Investigation of microsatellite loci for the identification of registered varieties of Perilla frutescens and a discussion on the ancestor species of P. frutescens.

Techniques for identifying varieties of crops used as spices and food additives have important implications for the safety of food production, prevention of false labeling, protection of breeders' rights, and prevention of theft or outflow to other countries. Presently, there are 16 varieties of Perilla frutescens in the variety registration system of the Ministry of Agriculture, Forestry and Fishes in Japan (Ministry of Agriculture, Forestry and Fisheries. Variety registration data search. http://www.hinshu2.maff.go.jp/ . Accessed 03 Nov 2022). One such variety is "Shimoadachi," which contains citral as a main essential oil component and has a lemon-like smell. To our knowledge, no other cultivars with similar characteristics in P. frutescens have been identified. Additionally, the registered variety "per-001" contains high contents of perillaldehyde and rosmarinic acid, with practical applications for herbal medicines and functional foods. Therefore, the development of variety identification techniques is necessary for stable production and protection. In this study, we investigated microsatellite loci for the accurate identification of registered varieties of red perilla. These loci provide a basis for breeding superior varieties of medicinal plants.

Mechanisms and Safety of Antidepressant-Like Effect of Nutmeg in Mice.

Nutmeg, a dried seed kernel of a tall evergreen Myristicaceae tree, is widely used as a spice and herbal medicine and is known to have antidepressant-like effects. This study evaluates the mechanisms underlying this antidepressant-like effect and safety of nutmeg n-hexane extract (NNE) in mice. Tail suspension and open field tests showed that NNE (10 mg/kg, per OS (p.o.)) significantly decreased the immobility time of mice without effecting their spontaneous locomotor activity. The reduction of immobility time of mice elicited by NNE was significantly inhibited by ketanserin (5-hydroxytryptamine (5-HT)2A/2C receptor antagonist), ondansetron (5-HT3 receptor antagonist), and yohimbine (α2 receptor antagonist). WAY100635 (5-HT1A receptor antagonist) tended to inhibit the effect of NNE but without significance. Testing according to the Organisation for Economic Co-operation and Development Guidelines, no mice died due to administrated NNE (2000 mg/kg, p.o.), and behavioral and weight changes were not seen in the acute toxicity test. In the Ames test, no increase in the number of revertant colonies for each bacterial strain test strains TA98 and TA100 by nutmeg powder was observed either with or without metabolic activity by S9 mix. These results suggest that NNE shows an antidepressant-like effect involving various serotonergic and noradrenergic nervous systems and maybe a highly safe natural preparation.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Caffeic acid and rosmarinic acid contents in genus Perilla.

Caffeic acid and rosmarinic acid are common components of Labiatae plants, such as shiso (Perilla frutescens Britton var. crispa W. Deane) and Boraginaceae plants. These compounds have various pharmacological activities, such as anti-inflammatory, anti-anxiety, and anti-depressive activities, but the content of these compounds in perilla has not been studied in detail. This study investigated the caffeic acid and rosmarinic acid contents of several pure strains in genus Perilla. Perilla plants cultivated under a certain set of conditions had different caffeic acid and rosmarinic acid contents. For example, their contents were higher in P. setoyensis ("Setoegoma"), suggesting that the genetic background of the species greatly affects caffeic acid and rosmarinic acid contents. Several strains of P. frutescens var. crispa were cultivated at the Experimental Station for Medicinal Plants, Graduate School of Pharmaceutical Sciences, Kyoto University and differences in their caffeic acid and rosmarinic acid contents were also observed. The total content of anthocyanins, which are closely related to the leaf color of perilla, was measured as cyanidin-3-glucoside equivalents, and a weak positive correlation was observed between the content of rosmarinic acid, and the total content of total anthocyanins. Furthermore, the results suggest that luminosity and photon flux density of light during cultivation can affect rosmarinic acid content.

Rosmarinic acid in Perilla frutescens and perilla herb analyzed by HPLC.

High-quality perilla leaves are defined as those having purple upper and lower surfaces and a pleasant smell. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not the content of rosmarinic acid. Rosmarinic acid is a common component of Labiatae plants such as shiso (Perilla frutescens Britton var. crispa W. Deane). Rosmarinic acid has been reported to exhibit anti-inflammatory and anti-oxidant activity but the factors affecting the content of rosmarinic acid in plants remain unknown. This study describes a simple and reproducible method for quantifying rosmarinic acid. We elucidated the main causes for the different rosmarinic acid contents of plants by examining various samples of perilla using the proposed method. Significant differences in rosmarinic acid content between varieties and cultivators were observed. The rosmarinic acid content was higher in green perilla compared with red perilla, in wild species compared with cultivated species, and in plants cultivated in outdoor nurseries compared with in indoor nurseries. The proposed quantitative method was used to examine the rosmarinic acid content in a Kampo formula, Hangekobokuto, and was found to be higher in decoctions prepared using the Kouge method compared with the typical preparation method. We examined the chlorophyll and caffeic acid contents of several samples and their relationship with the rosmarinic acid content.

Relationship of Asian Dust Events with Atmospheric Endotoxin and Protein Levels in Sasebo and Kyoto, Japan, in Spring.

Asian dust events are caused by dust storms originating from deserts in Mongolia and northern China, and these events are observed in Japan, mainly in spring. To explore the effect of Asian dust events on atmospheric endotoxin and protein levels, we collected the total suspended particles (TSP) in the spring months (March, April, and May) of 2015 in Sasebo and Kyoto, Japan, and assessed the levels of biological elements at both locations. At both locations, the daily concentrations of TSP, water-soluble Ca2+ (an indicator mineral of soil in dust), endotoxins, and proteins were found to be high during and after Asian dust events recorded by the Japan Meteorological Agency. The concentration of Ca2+ showed a strong positive correlation with the concentrations of TSP and endotoxin, while the concentration of protein was moderately positively correlated with Ca2+ in both Sasebo and Kyoto. There were large concentrations of endotoxins, and the fluctuation ranges were higher in Sasebo than in Kyoto. In contrast, protein concentrations showed low levels of fluctuation, and no major differences were found in the concentration at each location.

Ruthenium N-Confused Porphyrins: Selective Reactivity for Ambident 2-Heteroatom-Substituted Pyridines Serving as Axial Ligands.

Three types of ruthenium(II) N-confused porphyrin (NCP) complexes bearing an axial 2-thiopyridine, 2-pyridone, and 2-iminopyridine moiety at the inner carbon atom, respectively, were synthesized. The unique reactivity of the 2-substituted pyridine derivatives (2-X-pyridine; X=SH, OH, NH2 ) toward the inner carbon atom of the NCP allows the formation of two types of coordinated products (i. e., pyridine donor versus 2-heteroatom donors), as inferred from single-crystal X-ray structures. The selective reactivity was investigated by using density functional theory (DFT) calculations. Finally, the catalytic activity of these ruthenium complexes was demonstrated through the styrene oxidation reactions. As a result, the ruthenium(II) NCP complex bearing a 2-thiopyridine moiety, together with aqueous H2 O2 as an oxidant showed the highest selectivity for benzaldehyde (benzaldehyde/styrene oxide=20 : 1).

Seasonal Fluctuation of Endotoxin and Protein Concentrations in Outdoor Air in Sasebo, Japan.

To determine the levels of endotoxin, which is a major component of outer membrane of Gram-negative bacteria, and protein in the atmosphere in Sasebo, Japan, we measured these biological materials in fine (aerodynamic diameter ≤2.5 µm) and coarse (≥2.5 µm) particles collected for 81 weeks (September 2014 to May 2016). The monthly concentrations (i.e., the mean value of weekly concentrations for each month) of endotoxin were higher in coarse particles than in fine particles. Fluctuations in monthly endotoxin concentrations were large in both fine (0.0005-0.0208 EU/m3) and coarse (0.0032-0.1164 EU/m3) particles. Furthermore, the endotoxin concentrations in coarse particles were highest in October 2014 and 2015 as well as September 2014 (0.0407-0.1164 EU/m3). However, the monthly protein concentrations were higher in fine particles than in coarse particles. Compared to the endotoxin concentrations, the fluctuations in the monthly protein concentrations were smaller in both coarse and fine particles. To our knowledge, this study is the first to report long-term atmospheric concentrations of endotoxin and protein in Japan. Since the endotoxin concentrations in coarse particles were positively associated with the concentrations of Na+ and Cl-, it suggests the involvement of Gram-negative bacteria from seawater to the endotoxin levels in the atmosphere. For fine particles, the protein concentrations were positively associated with the concentrations of particles, NO3- and SO42-. These results suggest that combustion of organic materials, such as biomass burning, may be a contributor to atmospheric protein during this study period.

Spatial correlativity of atmospheric particulate components simultaneously collected in Japan.

The simultaneous sampling of total suspended particles was performed at 14 sites in Japan during July 2008-June 2009. The spatial correlativity of each particulate composition toward Osaka was obtained for nine selected sites to overview the chemical composition and geographical distribution of particulate components across a wide range of areas nationwide. The spatial correlatives of atmospheric particulate components were extended to an even wider range of areas up to 950 km distance (meso-alpha scale region, >200 km) for a far-reaching distance analysis unique in the literature. Overall, the spatial correlations of ionic species and both organic and elemental carbons were significant, suggesting their shared advections, including their long-range transport from East Asia. Although sulfate ions are widely dispersed across Japan, such is not necessarily correlated with organic and elemental carbon, possibly indicating that the sulfate emission source, including long-range transport, differs from that of carbonaceous particulates. By contrast, the characteristics of spatial correlatives of metallic constituents vary; for example, particulate Pb and Cd show a significantly wide range of spatial correlatives to Osaka, while Mn-though limited to cities neighboring Osaka-shows significant spatial correlations. Other metallic constituents showed no significant spatial correlatives, indicating the effects of local pollutants. Moreover, the extent of the spatial dispersion of the particulate components and the relationships among chemical components were analyzed via factor analysis to highlight the effects of long-range inflow and local original emissions. In this treatment, 13 particulate components among the 19 measured were implicated in long-range transport.

