Anti-inflammatory and bioavailability studies on dietary 3,5,4'-trihydroxy-6,7-methylenedioxyflavone-O-glycosides and their aglycone from indigo leaves in a murine model of inflammatory bowel disease.

Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities. We have recently demonstrated that indigo leaves contain high levels of flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) as an aglycone. In this study, we attempted to evaluate anti-inflammatory activities of TMF-O-glycosides and free TMF prepared from indigo leaves after extraction with hot water. Free TMF was found to appreciably down-regulate the gene expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, inducible nitric oxide, and tumor necrosis factor-α in cultured macrophage cells stimulated with lipopolysaccharide while up-regulating the expression of anti-inflammatory IL-10. However, no study has been conducted regarding in vivo anti-inflammatory activities of TMF-O-glycosides and free TMF until now. Here, we assessed in vivo anti-inflammatory effects of these dietary compounds on ulcerative colitis in a murine model of inflammatory bowel disease by the induction with dextran sulfate sodium (DSS). Histological evaluation revealed that both TMF-O-glycosides and free TMF effectively protected against DSS-induced ulcerative colitis. The analysis of digested products by liquid chromatography and mass spectrometry led us to detect free TMF as a predominant metabolite in the feces of mice fed with TMF-O-glycosides. Moreover, free TMF was later detected as glucuronyl conjugates of TMF in the liver of mice fed with both fractions. These results indicate the effective digestion of TMF-O-glycosides and the subsequent absorption of free TMF in the gut of mice for exerting anti-inflammatory effects. Taken together, our findings suggest that dietary TMF-O-glycosides could be promising natural sources for the utilization as herbal medicine and nutraceuticals to expect in vivo anti-inflammatory activities.

Suppression of Light-Induced Retinal Degeneration by Quercetin via the AP-1 Pathway in Rats.

We examined the cytoprotective effect of quercetin via activator protein (AP-1) and the heat shock protein 70 (Hsp70) pathway against light-induced retinal degeneration in rats. Quercetin was administered intraperitoneally to Sprague-Dawley rats for seven days before light exposure to intense white fluorescent light (3000 lux) for 24 h. Light-induced retinal damage was determined by the number of rows of photoreceptor cell nuclei, the microstructures of the rod outer segments and retinal pigment epithelium, and terminal deoxynucleotidyl transferase (TdT)-mediated 2'-Deoxyuridine-5'-triphosphate (dUTP) nick end labeling. To elucidate the cytoprotective mechanism of quercetin, expression levels were measured in the rat retinas of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative stress; Hsp70; and transcription factor AP-1 transcription activity. Pretreatment with quercetin inhibited light-induced photoreceptor cellular apoptosis and subsequent retinal degeneration in rats. 8-OHdG and Hsp70 protein expressions were up-regulated markedly by light exposure and suppressed by quercetin pretreatment. The results of an electrophoretic mobility shift assay showed that AP-1-binding activity was activated by light exposure, and binding of c-Fos and c-Jun, but not JunB, mediated the binding activity. Intraperitoneal administration of quercetin decreases photooxidative damage in the retina and mediates cytoprotection against light-induced photoreceptor cell degeneration in rats. Suppression of the heterodimeric combination of c-Jun and c-Fos proteins at the AP-1 binding site is highly involved in quercetin-mediated cytoprotection.

Constituents of flavonol O-glycosides and antioxidant activities of extracts from seeds, sprouts, and aerial parts of Polygonum tinctorium Lour.

Polygonum tinctorium Lour. (family Polygonaceae), known as indigo plant, has been useful as a medicinal or edible plant abundant in polyphenolic compounds. We have recently shown that flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) are predominant flavonoids in indigo leaves. However, no study has been performed regarding changes in the levels of flavonoid species during the germination and growth of indigo plant. Here, we attempted to determine the individual constituents of flavonol O-glycosides and the changes in their contents of the seeds, sprouts, and aerial parts. These results revealed that only the seeds predominantly contained flavonol O-(acetyl)-rhamnosides with quercetin or kaempferol as an aglycone. During the development of the sprouts and aerial parts, flavonol O-glycosides with TMF as an aglycone became mainly detectable and accounted for 79.4% and 74.9% of total flavonol O-glycosides from the extracts of aerial parts harvested in 2016 and 2017, respectively. Of the plant organs tested, the aerial parts exhibited the highest antioxidant activities concomitant with greatly increased levels of total polyphenols. Thus, we were able to conduct the identification and quantification of flavonol O-glycosides from the seeds, sprouts, and aerial parts of indigo plant and to evaluate antioxidant activities of their extracts. Taken together, our findings clearly provide the evidence that the aerial parts of indigo plant are a rich source of flavonol O-glycosides with TMF and exhibit much higher antioxidant activities, indicating the usefulness for the application to food and nutraceutical purposes.

