Pressurized liquid extraction of active ingredients (ginsenosides) from medicinal plants using non-ionic surfactant solutions

The feasibility of employing aqueous non-ionic surfactant solutions as an alternative solvent system in pressurized liquid extraction (PLE) is demonstrated for the first time using the roots of American ginseng as model solid samples. When compared to the use of pure water or methanol, the presence of a common non-ionic surfactant (Triton X-100) in water at a concentration above its critical micelle concentration was shown to enhance the amount of pharmacologically active ingredients (ginsenosides) extracted from ginseng roots. The advantages of using aqueous non-surfactant solutions were also demonstrated by comparing extraction performances between ultrasonic-assisted extraction and PLE methods. Furthermore, the combination of PLE and cloud point extraction was shown to be a new and effective approach for the rapid sample preconcentration of herbal materials prior to analysis by high-performance liquid chromatography.     

A photoreactive small-molecule probe for 2-oxoglutarate oxygenases

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable crosslinking group and an affinity-purification tag. Following studies with recombinant proteins, the probe was shown to crosslink to 2-OG oxygenases in human crude cell extracts, including to proteins at endogenous levels. This approach is useful for inhibitor profiling, as demonstrated by crosslinking to the histone demethylase FBXL11 (KDM2A) in HEK293T nuclear extracts. The results also suggest that small-molecule probes may be suitable for substrate identification studies.     

Alkoxy radical cyclizations onto silyl enol ethers relative to alkene cyclization, hydrogen atom transfer, and fragmentation reactions

This study examines the chemoselectivity of alkoxy radical cyclizations onto silyl enol ethers compared to competing cyclizations, 1,5-hydrogen atom transfers (1,5-HATs), and β-fragmentations. Cyclization onto silyl enol ethers in a 5-exo mode is greatly preferred over cyclization onto a terminal alkene. The selectivity decreases when any alkyl substitution is present on the competing alkene radical acceptor. Alkoxy radical 5-exo cyclizations displayed excellent chemoselectivity over competing β-fragmentations. Alkoxy radical 5-exo cyclizations onto silyl enol ether also outcompeted 1,5-HATs, even for activated benzylic hydrogen atoms. In tetrahydropyran synthesis, where 1,5-HAT has plagued alkoxy radical cyclization methodologies, 6-exo cyclizations were the dominant mode of reactivity. β-Fragmentation still remains a challenge for tetrahydropyran synthesis when an aryl group is present in the β position.     

Diagnosis of Helicobacter pylori infection.

A number of reliable methods are currently available for the diagnosis of Helicobacter pylori infection. These diagnostic tests can be classified into invasive methods that require endoscopy and gastric biopsy, and noninvasive methods. Invasive methods include gastric mucosal biopsies at endoscopy for bacteriologic culture, histology, and the rapid urease test. Noninvasive methods include the urea breath test and serologic tests. Each of these diagnostic tests has its advantages and disadvantages. Histologic examination remains the gold standard for diagnosis. It can also detect coccoidal forms of the bacteria and be used to assess the severity of gastritis. Culture of H pylori should be performed if antibiotic sensitivity of the organism is required. A rapid urease test is the quickest test for H pylori status. The urea breath test detects urease activity in the entire stomach, thus eliminating the possibility of a sampling error, which occurs in random gastric biopsies. Serologic tests using either ELISA or latex-agglutination methods are excellent for diagnosis of H pylori infection, but not useful for monitoring effects of therapy. Recently, the polymerase chain reaction has been applied to fixed-tissue biopsies, as well as body secretions in the diagnosis of H pylori infection.     

Metabolic studies of turinabol in horses

Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6beta-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6beta,16-dihydroxyturinabol (M3a, M3b) and 6beta,20-dihydroxyturinabol (M4). The metabolite 6beta-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation. For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6beta,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a-M6c and M7a-M7c), derived from D-ring hydroxylation and A-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48 h after administration.     

Synthesis of mono- and dihalogenated derivatives of (Me2S)2B12H10 and palladium-catalyzed boron-carbon cross-coupling reactions of the iodides with grignard reagents

