Lactococcus lactis SG-030. The purified glucooligosaccharides produced from Weissella cibaria YRK005 were used as prebiotics. RAW 264.7 cells were treated with synbiotics in two ways. One way was a simultaneous treatment with lactic acid bacteria and glucooligosaccharides. The other way was to pre-culture the lactic acid bacteria with glucooligosaccharides followed by treatment with synbiotic culture broth or synbiotic culture supernatant. In both cases, synbiotics synergistically increased NO production in RAW 264.7 cells. In addition, synbiotics treatment increased the expression of tissue necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase genes. Synbiotics also increased the expression of P38, extracellular signal-regulated kinases, c-Jun N-terminal kinases, phosphoinositide 3-kinase, and Akt proteins. The results confirmed that the synbiotics prepared in this study exhibited synergistic immunostimulatory activity.Much attention has been recently paid to the health benefits of synbiotics, a combination of probiotics and prebiotics. In this study, synbiotics were prepared by combining lactic acid bacteria with potential as probiotics and purified glucooligosaccharides, and their immunostimulatory activity was evaluated using RAW 264.7 macrophage cells. A lactic acid bacteria strain with high antioxidant activity, acid and bile salt tolerance, adhesion to Caco-2 cells, and nitric oxide (NO) production was selected as a potential probiotic strain. The selected strain, isolated from forsythia, was identified as  Recently, research on probiotics, prebiotics, and their combination, synbiotics, has been actively conducted along with microbiome research because of their beneficial health functions. In 2001, the FAO and WHO defined probiotics as live microorganisms which confer health benefits on the host when administered in adequate amounts . MoreovePrebiotics refers to some non-digestible but fermentable dietary carbohydrates that can selectively stimulate certain bacteria in the colon, such as bifidobacteria and lactobacilli, considered beneficial to humans. Short-chain carbohydrates are also referred to as indigestible oligosaccharides . PrebiotSynbiotics are a product consisting of a combination of probiotics and prebiotics and were defined as a mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and/or activating the metabolism of one or a limited number of health-promoting bacteria, and thus improving host welfare . With reIn synbiotic combination, the proper selection of probiotics and prebiotics is an important criterion. Each probiotic and prebiotics should be able to benefit host health even when used separately. Synbiotics likewise have several health effects, such as promoting the growth of probiotic bacteria in the gastrointestinal tract and improving the survival of beneficial microorganisms ,12. The Weissella cibaria YRK005 were purified, and their structure was identified to be glucooligosaccharides, one of the emerging prebiotics [Although there is growing interest in and studies on synbiotics, the current studies on synbiotics are limited to using natural carbohydrate sources from plants such as sugar cane fiber or well-established prebiotics such as fructooligosaccharides as prebiotics ,16. In tebiotics .Weissella cibaria YRK005 as prebiotics and potentially probiotic lactic acid bacteria Lactococcus lactis SG-030 as probiotics, and their immunostimulatory activity was evaluated by determining the level of nitric oxide production and the expression of genes encoding cytokines such as tissue necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase gene in RAW 264.7 macrophage cells. The expression level of genes involved in the MAPKs signaling pathways and PI3K/Akt signaling pathways such as P38, extracellular signal-regulated kinases, c-Jun N-terminal kinases, phosphoinositide 3-kinase, and Akt proteins was also evaluated.In this study, the synbiotics were prepared by combining purified glucooligosaccharides from w/v) NaCl), serially diluted with decimals, plated on PCA agar  with 0.005% bromocresol purple, and incubated anaerobically at 37 °C for 18 h. The pure isolated strains by selecting colonies with a different morphology from each plate were anaerobically cultured and maintained on lactobacilli de Man Rogosa Sharpe , consisting of 20 g glucose, 10 g peptone, 8 g meat extract, 4 g yeast extract, 5 g sodium acetate trihydrate, 2 g triammonium citrate, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, and 0.05 g manganous sulfate tetrahydrate, per liter, at 37 °C for 18 h.Fermented foods such as kimchi and salted fish, seafood, and plant flowers and fruits were collected from several local markets and fields in Korea to isolate lactic acid bacteria. Ten grams of each sample were suspended in sterilized saline (0.88% (Weissella cibaria YRK005 was isolated from young radish [Glucooligosaccharides-producing strain g radish . The strv/v) and incubated at 37 °C for 18 h. The culture broth was centrifuged at 21,200× g for 5 min to obtain the supernatant, and 10 μL of the supernatant was transferred into a 96-well plate. One hundred and ninety microliters of 0.1 mM DPPH were added and kept in the dark for 30 min at room temperature. The absorbance of the reaction mixture was measured at the wavelength of 517 nm using a spectrophotometer . A standard curve was prepared with 0–2 mM ascorbic acid. Distilled water was used as a control and 2 mM ascorbic acid was used for positive control. The antioxidant activity was expressed as a DPPH radical scavenging activity and calculated using Equation (1).The antioxidant activity of lactic acid bacteria was determined using 2,2-diphenyl-1-picrylhydrazyl . Two millimolar concentration of DPPH dissolved in methanol was diluted to 0.1 mM before use. Lactic acid bacteria culture grown in MRS was inoculated into MRS broth to achieve a final concentration of 1% (v/v) and incubated at 37 °C for 18 h. After centrifuging the culture at 21,200× g for 2 min, the obtained pellet was washed twice with sterilized saline. The washed cell pellet was resuspended in 4 mL of 100 mM glycine-HCl buffer (pH 2.5) and incubated at 37 °C for 2 h. The cells were spread onto MRS agar plate, incubated at 37 °C for 18 h, and the number of viable cells was counted. The cell pellet without incubation was used as a control. The acid tolerance of lactic acid bacteria was calculated using Equation (2).A colony of lactic acid bacteria was inoculated from an agar plate into MRS broth and incubated at 37 °C for 18 h. The culture was inoculated into 4 mL MRS broth to make a final concentration of 1% (v/v) and incubated at 37 °C for 18 h. The bacterial culture was spread onto MRS agar plate, incubated 37 °C for 18 h, and the viable cell number was counted. The bile salt tolerance was calculated using Equation (3).A single colony of lactic acid bacteria was inoculated into MRS broth and incubated at 37 °C for 18 h. Next, the culture was inoculated into MRS with or without 0.3% Bacto oxgall (BD) to make a final concentration of 1% (® HTB-37™) were purchased from American Type Culture Collection  and cultured in high-glucose Dulbecco’s modified eagle medium  containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum  at 37 °C in 5% CO2 incubator. Lactic acid bacteria were cultured in MRS broth until the number of viable cells was 1 × 108 CFU/mL. Five milliliters of bacterial culture were centrifuged at 15,920× g for 1 min, washed three times with Dulbecco’s phosphate-buffered saline , and resuspended with 1 mL DMEM containing 10% fetal bovine serum (CORNING). Caco-2 cells were seeded in a 24-well plate with 500 μL of DMEM containing 5 × 105 cells per well. The plate was pre-incubated at 37 °C in a 5% CO2 incubator for 20 h and washed twice with DMEM containing 10% fetal bovine serum. Two hundred microliters of lactic acid bacteria cells (1 × 108 CFU/mL) and 300 μL of DMEM containing 10% fetal bovine serum were added to Caco-2 cells and incubated at 37 °C in 5% CO2 incubator for 2 h. After incubation, the 24-well plate was washed twice with DPBS to remove lactic acid bacteria cells and dead caco-2 cells. Cells attached to the 24-well plate were detached using 200 μL of 0.25% Trypsin-EDTA (Gibco) and collected. Collected cells were washed twice with DPBS at 9420× g for 1 min, resuspended in 1 mL of 0.1% Triton X-100 (Sigma-Aldrich), 10-fold serially diluted in saline , spread on MRS agar plate, and the viable cell number of lactic acid bacteria were counted after incubation at 37 °C for 18 h. As a control, 200 μL of lactic acid bacteria cells and 800 μL of saline were used. Adhesion ability was calculated using Equation (4).Caco-2 cells (ATCChttps://apiweb.biomerieux.com/ (accessed on 10 May 2019).The carbohydrate utilization of lactic acid bacteria was examined using the API 50 CHL kit  following manufacturer’s instruction and the data were interpreted using Apiweb™ (bioMériuex) available at ® Genomic DNA extraction kit  following the manufacturer’s instructions. The PCR amplification of the 16S rRNA gene using universal primers 27F and 1492R, nucleotide sequencing of 16S rRNA gene, and phylogenetic tree analysis were carried out as described previously [Lactic acid bacteria genomic DNA was isolated using the Accuprep eviously ,19. ScanW. cibaria YRK005 in LM broth  containing 14.7% (w/v) sucrose and 13.5% (w/v) maltose as described previously [W. cibaria YRK005 was inoculated into 5 mL MRS broth and incubated anaerobically at 37 °C for 18 h. Three hundred microliters of the culture were inoculated into 30 mL MRS broth and incubated at 37 °C for 9 h. The culture was then inoculated into 2 L LM broth in a 3 L jar fermenter  and incubated without aeration at 30 °C for 6 h. The pH of the culture was adjusted to 5.0 during