Prevalence of Salmonella enterica Subspecies enterica in Red-Eared Sliders Trachemys scripta elegans Retailed in Pet Shops in Japan.

We investigated the prevalence of Salmonella in 227 small red-eared sliders (Trachemys scripta elegans) from 2006 to 2008. A total of 130 turtles (57.3%) tested positive for S. enterica subsp. enterica. Twenty-two serotypes including S. Montevideo, S. Newport, S. Pomona, S. Braenderup, S. Sandiego, and S. Litchfield were identified. Salmonella strains with closely related pulsed field gel electrophoresis (PFGE) patterns were isolated from several shops located in different areas from 2006 to 2008. Antimicrobial resistance was detected among strains of S. Montevideo, S. Newport, S. Braenderup, S. Sandiego, and S. Litchfield. The relatedness of antimicrobial resistance and PFGE profiles was not observed. The PFGE patterns of S. Poona strains isolated in 2006 and 2008 and the causative strains of turtle-associated salmonellosis in 2006 were identical. These results revealed a high prevalence of Salmonella enterica subsp. enterica in red-eared sliders retailed in Japan. In addition, genetically closely-related strains of turtle-associated Salmonella were repeatedly introduced into Japan over the study period and were distributed widely in Japan. These Salmonella strains present a risk of a widely disseminated outbreak of turtle-associated salmonellosis.

Protective Effect of Highly Polymeric A-Type Proanthocyanidins from Seed Shells of Japanese Horse Chestnut (Aesculus turbinata BLUME) against Light-Induced Oxidative Damage in Rat Retina.

Retinal tissue is exposed to oxidative stress caused by visible light. Light-damaged rat used in age-related macular degeneration (AMD) studies clarified that antioxidants decrease retinal light damage. Albino rats were exposed to 5000 Lux light for 12 h with oral administration of the polyphenolic compounds fraction (PF) from the seed shells of Japanese horse chestnut (30 mg/kg, 100 mg/kg, and 300 mg/kg body weight: BW). To evaluate the protective effects against light damage, electroretinograms (ERGs), the outer nuclear layer (ONL) thickness, the antioxidant activity of plasma, oxidized retinal lipids, and the detection of apoptosis were examined. To reveal their active compounds, PF were separated into an A-type proanthocyanidin (PAF) and a flavonol O-glycosides fraction. The protective effects of these fractions against light damage were compared by measuring the thickness of the ERGs and ONL. Compared with the negative control, the PF group (100 mg/kg and 300 mg/kg BW) significantly suppressed the decrease of the ERG amplitudes and ONL thickness. PF (300 mg/kg BW) induced the elevation of in vivo antioxidant activity, and the suppression of retinal lipid oxidation. PF administration also suppressed apoptotic cell death. The protective effects against light damage were attributable to the antioxidant activity of PAF. The light-induced damage of retinas was protected by oral administration of PF and PAF. Taken together, these compounds are potentially useful for the prevention of the disease caused by light exposure. HIGHLIGHTS: The protective effects of retinal damage by light exposure were evaluated using polyphenolic compounds from the seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) as an antioxidant. Decreases in the electroretinographic amplitude and outer nuclear layer thickness were suppressed by the polyphenolic compounds of the seed shells. Polyphenolic compounds from the seed shells of Japanese horse chestnut inhibited the oxidation of retinal lipids. Highly polymeric A-type proanthocyanidin from the seed shells protected the rat retina from light exposure damage by inhibiting oxidative stress and apoptotic mechanisms.

In Vitro Anti-inflammatory and Antioxidant Activities of 3,5,4'-Trihydroxy-6,7-Methylenedioxyflavone-O-Glycosides and Their Aglycone from Leaves of Polygonum tinctorium Lour.

