Subcritical water extraction (SWE) is an eco-friendly new extraction technology because it does not contain harmful organic solvent and has high extraction efficiency in a short time compared with conventional extraction methods. Blueberries (Vaccinium corymbosum) are widely known as superfood due to rich source of anthocyanin (malvidin-3-o-galgctoside) and antioxidant activity. In this study, optimal extraction condition of SWE from blueberries was determined and compared with the conventional extraction methods. SWE was carried out using a Dionex Accelerated Solvent Extractor (ASE, Model 350) under conditions of temperatures (110, 130, 150 and 170°C) and times (1, 3, 5 and 10 min). Total anthocyanin of SWE extracts was compared with hot water (60°C, 1 h) extract and pressed juice extract. The total anthocyanin content was determined by pH differential method. Considering both the extraction time and temperature conditions, the highest content of total anthocyanin content was 0.455 mg/g FW Vaccinium corymbosum at 130°C for 3 min. At high temperature and long extraction time, the anthocyanin in the blueberries will undergo thermal degradation due to low stability of anthocyanin at extreme condition. Besides, maximum yield of anthocyanin from blueberries using SWE was about 1.2 and 3.8 times more higher than hot water extract and pressed juice extract, respectively. Therefore, SWE is faster and more efficient method to extract anthocyanin from blueberries than conventional extraction methods. This study shows a possibility of SWE applied to food processing industry.

Citrus fruit is important source of flavonoids, mainly flavanones which are narirutin and hesperidin. Those citrus flavonoids have been found to have health-related properties including antioxidant, anticancer, and anti-inflammatory. The main purpose of this study was to verify that the extraction of narirutin and hesperidin from Citrus peel can be more effective by combining pulsed electric field (PEF) pre-treatment and subcritical water extraction (SWE). Citrus unshiu peels were treated with PEF under conditions of electric field strength (3 kV/cm) and times (1 and 2 min). Subsequent SWE was conducted by using a Dionex Accelerated Solvent Extractor (ASE, Model 350) at extraction temperature 170°C for 10 min. The total flavonoids content was measured by using the aluminum chloride colorimetric method and the antioxidant capacity was analyzed by the Ferric reducing antioxidant power (FRAP) assay using spectrophotometer. The concentrations of narirutin and hesperidin were increased as PEF pre-treatment time increased. The highest concentrations of narirutin and hesperidin were 13.41 mg narirutin/g dry citrus peel and 141.16 mg hesperidin/g dry citrus peel at PEF pre-treatment condition of 3 kV/cm and 2 min. The total flavonoids contents of the extracts increased 105.2% and 123.1% for citrus peel PEF treated at 1 and 2 min, respectively. In addition, compared to the untreated sample, PEF pre-treatments of 1 and 2 min increased the antioxidant capacity of the extracts 109.2% and 160.8%, respectively. Therefore, the results demonstrate the potential of PEF pre-treatment to improve the SWE of flavonoids from citrus unshiu peel.

Mitochondria participate in various intracellular metabolic pathways such as generating intracellular ATP, synthesizing several essential molecules, regulating calcium homeostasis, and producing the cell’s reactive oxygen species (ROS). Emerging studies have demonstrated newly discovered roles of mitochondria, which participate in the regulation of innate immune responses by modulating NLRP3 inflammasomes. Here, we review the recently proposed pathways to be involved in mitochondria-mediated regulation of inflammasome activation and inflammation: 1) mitochondrial ROS, 2) calcium mobilization, 3) nicotinamide adenine dinucleotide (NAD+) reduction, 4) cardiolipin, 5) mitofusin, 6) mitochondrial DNA, 7) mitochondrial antiviral signaling protein. Furthermore, we highlight the significance of mitophagy as a negative regulator of mitochondrial damage and NLRP3 inflammasome activation, as potentially helpful therapeutic approaches which could potentially address uncontrolled inflammation.

Background: Periodontitis is an inflammatory disease characterized by the breakdown of tooth-supporting tissues, leading to tooth loss. Aggregatibacter actinomycetemcomitans are major etiologic bacterium causing aggressive periodontitis. Ursodeoxycholic acid (UDCA), a hydrophilic gall bladder acid, has been used as an effective drug for various diseases related to immunity. The aim of this study was to investigate the effect of UDCA on the inflammatory response induced by A. actinomycetemcomitans. Methods: A human acute monocytic leukemia cell line (THP-1) was differentiated to macrophage- like cells by treatment with phorbol 12-mystristate 13-acetate (PMA) and used for all experiments. The cytotoxic effect of UDCA was examined by MTT assay. THP-1 cells were pretreated with UDCA for 30 min before A. actinomycetemcomitans infection and the culture supernatant was analyzed for various cytokine production by ELISA. The effect of UDCA on bacterial growth was examined by measuring optical densities using a spectrophotometer. Results: UDCA showed no cytotoxic effect on THP-1 cells, up to 80 μM Ed highlight: Please confirm technical meaning. UDCA pretreatment inhibited the A. actinomycetemcomitansinduced IL-1β, TNF-⍺, and IL-17A secretion in a dosedependent manner. UDCA also inhibited IL-21 production at 60 μM. The production of IL-12 and IL-4 was not influenced by A. actinomycetemcomitans infection. Conclusion: These findings indicate that UDCA inhibits the production of inflammatory cytokines involved in innate and Th17 immune responses in A. actinomycetemcomitansinfected THP-1- derived macrophages, which suggests its possible use for the control of aggressive periodontitis.