the fermentation. The culture was centrifuged at 13,500× g for 10 min  and the supernatant was concentrated under reduced pressure at 60 °C using a rotary evaporator .Glucooligosaccharides were produced by incubating eviously ,20. Brien-propyl alcohol (Sigma-Aldrich):distilled H2O. The developed TLC plate was dipped in 0.3%(w/v) N-(1-naphtyl) ethylenediamine dihydrochloride (Sigma-Aldrich) and 5%(v/v) sulfuric acid (Sigma-Aldrich) in methanol (Sigma-Aldrich) and then baked at 121 °C for 5 min [The concentrated supernatant was loaded onto Bio-gel P2 resin  in a Glass Econo-Column (1.5 × 120 cm) and the oligosaccharides were separated by gel permeation column chromatography . The flow rate was 30 mL/h and the eluents were fractionated using a fraction collector . The purified oligosaccharides were freeze-dried  and analyzed by thin-layer chromatography (TLC). Silica gel 60 F254  was used for TLC and the samples were developed twice using 2:5:1.5 nitromethane (Sigma-Aldrich): or 5 min .TM, ATCC, Rockville, MD, USA) in two ways to evaluate the immunostimulatory effect of synbiotics on RAW 264.7 cells. One way was the simultaneous treatment of lactic acid bacteria and oligosaccharides in RAW 264.7 cells. Strain SG-030 was grown on RAW 264.7 cells with a multiplicity of infection  of 2 . For this, 1.5 mL of lactic acid bacteria culture grown in MRS broth was centrifuged at 15,920× g for 1 min and the pellet was washed three times with DPBS (Welgene) and resuspended with 500 μL of DMEM containing 10% FBS and 1% penicillin-streptomycin (Gibco). Glucooligosaccharides from W. cibaria YRK005 were applied to RAW 264.7 cells at a final concentration of 0.5 μg/mL.Probiotic lactic acid bacteria and glucooligosaccharides were applied to RAW 264.7 macrophage cells, a mouse peritoneal macrophage cell line (TIB-71v/v) followed by incubation at 37 °C for 18 h. After incubation, the culture was divided into two preparations. One preparation was the culture broth itself. The other preparation was made by centrifuging the culture broth at 15,920× g for 1 min, and the supernatant was filtered with a sterilized PVDF filter .The other way was inoculating lactic acid bacteria culture to 0.1 μg/mL or 0.5 μg/mL oligosaccharides dissolved in modified MRS  at the concentration of 5% (5 cells per well) with 500 μL of DMEM. The plates were pre-incubated at 37 °C in a 5% CO2 incubator for 20 h. Lactic acid bacteria cells were added to RAW 264.7 cells suspensions to make MOI of 500. Meanwhile, the oligosaccharides were added to RAW 264.7 cells at each 0.1, 1, and 10 mg/mL concentration. Lipopolysaccharides (LPS) at a concentration of 1 μg/mL were used as a control. The plates were cultured at 37 °C for 24 h, and 100 μL of culture supernatant was mixed with 100 μL of Griess reagent -ethylenediamine dihydrochloride, and 2.5% phosphoric acid, Sigma-Aldrich) and allowed to react at room temperature and for 15 min. The produced NO was measured using a microplate reader  at a wavelength of 450 nm.Nitric oxide (NO) production by RAW 264.7 cells was measured using the Griess reagent. RAW 264.7 cells were attached in a 24-well plate  following the manufacturer’s instruction. cDNA was synthesized from the isolated RNA using Transcriptor First Strand cDNA Synthesis Kit  according to manufacturer’s instruction.Real-time PCR was used to measure inducible nitric oxide synthase (iNOS) gene expression levels and cytokine genes. The treated RAW 264.7 cells were washed with DPBS, and RNA was isolated using easy-BLUE® 96 (Roche) and FastStart Essential DNA Green Master kit (Roche). The sequences of primers  and incubated at 37 °C for 20 h. The method for sample treatment for protein extraction was the same as v/v) phosphatase inhibitor cocktail and 1% (v/v) protease inhibitor cocktail . Cells were lysed by reacting on ice for 10 min and centrifuged at 14,000× g for 10 min to obtain proteins. The extracted proteins were quantified using Pierce™ BCA protein assay kit .Western blot was conducted to analyze the changes in protein expressions in RAW 264.7 cells treated with synbiotics preparations. RAW 264.7 cells were plated in a 6-well plate , phospho-JNK, Akt, phospho-Akt  and anti-rabbit secondary antibodies for 30 min, respectively. The blots were visualized using ECL detection reagent on JESS. The intensity of bands was analyzed by Image J software [Western blot analysis was performed using an automated western blot analyzer . Total 16 μg of protein solution were separated by 12–230 kDa JESS separation module. After separation, proteins were blocked by antibody diluent followed by incubation with primary antibodies against p38, phospho-p38, extracellular signal-regulated kinases (ERK) 1/2, phospho-ERK 1/2, phosphoinositide 3-kinases (PI3K), phospho-PI3K, c-Jun software .t-test using SPSS 25 . Statistical significance between the multi groups was determined by one-way analysis of variance (ANOVA) and Duncan’s multiple range test at the level of p < 0.05.The data were obtained by three replicate experiments and expressed as mean ± standard deviation (SD). Experimental data were verified by the student’s W. cibaria D30 (67.81%) and DPPH radical scavenging activities of Lactobacillus plantarum C88 (53.05%) [L. plantarum MA2 exhibited resistance to oxidative stress induced by reactive oxygen species by expressing antioxidant enzymes such as super oxide dismutase, glutathione reductase, and NADH peroxidase [The lactic acid bacteria strains with high antioxidant activity, acid tolerance, bile salt tolerance, adhesion to Caco-2 intestinal cells, and NO production were screened among 972 lactic acid bacteria strains isolated in our laboratory to select lactic acid bacteria strains with potential probiotic properties. Among them, 32 strains evaluated by DPPH radical scavenging showed good antioxidant activity . Several(53.05%) ,24. Simiroxidase .Resistances to acid and bile stresses are critical properties for probiotic bacteria. The capability of probiotics to survive, at appropriate levels, in gastrointestinal (GI) tract conditions is important to use in the food industry because the GI tract is a major location that influences the cell viability of lactic acid bacteria ,27. In tLactobacillus sp. The NO production increased dose-dependent in most strains. NO is one of the free radicals produced from L-arginine by NO synthase reaction [NO production in RAW 264.7 macrophage cells treated with lactic acid bacteria, showing high antioxidant activity, acid and bile salt tolerance, and adhesion ability to Caco-2 intestinal cells, were also checked, and some strains showed high NO production. Chang et al.  showed treaction . When mareaction .Based on the evaluation of data in Forsythia koreana (Rehd.) Naka), was identified by phenotypic, biochemical, and molecular biological analyses. Strain SG-030 was Gram-positive and catalase-negative. When the morphology of strain SG-030 was observed using a scanning electron microscope, its size was determined to be 1.2–1.5 μm in length and 0.4–0.6 μm in diameter , or the supernatant of synbiotics culture (with 0.1 μg/mL oligosaccharides and L. lactis SG-030), the treatment with synbiotic culture broth increased NO production the highest among samples , or the supernatant of synbiotics culture (with 0.5 μg/mL oligosaccharides and L. lactis SG-030), the treatments with synbiotic culture broth and synbiotic supernatant synergistically increased NO production , there was a small amount of oligosaccharides in the supernatant due to the depletion caused by bacterial uptake. When the oligosaccharides were supplemented in a high amount (0.5 μg/mL), there were enough oligosaccharides to induce NO production. This showed that oligosaccharides had an important role in the synbiotic immunostimulatory activity.L. lactis SG-030 and oligosaccharides using real-time PCR  to stimulate PI3K/Akt and ERK1/2 and finally activated the secretion of other cytokines, thereby regulating immune response ,46,47 F.L. lactis SG-030 showed good probiotic properties and was chosen as a probiotic strain, although in vivo study on the probiotic property is needed. In addition, glucodoligosaccharides from W. cibaria YRK005 were used as prebiotics due to their growth promotion activity on some probiotic strains.The purpose of this study was to prepare synbiotics combined with a probiotic strain and prebiotics and evaluate their immunostimulatory activity using RAW 264.7 macrophage cells. The selection of suitable probiotics and prebiotics is an important criterion for the preparation of synbiotics. To do this, lactic acid bacteria, exhibiting probiotic properties such as high antioxidant activity, acid and bile acid tolerance, adhesion activity to intestinal cells, and NO production in RAW 264.7 macrophage cells, were screened. A lactic acid bacteria strain This study was designed to prepare synbiotics by combining the selected lactic acid bacteria and the purified glucooligosaccharides and confirm whether this combination produces promising synbiotics with immunostimulatory activity. The results showed that synbiotics prepared in this study had synergistic immunostimulatory activity when compared to the results of probiotic strain alone or oligosaccharides alone. Especially, since the treatment of RAW 264.7 cells with synbiotic culture broth induced the expression of cytokine genes and iNOS gene involved in the MAPKs signaling pathways and PI3K/Akt signaling pathways than that with synbiotic culture supernatant, it is necessary to carry out further study to explain this result. Nevertheless, the results in this study confirmed that synbiotics showed a synergistic effect on immunostimulatory activity, suggesting the possibility of the development of synbiotics as a resource for producing beneficial health food.


Iyengaria stellata, Spatoglossum asperum, Sargassum linearifolium, and Stoechospermum polypodioides—were collected from the Saurashtra Coast of the Arabian Sea. They were analyzed for metabolite profiling, biochemical activities , and total phenolic and flavonoid contents. A concentration-dependent antioxidant, reducing, and scavenging activities were observed for all four brown seaweeds. The S. asperum and I. stellata extracts showed maximum total antioxidant activity. S. asperum also showed high scavenging and reducing activities compared to other studied brown seaweeds. Further, S. asperum contained high total phenolic and flavonoid content compared to other brown seaweeds collected from the same coast. A multivariate correlation study confirmed a positive correlation between total phenolic and flavonoid contents, and biochemical activities  for all brown seaweeds. About 35% anti-proliferative activity was observed with S. asperum extract on Huh7 cells; in contrast S. polypodioide showed about 44% proliferation inhibition of Huh7 cells. Similarly, 26% proliferation inhibition of HeLa cells was observed with S. asperum extract. Overall, S. asperum possesses high total flavonoid and phenolic amounts, and showed potential antioxidant, scavenging and reducing characteristics. The study confirmed the nutraceutical potential of S. asperum and that it could be a promising functional food ingredient.Brown seaweeds have shown high potential of bioactivity and provide health benefits as an important functional food ingredient. Therefore, four abundantly growing tropical brown seaweeds— The ocean is an extensive biome, in which a variety of life forms, starting from single-celled organisms to different types of multicellular organisms, develop, proliferate and die each day. It is presumed that the first life on Earth developed in the ocean. Additionally, it can be stated that single-cell algae eventually evolved to form each lifeform present today . The macDue to the harsh environmental conditions of the oceans, seaweeds are exposed to different changes in climate, such as variations in temperature, mineral concentration, ultraviolet light exposure, and the concentration of dissolved gases, as well as attacks by pathogens and predators . Due to Researchers have been paying attention to the evolution of novel biologically active compounds from natural sources because nature has proved many times that it is an important resource of the compounds, which have biomedical properties ,15,16. GIyengaria stellata, Spatoglossum asperum, Sargassum linearifolium, Stoechospermum polypodioides) were harvested from the Arabian sea , India to analyze the total flavonoid and phenolic contents, scavenging, total antioxidant and reducing activities, and anti-proliferative activity on human cancer cell lines (HeLa and Huh7). The results confirmed the nutraceutical potential of the brown seaweeds [Metabolomics is a new approach in the ‘omics’ techniques, and it represents the metabolite activities in an organism at a particular time. Seaweeds are known to possess a variety of metabolic activities, and the study of their metabolomics might unfold different aspects of seaweeds’ properties, and also identify their nutraceutical application . Brown sseaweeds ,30,31,32Iyengaria stellata (Børgesen) Børgesen (IS), Sargassum linearifolium (Turner) C.Agardh (SL), Spatoglossum asperum J.Agardh (SA), and Stoechospermum polypodioides (J.V.Lamouroux) J.Agardh (SP) grow abundantly in the Saurashtra coast of the Arabian Sea. These four brown seaweeds were collected from the Veraval , Okha , and Adri  coasts [Brown seaweeds, namely ) coasts . Collectg at 4 °C for 10 min. Supernatant was collected, again extracted with chloroform and, upper-phase was aspirated, dried and derivatized. For the quantification of metabolites, ribitol/adonitol (6 μg) was added as an internal reference. Methoxyamine hydrochloride (40 µL) and N-Methyl-N-(trimethylsilyl) trifluoroacetamide (70 µL) were used for the derivatization. The derivatized samples were analyzed by gas chromatography and mass spectroscopy . Mass spectra peaks were recorded and compared with the NIST library for the identification. The quantification was performed using internal standard.Metabolites were isolated and quantified using optimized method ,34,35. Bv/v, 500 mL) on magnetic stirrer for overnight. The supernatant was collected after centrifugation of extraction mixture at 7000× g for 10 min. The extraction procedure repeated twice. All three supernatants were pooled and concentrated under 100–150 mbar at 37 °C using rotary evaporator and then lyophilized. The lyophilized extracts were stored at −20 °C for further use.About 10 g of dry seaweed samples (passed through sieve) was used for the extraction with aqueous methanol  concentrations of seaweed extracts or trolox (standard) was mixed with ABTS free radicals (1 mL), and was incubated for 90 min. The absorbance was read at 734 nm and the activity of samples was compared with standards and percentage inhibition was calculated [Total antioxidant activity was performed by estimating scavenging of ABTS free radicals, which were generated by mixing potassium persulphate (2.45 mM) with ABTS diammonium salt (7 mM), followed by 12–16 h incubation in dark room at room-temperature, and trolox was used as standard ,37,38. Tlculated .w/v, in methanol) and diluting it with methanol until 0.98 ± 0.02 absorbance was obtained at 517 nm [−1) concentrations of seaweed extracts or trolox (standard) and was incubated overnight at room temperature in the dark. Sample absorbance was taken at 517 nm, which was compared with standard curve to obtain final scavenging activity of the extract [DPPH  is a free radical. Evaluation of DPPH scavenging activity was carried out by preparing DPPH stock solution  were mixed with 2.5 mL of Folin–Coicalteu (FC) reagents (0.2 M) , incubated for 5 min and sodium carbonate  was added, which was further incubated (90 min) at room temperature in dark condition. Afterwards, absorbance of the reaction mixture was taken at 760 nm, and TPC of extracts was calculated as mg·mL−1 gallic acid per 100 mg of extract. For TFC, different concentrations of seaweed extracts (200–1000 μg·mL−1) were mixed with 0.3 mL NaNO2 , incubated at room temperature for 5 min and 0.3 mL AlCl3  and 2 mL NaOH (1 M) were added to the reaction mixture. Absorbance was read at 510 nm, and TFC was calculated using quercetin standard curve as mg·mL−1 quercetin per 100 mg of extract 13 [Total flavonoid (TFC) and phenolic (TPC) contents were determined with the help of standards  were mixed with 1 mL of phosphate buffer  and 1 mL of K3Fe(CN)6 (10 mg·mL−1) and was then incubated for 20 min at 50 °C in water bath . The reaction was ended by addition of 1 mL of TCA (100 mg L−1) in the reaction mixture. Supernatant was collected after centrifugation at 7000× g for 10 min at room temperature. Freshly prepared FeCl3  was mixed with collected supernatant and incubated for 10 min at room temperature. Absorbance was taken at 700 nm to calculate reducing power [The reducing power of seaweed extracts was calculated by using ascorbic acid as standard. Different concentrations of seaweeds extracts  and DMEM:F12  medium, respectively at 37 °C under 5% CO2 in the air. Both media were supplemented with heat inactivated Fetal Bovine Serum and antibiotic antimycotic solution . The cells were used only up to 20 passages for all the experiments. MTT was used to measure anti-proliferative activity [2. Seaweed extracts were added to 96 well-plate, followed by addition of PBS and at the final concentration of 10% and incubation for 2.5 h. Absorbance was taken at 570 nm and anti-proliferative activity was calculated using following equation and compared with control (cells growing without extract).The human cervical cancer cell line (HeLa) and human hepatoma cancer cell line (Huh7), purchased from National Centre for Cell Science (NCCS), Pune (India) were maintained in MEM .Data were shown as average (mean) ± SE (standard error of the mean). ANOVA  and Tukey’s honestly significant difference (HSD) were applied for statistical analysis and statistical differences were expressed with different letters at significance All seaweed extracts showed concentration-dependent antioxidant, scavenging, and reducing activities . The seaThe highest scavenging activity was observed with SA and SL, which was 53% with 600 μg extract-dose, while at a dose of 1000 μg, both showed 70% inhibition. With the 1000 μg dose, IS and SP showed only 60% and 56% scavenging activity, respectively B. SA als−1 half maximal effective concentration (EC50) for antioxidant activity followed by SA (320 ± 2 μg·mL−1), SL (340 ± 5 μg·mL−1) and SP (635 ± 5 μg·mL−1). Furthermore, SA showed about 610 ± 2 and 250 ± 3 μg·mL−1 EC50 dose for scavenging and reducing activities, respectively. Overall, SA showed the lowest EC50 dose , therefore showing maximum activities (Seaweed IS showed about 285 ± 10 μg·mLtivities .−1·g extract) followed by SL (14 ± 2 mg·mL−1·g extract), IS (11 ± 2 mg·mL−1·g extract) and SP (9 ± 2 mg·mL−1·g extract) (−1·g extract), followed by SP (295 ± 45 mg·mL−1·g extract), and IS (280 ± 50 mg·mL−1·g extract), whereas the lowest TFC (113 ± 17 mg·mL−1 quercetin per g extract) was observed in SL (High TPC was estimated with SA (15 ± 3 mg·mLextract) A. Similaed in SL B.A very strong (0.9–1.0) and strong correlations (0.7–0.9) were observed between TPC and biochemical characteristics  for SP and SL seaweed extracts, respectively . In contThe SP and SL seaweed extracts showed high inhibition of cell proliferation, approximately 44% and 41% for Huh7 and HeLa cells, respectively, compared to their controls, i.e., untreated cells (cells growing in their respective media without any addition of seaweed extract) . About 3−1 DW), followed by meso-erythritol (453 ± 46 µg·g−1 DW). Similarly, a commercially important low-calorie sugar, psicose (76 ± 17 µg·g−1 DW) was estimated in SP. A high sucrose level (1097 ± 76 µg·g−1 DW) was estimated in SL, followed by SP (506 ± 37 µg g−1 DW). A number of amino-acids were estimated in IS compared to the other three brown seaweeds. IS has the highest concentration of metabolites (about 10 mg·g−1) followed by SL (2 mg·g−1), SP (1.7 mg·g−1) and SA (1 mg·g−1).A total of 68 different types of metabolites were quantified in the brown seaweeds . These m2 = 0.395), reducing (R2 = 0.161) and anti-proliferative activity of HeLa cells (R2 = 0.158). However, a strong correlation was found with the proliferation inhibition of Huh7 cells (R2 = 0.853). Further, the antioxidant activity did not show any correlation (0.0–0.1 coefficient) with TFC (R2 = 0.027); however, a moderate correlation with TPC (R2 = 0.466) was found. A very strong correlation (R2 = 0.978) was noticed between scavenging activity and TPC; however, a strong correlation was found for reducing activity with scavenging activity (R2 = 0.865) and TPC (R2 = 0.877). Further, there was no correlation of TFC with antioxidant (R2 = 0.027), reducing (R2 = 0.002) activity, TPC (R2 = 0.038) and proliferation inhibition of Huh7 cells (R2 = 0.0.29); however, it was moderately correlated with inhibition of HeLa cells (R2 = 0.623), and showed weak correlation with scavenging activity (R2 = 0.102). TPC showed a moderate correlation with the anti-proliferative activity of HeLa cells (R2 = 0.537), while showing a weak correlation with the anti-proliferative activity of Huh7 cells (R2 = 0.125). All activities  showed a proportional correlation (Pearson correlation matrix with positive sign) to each other, and also with TFC and anti-proliferation of Huh7 cells. Interestingly, TPC showed an inversely proportional correlation (Pearson correlation matrix with –ve sign) with all activities except for the anti-proliferation of HeLa cells . The proliferation inhibition of HeLa cells was inversely proportionally correlated with all activities , TFC and TPC (2 = 0.038) was observed between TPC and TFC.A cumulative correlation and principal component analysis (PCA) were conducted for the selected tropical brown seaweeds in a combination of biochemical  activities, contents (TFC and TPC), and anti-proliferative activity . Overall and TPC . No corrThe PCA showed a statistical difference among different bioactivities, contents (TFC and TPC), and proliferative inhibition activities for the tropical brown seaweeds . The bipSeaweeds have been used as food from ancient times in Asian countries, such as China, Japan and Korea; however, they were only used for industrial purposes in Western countries. Algae and their extracted constituents were included as novel foods and novel food ingredients in January 1997 as CE 258/97 regulation of the European Parliament and of the Council. The food industry has been allowed to use seaweeds in their raw or processed form as ingredients in their formulations or as additives. Hence, there has been an increase in awareness regarding the use of seaweeds and their components in pharmaceuticals, cosmetics, nutraceuticals and biotechnological applications .50 was observed with extract from Iyengaria stellata, followed by an extract from Spatoglossum asperum for total antioxidant activity. Similarly, the extracts obtained from S. asperum showed the lowest dose of EC50 for scavenging and reducing activities (S. asperum were highlighted for their high potential total antioxidant, scavenging and reducing activities compared to their respective standards [In the present study, all seaweed extracts showed dose-dependent biochemical activities , and thetivities . The mettandards .S. asperum (Spatoglossum to the presence of flavonoids [Suaeda species, differential accumulation of certain metabolites, such as amino acids, sugars, and organic acids, was shown under varying stress conditions [High total phenolic content and total flavonoid were observed in the brown seaweed  asperum  compared asperum . Similar asperum . Phaeophavonoids . Earlieravonoids ,51. Somenditions . The accnditions . The higS. asperum showed 35% anti-proliferation of Huh7 cells and 26% of HeLa cells (S. asperum contained high TPC content and, therefore, high anti-proliferation of HeLa cells (S. asperum is considered a potential source of natural antioxidant molecules and different bioactive metabolites [Phenolic and flavonoid compounds from marine algae are well known for their anti-proliferative activities ,53,54,55La cells . SelecteLa cells . PCA conLa cells . The ediabolites ,56,57.S. asperum showed potential antioxidant, scavenging and reducing activities among studied, tropical, abundantly grown brown seaweeds from the same coast. The study confirms the nutraceutical potential of the brown seaweed, S. asperum, and also recommends brown seaweeds to be a part of daily diet because of their beneficial health properties.In conclusion,


Experimental data can be satisfactorily explained by assuming, for all investigated cations, the formation of a mono-complex (LnL+) and a bis-complex (LnL2−). Differences were found between the formation constants of complexes of different Ln3+ cations, which can be correlated with ionic radius. Since gadolinium is the element that raises the most concern among lanthanide elements, its effects on organisms at different levels of biological organization were explored, in the presence and absence of harzianic acid. Results of ecotoxicological tests suggest that harzianic acid can decrease gadolinium biotoxicity, presumably because of complex formation with Gd3+.Rare-earth elements are emerging contaminants of soil and water bodies which destiny in the environment and effects on organisms is modulated by their interactions with natural ligands produced by bacteria, fungi and plants. Within this framework, coordination by harzianic acid (H Fungi are particularly prolific sources of secondary metabolites, which are low molecular weight compounds fulfilling very different functions  and multiple bioactivities . The staggering variation in the chemical structure of these compounds explains their broad range of activities and functions ,5,6,7,8.Trichoderma + (m/z 388.1730). Furthermore, mass peaks attributable to ions that contain both Ln3+ cations and harzianic acid, normally in a 1:2 metal-to-ligand ratio, were also observed. The most abundant ions in the mass spectrum (MS) of each solution are reported in 3+ cations and harzianic acid was gathered by employing circular dichroism (CD) and UV–Vis spectroscopy. Further evidence for bonding interactions occurring between Ln+], was measured with a pH indicator glass electrode calibrated to respond to free proton concentration in the CH3OH/0.1 M NaClO4 (50/50 w/w) solvent. Thus, in the following sections, the standard symbol pH merely indicates the anti-logarithm of the molar concentration of solvated protons in the tested solutions .To this end, CD and UV–Vis spectra were acquired, at 25 °C, on solutions which analytical composition was accurately known by preparation, where free proton concentration,  in the present case) in a reference solution of pH = 7.4, where the total concentrations of the metal cation and of the ligand are, respectively 1.0 × 10−6 M and 1.0 × 10−3 M. By consequence, a larger pM value corresponds to a lower concentration of the free metal ion in solution at equilibrium and, in abstract, to a higher capability of the ligand to complex the metal cation. pM can be used to compare the binding ability of different ligands toward a given metal cation or, vice versa, to reveal differences in affinity of a given ligand for different cations irrespective of the stoichiometry of the formed complexes ). In fact, to help in the interpretation of spectra, solutions in each set were planned to have the same ligand-to-metal ratio , respectively, very close to 1 for the first set and very close to 2 for the second set (4)3 or Ln(Cl)3 (4 (3OH/0.1 M NaClO4 (50/50 w/w) solvent; and a potentiometric apparatus constituted by a multi-neck titration vessel equipped with a Metrohm AG  60102-100 pH sensitive glass electrode (GE) and an Ag/AgCl(s)/0.1 M NaCl/ double junction reference electrode (RE).Acquisition workflow for CD and UV-Vis measurements on solutions of rare-earth cations and harzianic acid contemplates the acquisition, for each element, of two sets of CD and UV-Vis spectra on solutions of accurately known analytical composition , the solution in the potentiometric vessel was alkalimetrically titrated by stepwise addition of accurately measured volumes of the 3+ and of the In order to evaluate the constants 3+ and the 3+, −5 M and ≈4 × 10−5 M, while values of −4 M and ≈9 × 10−5 M.Finally, appropriate volumes of solution were withdrawn from the titration vessel and submitted for CD and UV measurements at 25.0 °C. By this procedure, using fixed volumes of the UV–Vis spectra were recorded by Cary model 5000 Spectrophotometer by Varian C. , from 200 to 600 nm  at 25.0 °C, under a constant flow of nitrogen.The far UV–CD spectra were recorded with a JASCO CD spectrometer model J-715 , from 250 to 500 nm  at 25.0 °C, under a constant flow of nitrogen.A. fischeri, R. subcapitata, and D. magna were carried out on solutions of different concentrations of Gd and harzianic acid prepared in ISO medium [3+ + harzianic acid, a test solution containing GdCl3 (3.7 µM) and harzianic acid (0.7 µM) was prepared in ISO medium [A. fischeri (NRRL-B-11177) according to ISO 11348 [Toxicity tests with O medium . ConcernO medium . AccordiSO 11348 . The lumR. subcapitata was carried out according to ISO 8692 [4 cell mL−1. The specific growth inhibition rate was calculated considering 3 replicates exposed at 20 ± 1 °C for 72 h under continuous illumination (6000 l×). Effect data are expressed as a percentage of growth inhibition.The growth inhibition test with ISO 8692 . The iniw/v). The significance of differences between average values of different experimental treatments was assessed by the analysis of variance (ANOVA) considering a significance threshold level always set at 5%. When ANOVA revealed significant differences among treatments, post hoc tests were carried out with Tukey’s test. Statistical analyses were performed using Microsoft® Excel 2013/XLSTAT©-Pro .Newborn daphnids (<24 h old) were exposed to the substrate according to the ISO 6341 . Toxicit3OH/0.1 M NaClO4 (50/50 w/w) solvent. Harzianic acid can form mono- (LnL+) and bis-complexes (LnL2−) with the considered rare-earth cations. As expected, the formation constants of LnL+ complexes (β11) increased as the ionic radius decreased, but an opposite trend was observed for the stepwise formation constants K12 = (β12/β11), which increased with ionic radius. Due to this trend, lanthanum had the highest value of the K12 (=11.2) stepwise formation constant and the lowest value of the first formation constant β11 (=6.3). Thus, the LaL+ complex is always a minor species, and the bis-complex, LaL2−, seems to be formed in a single step directly from La3+ through the reaction La3+ + 2L2− ⇌ LaL2− (β12 = 17.46). Furthermore, among the investigated Ln3+ cations, harzianic acid most efficiently complexed La3+. In fact, according to the calculated pM values, lanthanum appeared to be more efficiently complexed by harzianic acid in the CH3OH/0.1 M NaClO4 (50/50 w/w) solvent than by EDTA in water at pH = 7.4. Mass spectrometric, circular dichroism, UV–Vis spectrophotometric, and pH measurements were combined to shed light on the interactions of the fungal metabolite harzianic acid with selected rare-earth  tripositive cations in a CH3+–harzianic acid complexes.Finally, ecotoxicological tests suggest that gadolinium biotoxicity decreased with the presence of harzianic acid in the chemical environment, presumably because of the formation of Gd


Isorhamnetin reduced adenosine triphosphate levels (ATP) in platelets stimulated by collagen and TRAP-6. We also evidenced that isorhamnetin’s antiplatelet activity was related to the inhibition of mitochondrial function without effect on reactive oxygen species (ROS) levels. Additionally, we investigated isorhamnetin’s effect on thrombus formation in vitro under flow conditions on the damaged vessel wall. In this context, we demonstrate that isorhamnetin at 20 µM induced a significant inhibition on platelet deposition, confirming its antithrombotic effect. Our findings corroborate the antiplatelet and antithrombotic potential of isorhamnetin present in many foods of daily consumption.With the diet, we ingest nutrients capable of modulating platelet function, which plays a crucial role in developing cardiovascular events, one of the leading causes of mortality worldwide. Studies that demonstrate the antiplatelet and antithrombotic potential of bioactive compounds are vital to maintaining good cardiovascular health. In this work, we evaluate the flavonol isorhamnetin’s antiplatelet effect on human platelets, using collagen, thrombin receptor activator peptide 6 (TRAP-6), and phorbol myristate acetate (PMA) as agonists. Isorhamnetin induced a significant inhibition on collagen- and TRAP-6-induced platelet aggregation, with half-maximum inhibitory concentration (IC The number of deaths from diet-related cardiovascular events increased between 2010 and 2016 . A balan16H12O7) is a methylated flavonol present in leaves, flowers, and fruits of many plants [Phaseolus vulgaris L., belonging to the group of legume plants consumed in the five continents as an essential component of the diet [Isorhamnetin  and arachidonic acid. The antiplatelet mechanism was related to activation of protein kinase B (AKT), which decreases the activation of platelets [Epidemiological and clinical studies have shown that beans’ consumption is inversely related to coronary artery diseases and the risk of cardiovascular events ,13. Varilatelets .Isorhamnetin has been used in traditional medicine to prevent and treat various diseases ,15 due tIn the context of cardioprotective potential, isorhamnetin protects against cardiac hypertrophy by blocking the PI3K-AKT pathway ,15. The A causal factor in cardiovascular disorders is platelet activation. Platelets play a fundamental role in thrombus formation, atherogenesis, and atherosclerotic lesions progression . The inhThrombin 6 receptor activating peptide (TRAP-6), collagen, and phorbol myristate acetate (PMA), were obtained from Sigma-Aldrich . Antimycin (AA), citrate-dextrose solution, mepacrine, dihydroethidium (DHE), intracellular calcium fluorescence indicator (Fluo-3-AM), and trifluoromethoxyphenylhydrazone (FCCP) were also obtained from Sigma-Aldrich . FITC annexin V apoptosis, PE mouse IgG1 isotype control, and FITC mouse anti-human CD61 were obtained from BD Biosciences . Isorhamnetin was obtained from Cayman Chemical, USA. All assays incorporated as vehicle control dimethyl sulfoxide (DMSO) 0.2%.v/v and then centrifuged at room temperature for 10 min at 240× g to obtain platelet-rich plasma (PRP). PRP was centrifuged (800× g) at room temperature for 8 min [v/v). The platelets were washed again by centrifugation at 800× g for 8 min [Platelets were obtained from six healthy volunteers (men and women) who did not consume medication for two weeks. Donors signed the informed consent according to the protocol approved by the Scientific Ethics Committee of the University of Talca (protocol no. 19/2018), following the Declaration of Helsinki . Blood wor 8 min . Then plor 8 min . The plaor 8 min . Flow cy8 platelets/mL) were incubated with isorhamnetin  for 10 min at 37 °C. Subsequently, platelets were centrifuged (800× g) for 8 min, and the supernatant was analyzed with the lactate dehydrogenase (LDH) cytotoxicity assay kit . A microplate reader  was used to measure the reaction’s absorbance at 490 nm. The positive control was Triton X-100 (10%) [Washed platelets (3 × 1000 (10%) .8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) plus isorhamnetin  or vehicle . Similar concentrations in vitro have been evaluated in other studies [50) was obtained from isorhamnetin’s concentration curves .Inhibition of platelet aggregation was evaluated by a turbidimetric method using a lumi-aggregometer  ,26. Wash studies . Platele studies . The pla studies . The con8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) and subsequently with isorhamnetin  for 10 min at 37 °C. In addition, PS was induced in activated conditions by collagen (1 mg/mL)/TRAP-6 (5 µM). The samples were then diluted with annexin V-binding buffer and pre-incubated in the dark with annexin V-FITC and anti-CD61-PE for 30 min. Samples were examined on an Accuri C6 flow cytometer .Externalization of phosphatidylserine (PS) in platelets was determined by flow cytometry, following the methodology described by Mendez et al. 2020, with slight modifications ,29. Wash8 platelets/mL) were incubated for 5 min with CaCl2 (2 mM) and isorhamnetin . The luciferin (Chrono-Lume) reagent and the platelet agonists (TRAP-6 5 µM and collagen 1 µg/mL) were added consecutively. ATP secretion (100%) was obtained with the vehicle (DMSO 0.2%) plus the platelet agonists. Adenosine (10 µM) was used as a positive control of inhibition.Extracellular adenosine triphosphate (ATP) was determined using Chrono-lume  . Washed 8 platelets/mL) were incubated with Fluo-3-AM (0.4 µM) at room temperature for 30 min as previously described by Mendez et al. 2020 [7 platelets/mL) with Tyrode’s buffer without calcium, and the samples were incubated with the negative control, FCCP (1 µM) or isorhamnetin  for 10 min at 37 °C. The effect of isorhamnetin on the intracellular calcium levels was calculated relative to the vehicle (DMSO 0.2%) using the Accuri C6 flow cytometer .Washed platelets  with DHE (10 µM) in the presence of isorhamnetin  and CaCl2 (2 mM) for 30 min at 37 °C. Antimycin 10 µM was a positive control. ROS formation was analyzed by Accuri C6 flow cytometer  [Reactive oxygen species production (ROS) was determined in washed platelets  ,30.2 (2 mM) was added to the washed platelets (5 × 107 platelets/mL). Then, TMRM (100 nM) was added and incubated with DMSO 0.2%, isorhamnetin , or FCCP 1 µM at 37 °C for 30 min. The samples were studied in the Accuri C6 flow cytometer  [Mitochondrial membrane potential (Δψm) was assessed with the permeating cellular dye tetramethylrhodamine methyl ester perchlorate (TMRM) as before described . CaCl2  .y-axis. The data were linearized, and regression analysis was calculated .The effect of isorhamnetin (20 µM) on platelet adhesion and thrombus formation was evaluated under controlled blood flow conditions in the Badimon Perfusion Chamber ,33. The 50 was calculated from the dose-response curves of isorhamnetin. Results were examined by analysis of variance (ANOVA) and Tukey’s post hoc test to determine the significant differences between samples [t-test. p values < 0.05 were considered statistically significant.Data were represented as the mean ± standard error of the mean (SEM) of the experiments and were analyzed using Prism 6.0 software . The IC samples . DiffereLDH is released from the cell because of damage or lysis. In this context, isorhamnetin’s cytotoxic effects were evaluated at 10, 50, and 100 µM. It was observed that isorhamnetin at none of the concentrations evaluated induced cytotoxic activity A. The PS50 of 8.1 ± 2.6 µM) had a powerful concentration-dependent antiplatelet effect when platelet aggregation was induced by collagen. When TRAP-6 was used as an agonist, the compound inhibited platelet aggregation with IC50 of 16.1 ± 11.1 µM. While PMA-induced platelet aggregation had a lower activity (IC50 > 100 µM) and at 100 µM inhibited PMA-induced platelet aggregation by about 30 ± 6%. Based on these results, we decided to continue the antiplatelet studies with isorhamnetin 1, 10, and 20 µM.The efficacy of isorhamnetin  to inhibit platelet aggregation was evaluated by turbidimetry stimulated by collagen (1 µg/mL), TRAP-6 (5 µM), and PMA (100 nM) A–C. The Platelet activation is stimulated by platelet secretion products, such as ATP. About the control, isorhamnetin at 20 µM lowers ATP levels to 0.28 ± 0.03 and 0.28 ± 0.05 (folds of control) in collagen- and TRAP-6-stimulated platelet, respectively A. MeanwhThe association between developing CVD and mitochondrial damage is well-known. We study if the isorhamnetin has an antiplatelet activity through the inhibition of mitochondrial bioenergetics. In this context, we observed that isorhamnetin at 1, 10, and 20 µM significantly decreases the membrane potential compared to the control B.Platelet activation is regulated by the endogenous generation of ROS induced by a wide variety of stimuli, for example, at the mitochondrial level by antimycin A. In our study, isorhamnetin was shown not to affect ROS C.2+ levels relative to baseline  and at high shear rate . Isorhamnetin at 20 µM induced a significant reduction in platelet deposition versus an untreated blood sample  at both Platelets are the smallest blood cells, and their activation at sites of the vascular lesion is essential for thrombus formation . The treIsorhamnetin has broad pharmacological effects, such as anti-osteoporosis , anti-inLDH activity can be used as an indicator of cell membrane integrity and, therefore, a measure of cytotoxicity . IsorhamPlatelet agonists activate different pathways in signal transductions. TRAP-6 acts as a PAR1 agonist in platelet activation ,47; glycMitochondria via ATP production are involved as central drivers in platelet activation. In this context, inhibition of mitochondrial function has been reported as an antiplatelet target via a decrease of ATP levels ,54. We dIt has been described that atherothrombosis is initiated by collagen exposure when endothelial damage occurs, and this triggering platelet activation/adhesion ,55. In tAlthough this study has some limitations since the effects of isorhamnetin on platelet aggregation are more complex in vivo conditions, our results support scientific evidence that isorhamnetin helps reduce platelet activation and thereby thrombotic and cardiovascular risk.Diet and nutrition influence the progression and/or treatment of CVD. Isorhamnetin may be a promising scaffold compound to develop new antiplatelet agents with specific action on thrombotic diseases. Isorhamnetin did not induce cytotoxic effects. Its antiplatelet potential was significant in collagen-stimulated platelet aggregation. Consecutively, it induced a fall in membrane potential and a decrease in ATP levels. The evidence presented in this work suggests that this flavonoid may play an important role in health maintenance and possibly protect against cardiovascular disease associated with the inhibition of platelet function and a possible reduction in the risk of thrombosis. Certainly, future studies, such as absorption and metabolism, are needed to translate the antiplatelet activity of in vitro dose–response to in vivo concentrations (human).


Background: Ageing and chronic kidney disease (CKD) affect pharmacokinetic (PK) parameters. Since mechanisms are related and remain unclear, cytochrome P450 (CYP) 3A and drug transporter activities were investigated in the elderly with or without CKD and compared to healthy adults using a microdose cocktail.Methods: Healthy young participants (n = 20), healthy elderly participants (n = 16) and elderly patients with CKD (n = 17) received, in study period 1, a single dose of microdose cocktail probe containing 30 µg midazolam, 750 µg dabigatran etexilate, 100 µg atorvastatin, 10 µg pitavastatin, and 50 µg rosuvastatin. After a 14-day wash-out period, healthy young participants continued to study period 2 with the microdose cocktail plus rifampicin. PK parameters including area under the plasma concentration-time curve (AUC), maximum plasma drug concentration (Cmax), and half-life were estimated before making pairwise comparisons of geometric mean ratios (GMR) between groups.Results: AUC and Cmax GMR  of midazolam, a CYP3A probe substrate, were increased 2.30 (1.70–3.09) and 2.90 (2.16–3.88) fold in healthy elderly and elderly patients with CKD, respectively, together with a prolonged half-life. AUC and Cmax GMR (95%CI) of atorvastatin, another CYP3A substrate, was increased 2.14 (1.52–3.02) fold in healthy elderly and 4.15 (2.98–5.79) fold in elderly patients with CKD, indicating decreased CYP3A activity related to ageing. Associated AUC changes in the probe drug whose activity could be modified by intestinal P-glycoprotein (P-gp) activity, dabigatran etexilate, were observed in patients with CKD. However, whether the activity of pitavastatin and rosuvastatin is modified by organic anion transporting polypeptide 1B (OATP1B) and of breast cancer resistance protein (BCRP), respectively, in elderly participants with or without CKD was inconclusive.Conclusions: CYP3A activity is reduced in ageing. Intestinal P-gp function might be affected by CKD, but further confirmation appears warranted.Clinical Trial Registration:http://www.thaiclinicaltrials.org/  Chronic kidney disease (CKD) is a significant public health problem worldwide, impacting national health expenditures and patient morbidity and mortality . The oveCytochrome P450 3A (CYP3A), the most abundant isozyme in the liver and intestine , plays aClinical studies using probe substrate cocktails are recognized by both the United States Food and Drug Administration (United States FDA) and the European Medicines Agency (EMA) to evaluate the activity of multiple CYP enzymes and drug transporters simultaneously . A cocktThis study aimed to evaluate age- versus CKD-related changes in the activity of CYP3A and the drug transporters, P-gp, OATP, and BCRP, using a microdose cocktail in the elderly with and without CKD compared to young healthy participants.This study was a clinical pharmacokinetic study . The study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Written informed consent was obtained from all participants before the start of the study. The nomenclature of enzymes and drug transporters conformed to the International Union of Basic and Clinical Pharmacology/British Pharmacological Society (IUPHAR/BPS) Guide to Pharmacology Nomenclature Classification .2, group 2  aged ≥60 years with eGFR >60 ml/min/1.73 m2, and group 3 (elderly patients with CKD) aged ≥60 years with eGFR 15–60 ml/min/1.73 m2, diagnosed as patients with CKD by attending nephrologists. The pre-specified inclusion/exclusion criteria were fully described in Participants were classified into three groups; group 1  aged 20–40 years with estimated glomerular filtration rate (eGFR) ≥90 ml/min/1.73 mWe assumed that the area under the plasma concentration-time curve (AUC) of MDZ between healthy young participants and the elderly participants would differ by 70% with a common standard deviation of 11.84, based on a previous study using the same microdose cocktail . Under tHealthy young participants were recruited from the volunteers’ database of Maha Chakri Sirindhorn Clinical Research Center under the Royal Patronage, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Healthy elderly participants were recruited from out-patients of the Comprehensive Geriatric Clinic, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand. Elderly patients with CKD were recruited from an out-patient of the Nephrology Clinic, King Chulalongkorn Memorial Hospital, by nephrologists in the study team.All participants were advised to refrain from xanthine-containing beverages, citrus juice, herbal and dietary supplements, and products containing St. John’s wort for 72 h before study start, until study conclusion. In addition, healthy young and healthy elderly participants abstained from any drug intake for at least two weeks before the study date. Elderly patients with CKD continued their medications as usual, except for PTV, ATV, or RSV, which were stopped or switched to the equivalent dose of simvastatin at least two weeks before the study date. The concomitant drugs in elderly patients with CKD were also examined for potential drug-drug interaction with probe substrates in the microdose cocktail.The powder formulation of microdose cocktail containing 30 µg of MDZ, 750 µg of DABE, 10 µg of PTV, 100 µg of ATV, and 50 µg of RSV, was prepared by a single pharmacist trained in the study procedures at the Pharmacy Department, King Chulalongkorn Memorial Hospital. The microdose cocktail was dissolved with 10 ml of water into a solution before immediate oral administration.All participants received a single oral dose of the microdose cocktail after 10 h of an overnight fast (study period 1). Venous blood samples were collected into EDTA tubes at predose (0) and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h post-dose. Urine samples were collected at predose (0) and then between 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–36 h, and 36–48 h post-dose. Plasma and urine samples were aliquoted and kept at −80°C until analysis. Samples for statins analysis were treated immediately with 1 M ammonium buffer (pH 5) at a ratio of 5/100 (buffer/plasma) to prevent interconversion. All participants stayed at the clinical study site for at least 12 h post-dose and returned for subsequent blood sample collections.After a 2-week wash-out period, the healthy young participants continued to study period 2. On this occasion, participants received another single oral microdose cocktail together with 450 mg of rifampicin (RIF), a well-known OATP1B, BCRP, and intestinal P-gp inhibitor, to evaluate the magnitude of drug-drug interactions between RIF and microdose cocktail in Thai subjects, as compared to the earlier study in Caucasian . Blood aIt has to be noted that during the final part of the study period, the MDZ tablet was not available in Thailand. Hence, an intravenous form of MDZ was used in 4 elderly patients with CKD instead. After dehydrating, the solution was prepared to power for the preparation of the microdose cocktail. Despite limited power, pharmacokinetic parameters between powder and solution of MDZ were similar .Plasma concentrations of MDZ, dabigatran (DABI) which is an active metabolite of DABE , PTV, piMDZ, statins, and RIF were measured using reverse-phase liquid chromatography. Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection employing a turbo ion spray interface in positive ion mode was used for DABI. The lower limit of quantitation (LLOQ) was 1 pg/ml for MDZ, 20 pg/ml for DABI, and 5 pg/ml for statins and their metabolites.SLCO1B1) genotypic test results, as statins levels are affected by single nucleotide polymorphisms (SNPs) in drug transporters (SLCO1B1 and ATP Binding Cassette Subfamily G2 (ABCG2) genes to adjust for this possible confounding factor (SLCO1B1 c.521T > C (rs4149056), c.388A > G (rs2306283), g.-11187G > A (rs4149015), and ABCG2 c.421C > A (rs2231142) of all study participants have previously been reported (CYP3A5*3 c.6986A > G (rs776746) was also carried out in all participants. CYP3A5*3 in this study.The Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline recommends simvastatin drug monitoring and dose adjustment in patients based on solute carrier organic anion transporter 1B1  and time to maximum plasma concentration (Tmax) were directly obtained from the individual plasma drug concentration profiles versus the time curve. The area under the plasma concentration-time curve from time zero to the last point with measurable concentration (AUC0-last) was calculated using the log-linear trapezoidal rule. AUC0-inf was obtained from the summation of AUC0-last and the last observed quantifiable concentration (Clast)/elimination rate constant (Kel). Kel was estimated by log-linear least square regression of the terminal part of the plasma concentration versus time curve. Half-life (T1/2) was calculated using a natural logarithm of 2 (ln2) divided by Kel. Oral clearance  was also calculated as dose divided by AUC0-last. The cumulative amount in urine (Ae) was measured and divided by AUC0-last to estimate renal clearance (CLR) in DABI and RSV. Additionally, the theoretical AUC0-last ratios (AUCR) in AUC0-last of healthy elderly and elderly patients with CKD over AUC0-last of healthy young participants were calculated for DABI and RSV, assuming no change in bioavailability (F). The calculated AUCRs were compared to the observed AUCRs to confirm the effects of ageing and/or CKD on intestinal P-gp and BCRP functions.Pharmacokinetic data were analyzed using noncompartmental methods. Maximum plasma concentration . Graphs were created by GraphPad Prism version 8.0 . Demographic data are presented as median  or frequency (%) for continuous and categorical data, respectively. A Kruskal-Wallis test analyzed formal comparisons of continuous characteristics between groups, and when differences were found, pairwise comparisons were further investigated with a Wilcoxon rank-sum test. Finally, categorical characteristics were compared with a Chi-square or Fisher’s exact test as appropriate.0-last, AUC0-inf, Cmax, CL/F, and CLR. Regression techniques with an outcome of the natural logarithm of each parameter were developed for these parameters, and pairwise comparisons were made between groups. The relevant model parameters were then exponentiated to obtain geometric mean ratios (GMR) and corresponding 95% confidence interval (95%CI). Tmax and T1/2 were reported as median (IQR), and pairwise comparisons were made with a Kruskal-Wallis test/Wilcoxon test for demographic parameters. Pre-RIF versus post-RIF comparisons in healthy young participants were assessed using Generalized Estimating Equation (GEE). All p-values were adjusted for multiple comparisons using a Bonferroni correction. Multivariable models were used to adjust group differences for potential confounders, including gender, body weight, alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, albumin, and genotype of SLCO1B1 and ABCG2 genes.The geometric mean (GM) with 95% confidence interval (95%CI) was calculated for AUCBaseline characteristics between three groups of participants were comparable except for age, body weight, and renal function . The med0-last, AUC0-inf, and Cmax of MDZ were significantly increased in healthy elderly  and elderly patients with CKD , as compared to healthy young adults. T1/2 was also prolonged in healthy elderly and elderly patients with CKD. No significant differences were seen between the groups of healthy elderly and elderly patients with CKD , while elderly patients with CKD showed a marked increase in drug exposure  when compared to healthy young controls. Elderly patients with CKD had higher AUC0-last and AUC0-inf than healthy elderly (GMR (95% CI): 2.65 (1.91–3.69) and 2.91 (2.14–3.95), respectively), but no differences were observed in Cmax. The theoretical AUCR of DABI was increased with the same magnitude, as seen in the observed AUCRs in healthy elderly (1.70 fold) but not for elderly patients with CKD (2.84 fold). Of note, both healthy elderly and elderly patients with CKD had a reduction in DABI CLR by 40 and 80%, respectively, but only the elderly patients with CKD had a very prolonged DABI T1/2  and PTV-lactone , as compared to healthy young adults  and more prominently increased in elderly patients with CKD  as compared to healthy young participants  and Cmax (1.63 fold) of RSV when compared to healthy young controls (R of RSV was considerably low in elderly patients with CKD (80% reduction) compared to healthy elderly  showed increased AUC0-last and Cmax of RSV than ABCG2 wild type , AUC0-inf (2.57 fold), and Cmax (1.83 fold) in elderly patients with CKD as compared to healthy young participants (CPY3A5 genetic variants (6968AG and 6986GG) and wild type (6986AA) were also compared in healthy young participants. There were no differences observed between them  had increased AUC and Cmax of MDZ (GMR∼2.0–3.0) when compared to healthy young participants with wild-type CYP3A5 , intestinal BCRP, and possibly, to a minor extent, intestinal P-gp . Here, w4-OH-ATV . A reducis study  was in lis study  is similis study .max of ATV were associated with the severity of renal impairment . No significant difference of unbound fraction amongst the three studied groups was found for all drugs used in this cocktail (data not shown). These data suggested a negligible effect of plasma protein binding in our study, and consequently, similar conclusions would be expected with unbound drug pharmacokinetics. Currently, efforts are underway to develop a PBPK model using data from this study to delineate the effect of ageing and CKD on the underlying mechanisms related to drug metabolizing enzymes and drug transporters in elderly patients with CKD.It is also worth noting that our study did not directly measure unbound plasma concentrations due to the assay sensitivity limitation. However, plasma protein binding was indirectly determined by This study has limitations. In general, gender could affect the pharmacokinetics of drugs because of the differences in their physiology. For instance, women usually have lower body weight, lower muscle mass, and less intestinal enzymatic activity than men . AlthougAdditionally, further studies with larger cohort sizes that stratify based on CKD severity, together with patients on dialysis and RIF co-administration in all study populations, may be warranted. Population pharmacokinetic studies would be helpful to elucidate if and how other factors affect the pharmacokinetics of the probe drugs profiles. Finally, PBPK studies using data from this study would provide more information to inform predictions of changes in drug metabolizing enzymes and drug transporter activity in elderly patients with CKD.max were conflicting, this has to be confirmed in further studies. Health care providers should be aware of the potential consequences of administering CYP3A and intestinal P-gp substrates or inhibitors to these target populations.In conclusion, the microdose cocktail approach provides a valuable and safe screening tool to determine pharmacokinetic parameter alterations showing reduced CYP3A activity in the elderly. Intestinal P-gp activity might be reduced in CKD, but as the results in AUCR and C


The primary endpoint was the percentage of patients with complete thrombus resolution by TEE imaging at 4 weeks. The mean age of the study population was 68.3 ± 10.8 years with a female population of 16%. AF was permanent in all cases, with a mean arrhythmia duration of 4.3 ± 1.7 years. CHA2DS2-VASc and HAS-BLED scores were 3.2 ± 1.5 and 1.9 ± 1.1, respectively. We were able to demonstrate a complete thrombus resolution in 14 patients (56%) at 4 weeks. In patients with residual atrial thrombosis (n = 11), we observed a 15.4 ± 14.9% reduction in the thrombus area from baseline. As compared with patients without thrombus dissolution, those with thrombus resolution had a numerically lower-indexed LA diameter (27.9 ± 9.3 vs 34.8 ± 16.1 mm/m2), a smaller maximum thrombus area at baseline (45.5 ± 44.6 vs 63.9 ± 43.5 mm2), a higher left ventricular ejection fraction (47.4 ± 21.0% vs 38.4 ± 20.6%) and higher maximum LAA flow velocities (26.3 ± 15.2 vs 19.3 ± 10.0 cm/s). Figures on the percentage of thrombus resolution in this study are comparable to those reported in the literature for the other OACs. We conclude that, in patients with AF, the use of edoxaban is associated with a >50% resolution of atrial thrombus at 4 weeks, similar to studies using VKAs and the other NOACs .Available evidence on left atrial (LA) thrombus dissolution in patients with atrial fibrillation (AF) largely refers to the use of vitamin K antagonist oral anticoagulants (VKAs), showing >50% thrombus resolution over a 4-week to 12-month treatment period. Available data on non-vitamin K antagonist anticoagulants (NOACs) in this setting are limited and derive from isolated case reports or observational small-sized investigations with dabigatran, rivaroxaban or apixaban. The aim of this study was to investigate the extent of thrombus resolution with edoxaban therapy in patients with AF and LA thrombosis. We conducted a prospective, observational, open-label pilot study in seven Italian institutions. We included a total of 25 patients with non-valvular AF and LA ) thrombosis, documented by transesophageal echocardiography (TEE). All patients received edoxaban OD treatment ( The prevalence of left atrial (LA) thrombosis in patients with atrial fibrillation (AF) is relatively low   and depeIn randomized phase III trials on patients with non-valvular AF, compared with the VKA warfarin, the non-vitamin K antagonist anticoagulants (NOACs) were overall at least non-inferior to warfarin for the incidence of the primary outcome, including stroke or peripheral embolism, with a better safety profile, mainly due to a significant reduction in intracranial bleeding . Based oThis was a non-controlled, observational, prospective, open-label pilot study, performed in seven Italian centers ; Campus Bio-Medico University Hospital of Rome; Cardiology Clinic, University of Padua; “Madonna del Soccorso” Hospital, S. Benedetto del Tronto; “Sant’Anna e San Sebastiano” Hospital, Caserta; Monaldi Hospital of Naples; and F. Miulli Regional General Hospital, Acquaviva delle Fonti). Patients with non-valvular AF and LA or LAA thrombosis documented at transesophageal echocardiography (TEE) were included.The primary aim of the study was to document the extent of LA/LAA thrombus resolution upon edoxaban treatment in patients with AF. A secondary aim was to collect information for the planning and design of a larger investigation directly comparing edoxaban vs warfarin in the same clinical setting.2DS2-VASc score >1; and LA or LAA thrombosis documented at TEE imaging, performed prior to cardioversion/ablation or, in the case of a new AF episode, of undetermined duration.Specific inclusion criteria considered at the screening visit were the following: age ≥ 18 years; AF documented by electrocardiographic evidence  <30 days before the enrollment; a CHASubjects with newly diagnosed AF were eligible, provided that there was evidence that AF was “non-valvular” and there was electrocardiographic evidence for AF on two occasions 24 h apart .Cardiac-related conditions: hemodynamically significant mitral valve stenosis; mechanical or biological prosthetic heart valve ; transient AF caused by a reversible disorder ; known presence of atrial myxoma or left ventricular thrombus; active endocarditis.Bleeding risk-related criteria: active internal bleeding; history of conditions associated with increased bleeding risk, including, but not limited to: major surgical operation or trauma < 30 days before enrolment; clinically significant gastrointestinal bleeding < 6 months before enrolment; previous intracranial, intraocular, spinal, non-traumatic intra-articular bleeding; chronic hemorrhagic disorders; any neoplasm; arterio-venous malformation or aneurysm; a platelet count < 90,000/µL at the screening visit; sustained uncontrolled arterial hypertension (systolic blood pressure ≥ 180 mmHg or diastolic blood pressure ≥ 100 mmHg); severe, disabling stroke (with modified Rankin score of 4 to 5 after <3 months or any stroke < 14 days); transient ischemic attack (TIA) <3 days before enrolment.Concomitant or intercurrent therapies: treatment with any NOAC or VKA with optimal international normalized ratio (INR) control, defined as all INR values between 2 and 3 in the previous 30 days; aspirin > 160 mg daily; aspirin plus a P2Y12 inhibitor < 5 days before enrolment; intravenous antiplatelet drugs < 5 days before enrolment; fibrinolytic drugs < 10 days before enrolment; anticipated need for therapy with a non-steroidal anti-inflammatory agent in the next 4 weeks; treatment with a strong inhibitor of cytochrome P450 or inhibitors/inducers of P-glycoprotein, such as ritonavir, lopinavir, telaprevir, and indinavir, or planned treatment with such drugs during the study; other indications for anticoagulant therapy.Other concomitant or intercurrent conditions: hypersensitivity or intolerance to the study drug, including excipients; women of childbearing potential who did not want to adopt a contraceptive method during the study period and the next 4 weeks; breast-feeding women during the study period and over the next 4 weeks; anemia (hemoglobin < 10 g/dL) at the screening visit; known significant liver disease , or Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) > 2 × upper limit of normal (ULN) or total bilirubin > 1.5 × ULN; severe or end-stage renal disease .Exclusion criteria were:2DS2-VASc score), bleeding risk (by HAS-BLED score), vital signs, electrocardiographic findings, transthoracic echocardiography (TTE) and TEE parameters, laboratory test results  and medications—were collected. All patients underwent an ambulatory follow-up visit at 4 weeks, when the investigator-assessed clinical status, incidence of side effects or adverse events after enrollment, and therapeutic changes and compliance to edoxaban treatment were recorded. At that time, a TEE was performed to evaluate changes in LA/LAA thrombosis. The timing of repeat TEE after 4 weeks of edoxaban treatment was consistent with guideline recommendations and reported clinical experience [ClinicalTrials.gov with identifier number NCT034899395.Enrolled patients received 60 mg of edoxaban once a day, with an open-label design, for at least 8 weeks (up to follow-up completion); edoxaban dose was to be, however, reduced to 30 mg daily in patients with CrCL 15–49 mL/min, body weight ≤ 60 kg or concomitant use of the following P-glycoprotein inhibitors: cyclosporine, dronedarone, erythromycin or ketoconazole. An electronic case report form was used, where a unique pseudo-anonymized code was assigned to each patient, and individual data—including baseline patients’ demographic details, comorbidities, thromboembolic risk , Declaration of Helsinki and national regulations. The protocol was approved by Ethics Committees at each center. This was an investigator-initiated study, funded by Daiichi Sankyo Italy; its conduct was followed by an independent contract research organization .The primary efficacy outcome was the percentage of patients after 4 weeks of edoxaban treatment with complete thrombus resolution by TEE imaging, using the following probe angulations: 0°, 45° to 60°, and 90°.The percent variation of thrombus area at 4 weeks by TEE ;The incidence of thromboembolic events at 4 and 8 weeks .Secondary endpoints were: The occurrence of bleeding events was assessed at 4 and 8 weeks (by a telephone interview). Major bleeding was defined as fatal bleeding; and/or symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intraarticular or pericardial bleeding, or intramuscular with compartment syndrome; and/or bleeding causing a fall in hemoglobin level of 20 g/L (1.24 mmol/L) or more, or leading to transfusion of ≥2 units of whole blood or red blood cells . Any hemThere was no previous study conducted with edoxaban on LA/LAA thrombosis resolution, so this was therefore intended as a pilot study. For this reason, the sample size was determined empirically according to previous case reports with other NOACs in the same clinical setting . On thisp-value < 0.05 was considered statistically significant. All analyses were performed with the SPSS 24.0 software .Continuous data are reported as mean ± standard deviation and were compared by the Student t-test. Categorical variables are indicated as number (percentage), and proportions were compared by the chi-squared test. A two-sided 2DS2-VASc and HAS-BLED scores were 3.2 ± 1.5 and 1.9 ± 1.1, respectively. A 60 mg daily dose of edoxaban was given to 23 patients, and a 30 mg dose in the remaining 2, both having a CrCl 30–49 mL/min. In all cases, thrombosis at baseline was located in the LAA, being ovoid in 14 patients and pedunculated in 11. Maximum thrombus area was 70.2 ± 59.8 mm2.The main baseline characteristics in the 25 patients are indicated in n = 11), a 15.4 ± 14.9% reduction in the thrombus area from baseline was observed. As compared with patients without thrombus resolution, those with thrombus resolution showed a numerically lower indexed LA diameter (27.9 ± 9.3 vs 34.8 ± 16.1 mm/m2), smaller maximal thrombus area at baseline (45.5 ± 44.6 vs 63.9 ± 43.5 mm2), higher left ventricular ejection fraction (47.4 ± 21.0% vs 38.4 ± 20.6%) and higher LAA maximum flow velocities (26.3 ± 15.2 vs 19.3 ± 10.0 cm/s) , no patient had a stroke, TIA, systemic embolism or major bleeding. A minor bleeding  occurred in one patient more than 40 days after enrollment.The follow-up duration for TEE evaluation was 29.4 ± 4.0 days. A full therapeutic compliance with edoxaban was demonstrated in all patients. Complete thrombus resolution by TEE imaging was demonstrated in 14 patients (56%). In patients with residual atrial thrombosis (.0 cm/s) . None ofWe performed an extended clinical follow-up on all patients, at a mean of 25 ± 13 months. Here, no stroke/TIA/systemic embolism occurred, three patients died , and two patients had a major bleeding (on edoxaban treatment). Of the 11 patients with residual atrial thrombus at 4 weeks, three patients were switched to warfarin. In these, TEE, repeated after a mean of 2 months, revealed thrombus resolution. Eight patients continued edoxaban treatment. Among these, TEE was repeated in four patients after a mean of 3 months, demonstrating thrombus resolution, whereas the remaining four patients did not undergo further TEE examination and experienced no clinical events.This is the first study specifically focused on investigating, in a prospective fashion, the efficacy of edoxaban in resolving atrial thrombosis in patients with AF. We found that the use of this NOAC was associated with complete thrombus resolution in 56% of cases at 4 weeks.In patients with AF, atrial paralysis and blood stasis, coupled with slow LAA flow  and atri2DS2-VASc score, prevalence of hypertension, edoxaban dose and spontaneous echo-contrast grade at the time of enrolment were similar. Previous data demonstrated a higher degree of thrombin generation in diabetic patients [In our population with AF and LAA thrombosis, edoxaban was given for 4 weeks and then TEE was repeated by protocol in all patients. Compliance (adherence and persistence) to the study therapy was 100%. A complete thrombus resolution was obtained in 14/25 patients (56%). Moreover, in patients without thrombus resolution, a 15 ± 15% reduction in the thrombus area from baseline was demonstrated. The above-mentioned percentage of thrombus resolution with edoxaban observed in the present investigation is comparable to that previously reported in larger studies with VKAs  or with patients ; howeverThe present study has to be considered in light of its limitations. It is an exploratory pilot investigation enrolling a low number of patients. Its nature precluded analyses aimed at investigating independent predictors of the lack of thrombus resolution while on edoxaban treatment. By study design, no control group was included and no direct comparison between edoxaban and VKAs was performed, as our goal was also to provide data for the planning and design of a larger trial, directly comparing edoxaban vs warfarin in the same type of population. Notably, the ongoing RE-LATED AF–AFNET 7 trial has been conceived to provide a head-to-head comparison of dabigatran 150 mg BID vs phenprocoumon, with the percentage of atrial thrombus dissolution at 3–6 weeks being the main endpoint . FinallyIn conclusion, our multicenter observational study on patients with AF and atrial thrombosis indicates that the use of edoxaban is associated with percentages of thrombus resolution at 4 weeks similar to those described using VKAs and the other NOACs  ,25. We b