Polygonum tinctorium Lour (indigo plant) has been regarded as a useful medicinal plant for traditional herbal medicine. The polyphenolic fraction of indigo leaves exhibited anti-inflammatory activities as determined by the suppressed synthesis of nitric oxide (NO) in cultured RAW264 macrophage cells. The acid hydrolysate of the fraction showed much more potent effect than the unhydrolyzed one. In sharp contrast, those fractions of indigo stems had almost no effect. 3,5,4'-Trihydroxy-6,7-methylenedioxyflavone (TMF)-O-glycosides and tryptanthrin were detected exclusively in the extracts of the leaves. The isolated flavonol species were furthermore tested for their anti-inflammatory activities against the synthesis of NO and prostaglandin E2 in the cultured macrophage cells. More potent anti-inflammatory effects were recognized with different aglycones of flavonols than their flavonol O-glycosides. Although the inhibitory effects of TMF were less effective than those of tryptanthrin, the levels of flavonol O-glycosides with TMF were much more abundant than those of tryptanthrin in the leaves. Oral administration of the fraction containing flavonol O-glycosides with TMF into mice revealed the detection of free TMF in the blood circulation, indicating that the aglycone moiety can be cleaved by digestive enzymes and absorbed in the gut. Alternatively, the assay of hydrophilic oxygen radical absorbance capacity revealed that the isolated species of flavonol O-glycosides with TMF and their aglycone had appreciable antioxidant activities. Taken together, our findings suggest that the predominant flavonol O-glycosides with TMF as an aglycone could be promising natural agents for the application to herbal medicine, nutraceuticals, and food additives.

Antioxidant activities and structural characterization of flavonol O-glycosides from seeds of Japanese horse chestnut (Aesculus turbinata BLUME).

We attempted to evaluate the contents and distribution of antioxidants in the whole seeds, seed shells, and peeled seeds of the Japanese horse chestnut. The seed shells exhibited the highest antioxidant activities due to the presence of highly polymeric proanthocyanidins as we have reported recently. On the other hand, the peeled seeds predominantly contained flavonols such as quercetin and kaempferol at a high level of 66.7% of total polyphenols, also contributing to the predominant antioxidant activities. The instrumental analysis of the extract from the whole seeds revealed the identification of eight flavonol O-glycosides, including six compounds with quercetin and two species with kaempferol as aglycones. The isolated species exhibited different antioxidant activities depending on the types of aglycones, glycosides, and acylated moieties. The results indicate that the peeled seeds are a good source of flavonol O-glycosides serving as antioxidants to be used for food additives and dietary supplements.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

Identification of new flavonol O-glycosides from indigo (Polygonum tinctorium Lour) leaves and their inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA reductase.

Indigo plant (Polygonum tinctorium Lour) has been utilized as a medicinal plant with a variety of biological activities. We have recently detected higher levels of flavonoids in indigo leaves. This study was undertaken to conduct the simultaneous analysis of those flavonoids using total extracts from indigo leaves by ultra-performance liquid chromatography-electrospray ionization-time-of-flight/mass spectrometry(E) (UPLC-ESI-TOF/MS(E)). The analysis by UPLC-ESI-TOF/MS(E) allowed us to determine 11 peaks of flavonoid species. The chemical structures of these compounds were identified as flavonol O-glycosides with different types of aglycones by the combination of spectroscopic and chemical methods. The predominant compounds were flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone as an aglycone. Of these, three compounds were elucidated as new compounds. All the isolated flavonol O-glycosides exhibited the inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA reductase in a dose-dependent manner with different potencies. Taken together, our results suggest the potential usefulness of the major flavonol O-glycosides from indigo leaves in controlling cholesterol biosynthesis.

Turtle-Associated Salmonella Infections in Kanagawa, Japan.

In this paper, we examine 2 case reports for different reptile-related Salmonella enterica subspecies enterica serotypes. In case 1, a 5-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Poona. The suspected source of infection was a turtle kept at the patient's home. In case 2, a 4-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Abony. The Pulsed-field gel electrophoresis analysis suggested that a tortoise kept at the patient's home was the source of infection. This paper presents a review of the literature and an examination of cases regarding turtle-associated salmonellosis in Japan.