Background: Periodontitis is generally a chronic disorder characterized by the breakdown of tooth-supporting tissues. P. gingivalis, a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Frequently, P. gingivalis infection leads to cell death. However, the correlation between P. gingivalis–induced cell death and periodontal inflammation remains to be elucidated. Among cell deaths, the death of immune cells appears to play a significant role in inflammatory response. Thus, the aim of this study was to examine P. gingivalis–induced cell death, focusing on autophagy and apoptosis in THP-1 cells. Methods: Human acute monocytic leukemia cell line (THP-1) was used for all experiments. Autophagy induced by P. gingivalis in THP-1 cells was examined by Cyto ID staining. Intracellular autophagic vacuoles were observed by fluorescence microscopy using staining Acridine orange (AO); and 3-methyladenine (3-MA) was used to inhibit autophagy. Total cell death was measured by LDH assay. Cytokine production was measured by an ELISA method. Results: P. gingivalis induced autophagy in an MOI-dependent manner in THP-1 cells, but 3-MA treatment decreased autophagy and increased the apoptotic blebs. P. gingivalis infection did not increase apoptosis compared to the control cells, whereas inhibition of autophagy by 3-MA significantly increased apoptosis in P. gingivalis-infected THP-1 cells. Inhibition of autophagy by 3-MA also increased total cell deaths and inflammatory cytokine production, including IL-1β and TNF-⍺. Conclusion: P. gingivalis induced autophagy in THP-1 cells, but the inhibition of autophagy by 3-MA stimulated apoptosis, leading to increased cell deaths and pro-inflammatory cytokines production. Hence, the modulation of cell deaths may provide a mechanism to fight against invading microorganisms in host cells and could be a promising way to control inflammation.

This experiment was carried out to investigate the effect of harvesting time of Italian ryegrass (Lolium multiflorum Lam; IRG) in spring on dry matter (DM) yield. IRG cultivars ‘Kowinearly’ and ‘Greenfarm’ were seeded at 50 kg/ha and grown on rice paddy fields. There was no difference in growth characteristics between both varieties before winter. However, cold tolerance of Kowinearly was higher than that of Greenfarm, as demonstrated by post-wintering growth characteristics. The heading date of both varieties was affected by the spring weather. The IRG was harvested three times at 5-day intervals beginning from 9 May. The DM yield of Greenfarm was 6,306; 7,335; and 8,109 kg/ha, and that of Kowinearly was 7,498; 9,196; and 10,449 kg/ha at the three consecutive harvests. The delay of the harvesting time for 5 and 10 days increased the DM yield of Greenfarm by 16% and 29% and that of Kowinearly by 23% and 39%, respectively, compared to the yield at first harvest (p < 0.05). Therefore, IRG harvest later than early to mid-May is expected to increase productivity. The feed values of Greenfarm were: 12.2% of crude protein (CP), 34.5% of acid detergent fiber (ADF), 57.7% of neutral detergent fiber (NDF), 61.6% of total digestible nutrients (TDN), and 72.3% of in vitro DM digestibility (IVDMD). For Kowinearly, these values were 16.4% of CP, 30.4% of ADF, 52.7% of NDF, 64.9% of TDN and 79.0% of IVDMD.

The present study investigated the effects of different seeding rates on growth characteristics and seed productivity of the “Kowinearly” cultivar of Italian ryegrass (Lolium multiflorum Lam.) in paddy fields. Sowing Kowinearly in paddy fields under growing rice at the rates of 20, 30, 40, and 50 kg/ha resulted in seed yields of 1.57, 1.92, 2.06, and 2.09 ton/ha, respectively. Seed yield of Kowinearly was the highest at a seeding rate of 50 kg/ha (p<0.05), at which the cultivar was able to survive in winter and the weed ratio was low. Under these conditions, most growth characteristics such as winter survival (85%), weed ratio (10%), stems per square meter (1,006) were superior than those sown at other seeding rates (p<0.05). In addition, it has been reported that the economic efficiency of the 50 kg/ha seeding rate was higher than that of the other seeding rates.

Interleukin-1b (IL-1β), a proinflammatory cytokine, regulates the innate immune responses against bacterial infection. Mature IL-1β is produced from pro-IL-1β by activated caspase-1, which in turn is activated by the inflammasome complex formation. In this study, we compared the inflammasome mRNA expression induced by S. sanguinis, S. oralis, F. nucleatum and P. intermedia. Among the tested bacteria, S. sanguinis induced the highest IL-1β secretion. S. oralis, F. nucleatum and P. intermedia induced very weak IL-1β secretion. S. sanguinis mostly induced the NLRP3 mRNA expressions. Although F. nucleatum did not induce high IL-1β secretion, it induced high expression levels of AIM2, NLRP2, and NLRP3. No specific inflammasomes were induced by S. oralis and P.intermedia. Studying the inflammasome complex activation induced by oral bacteria may thus enhance our understanding of the pathogenesis of oral diseases.

Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.