Chrysanthemum indicum suppresses adipogenesis by inhibiting mitotic clonal expansion in 3T3-L1 preadipocytes.

Herbs have been of interest to treat diseases, including obesity, owing to their various bioactive constituents that exhibit therapeutic and prophylactic properties. The present study examined the anti-adipogenic effects and mechanisms of Chrysanthemum indicum aqueous extract (CAE) in 3T3-L1 preadipocytes. CAE comprises 1,3-dicaffeoylquinic acid, chlorogenic acid, kaempferol-3-O-glucoside, caffeic acid, and apigenin, which were corresponded with previous reports. CAE inhibited the accumulation of lipid droplets and significantly alleviated the expression of lipogenesis- and adipogenesis-associated biomarkers. Treatment with CAE inhibited the mitotic clonal expansion (MCE), corroborated by cell cycle arrest at the G0 /G1 phase, and mitigated the expression of cell cycle progression-associated proteins and in addition to phosphorylation of MCE-promoting transcription factors. Moreover, CAE downregulated the activation of Akt and extracellular signal-regulated kinase 1/2 signaling pathways. In summary, CAE facilitates adipogenic inhibition during the early phase of differentiation, especially MCE, and its phenolic compounds can contribute to its anti-obesogenic properties. PRACTICAL APPLICATIONS: Chrysanthemum indicum has been mainly used as traditional herbal tea and drinks. Chrysanthemum indicum aqueous extract (CAE) inhibits adipogenesis by suppressing mitotic clonal expansion during the early phase of differentiation in 3T3-L1 preadipocytes. 1,3-Dicaffeoylquinic acid, chlorogenic acid, kaempferol-3-O-glucoside, caffeic acid, and apigenin were detected in CAE. Based on these findings, CAE can be used as nutraceutical agents for prevention and treatment of obesity.

LC-ESI-MS/MS Simultaneous Analysis Method Coupled with Cation-Exchange Solid-Phase Extraction for Determination of Pyrrolizidine Alkaloids on Five Kinds of Herbal Medicines.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with potential hepatic and carcinogenic diseases in humans and animals. The concern over PAs has increased as the consumption of herbal medicines has increased. OBJECTIVE: This study aimed to develop and validate a sensitive analytical method to determine 28 PAs in five herbal medicines using liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). Additionally, this study identified and quantified the amount of PAs in 10 samples of each herbal medicine. METHODS: The pretreatment in the proposed LC-MS/MS analysis comprised solvent extraction using 0.05M H2SO4 in 50% methanol and clean-up step using an mixed-mode cationic exchange (MCX)-solid-phase extraction (SPE) cartridge. The PA contents in herbal medicines were measured by using the developed method. RESULTS: The proposed method had recoveries ranging from 72.5-123.7% for the Atractylodis Rhizoma Alba, 70.6-151.7% for Alba Chrysanthmi Flos, 80.6-130.9% for Leonuri Herba, 70.3-122.9% for Gastrodiae Rhizoma, and 67.1-106.9% for Glycyrrhizae Radix. Even though a few samples showed recoveries in unsatisfactory values, the proposed method indicated entirely sufficient recoveries and precision in most samples. In monitoring results, only Leonuri Herba contained two PAs, which indicated Retrorsine (4/10) of 84.7-120.9 µg/kg and Senkirkine (10/10) of 60.9-170.7 µg/kg. CONCLUSION: The results obtained from this study demonstrate that the proposed method is fit for purpose to determine 28 PAs in herbal medicines. Therefore it could serve as a regulatory method capable of being used for controlling the risks of PAs in certain medicinal plants and dietary supplements. HIGHLIGHTS: An LC-MS/MS method for the determination of 28 pyrrolizidine alkaloids in herbal medicines was developed and validated through this study. The proposed method is considered as an useful method for monitoring pyroolizidine alkaloids in herbal medicines.

Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals.

In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCETM.

The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage.

Ezrin as a complementary marker in ocular toxicity assessment using a three-dimensional reconstructed human corneal-like epithelium model, EpiOcular™.

INTRODUCTION: A variety of in vitro tests to replace the Draize test have been developed; however, there is no available method for assessing the full spectrum of Globally Harmonized System (GHS) categories. Human cornea-like three-dimensional (3D) reconstructed tissue models are the most promising in vitro systems. The objective of this study was to evaluate the ocular toxicity of 11 test substances using the EpiOcular™ model after performing proficiency tests. We further evaluated the effectiveness of ezrin staining as a complementary marker in histological analysis to overcome the limitation of eye irritation tests using 3D reconstructed human corneal epithelium models. METHODS: The assessment of ocular toxicity was performed by the suggested OECD TG 492 procedure. After treatment with proficiency test chemicals and 10 test substances, EpiOcular™ tissue models were stained with hematoxylin and eosin and ezrin, and the histological changes were observed by immunofluorescence microscopy. RESULTS: The ocular toxicity assessment of 10 test chemicals using the EpiOcular™ eye irritation test were in accordance with the UN GHS classification of test chemicals. Histological analysis of ezrin staining showed that the cell membranes of models treated with 10 out of 11 non-irritant chemicals were maintained, whereas those of models treated with 14 eye irritant substances resulted in the apparent translocation of ezrins from the cell membrane to the cytoplasm or nucleus by destruction of cell membrane. DISCUSSION: Ezrin may be used as a complementary marker to more accurately assess ocular toxicity using 3D reconstructed human cornea-like epithelium models.

Analysis of Insecticides in Dead Wild Birds in Korea from 2010 to 2013.

Wild birds are exposed to insecticides in a variety of ways, at different dose levels and via multiple routes, including ingestion of contaminated food items, and dermal, inhalation, preening, and embryonic exposure. Most poisoning by insecticides occurs as a result of misuse or accidental exposure, but intentional killing of unwanted animals also occurs. In this study, we investigated insecticides in the gastric contents of dead wild birds that were suspected to have died from insecticide poisoning based on necropsy. The wild birds were found dead in various regions and locations such as in mountains, and agricultural and urban areas. A total of 182 dead wild birds of 27 species were analyzed in this study, and insecticide residue levels were determined in 60.4% of the total samples analyzed. Monocrotophos and phosphamidon were the most common insecticides identified at rates of 50.0% and 30.7% of the insecticide-positive samples, respectively. Other insecticides identified in dead wild birds included organophosphorous, organochlorine and carbamate insecticides. However, there was limited evidence to conclusively establish the cause of death related to insecticides in this study. Nevertheless, considering the level of insecticide exposure, it is speculated that the exposure was mainly a result of accidental or intentional killing, and not from environmental residue.

Effects of Plant-Derived Extracts, Other Antimicrobials, and Their Combinations against Escherichia coli O157:H7 in Beef Systems.

The antimicrobial effects of thyme oil (TO), grapefruit seed extract (GSE), and basil essential oil, alone or in combination with cetylpyridinium chloride (CPC), sodium diacetate, or lactic acid, were evaluated against Escherichia coli O157:H7 in a moisture-enhanced beef model system. The model system was composed of a nonsterile beef homogenate to which NaCl (0.5%) and sodium tripolyphosphate (0.25%) were added, together with the tested antimicrobial ingredients. Beef homogenate treatments were inoculated (ca. 3 log CFU/ml) with rifampin-resistant E. coli O157:H7 (eight-strain mixture) and incubated at 15 °C (48 h). The most effective individual treatments were TO (0.25 or 0.5%) and GSE (0.5 or 1.0%), which immediately reduced (P < 0.05) pathogen levels by ≥ 3.4 log CFU/ml. Additionally, CPC (0.04%) reduced initial E. coli O157:H7 counts by 2.7 log CFU/ml. Most combinations of the tested plant-derived extracts with CPC (0.02 or 0.04%) and sodium diacetate (0.25%) had an additive effect with respect to antibacterial activity. In a second study, antimicrobial interventions were evaluated for their efficacy in reducing surface contamination of E. coli O157:H7 on beef cuts and to determine the effect of these surface treatments on subsequent internalization of the pathogen during blade tenderization. Beef cuts (10 by 8 by 3.5 cm) were inoculated (ca. 4 log CFU/g) on one side with the rifampin-resistant E. coli O157:H7 strain mixture and were then spray treated (20 lb/in(2), 10 s) with water, GSE (5 and 10%), lactic acid (5%), or CPC (5%). Untreated (control) and spray-treated surfaces were then subjected to double-pass blade tenderization. Surface contamination (4.4 log CFU/g) of E. coli O157:H7 was reduced (P < 0.05) to 3.4 (5% CPC) to 4.1 (water or 5% GSE) log CFU/g following spray treatment. The highest and lowest transfer rates of pathogen cells from the surface to deeper tissues of blade-tenderized sections were obtained in the untreated control and CPC-treated samples, respectively.

Analysis method for determination of nisin A and nisin Z in cow milk by using liquid chromatography-tandem mass spectrometry.

Nisin, a polypeptide with antimicrobial properties, is known as a natural preservative. It is used in various foods, including dairy products. This study validated a novel procedure using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of nisin A and nisin Z in cow milk. An extraction solution of 0.1 M acetate buffer containing 1 M NaCl (pH 2.0) and MeOH (1:1) was used to extract nisin A and nisin Z from milk samples. After the addition of extraction buffers, the samples were homogenized and centrifuged. The supernatant was filtered and injected for LC-MS/MS analysis. The linearity of the analytical method had a high correlation coefficient (r≥0.9987). The limits of quantitation of nisin A and nisin Z were approximately 12.9 and 10.9 µg/kg, respectively. The accuracy of the analytical method in milk ranged from 90.6 to 103.4% for nisin A and from 83.8 to 104.4% for nisin Z. The coefficient of variation values of intra- and interday in milk determined to be less than 5% in both nisin A and nisin Z. Because the proposed method has comparatively high recovery and low coefficient of variation, it seems appropriate for the determination of nisin A and nisin Z in milk samples. As the quantification of nisin A and nisin Z in milk samples by using LC-MS/MS has only been rarely reported until now, this study provides a meaningful technological advance for the dairy industry.

Determination of organophosphorus pesticides in stomach contents of postmortem animals by QuEChERS and gas chromatography.