Identification and quantitative analysis of polyphenolic compounds from the indigo plant (Polygonum tinctorium Lour).

The indigo plant (Polygonum tinctorium Lour) has been used traditionally as a medicinal plant with a variety of biological effects. Of these, polyphenolic ingredients are postulated to contribute to these activities. However, the identification and quantification of polyphenolic compounds in indigo plants have not been conducted comprehensively until now. This study was undertaken to identify the related ingredients by combined instrumental analyses using ultra-performance liquid chromatography electrospray-ionisation mass spectrometry and gas chromatography-mass spectrometry after the extracts of plant tissues were fractionated by absorption column chromatography. These analyses allowed the identification of kaempferol, quercetin-3-O-glucuronide, quercetin, kaempferol-3-O-glucopyranoside, caffeic acid, chlorogenic acid and tentative 3,5,4'-trihydroxy-6,7-methylenedioxyflavone. Furthermore, predominant polyphenolic compounds were quantified by reverse-phase high-performance liquid chromatography and capillary gas chromatography, revealing the higher proportions of kaempferol, quercetin-3-O-glucuronide and quercetin among them. The results indicate that the indigo plant is a promising source for flavonoids and the related compounds with beneficial medicinal effects.

Prevalence of Salmonella in wild snakes in Japan.

A total of 87 wild snakes of 6 species in 2 families collected in Japan were examined for the presence of Salmonella. The prevalence of Salmonella was 58.6%, and that of Salmonella enterica subspecies I, which includes most human pathogenic serotypes, accounted for 12.6%. S. enterica subspecies I was isolated from Japanese grass snakes and Japanese four-striped snakes, and the isolates belonged to 6 serotypes: S. enterica subspecies enterica serotypes Eastbourne, Mikawashima, Narashino, Newport, Saintpaul, and Thompson. The prevalence of S. enterica subspecies IIIb was higher (41.4%) than that of S. enterica subspecies I, and it was isolated from 4 snake species. The prevalence of Salmonella enterica subspecies and isolation of serotypes that are commonly detected in reptiles and human salmonellosis suggest that wild snakes may become a source of Salmonella infection.

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.

A collaborative study on a method to detect Salmonella in food.

Fourteen laboratories with expertise in Salmonella detection in food joined in a collaborative study. The laboratories performed qualitative analyses of ground pork samples using the proposed detection method. Salmonella Typhimurium (hydrogen sulfide-producing strain) and Salmonella Senftenberg (hydrogen sulfide-nonproducing strain) were used for inoculation. Three levels of Salmonella contamination were used for the study (0, 1-10, and 11-100 cfu/25 g). We evaluated the presence of Salmonella in each sample and the serological O group. Unmarked samples delivered to the laboratories were accurately judged to be inoculated or not inoculated with Salmonella at a 99.8% (419/420) detection rate in this collaborative study. The proposed method is suitable as a standard method to detect Salmonella in food.

Bathwater-associated cases of legionellosis in Japan, with a special focus on Legionella concentrations in water.

To evaluate the relationship between the incidence of legionellosis and Legionella concentrations in bathwater, we sent a questionnaire to 76 prefectural and municipal public health laboratories in Japan and found that 35 had encountered cases of legionellosis and had implemented investigations to determine the sources of the infections. Based on the results of the questionnaire, we were able to analyze various characteristics of the patients, of the facilities that were thought to be associated with the cases, and of the species and serogroups of the isolates and concentrations of Legionella. Ninety-six cases were included in this study. The median age was 67 years (range, 13-89 years). The most prevalent underlying medical condition among patients was diabetes, and the second most prevalent was high blood pressure. Concentrations of Legionella in bathwater ranged from 10 to 160,000 CFU/100 ml. Ten episodes were selected in which causative strains were found in the suspected source environment, and were then confirmed by pulsed-field gel electrophoresis analysis, enabling us to provide an estimated infectious concentration range of Legionella of 90 to 140,000 CFU/100 ml. It was thus suggested that the current Japanese regulatory safety level for Legionella in bathwater, which is set below the detection limit of culture techniques (10 CFU/100 ml), should be appropriate to prevent bathwater-associated legionellosis. In tandem with the above-mentioned research, a review of literature concerning bathwater-associated legionellosis and typical cases was undertaken.