolism studies of DMT and five of its structural analogues with different substituents at the 17α position (R ? H, ethyl, vinyl, ethynyl and ²H₃‐methyl). In addition, the in vivo metabolism of desoxy‐vinyltestosterone ( DVT ) in horses will be presented. The in vitro studies revealed that the metabolic pathways of DMT and its analogues occurred predominantly in the A‐ring by way of a combination of enone formation, hydroxylation and reduction. Additional biotransformation involving hydroxylation of the 17α‐alkyl group was also observed for DMT and some of its analogues. The oral administration experiment revealed that DVT was extensively metabolised and the parent drug was not detected in urine. Two in vivo metabolites, derived respectively from (1) hydroxylation of the A‐ring and (2) di‐hydroxylation together with A‐ring double‐bond reduction, could be detected in urine up to a maximum of 46 h after administration. Another in vivo metabolite, derived from hydroxylation of the A‐ring with additional double‐bond reduction and di‐hydroxylation of the 17α‐vinyl group, could be detected in urine up to a maximum of 70 h post‐administration. All in vivo metabolites were excreted mainly as glucuronides and were also detected in the in vitro studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Growth and kinetics of in vitro poly([R]‐(–)‐3‐hydroxybutyrate) granules interpreted as particulate polymerization with coalescence

Transmission electron microscopy (TEM) and Cryo‐TEM were used to study the growth kinetics of poly(3‐hydroxybutyrate) granules produced by in vitro polymerization. The in vitro formation of poly(3‐hydroxybutyrate) uses a recombinant form of the PHA synthase to polymerize [R]‐(–)‐3‐hydroxybutyryl‐CoA. Since the in vitro reaction contains only synthase and monomer, it is a simpler system than the in vivo biosynthesis of poly(3‐hydroxybutyrate). TEM and Cryo‐TEM were used in conjunction with image analysis to examine the granules that were formed in the in vitro reaction. The in vitro reaction yielded spherical granules of rapidly increasing size; the initially observed granules were already larger than 0.1 μm. While the average granule diameter and volume increased with reaction time, the number of granules decreased throughout the reaction due to coalescence. Basic kinetic parameters, including K_M and V_max were determined and compared to those reported for the in vivo biosynthesis of poly(3‐hydroxybutyrate). In addition it was found that the granules formed by this process were essentially noncrystalline. A computer simulation of the reaction, based on initial formation of relatively large microporous granules that consolidate by expulsion of water during polymerization, accounted for the shape of the kinetic curves.     

Synthesis and antibacterial activities of novel oxazine and thiazine ring-fused tricyclic quinolonecarboxylic acids: 10-(Alicyclic amino)-9-fluoro-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acids and the corresponding 1-thia congeners

Several analogs 4 and 5 of Ofloxacin ( 1 ) which contain the oxazine and thiazine rings fused with a quinolone carboxylic acid moiety, respectively, were prepared and their in vitro and in vivo antibacterial activities were compared with those of 1 and its previously prepared 3-exo-methylene analogs 2 and 3 . Unlike 1, 2 , and 3 , analogs 4 and 5 possess an antiaromatic oxazine and thiazine moiety and show markedly lower antibacterial activities. Alteration of their C-10 amino-substituent groups from piperazine to azetidine significantly improved the in vitro antibacterial activities, particularly in the case of the thiazine derivative 5 , but not the in vivo ones. The antibacterial activities of these three types of tricyclic quinolonecarboxylic acids are briefly discussed on the basis of the molecular properties revealed by molecular orbital calculation. The molecular dipole moment was suggested to be one possible factor controlling the binding affinity of these compounds with DNA gyrase.