Impact of phenolic compounds in the acyl homoserine lactone-mediated quorum sensing regulatory pathways.

Quorum sensing (QS) is a cell density-dependent regulation of virulent bacterial gene expression by autoinducers that potentially pertains in the epidemic of bacterial virulence. This study was initially designed to evaluate the effect of 5 phenolic compounds in the modulation of QS and virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa, and to determine the mechanisms of their effects. Biosensor strains were used to assess antibacterial and anti-QS effect of these compounds. Only methyl gallate (MG) among these compounds demonstrated profound anti-QS effect in the preliminary study, and thus only MG was utilized further to evaluate the effects on the synthesis and activity of acyl homoserine lactone (AHL) in C. violaceum and on the modulation of biofilm, motility, proteolytic, elastase, pyocyanin, and rhamnolipid activity in P. aeruginosa. Finally, the effect of MG on the expression of QS-regulated genes of P. aeruginosa was verified. MG suppressed both the synthesis and activity of AHL in C. violaceum. It also restricted the biofilm formation and other QS-associated virulence factor of P. aeruginosa. MG concentration-dependently suppressed the expression of lasI/R, rhlI/R, and pqsA of P. aeruginosa and was non-toxic in in vitro study. This is the first report of the anti-QS mechanism of MG.

In vitro prebiotic effects and quantitative analysis of Bulnesia sarmienti extract.

Prebiotics are used to influence the growth, colonization, survival, and activity of probiotics, and enhance the innate immunity, thus improving the health status of the host. The survival, growth, and activity of probiotics are often interfered with by intrinsic factors and indigenous microbes in the gastrointestinal tract. In this study, Bulnesia sarmienti aqueous extract (BSAE) was evaluated for the growth-promoting activity of different strains of Lactobacillus acidophilus, and a simple, precise, cost-effective high-performance liquid chromatography (HPLC) method was developed and validated for the determination of active prebiotic ingredients in the extract. Different strains of L. acidophilus (probiotic) were incubated in de Man, Rogosa, and Sharpe (MRS) medium with the supplementation of BSAE in a final concentration of 0.0%, 1.0%, and 3.0% (w/v) as the sole carbon source. Growth of the probiotics was determined by measuring the pH changes and colony-forming units (CFU/mL) using the microdilution method for a period of 24 hours. The HPLC method was designed by optimizing mobile-phase composition, flow rate, column temperature, and detection wavelength. The method was validated according to the requirements of a new method, including accuracy, precision, linearity, limit of detection, limit of quantitation, and specificity. The major prebiotic active ingredients in BSAE were determined using the validated HPLC method. The rapid growth rate of different strains of L. acidophilus was observed in growth media with BSAE, whereas the decline of pH values of cultures varied in different strains of probiotics depending on the time of culture. (+)-Catechin and (-)-epicatechin were identified on the basis of their retention time, absorbance spectrum, and mass spectrometry fragmentation pattern. The developed method met the limit of all validation parameters. The prebiotic active components, (+)-catechin and (-)-epicatechin, were quantified as 1.27% and 0.71% (w/w), respectively, in crude extract, and 6.36±0.06 µg/mL and 4.47±0.41 µg/mL (mean±standard deviation), respectively, in a prebiotic capsule of BSAE by HPLC analysis. BSAE contains the active components of prebiotics and enhances the growth of L. acidophilus.

Hypolipidemic and Hepatic Steatosis Preventing Activities of the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Higher Basidiomycetes) Ethanol Extract In Vivo and In Vitro.

Obesity, a rapidly growing threat to human health worldwide, is responsible for a large proportion of the total burden of disease. Therefore, obesity control could be a vital scheme to prevent many diseases. The aim of this study was to examine the activities and mechanism of Auricularia auricula-judae 70% ethanol extract (AAE) in preventing hypolipidemic and hepatic steatosis. A normal diet (ND) and a high-fat diet (HFD) with or without 0.1% (w/w), 0.3% (w/w), and 1% (w/w) AAE were given to male C57BL/6 mice. Plasma lipids and liver enzymes were measured and tissue sections of liver were examined. Further mechanistic studies of mouse 3T3-L1 adipocytes were performed in vitro by verifying triglyceride, glycerol, and glycerol-3-phosphate dehydrogenase activity and messenger RNA expression of adipogenic and lipogenic genes using reverse transcriptase polymerase chain reaction amplification. Body weight and adipose tissue mass were significantly reduced in mice fed an ND and a HFD plus AAE compared with mice fed an HFD. In AAE-supplemented groups, plasma lipids and liver enzymes decreased dose-dependently. AAE suppressed the expression of adipogenic/lipogenic genes (PPARγ, C/EBPα, FAS) in 3T3-L1 cells without cytotoxicity. These findings suggest that AAE may reduce the risk of hepatic steatosis by modulating plasma lipids via the regulation of adipogenic/lipogenic transcriptional factors. AAE may have interesting applications to improve plasma lipids and liver enzymes.