The quick, easy, cheap, effective, rugged and safe (QuEChERS) method for sample preparation was applied to determine seven organophosphorus pesticides (OPs) in stomach contents of poisoned postmortem animals. The pesticides consisted of diazinon, edifenphos, ethyl p-nitrophenyl phenylphosphonothioate, fenitrothion, monocrotophos, parathion and phosphamindon, and tested samples included stomach contents from postmortem animals of cattle, goat, dog, cat, birds, deer and rabbit. The pesticides were spiked into the samples which were found to be negative through previous pesticide poisoning analysis, and the pesticides were extracted and cleaned up based on the QuEChERS process and then they were analyzed using gas chromatography (GC)-flame photometric detector (FPD) or GC-nitrogen-phosphorus detector (NPD) with a DB-5 column. Limits of detection ranged from 0.27 to 0.41 mg/kg for the seven pesticides. The mean recoveries ranged from 80 to 99% in GC-FPD and 83 to 90% in GC-NPD. The coefficients of variation were <10% for all analytes and sample matrix combinations except for phosphamidon and edifenphos in dog stomach contents. This study demonstrated that the method using QuEChERS and GC-FPD and/or GC-NPD is very effective to analyze the OPs in the stomach contents of postmortem animals.

Determination of Tinopal CBS-X in rice papers and rice noodles using HPLC with fluorescence detection and LC-MS/MS.

To date there have been no reports of methods to determine Tinopal CBS-X. We developed a rapid and simple method to determine the Tinopal CBS-X content in rice noodles and rice papers using HPLC equipped with fluorescence detection. Heating the rice noodles and rice papers to 80°C after adding 75% methanol solution induced the release of Tinopal CBS-X from processed rice products. Tinopal CBS-X was separated using an isocratic mobile phase comprising 50% acetonitrile/water containing 0.4% tetrabutyl ammonium hydrogen sulphate at pH 8.0. The samples suspected to be positive by HPLC analysis were then confirmed by LC-MS/MS analysis. This study also investigated the Tinopal CBS-X content of three rice noodle products and two rice papers. The limits of quantification for rice papers and rice noodles were 1.58 and 1.51 µg kg(-1), respectively, and their correlation curves showed good linearity with r(2) ≥ 0.9997 and ≥ 0.9998, respectively. Moreover, rice papers had recoveries of 70.3-83.3% with precision ranging from 5.0% to 7.9%, whereas rice noodles had slightly lower recoveries of 63.4-78.7% and precisions of 8.5-11.5%. Only one rice noodle product contained Tinopal CBS-X, at around 2.1 mg kg(-1), whereas it was not detected in four other samples. Consequently, Tinopal CBS-X from rice noodles and rice papers can be successfully detected using the developed pre-treatment and ion-pairing HPLC system coupled with fluorescence detection.

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis.

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit ß, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, ß-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.

Advanced multiplex PCR assay for differentiation of Brucella species.

Two new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.

Evaluation of brining ingredients and antimicrobials for effects on thermal destruction of Escherichia coli O157:H7 in a meat model system.

Escherichia coli O157:H7 may become internalized during brine injection of meat. This study evaluated the effect of brining ingredients on E. coli O157:H7 in a meat model system after simulated brining, storage, and cooking. Fresh knuckles (5.3 +/- 2.4% fat) or beef shoulder (15.3 +/- 2.2% fat) were ground individually, mixed with an 8-strain composite of rifampicin-resistant E. coli O157:H7 (7 log CFU/g) and brining solutions. Treatments included no brining, distilled water, sodium chloride (NaCl, 0.5%), sodium tripolyphosphate (STP, 0.25%), sodium pyrophosphate (SPP, 0.25%), NaCl + STP, NaCl + SPP, NaCl + STP + potassium lactate (PL, 2%), NaCl + STP + sodium diacetate (SD, 0.15%), NaCl + STP + PL + SD, NaCl + STP + lactic acid (0.3%), NaCl + STP + acetic acid (0.3%), NaCl + STP + citric acid (0.3%), NaCl + STP + EDTA (20 mM) + nisin (0.0015%) or pediocin (1000 AU/g), NaCl + STP + sodium metasilicate (0.2%), NaCl + STP + cetylpyridinium chloride (CPC; 0.5%), and NaCl + STP + hops beta acids (0.00055%). Samples (30 g) were analyzed for pH, and total microbial and rifampicin-resistant E. coli O157:H7 (inoculum) populations immediately after mixing, storage (24 h at 4 degrees C), and cooking to 65 degrees C. Fat and moisture contents and water activity were measured after storage and cooking only; cooking losses also were determined. The effect of beef type on microbial counts, pH, and water activity was negligible. No reductions in microbial counts were obtained by the brining solutions immediately or after storage, except for samples treated with CPC, which reduced (P < 0.05) pathogen counts after storage by approximately 1 log cycle. Cooking reduced pathogen counts by 1.5 to 2.5 logs, while CPC-treated samples had the lowest (P < 0.05) counts compared to any other treatment. These data may be useful in developing/improving brining recipes for control of E. coli O157:H7 in moisture-enhanced beef products.