Induction effect of coadministration of soybean isoflavones and sodium nitrite on DNA damage in mouse stomach.

We have already found that nitrite-treated isoflavones exhibit genotoxic activities toward Salmonella typhimurium TA 100 and 98 strains (submitted: nitrite-treated genistein). However, we have not demonstrated genotoxic activity induced by simultaneous treatment with isoflavones and NaNO(2)in vivo. In the present study, we examined whether coadministration of isoflavones (such as daidzein and genistein) and NaNO(2) induces DNA damage in the stomach of ICR male mice. Mice were coadministered with isoflavones (1mg/kg body weight) and NaNO(2) (10mg/kg body weight), and dissected to collect tissues at 1, 3, and 6h after administration. We used comet assay combined with repair enzyme formamidopyrimidine-N-glycosylase (FPG) to detect FPG-sensitive sites. An HPLC-ECD system was employed to determine 8-oxo-2'-deoxyguanosine (8-oxodG) in the stomach. In addition, we observed leukocyte infiltration by histopathological investigation, and measured total superoxide dismutase (SOD) in the stomach. We confirmed that oxidative DNA damage in the stomach was significantly increased by coadministration. Total SOD activities were also significantly stimulated by coadministration. However, the induction of inflammation in the stomach was not found. These data suggest that coadministration of isoflavones and NaNO(2) can cause DNA damage in the stomach because of the formation of radicals.

Leaf extract of Wasabia japonica relieved oxidative stress induced by Helicobacter pylori infection and stress loading in Mongolian gerbils.

Infection with Helicobacter pylori (H. pylori) can induce gastric disorders, and though its presence cannot explain disease pathogenesis and does not have associations with other factors, it is well known that H. pylori infection causes stomach inflammation following oxidative stress. We examined the suppressive effects of a leaf extract of Wasabia japonica on H. pylori infection and on stress loading in Mongolian gerbils. Following oral administration of wasabi extract of 50 and 200 mg/kg B.W./d for 10 d, the animals were exposed to restraint stress for 90 and 270 min. As for the results, the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the stomach and oxidative DNA damage in peripheral erythrocytes at 270 min significantly increased. That elevation was significantly suppressed by the addition of the leaf extract. We concluded that the simultaneous loading of H. pylori infection and physical stress loading might induce oxidative DNA damage additively, while a leaf extract attenuated this DNA damage in the stomach as well as the peripheral erythrocytes.

Genotoxicity and estrogenic activity of 3,3'-dinitrobisphenol A in goldfish.

3,3'-Dinitrobisphenol A (dinitro-BPA) is formed in a mixture of bisphenol A (BPA) and nitrite under acidic conditions. It shows genotoxicity in male ICR mice on a micronucleus test, but its estrogenic activity has not been examined in vivo. We examined its estrogenic activity using goldfish (Carassius auratus) by measuring plasma levels of vitellogenin (VTG) by the ELISA method. Expression of VTG didn't increase in the plasma of goldfish intraperitoneal injected with dinitro-BPA at a dose of 10 mg/kg of body weight. We also examined the genotoxicity of dinitro-BPA by single-cell gel electrophoresis (comet assay) and a micronucleus test using goldfish. The DNA tail moment of blood cells increased after intraperitoneal injection of dinitro-BPA. Dinitro-BPA at the same dose significantly increased micronucleus frequency in gills of goldfish. On the other hand, BPA did not significantly increase the frequency of micronucleated cells. In conclusion, we found that dinitro-BPA did not show estrogenic activity, but had genotoxic potency stronger than that of BPA.

Application of a new bioassay technique using goldfish for assessment of water toxicity.

There are a variety of chemicals in aquatic environment, so it is important to assess the toxicity. The biomarkers such as induction of DNA damage, micronuclei, vitellogenin, and hepatic P450 in fish are known to be effective for monitoring genotoxic and/or estrogenic chemicals. However, there is little study to use these biomarkers in same fish. Goldfish (Carassius auratus) is widely used and is suitable in size to collect blood or organs. In this study, validity of multiple-biomarkers in goldfish was checked using standard chemicals and applied in the river water. Ho River, which flows through the textile dyeing factory in Shizuoka Prefecture, Japan, was reported to show genotoxicity toward Salmonella typhimurium TA98 and YG1024. When the goldfish were exposed to Ho River, DNA damage, estrogenic activity, and CYP1A induction were observed. Through the study, it was assumed that not only mutagens/carcinogens but also endocrine disrupting chemicals and poly aromatic hydrocarbons were present in Ho River. Therefore, chemical identification should be required. We could evaluate both genotoxicity and estrogenic activity simultaneously, so goldfish might be a good experimental model for estimation of chemical contamination levels in aquatic environment.

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish.

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite.

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay.

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.