Modulation of quorum sensing-controlled virulence factors by Nymphaea tetragona (water lily) extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Nymphaea tetragona is a widely distributed ornamental species with ethnomedicinal uses in the treatment of diarrhea, dysentery, eruptive fevers, and infections. The anti-infectious activities of this herb have already been assessed to clarify its traditional use as a medicine. AIM OF STUDY: In this study, we aimed to verify the inhibitory effects of N. tetragona 50% methanol extract (NTME) on quorum sensing (QS)-controlled virulence factors of bacteria since QS and its virulence factors are novel targets for antimicrobial therapy. MATERIALS AND METHODS: The antibacterial activity of this extract was evaluated against Chromobacterium violaceum and Pseudomonas aeruginosa. The inhibition of the violacein pigment of C. violaceum by NTME was determined qualitative and quantitative using standard methods. The effects of NTME on swarming motility, biofilm viability, pyocyanin production, and LasA protease activity were evaluated using P. aeruginosa. Finally, the in vitro and in vivo cytotoxicity of NTME were verified by MTT assay and oral administration to rats, respectively. RESULTS: The extract had concentration-dependent antibacterial activity against gram-negative bacteria. NTME at 1/2× minimum inhibitory concentration (MIC), 1× MIC and 2× MIC significantly lowered the levels of violacein of C. violaceum compared to that of the control. The swarming motility of P. aeruginosa was inhibited by ≥70% by treatment with 1/2× MIC of NTME. There were remarkable reductions in pyocyanin production and LasA protease activity in the overnight culture supernatant of P. aeruginosa supplemented with NTME when compared with that of the untreated control. The confocal micrographs of 24h biofilms of P. aeruginosa exposed to NTME exhibited a lower number of live cells than the control. No toxic effect was observed in in vitro and in vivo cytotoxicity assays of NTME. CONCLUSIONS: NTME was demonstrated to have significant concentration-dependent inhibitory effects on quorum sensing-mediated virulence factors of bacteria with non-toxic properties, and could thus be a prospective quorum sensing inhibitor.

Dichlormethane extract of the jelly ear mushroom Auricularia auricula-judae (higher Basidiomycetes) inhibits tumor cell growth in vitro.

In this study, a dichloromethane fraction (DCMF) from 70% Auricularia auricula-judae ethanol extract showed the highest level of antitumor activity compared to other solvent fractions (ethyl acetate, butanol, and water). The DCMF was found to have more potent antitumor activity against broncheoalveolar cancer (half maximal inhibitory concentration = 57.2 µg/mL) and gastric cancer cells (half maximal inhibitory concentration = 73.2 µg/mL) compared to the other solvent fractions, although all fractions inhibited the proliferation of the tumor cells in a dose-dependent manner. We further analyzed the DCMF composition by gas chromatography-coupled mass spectroscopy. Based on the results of this analysis, an antitumor active component (diazane) was identified in the DCMF. However, we found that diazane alone had a lower level of antitumor activity than the DCMF. These findings indicate that other unknown components of the DCMF also are responsible for the cytotoxic effects of DCMF against tumor cells. Semiquantitative reverse transcription polymerase chain reaction analysis demonstrated that DCMF induced cytotoxicity or tumor cell apoptosis as a result of the downregulation of Bcl-2 expression and p53 overexpression. Taken together, our study results demonstrated that the DCMF may be used as a functional additive for enhancing antioxidant activities and suppressing tumor growth in the body.

Comparative antitumor activity of jelly ear culinary-medicinal mushroom, Auricularia auricula-judae (Bull.) J. Schrot. (higher basidiomycetes) extracts against tumor cells in vitro.

