Rapid method for the simultaneous detection of boar taint compounds by means of solid phase microextraction coupled to gas chromatography/mass spectrometry.

Because of animal welfare issues, the voluntary ban on surgical castration of male piglets, starting January 2018 was announced in a European Treaty. One viable alternative is the fattening of entire male pigs. However, this can cause negative consumer reactions due to the occurrence of boar taint and possibly lead to severe economic losses in pig husbandry. In this study, headspace solid phase microextraction (HS-SPME) coupled to GC-MS was used in the development and optimization of a candidate method for fast and accurate detection of the boar taint compounds. Remarkably fast extraction (45s) of the boar taint compounds from adipose tissue was achieved by singeing the fat with a soldering iron while released volatiles were extracted in-situ using HS-SPME. The obtained method showed good performance characteristics after validation according to CD 2002/657/EC and ISO/IEC 17025 guidelines. Moreover, cross-validation with an in-house UHPLC-HR-Orbitrap-MS method showed good agreement between an in-laboratory method and the new candidate method for the fast extraction and detection of skatole and androstenone, which emphasizes the accuracy of this new SPME-GC-MS method. Threshold detection of the boar taint compounds on a portable GC-MS could not be achieved. However, despite the lack of sensitivity obtained on the latter instrument, a very fast method with run-to-run time of 3.5min for the detection of the boar taint compounds was developed.

Nodulisporiviridins A-H, Bioactive Viridins from Nodulisporium sp.

Eight new viridins, nodulisporiviridins A-H (1-8), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (No. 65-17-2-1) that was fermented with potato-dextrose broth. The structures were determined using spectroscopic and X-ray crystallographic analysis. Nodulisporiviridins A-D (1-4) are unique viridins with an opened ring A. The Aß42 aggregation inhibitory activities of 1-8 were evaluated using a thioflavin T (ThT) assay with epigallocatechin gallate (EGCG) as the positive control (EGCG IC50 of 0.5 µM). Nodulisporiviridin G (7) displayed potent inhibitory activity with an IC50 value of 1.2 µM, and the preliminary trend of activity of these viridins as Aß42 aggregation inhibitors was proposed. The short-term memory assay on an Aß transgenic drosophila model of Alzheimer's disease showed that all eight compounds improved the short-term memory capacity, with potencies close to that of the positive control (memantine).

Nodulisporisteriods A and B, the first 3,4-seco-4-methyl-progesteroids from Nodulisporium sp.

Two new 4-methyl-progesteroids, nodulisporisteriod A (1) and nodulisporisteriod B (2), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (No. 65-17-2-1), along with two related metabolites, demethoxyviridin (3) and inoterpene B (4). Their structures were determined by detailed spectroscopic analyses, X-ray crystallographic analysis and comparison of the NMR data with those of the closely related compounds previously reported. Nodulisporisteriod A (1) and nodulisporisteriod B (2) possess new carbon skeletons, which are the first cases of fission at C-3,4 in 4-methyl-progesteroids. A hypothetical biosynthetic pathway for 1 and 2 was proposed. Moreover, the Aß42 aggregation inhibitory activities of 1-4 were evaluated using standard thioflavin T (ThT) fluorescence assay with epigallocatechin gallate (EGCG) as positive control. Demethoxyviridin (3) displayed anti-Aß42 aggregation activity with IC50 value of 13.4µM.

Restricted-access nanoparticles for magnetic solid-phase extraction of steroid hormones from environmental and biological samples.

Restricted-access materials based on non-ionic surfactant-coated dodecyl-functionalized magnetic nanoparticles were prepared and applied to extract steroid hormones from environmental and biological samples. The magnetic nanoparticles were synthesized by co-precipitation, and were functionalized with dodecyltriethoxysilane, giving dodecyl-grafted magnetic nanoparticles (C12-Fe3O4). They were further modified with different non-ionic surfactants by self-assembly adsorption. Several types of non-ionic surfactants, Tween-20, 40, 60 and 85, and Span-40, 60 and 80, were investigated as the coatings. Tween surfactants coated C12-Fe3O4, named as TW-20 (40, 60, 85)-C12, exhibited good dispersibility in aqueous solution, which was a preferred character in extraction; besides, TW-20-C12 and TW-40-C12 showed good anti-interference ability and satisfactory reproducibility when they were used as magnetic solid-phase extraction (MSPE) sorbents. The factors that may influence the extraction, including the amount of magnetic nanoparticles, extraction and desorption time, the amount of salt addition, the type and volume of desorption solvent, the volume of methanol addition and pH of sample solution, were investigated in detail. High performance liquid chromatography-UV detection was employed for analysis of target analytes (steroid hormone compounds). The developed method was successfully used for the determination of the target analytes in environmental and urine samples. Both tested materials afforded good recovery, satisfactory reproducibility and low limits of detection for environmental samples, which indicates that the materials possessed anti-interference ability. However, compared to TW-40-C12, TW-20-C12 nanoparticles provided better recovery in relatively complex biological samples, which may indicate that the latter one is more appreciated in complex samples.

Development of a candidate reference method for the simultaneous quantitation of the boar taint compounds androstenone, 3α-androstenol, 3ß-androstenol, skatole, and indole in pig fat by means of stable isotope dilution analysis-headspace solid-phase microextraction-gas chromatography/mass spectrometry.

The steroidal pig pheromones androstenone (5α-androst-16-en-3-one), 3α-androstenol (5α-androst-16-en-3α-ol), and 3ß-androstenol (5α-androst-16-en-3ß-ol) as well as the heterocyclic aromatic amines skatole and indole, originating from microbial degradation of tryptophan in the intestine of pigs, are frequently recognized as the major compounds responsible for boar taint. A new procedure, applying stable isotope dilution analysis (SIDA) and headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) for the simultaneous quantitation of these boar taint compounds in pig fat was developed and validated. The deuterated compounds androstenone-d(3), 3ß-androstenol-d(3), skatole-d(3), and indole-d(6) were synthesized and successfully employed as internal standards for SIDA. The new procedure is characterized by a fast, simple, and economic sample preparation: methanolic extraction of the melted fat followed by a freezing and an evaporation step allows for extraction and enrichment of all five analytes. Additional time-consuming cleanup steps were not necessary, as HS-SPME sampling overcomes fat-associated injector and column contamination. The method has been validated by determining intra- and interday precision and accuracy as well as the limit of detection (LOD) and limit of quantitation (LOQ). Additionally, a cross-validation for androstenone, skatole, and indole was carried out comparing the results of 25 back fat samples obtained simultaneously by the new SIDA-HS-SPME-GC/MS procedure with those obtained in separate GC/MS and high-performance liquid chromatography fluorescence detection (HPLC-FD) measurements. The cross-validation revealed comparable results and confirms the feasibility of the new SIDA-HS-SPME-GC/MS procedure.

Steroids, alkaloids, and coumarins from Gelsemium sempervirens.

The 95 % ethanol extract of Gelsemium sempervirens showed inhibitory activity against human DNA topoisomerase I (Topo I). Phytochemical investigations of this active extract resulted in the isolation and identification of three new steroids ( 1 - 3), together with eight known compounds 12 beta-hydroxy-5 alpha-pregn-16-ene-3,20-dione ( 4), gelsemine ( 5), sempervirine ( 6), scopoletin ( 7), 7- O- beta- D-glucopyranosylscopoletin ( 8), 7- O- beta- D-apiofuranosyl-(1-->6)- beta- D-glucopyranosylscopoletin ( 9), uvaol ( 10), and 2-(4-hydroxyphenyl)ethyl heptadecanoate ( 11). The structures of the new steroids were determined by extensive NMR and HR-ESI-MS analyses as 21-hydroxy-5 alpha-pregn-16-ene-3,20-dione ( 1), 3-oxoandrosta-16-ene-17-carboxylic acid ( 2), and 3-oxoandrosta-4,16-diene-17-carboxylic acid ( 3). This study suggests that sempervirine ( 6) intercalates to DNA and also inhibits Topo I through modulating the enzyme activity with an IC (50) of 54.5 +/- 15.9 muM.

Characterization of hydrocortisone biometabolites and 18S rRNA gene in Chlamydomonas reinhardtii cultures.

A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 degrees C for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17 beta-Dihydroxyandrost-4-en-3-one (2), 11 beta-hydroxyandrost-4-en-3,17-dione (3), 11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one (4) and prednisolone (5) were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans.

