Evaluation of Aculeatin and Toddaculin Isolated from Toddalia asiatica as Anti-inflammatory Agents in LPS-Stimulated RAW264 Macrophages.

Anti-inflammatory activity of aculeatin and toddaculin, which are coumarins with a similar structure isolated from Toddalia asiatica (L.) LAM., was evaluated using lipopolysaccharide (LPS)-stimulated RAW264 mouse macrophage cells. Both aculeatin and toddaculin significantly inhibited mRNA expression of inflammatory mediators and nitric oxide production. Furthermore, Toddaculin suppressed LPS-induced phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2 and inhibited LPS-induced activation of nuclear factor-kappaB (NF-κB). However, aculeatin did not exhibit such effects, suggesting that aculeatin and toddaculin suppress LPS-induced inflammation of RAW264 cells via different mechanisms. The cellular uptake of these compounds was also evaluated. Toddaculin was detected in RAW264 cells after 4 and 24 h. However, aculeatin levels were not observed in RAW264 cells at all incubation intervals. These results indicate that de-epoxidation of a prenyl group can increase hydrophobicity of molecule and is thought to accelerate cellular uptake and/or interactions with the phospholipid bilayers of cell membranes.

Novel Method to Quantify ß-Glucan in Processed Foods: Sodium Hypochlorite Extracting and Enzymatic Digesting (SEED) Assay.

Some ß-glucans have attracted attention due to their functionality as an immunostimulant and have been used in processed foods. However, accurately measuring the ß-glucan content of processed foods using existing methods is difficult. We demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay, in which ß-glucan is extracted using sodium hypochlorite, dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into ß-glucan fragments using Westase which is an enzyme having ß-1,6- and ß-1,3 glucanase activity. The ß-glucan fragments are further digested into glucose using exo-1,3-ß-d-glucanase and ß-glucosidase. We measured ß-glucan comprising ß-1,3-, -1,6-, and -1,(3),4- bonds in various polysaccharide reagents and processed foods using our novel method. The SEED assay was able to quantify ß-glucan with good reproducibility, and the recovery rate was >90% for food containing ß-glucan. Therefore, the SEED assay is capable of accurately measuring the ß-glucan content of processed foods.

Synthesis of novel 5,6-dehydrokawain analogs as osteogenic inducers and their action mechanisms.

An imbalance between bone resorption by osteoclasts and bone formation by osteoblasts can cause bone loss and bone-related disease. In a previous search for natural products that increase osteogenic activity, we found that 5,6-dehydrokawain (1) from Alpinia zerumbet promotes osteoblastogenesis. In this study, we synthesized and evaluated series of 5,6-dehydrokawain analogs. Our structure-activity relationships revealed that alkylation of para or meta position of aromatic ring of 1 promote osteogenic activity. Among the potential analogs we synthesized, (E)-6-(4-Ethylstyryl)-4-methoxy-2H-pyran-2-one (14) and (E)-6-(4-Butylstyryl)-4-methoxy-2H-pyran-2-one (21) both significantly up-regulated Runx2 and Osterix mRNA expression at 10µM. These osteogenic activities could be mediated by bone morphogenetic protein (BMP) and activation of p38 MAPK signaling pathways. Compounds 14 and 21 also inhibited RANKL-induced osteoclast differentiation of RAW264 cells. These results indicated that novel 5,6-dehydrokawain analogs not only increase osteogenic activity but also inhibit osteoclast differentiation, and could be potential lead compounds for the development of anti-osteoporosis agents.

[Analytical method of cereulide in processed cereal-based foods and milk powder by LC-MS/MS].

An LC-MS/MS method for analysis of cereulide, an emetic toxin produced by Bacillus cereus, was developed. Cereulide was extracted from samples, fried rice, pan-fried noodles, red bean paste and baby formula, with methanol and purified using Oasis HLB cartridges. LC separation was performed on a C18 column with a mixture of formic acid solution and methanol containing ammonium formate as a mobile phase, and the mass spectrometer was operated in the positive electrospray ionization mode. Performance evaluation showed that trueness was higher than 70% and repeatability and reproducibility were within 10%. The limits of quantification were lower than 1 µg/kg.

[Determination of vedaprofen in livestock products and seafoods by liquid chromatography-tandem mass spectrometry].

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the trace residue determination of vedaprofen (VPF) in livestock products and seafoods. VPF was extracted from each sample with acidified acetone, and the crude extract was re-extracted with ethyl acetate and NaCl solution. Clean-up was performed using a weak anion exchange cartridge (Bond Elut DEA). The LC separation was performed on a C18 column using acetonitrile-0.0025 mol/L formic acid (3 : 2) as the mobile phase and MS was run in the negative ion electrospray ionization mode. The calibration curve was linear in the range of 0.001-0.1 µg/mL VPF. The mean recoveries from equine muscle, cattle muscle, cattle liver, cattle fat, salmon, eel, corbicula, milk, egg and buckwheat honey were 72-94%, and the relative standard deviations (RSDs) were 1.1-2.0%. Limits of quantitation (LOQs) ranged from 0.001 to 0.007 µg/g.

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

Determination of carbendazim, thiophanate, thiophanate-methyl and benomyl residues in agricultural products by liquid chromatography-tandem mass spectrometry.

A simple and reliable liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for carbendazim (MBC), thiophanate (TE), thiophanate-methyl (TM) and benomyl (BM) in agricultural products. These compounds were extracted from agricultural products with methanol after addition of sodium L-ascorbate. BM was hydrolyzed to MBC during the extraction with methanol. TE and TM were cyclized to ethyl 2-benzimidazole carbamate (EBC) and MBC by refluxing at 120 °C for 30 min with copper acetate in 50% acetic acid. MBC and EBC were cleaned up by an n-hexane wash and extraction with ethyl acetate and determined by LC-MS/MS. The mean recoveries from 10 agricultural products were in the range of 75.8-100.0%, and the relative standard deviations of 5 experiments were in the range of 1.5-9.2% at concentrations equal to the maximum residue limits (MRLs). The calibration curves were made by using commercial MBC and EBC as reference analytical standards without refluxing. The quantification limits were 0.01 mg/kg (as MBC), which is the uniform limit in the positive list system for agricultural chemical residues in foods in Japan.

Development of green onion and cabbage certified reference materials for quantification of organophosphorus and pyrethroid pesticides.

Green onion and cabbage certified reference materials for the analysis of pesticide residues were issued by the National Metrology Institute of Japan, part of the National Institute of Advanced Industrial Science and Technology. Green onion and cabbage samples were grown so as to contain several kinds of organophosphorus and pyrethroid pesticides, and those were collected from a field in the Kochi Prefecture in Japan. The certification was carried out by using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides (diazinon, fenitrothion, cypermethrin, etofenprox, and permethrin for green onion and chlorpyrifos, fenitrothion, and permethrin for cabbage) were obtained by isotope dilution mass spectrometry. Certified values of target pesticides were 0.96-13.9 and 2.41-6.9 mg/kg for green onion and cabbage, respectively. These are the first green onion and cabbage powder certified reference materials in which organophosphorus and pyrethroid pesticides are determined.

Determination of dithiocarbamates and milneb residues in foods by gas chromatography-mass spectrometry.

A highly sensitive gas chromatographic-mass spectrometric (GC-MS) method was developed for dithiocarbamates (DTCs) and milneb in foods. DTCs and milneb were extracted from foods with cysteine-EDTA solution as sodium salts, and methylated with methyl iodide. Methyl derivatives of DTCs and milneb were cleaned up on a neutral alumina mini column and determined by GC-MS. The mean recoveries of DTCs and milneb were in the range of 72-120%, except for methiram. The quantification limits were 0.01 mg/kg (as CS(2)) in foods except tea (0.1 mg/kg as CS(2)). The developed method was applied to 10 compounds (4 dimethyldithiocarbamates, 3 ethylenebisdithiocarbamates, polycarbamates, propineb and milneb).

[Determination of bicozamycin in livestock products and seafoods by liquid chromatography-tandem mass spectrometry].

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for trace residue determination of bicozamycin (BZM) in livestock products and seafoods. BZM was extracted from a sample with acetonitrile-water (4 : 1), followed by a two-stage SPE enrichment and cleanup. The first stage involved a styrene-divinylbenzene copolymer cartridge (GL-Pak PLS-2), and the second stage involved a divinylbenzene-N-vinylpyrrolidone copolymer cartridge (Oasis HLB). The LC separation was performed on a C18 column using 0.01% formic acid-methanol (8 : 2) as the mobile phase and MS detection with negative ion electrospray ionization. The mean recoveries from swine muscle, liver, yellowtail, and milk fortified at the minimum residue limit (MRL) levels and 0.01 microg/g were >70%, and the relative standard deviations (RSDs) were <20%. Limits of quantitation (LOQs) ranged from 0.002 to 0.005 microg/g.

Determination of chloramphenicol residues in bee pollen by liquid chromatography/tandem mass spectrometry.

A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.

[Simultaneous determination of sedecamycin and terdecamycin in livestock products by LC/MS].

A simple and rapid LC/MS method for simultaneous determination of sedecamyin (SCM) and terdecamycin (TDM) in livestock products has been developed. SCM and TDM were extracted with acetonitrile. The extract was washed with n-hexane and then evaporated to dryness. The residue was dissolved in methanol, and injected into the LC/MS. The mass spectrometer was operated in the positive electrospray ionization (ESI) mode. LC separation was performed on a high-pH-resistant C18 column with 10 mmol/L carbonic acid-ammonia buffer (pH 10.0)-acetonitrile as a mobile pahse. The recoveries from swine muscle and liver fortified at the levels of 0.01 and 0.05 microg/g were 77-88%, and those from poultry muscle and liver fortified at the levels of 0.01 and 0.3 microg/g were 51-93%. The quantitation limits of SCM and TDM were 0.008 microg/g and 0.005 microg/g, respectively.

Contamination levels of PCDDs, PCDFs and Co-PCBs in commercial baby foods in Japan.

To examine dioxin contamination in commercial baby foods in Japan, congener analyses of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs) were performed on 102 varieties of baby foods obtained from supermarkets in 2001-2002. The toxic equivalent quantity (TEQ) levels for dioxins in samples ranged from < 0.001 to 0.135 pg-TEQ/g wet weight when undetected or trace levels of congeners were taken as zero. Among 102 samples tested, 26 samples exceeded 0.010 pg-TEQ/g. The highest TEQ value was for "sardine, vegetables" (0.135 pg-TEQ/g), followed by "Japanese radish (daikon), sardine" (0.080 pg-TEQ/g). Thus, dioxins were detected at low levels in baby foods containing animal products such as fishes and/or dairy products.