The present study compares the antitumor activity of extracts from Auricularia auricula-judae, Phellinus gilvus, Ganoderma lucidum, and 100 Korean wild plants in the P388D1 macrophage cell line. The antitumor activity of A. auricula-judae extract (44.21%) did not differ significantly (P < 0.05) from those of Ph. Gilvus (39.46%) and G. lucidum (36.64%) at 1 mg/mL of concentration. Among 100 wild plants, Morus bombycis f. kase, Draba nemorosa var. hebecarpa, Sedum oryzifolium, Lotus corniculatus var. japonicus, and Auricularia auricula-judae 70% ethanol extracts inhibited the viability of tumor cells by 41.85%, 37.31%, 30.29%, 31.98%, and 25.40% at 3 mg/mL of concentration, while inhibition concentration (IC50) values were 1.81, 1.49, 1.05, 1.10, and 0.72 mg/mL, respectively. In Sarcoma 180, NCI H358, and SNU 1 cell lines, the inhibitory activities of A. auricula-judae extract were 65.71%, 69.76%, and 68.01%, respectively. Taken together, the results obtained from the present study indicated that four plant extracts (4% of tested wild plants) and A. auricula-judae extract with similar levels of Ph. Gilvus and G. lucidum extracts may be new potential antitumor agents.

Immunomodulatory activities and subacute toxicity of a novel ß-glucan from Paenibacillus polymyxa JB115 in rats.

Subacute toxicity and immunopharmacological activities of ß-glucan from P. polymyxa JB115 was evaluated in a 28-day feeding study in rats. The white blood cell count, red blood cell count, hematocrit, hemoglobin, thrombocytes (THR) and thrombocytocrit were significantly higher in male fed with ß-glucan than control rats and the insignificant lower eosinophil count, mean corpuscular volume, mean cell hemoglobin and uninfected THR (uTHR) levels were observed in male whereas no marked changes in female rats. No other significant differences in serum chemistry and liver, kidney, and spleen weights were observed. The pathological changes and other abnormal indicators were not detected in urine. Female rats fed with diet supplemented with 0.01% ß-glucan also showed marked increase in the percentage of blood cytotoxic T-lymphocytes compared to that of the control group while not significant differences in the percentage of blood B-lymphocytes. No adverse effects on general condition and behavior, growth, feed and water consumption and feed conversion efficiency were found. The results suggest that consumption of the novel ß-1, 3/1, 6-glucan from P. polymyxa JB115 was not associated with any obvious toxic effects in rats, indicating its safety as a potential immunostimulant or as an adjuvant of some animal vaccines.

Anti-inflammatory Activity of Dichloromethane Extract of Auricularia auricula-judae in RAW264.7 Cells.

The present study investigated the anti-inflammatory effects of dichloromethane extract of Auricularia auricula-judae. Dichloromethane extract of Auricularia auricula-judae inhibited Lipopolysaccharide (LPS) -induced nitric oxide (NO) production significantly in a dose-dependent manner in the concentration ≥10 µg/ml (p < 0.05) . Furthermore, RT-PCR results of this study indicated that the extract markedly reduced the expressions of inflammatory cytokines (IL-6, TNF-α and IL-1ß) mRNA in LPS-treated murine RAW 264.7 macrophages, which could possibly ameliorate the inflammation. Nevertheless, dichloromethane extract of Auricularia auricula-judae did not show complete inhibition of IL-6 mRNA expression. The inhibition of IL-1ß cytokine at protein level was also observed in a dose dependent manner. In conclusion,the current study revealed the previously unknown effect of dichloromethane ethyl extract of Auricularia auricula-judae inhibitions of the production of NO, IL-6, TNF-α and IL-1ß in LPS-stimulated macrophages.

Studies on the Dermal and Ocular Irritation of Prodigiosin Isolated from Zooshikella rubidus.

This study was carried out to investigate the irritation of the prodigiosin isolated from Zooshikella rubidus on the skin and eyes in New Zealand white rabbits. The tests were performed on the basis of Korea Food and Drug Administration (KFDA) guidelines. Prodigiosin induced severe eye irritation at high concentration (0.5 g/site/ml) but there was no eye irritation at low concentration (0.3 mg/site/ ml). The primary irritation index was calculated from higher concentration (0.5 g/site/ml) to lower concentration (0.3 mg/site/ml). There were found non-irritation or induced mild irritation at lower concentration of prodigiosin application. On the basis of this study, it could be concluded that the prodigiosin may be non-irritant to mild irritant of usual application at lower concentration (0.3 mg/site) resulting it is safe and useful in dyeing technology of fabrics.