Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Ethanolamine utilization supports Clostridium perfringens growth in infected tissues.

Clostridium perfringens possesses the ethanolamine (EA) utilization (eut) system encoded within the eut operon, which utilizes the EA as a carbon, nitrogen and energy source. To determine the role of the eut system in C. perfringens growth, an in-frame deletion of the eutABC genes was made in strain HN13 to generate the eutABC-deleted mutant strain HY1701. Comparison of HN13 and HY1701 growth in media supplemented with 1.0% glucose and/or 1.0% EA showed that glucose enhanced the growth of both strains, whereas EA enhanced HN13 growth, but not that of HY1701, indicating that the eut system is necessary for C. perfringens to utilize EA. The two-component regulatory system EutVW is needed to induce eut gene expression in response to EA whereas the global virulence regulator VirRS differentially controlled eut gene expression depending on glucose and EA availability. To assess the role of the eut system in vivo, an equal number of HN13 and HY1701 cells were injected into the right thigh muscles of mice. Mice infected with HY1701 showed fewer symptoms than those injected with HN13. The mortality rate of mice infected with HY1701 tended to be lower than for mice infected with HN13. In addition, in infected tissues from mice injected with a mixture of HN13 and HY1701, HN13 outnumbered HY1701. PCR screening demonstrated that C. perfringens isolated from gas gangrene and sporadic diarrhea cases carried both eut genes and the perfringolysin O gene (pfoA) as well as the phospholipase C gene (plc). However, pfoA was not detected in isolates from food poisoning patients and healthy volunteers. Culture supernatants prepared from HN13 grown in media containing 7.5% sheep red blood cells induced significantly higher eutB expression levels compared to those from plc- and/or pfoA-deletion mutants. Together, these results indicate that the eut system plays a nutritional role for C. perfringens during histolytic infection.

Cleansing effect of acidic L-arginine on human oral biofilm.

BACKGROUND: Dental plaque formed on tooth surfaces is a complex ecosystem composed of diverse oral bacteria and salivary components. Accumulation of dental plaque is a risk factor for dental caries and periodontal diseases. L-arginine has been reported to decrease the risk for dental caries by elevating plaque pH through the activity of arginine deiminase in oral bacteria. Here we evaluated the potential of L-arginine to remove established oral biofilms. METHODS: Biofilms were formed using human saliva mixed with Brain Heart Infusion broth supplemented with 1 % sucrose in multi-well plates or on plastic discs. After washing the biofilms with saline, citrate (10 mM, pH3.5), or L-arginine (0.5 M, pH3.5), the retained biofilms were analyzed by crystal violet staining, scanning electron microscopy, and Illumina-based 16S rDNA sequencing. RESULTS: Washing with acidic L-arginine detached oral biofilms more efficiently than saline and significantly reduced biofilm mass retained in multi-well plates or on plastic discs. Illumina-based microbiota analysis showed that citrate (pH3.5) preferentially washed out Streptococcus from mature oral biofilm, whereas acidic L-arginine prepared with 10 mM citrate buffer (pH3.5) non-specifically removed microbial components of the oral biofilm. CONCLUSIONS: Acidic L-arginine prepared with citrate buffer (pH3.5) effectively destabilized and removed mature oral biofilms. The acidic L-arginine solution described here could be used as an additive that enhances the efficacy of mouth rinses used in oral hygiene.

Bolus ingestion of individual branched-chain amino acids alters plasma amino acid profiles in young healthy men.

Physiological conditions in humans affect plasma amino acid profiles that might have potential for medical use. Because the branched-chain amino acids (BCAAs) leucine, isoleucine and valine are used as medicines and supplements, we investigated the acute effects of individual BCAAs (10-90 mg/kg body weight) or mixed BCAAs ingested as a bolus on plasma amino acid profiles in young healthy men. Plasma leucine levels rapidly increased and peaked around 30 min after leucine ingestion. Concentrations of plasma isoleucine, valine and phenylalanine subsequently decreased after ingestion, and those of methionine and tyrosine tended to decrease. The effects of ingested leucine on other plasma amino acids were biphasic, being higher at lower doses (10-20 mg/kg body weight). Isoleucine or valine intake also caused corresponding plasma amino acid concentrations to rapidly elevate, and peaks at 30-40 min after ingestion were much higher than that of plasma leucine after leucine ingestion. However, the increase in plasma isoleucine and valine concentrations essentially did not affect those of other plasma amino acids. The rate of decline among peak plasma BCAA concentrations was the highest for leucine, followed by isoleucine and valine. Oral mixed BCAAs promoted the decline in plasma isoleucine and valine concentrations. These results suggest that plasma leucine is a regulator of the plasma concentrations of BCAAs, methionine and aromatic amino acids.

Effect of Kampo medicine "Dai-kenchu-to" on microbiome in the intestine of the rats with fast stress.