Two series of compounds, 9-X-1,7-(Me(2)S)(2)B(12)H(9) and 9,10-X(2)-1,7-(Me(2)S)(2)B(12)H(8) (X = Cl, Br, I), have been synthesized from reactions of 1,7-(Me(2)S)(2)B(12)H(10) with various halogenating reagents. In addition, reactions of 1,7-(Me(2)S)(2)B(12)H(10) with 2,4-(NO(2))(2)C(6)H(3)SCl and PhSeBr resulted in 9-(2',4'-(NO(2))(2)C(6)H(3)S)-1,7-(Me(2)S)(2)B(12)H(9) and 9,10-(PhSe)(2)-1,7-(Me(2)S)(2)B(12)H(8), respectively. X-ray studies of the dibromo, monoiodo, and aryl thioether derivatives show that electrophilic substitution in 1,7-(Me(2)S)(2)B(12)H(10) takes place at positions 9 and 10, as in the case of the meta-carborane 1,7-C(2)B(10)H(12). From 1,12-(Me(2)S)(2)B(12)H(10) the halides 2-X-1,12-(Me(2)S)(2)B(12)H(9) (X = Br, I) were prepared. For both 1,7- and 1,12-(Me(2)S)(2)B(12)H(10) the best iodination results were obtained using iodine monochloride in refluxing acetonitrile. In the presence of 5 mol % (PPh(3))(2)PdCl(2) the iodides 9-I-1,7-(Me(2)S)(2)B(12)H(9), 2-I-1,12-(Me(2)S)(2)B(12)H(9), and 9,10-I(2)-1,7-(Me(2)S)(2)B(12)H(8) react with RMgX (R = Me, Ph, Bn; X = Cl, Br) in THF to yield the corresponding B-alkyl- and B-aryl-substituted products in good yields without using CuI as a cocatalyst. The bromo derivative 9-Br-1,7-(Me(2)S)(2)B(12)H(9) did not react under similar conditions. No interference from the nearby Me(2)S substituent was observed in palladium-catalyzed substitution of iodide in 2-I-1,12-(Me(2)S)(2)B(12)H(9). Presumably due to the intramolecular activation of an aryl C-H bond of the benzyl substituent in the intermediate palladium complex, the yield of 9,10-Bn(2)-1,7-(Me(2)S)(2)B(12)H(8) was significantly lower than those of the dimethyl and diphenyl derivatives. The molecular structures of 9-R-1,7-(Me(2)S)(2)B(12)H(9) (R = Ph, Bn) and 2-Bn-1,12-(Me(2)S)(2)B(12)H(9) were obtained by single-crystal X-ray analysis.     

Method extension study to validate applicability of AOAC Official Method 996.14 Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Assurance Listeria Polyclonal Enzyme Immunoassay (EIA) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 23 collaborators, representing government agencies, as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 550 test portions and controls was analyzed and confirmed, of which 207 were positive and 336 were negative by both methods. Six test portions were positive by culture, but negative by the EIA. Three test portions were negative by culture, but positive by the EIA. Two test portions were negative by EIA and by culture, but confirmed positive when EIA enrichment broths were subcultured to selective agars. The data reported here indicate that the Assurance Listeria EIA method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

Detection of Salmonella in fresh cheese,poultry products,and dried egg products by the ISO 6579 Salmonella culture procedure and the AOAC official method: collaborative study

Three food types were analyzed for the presence of Salmonella by the AOAC culture method and by the International Organization for Standardization (ISO 6579:2002) culture method. Paired test portions of each food type were simultaneously analyzed by both methods. A total of 21 laboratories representing federal government agencies and private industry, in the United States and Europe, participated in this interlaboratory study. Foods were artificially contaminated with Salmonella and competing microflora if naturally contaminated sources were not available. No statistical differences (p < 0.05) were observed between the AOAC and ISO culture methods for fresh cheese and dried egg products. A statistically significant difference was observed for one of the 2 lots of poultry from the first trial. The poultry meat used in this run was radiation sterilized, artificially contaminated with Salmonella and competitive flora, and then lyophilized. A second trial was conducted with 2 separate lots of raw ground chicken that were naturally contaminated. The results from the second trial showed no statistical difference between the 2 culture methods. A third trial involving 4 laboratories was conducted on 2 separate lots of naturally contaminated raw poultry. Again, no statistically significant differences occurred. It is recommended that ISO 6579:2002 culture method for Salmonella be adopted Official First Action for the analysis of fresh cheese, fresh chilled and frozen poultry, and dried egg products.     

Method extension study to validate applicability of AOAC Official Method 997.03 visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Visual Immunoprecipitate assay (VIP) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 27 laboratories, representing government agencies as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 615 test portions and controls was analyzed and confirmed, of which 227 were positive and 378 were negative by both methods. Nine test portions were positive by culture, but negative by the VIP. Five test portions were negative by culture, but positive by the VIP. Four test portions were negative by VIP and by culture, but confirmed positive when VIP enrichment broths were subcultured to selective agars. The data reported here indicate that the VIP method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

Lacticin 481 synthetase phosphorylates its substrate during lantibiotic production

Lacticin 481 synthetase (LctM) catalyzes the ATP-dependent conversion of a ribosomally synthesized peptide to a polycyclic thioether antibiotic. It is a bifunctional enzyme that dehydrates four Ser/Thr residues to the corresponding dehydro amino acids and catalyzes the conjugate addition of Cys residues to these dehydro residues in a regio- and stereoselective process. We show here that incubation of truncated substrates with LctM results in products that are phosphorylated in the region of dehydration. Furthermore, synthetic peptides containing phosphorylated Ser and/or Thr residues are accepted by the enzyme as substrates resulting in the elimination of phosphate and dehydro amino acid production. This activity is only observed if ADP is added as cosubstrate. These results argue strongly that the enzyme utilizes ATP to phosphorylate the Ser/Thr residues that are targeted for dehydration. ATP does not appear to be required for peptide translocation or cyclization.