Microbial transformation of adrenosterone (1) by suspended-cell cultures of the filamentous fungus Cunninghamella elegans resulted in the production of five metabolites 2-6, which were identified as 9alpha-hydroxyadrenosterone (2), 11-ketotestosterone (3), 6beta-hydroxyadrenosterone (4), 9alpha-hydroxy-11-ketotestosterone (5), and 6beta-hydroxy-11-ketotestosterone (6). Structures of new metabolites 2, 5, and 6 were established by single-crystal X-ray diffraction analysis.

Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum.

Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was, isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11beta-hydroxytestosterone, 11beta-hydroxyandrost-4-en-3,17-dione and 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.

[Biotransformation of dehydroepiandrosterone by hairy root cultures of Anisodus tanguticus].

AIM: To modify the structure of dehydroepiandrosterone (DHEA). METHODS: Using hairy root cultures of Anisodus tanguticus to perform biotransformation of DHEA, using chromatographic and spectral techniques to isolate and identify the products. RESULTS: (1) The MS medium without plant hormone was suitable for the growth of the hairy root. (2) DHEA was converted into five products: androst-4-ene-3, 17-dione (I); 6alpha-hydroxyandrost-4-ene-3, 17-dione (II); 6alpha, 17beta-dihydroxyandrost-4-ene-3-one (III); androst-4-ene-3, 6, 17-trione (IV) and 17beta-hydroxyandrost-4-ene-3-one (V). CONCLUSION: It is the first time to use hairy root cultures of Anisodus tanguticus for the biotransformation of DHEA and five DHEA-related compounds were obtained.

New compounds from an extract of Vernonia colorata leaves with anti-inflammatory activity.

Bioassay-directed fractionation of an anti-inflammatory CHCl(3)-MeOH (9:1) extract of leaves of Vernonia colorata, using a carrageenan-induced rat paw model, led to the isolation of six new compounds (1-6). These were assigned as two new androst-8-en glycosides, 3-O-[beta-d-galactopyranosyl-(1-2)-[beta-d-glucopyranosyl-(1-->6)]-beta-d-glucopyranoside]-5alpha,14alpha-androst-8-ene (1) and 3-O-[beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranoside]-5alpha,14alpha-androst-8-ene (2), two new stigmastane-type glycosides, 3beta,21,24-trihydroxy-21,23;22,28;26,28-triepoxy-5alpha-stigmasta-8(9),14(15)-dien-3-O-beta-d-galactopyranosyl-(1-->2)-beta-d-glucopyranoside (3) and 3beta,21,24-trihydroxy-21,23;22,28;26,28-triepoxy-5alpha-stigmasta-8(9),14(15)-dien-3-O-beta-d-galactopyranosyl-(1-->2)-beta-d-(6-acetyl)glucopyranoside (4), and two new stigmastane-type steroids, 3beta,25,29-trihydroxy-5alpha-stigmasta-8(9),14(15),24Z(28)-triene (5) and 3beta,23,25-trihydroxy-24,28-epoxy-5alpha-stigmasta-8(9),14(15)-diene (6). The structures of 1-6 were elucidated by spectral and chemical studies. Compounds 1-6 were tested for the anti-inflammatory activity, but all were inactive or weakly inactive as anti-inflammatory agents.

Systematic optimisation of high-performance liquid chromatographic separation by varying the temperature, gradient, and stationary phase.

Optimisation of the separation of a synthetic drug mixture by HPLC is performed by changing both continuous variables, i.e. mobile phase composition and temperature, and categorical variables, here the stationary phase. The retention of solutes is described on the basis of a general linear model in which the different columns are modelled by indicator variables. From the solute-specific retention models the global separation optimum is evaluated on the basis of multidimensional window diagrams using relative retentions of all peak pairs as the figure-of-merit.

Isolation and characterization of viridin, a new 65 kDa antifungal protein from the mould Trichoderma viride.

A new extracellular antifungal protein with a yield of 10 mg per liter was isolated from the culture medium of the mould Trichoderma viride. The protein, which we named viridin, was purified by carboxymethyl-cellulose cation-exchange chromatography and Superose 12 HR 10/30 high-performance liquid chromatography. Viridin, a basic protein of approximately 65 kDa as determined by SDS-PAGE, inhibits the growth of the cotton pathogen Verticillum dahliae, the IC50 being 6 microM.