PURPOSE: Diversity of gut microbiome has been recently reported to be lost in inflammatory bowel disease. We have previously reported that the Dai-kenchu-to (DKT) prevented the bacterial translocation through suppression of cytokine and apoptosis in rat's fast stress model. The aim of this study was to evaluate the effect of DKT on maintenance of microbial diversity in rat's intestine with inflammation. METHOD: Wister rats were received the fast stress for 5 days. In DKT group, rats were administered with DKT (300 mg/kg/day) during the fast stress (DKT-group). The gut microbiomes were analyzed at before- and after- fast stress, and the effect of DKT for on microbial diversities of the gut were evaluated by the PCR-clone library method targeting the 16 S ribosomal RNA gene. RESULT: In Control-group, Erysipelotrichaceae increased to 86% in after fast stress, OTU of before-fast stress was 111 and after fast stress was only 9 (changing rate: 58%). The diversity of microbiome was severely decreased. On the other hand, in DKT-group, diversity of microbiome was kept after fast stress (Lachnospiraceae: Ruminococcaceae: Coriobacteriales 54%, 22%, 5%), Operational taxonomic units of before fast stress was 52 and after fast stress was 55 (changing rate: 6%). Family Lachnospiraceae which includes butyrate-producing Clostridia (Clostridium IV and XIVa). CONCLUSION: DKT prevented the reduction of diversity of microbiome in rat's fast stress model. Our data suggested the new anti-inflammatory mechanism of DKT through gut microbiome.

Inhibitory effects of caraway (Carum carvi L. ) and its component on N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenicity.

To elucidate the mechanism of antimutagenicity of caraway, we examined the effects of caraway seed extract on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in DNA methyltransferase-deficient Salmonella typhimurium strains, O6-methylguanine DNA adduct formation, and thiol content in S. typhimurium cells. MNNG was highly mutagenic for ogt- strains YG7104 (ogt- ada+) and YG7108 (ogt- ada-), and it showed slightly higher mutagenicity in strain YG7100 (ogt+ ada-) than in strains TA100 and TA1535. Hot water extract of caraway seeds inhibited MNNG-induced mutation only in the ogt+ strains. In the presence of caraway extract, O6-methylguanine DNA adducts in strain YG7100 were decreased in proportion to the decrease of MNNG-induced mutagenesis. Although MNNG is known to degrade in the presence of thiols to produce methyl cation which can react with DNA, caraway had no effect on cellular concentrations of acid-soluble thiols. These results indicate that caraway does not directly inactivate MNNG and that Ogt-O6-methylguanine-DNA methyltransferase may be involved in the antimutagenic activity of caraway.

Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells.

Asiatic acid is a pentacyclic triterpene contained in medicinal plants. The cytotoxic effect of this compound and its augmentative effect on the anticancer drug irinotecan hydrochloride (CPT-11) were investigated in the human colon adenocarcinoma cell line HT-29. Asiatic acid dose-dependently showed cytotoxicity in HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were observed in a dose-dependent manner. A caspase-3 inhibitor suppressed the DNA ladder formation in a concentration-dependent manner. Bcl-2 and Bcl-XL proteins were decreased by asiatic acid treatment. These results indicate that asiatic acid induced apoptosis in HT-29 cells via caspase-3 activation. Cytotoxic effects of combined treatment with CPT-11 and asiatic acid on HT-29 cells were further examined. Simultaneous treatment or sequential exposure first to asiatic acid and then to CPT-11 showed an additive effect. Synergism was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid can be used as an agent for increasing sensitivity of colon cancer cells to treatment with CPT-11 or as an agent for reducing adverse effects of CPT-11.

Role of dietary phosphorus in the progression of renal failure.

Dietary phosphorus is thought to be a factor that impairs the residual renal function in patients with chronic renal failure. To determine the effect of dietary phosphorus on the prognosis of chronic renal failure, low-phosphorus milk was prepared from normal cow's milk using boehmite, a synthetic phosphate-ion absorbent. Regular diet, normal cow's milk, and low-phosphorus milk were then given to 5/6-nephrectomized rats and the serum levels of inorganic phosphorus, calcium, creatinine, and blood urine nitrogen in the rats in each group were compared. The serum levels of inorganic phosphorus and calcium were not different among the groups, despite a significant difference in phosphorus intakes. On the other hand, serum levels of creatinine (Cr) and blood urine nitrogen (BUN) in the rats fed low-phosphorus milk were significantly lower (Cr, 0.54+/-0.054mg/dl; BUN, 29.2+/-3.90mg/dl) than those in the rats fed a regular diet (Cr, 0.64+/-0.057mg/dl; BUN, 37.4+/-3.55mg/dl) or normal milk (Cr, 0.61+/-0.040mg/dl; BUN, 34.5+/-3.59mg/dl). No beneficial effect of protein restriction was observed when residual renal functions in rats fed a regular diet and those fed normal milk were compared. The results suggest that dietary phosphorus plays a major role in the progression of renal failure.

Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats.

An 80% ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against 2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4-67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9 H-pyrido[2,3-b]indole, benzo[a]pyrene,N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract itself showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0 g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon.