In vitro antimicrobial activities of 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein against Streptococcus mutans.

The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microscopy. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 µg/ml, which was equivalent to 2 µg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 µg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries.

Protective effects of resveratrol oligomers from Vitis amurensis against sodium nitroprusside-induced neurotoxicity in human neuroblastoma SH-SY5Y cells.

Nitric oxide (NO) induces apoptosis in neuronal cells, and has been implicating in a variety of neuronal pathological process. Thus, there is much interest in identifying natural substances which have protective effects against damage induced by nitrosative stress. The roots of Vitis amurensis have been used as traditional medicine and contain structurally diverse resveratrol oligomers with various biological activities. However, there have been few studies on the protective effect of resveratrol oligomers against neurotoxic reactive nitrogen species. In this study, we evaluated the protective effects of two resveratrol oligomers from V. amurensis, vitisin A and heyneanol A, against NO-induced toxicity. Additionally, their antioxidant activities were determined by measuring NO and hydroxyl radical scavenging ability. Both vitisin A and heyneanol A reduced cell death and DNA fragmentation induced by sodium nitroprusside in SH-SY5Y cells. The present study indicates that radical scavenging activities of vitisin A and heyneanol A contribute to protecting neuronal cells against nitrosative stress.

Composition of the extracellular matrix of lymphatic novel threadlike structures: is it keratin?

Background. The lumen of novel threadlike structures (NTSs) is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM). We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs. Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM), electrospray ionization-mass spectrometry, and proteomic analysis. Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be ~100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase. Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.

The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures.

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 µg/ml for glucose or 16 µg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.

Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase.

Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100µg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10µg/ml with an ED(50) value of 2µg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K(+) and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium.

Cytoprotective effect of lignans from Forsythia suspensa against peroxynitrite-induced LLC-PK1 cell damage.

There is mounting evidence that peroxynitrite (ONOO(-)) is closely related to the pathogenesis of various diseases. As a pharmacological strategy aimed at preventing ONOO(-)-mediated toxicity, the protective activity of Forsythia suspensa (Thunb.) Vahl (Oleaceae) against ONOO(-)-induced cellular damage was investigated and its active components identified. After bioactivity-guided fractionation of its methylene chloride fraction, two tetrahydrofurofuran lignans were isolated, namely phillygenin and 8-hydroxypinoresinol. The protective effects of these lignans against ONOO(-)-induced cell death were evaluated using renal epithelial cell LLC-PK1. Phillygenin and 8-hydroxypinoresinol significantly reduced the cell injury by 3-morpholinosydnonimine (SIN-1), a ONOO(-) generator. The hydroxy substituents on the phenyl moieties may contribute to the antioxidant activities of these lignans. These results suggest that phillygenin and 8-hydroxypinoresinol may be useful for the therapeutic or preventive applications in treating ONOO(-)-related diseases.