S: 30 × 15 m) and large (SSGL: 40 × 20 m) pitches; both SSGs comprised of four 4 min periods, interspersed by 5 min of passive recovery. Players’ mean heart rate (170 ± 8 and 171 ± 7 bpm), peak heart rate (184 ± 7 and 184 ± 5 bpm), and blood lactate concentration (11.3 ± 4.7 and 11.8 ± 4.6 mmol/L) were similar in SSGS and SSGL, respectively. The total distance covered (1984 ± 166 m and 1702 ± 80 m) and the distance covered during quick (860 ± 112 m and 696 ± 69 m) and high-intensity running (439 ± 95 and 255 ± 44) in SSGL were significantly longer than those in SSGS (p < 0.05). Conversely, the number of accelerations (45 ± 3 and 41 ± 3) and decelerations (44 ± 3 and 40 ± 4), catching errors (2 ± 1 and 1 ± 1), and turnovers (8 ± 2 and 6 ± 2) in SSGS were significantly greater than those in SSGL (p < 0.05). This study suggests that ultimate Frisbee SSG provides high-intensity training, which stimulates the glycolytic pathway. Furthermore, manipulating SSG pitch size effectively modulates the physical demands of SSG.Small-sided games (SSGs) are common drills used in various team sports, but the exercise intensity in ultimate Frisbee SSG has not yet been investigated. To clarify the physical, physiological, and technical demands of ultimate Frisbee SSG, we investigated the influence of pitch size on exercise intensity during SSG. Nine male college ultimate Frisbee players played (3 vs. 3) SSG on small (SSG Ultimate Frisbee is a noncontact, team-based sport played by millions of people across approximately 50 countries ,2. Sex-sThe physical and physiological demands on players during ultimate Frisbee games are considered to be comparable with those in various intermittent team sports, such as soccer  and rugbTraining drills that are conducted with a lower number of players and on a smaller area pitch than those used in regular games, known as small-sided games (SSGs), are commonly used to develop players’ endurance, agility, technique, and tactical abilities in team sports ,9. An unThe manipulation of variables, such as pitch size, number of players, and game duration can modulate exercise intensity in SSG in various team sports . In partThe purpose of the present study was to clarify physical, physiological, and technical demands on players during ultimate Frisbee SSG. We investigated the influences of manipulating pitch size and playing multiple bouts  on the physical, physiological, and technical demands on players. We hypothesized that with a larger pitch size, distances covered  would be greater, and that HR and BLa would be higher. Additionally, we hypothesized that technical errors would increase during repeated bouts of SSG.Fifteen members of the Chukyo University Ultimate Frisbee Club volunteered to participate in this study. SSG sessions were conducted in a random order, with two pitch sizes , during the club’s preseason preparation phase (late February and March). Of the 15 players, 9 players played in SSG on both pitch sizes, and 6 players played SSG on only one pitch size; analysis was conducted using data from the 9 participants  who participated in SSG sessions on both pitch sizes. All players were intercollege level athletes who trained 4 sessions per week for more than 2 h per session and had at least 1 year of ultimate Frisbee training (including SSG). All participants were notified of the research procedures and the potential benefits and risks, and appropriate written informed consent was obtained from all participants. The study was approved by the research ethics committee of Chukyo University in conformity with the Declaration of Helsinki (No. 2020-44).S, 30 × 15 m) and large  pitches both had length-to-width ratios of 2:1; the end zones were 7 × 15 m and 8 × 20 m, respectively. SSG sessions comprised four 4 min periods, interspersed by 5 min of passive recovery, played with three players per team (3 vs. 3). A previous study recommended the use of a format with four 4 min SSG, in order to elicit high exercise intensity [SSG took place on a natural lawn pitch at the same time of day (10:00–11:00 a.m.); each session was separated by at least 48 h. The small  and global positioning system (GPS) unit throughout each SSG session. HR monitors were synchronized with the GPS units; the sampling rate was 18.18 Hz. Each player’s mean and peak HR were calculated for each period of SSG, and the relative value to age-predicted maximum HR (%HRmax) was calculated as follows : %HRmax 2), moderate acceleration (2–3 m/s2), low acceleration (1–2 m/s2), low deceleration (−1 to −2 m/s2), moderate deceleration (−2 to −3 m/s2), and hard deceleration (<−3 m/s2) [Participants’ GPS units  measured movement during SSG sessions. The GPS unit was placed on the back between the shoulders to obtain optimal satellite signals. Each GPS unit acquired data at 18.18 Hz, and the distance covered, speed, and acceleration were determined. The total distance covered, distance covered in five speed categories, number of accelerations and decelerations, and peak speed were calculated for each period of SSG. Five speed categories were established: walking 0–4 km/h), jogging (4–8 km/h), quick running (8–14 km/h), high-intensity running (14–22 km/h), and sprinting (>22 km/h). These speed categories are comparable with those used in a previous study, that examined physical demands during a competitive ultimate Frisbee game [ km/h, jo−3 m/s2) ,18.3,1) [All SSGs were recorded using digital video cameras  that were positioned around the pitch area. A hand notation system was used to assess the technical actions and errors of each player during SSG. The number of passes , percentage of successful passes, number of catching errors by the receiver, number of turnovers in which each offensive player was involved, and number of interceptions made by each defensive player were counted for each period of the SSG by two researchers experienced in ultimate Frisbee. The test–retest reliability of this system was checked using intraclass correlation coefficient  . Test–reS and SSGL) × period ). Because the BLa of some players could not be measured after periods 1 and 3, BLa was compared using only data from periods 2 and 4. Post hoc analyses were performed using Bonferroni correction when a significant main effect or interaction was detected. For post hoc analyses, Cohen’s d effect size (ES) was calculated and assessed as small (0.2–0.5), moderate (0.5–0.8), or large (>0.8) [p < 0.05. All statistical analyses were performed using statistical software .All data are presented as mean ± standard deviation. Data were compared using two-way repeated-measures analysis of variance (pitch size (SSGe (>0.8) . StatistS sessions and 171 ± 7 bpm (86 ± 4 %HRmax) in SSGL sessions; peak HR reached 184 ± 7 bpm (92 ± 4 %HRmax) in SSGS sessions and 184 ± 5 bpm (92 ± 3 %HRmax) in SSGL sessions. Mean and peak HR in each period showed no significant difference between SSGS and SSGL . Peak HR was significantly lower in period 1 than in period 2  and period 3 . There was no significant difference in BLa or RPE between SSGS and SSGL , period 2 , and period 3   in SSGand SSGL . Mean HRand SSGL . RPE was = 0.61) .p < 0.05) for distances covered during jogging and quick running, but not total distance covered; distances covered during walking, high-intensity running, and sprinting; or peak speed. Total distance per session was 1702 ± 80 m in SSGS and 1984 ± 166 m in SSGL; the total distance covered was significantly longer in SSGL than in SSGS   and period 3   . The distance covered while jogging was significantly longer in SSGS than in SSGL in period 4  and per full session  , period 2 , and period 4  and per full session . The distance covered per session during high-intensity running was significantly longer in SSGL than in SSGS  (S and SSGL in the distance covered while sprinting (L (22.7 ± 2.4 km/h) than in SSGS (22.0 ± 1.7 km/h) .The pitch size × period interaction was significant ( = 2.17) . The dis = 0.86) . The dis = 1.94) . The dis = 1.05) . The dis = 2.45) . There wprinting . The peaS: 45 ± 3, SSGL: 41 ± 3, Corrected-p < 0.01, ES = 1.48) and low , and the number of decelerations in all categories  and low , were significantly greater in SSGS than in SSGL sessions . The number of high accelerations  and moderate accelerations , and the number of hard decelerations  and moderate decelerations  were not significantly different between SSGS and SSGL  and turnovers per session  were significantly greater in SSGS than in SSGL . Mean HR, peak HR, and BLa were similar between SSGS and SSGL (p > 0.05). The total distance covered and the distance covered during quick running and high-intensity running were significantly longer in SSGL than in SSGS (p < 0.05). The number of accelerations, decelerations, catching errors, and turnovers were significantly greater in SSGS than in SSGL (p < 0.05). To the best of the authors’ knowledge, this is the first study to compare exercise intensity in ultimate Frisbee SSG using two different pitch sizes.The present study investigated the physical, physiological, and technical demands on players in ultimate Frisbee SSG using two pitches of different sizes  than those elicited in regular games [Although HR responses were similar when playing SSG on both small and large pitches , and regar games ,22,23. Tar games .L (40 × 20 m) than in SSGS (30 × 15 m)  . In SSGLs sports ,25,26,27s sports . The useL and SSGS  because of spatial constraints caused by reducing the space to attack [However, HR, BLa, and RPE were not significantly different between SSGand SSGS , Table 1 in SSGL ; not onlo attack ,30. Our In this study, mean and peak HR were significantly lower in period 1 than in period 2 , and disUltimate Frisbee players can be divided into two playing positions (cutters and handlers) . Cuttersn = 9) was small, which may preclude generalization of these results. We did not measure the fitness level of each player, and the maximum HR of each player was not determined using an incremental treadmill test or field test, as it was estimated based on age. Each player performed only one SSGS and one SSGL session. As the physiological, physical, and technical demands during SSG might be affected by several factors, such as the wind, more than two sessions would be preferable to improve the reliability of the data. Furthermore, as we conducted only two trials (SSGS and SSGL), future studies should systematically examine the influences of pitch size on the exercise intensity during SSG with increased numbers of pitch sizes .This study has some limitations. The sample size in this study . Ultimate Frisbee SSG elicited a high BLa . In terms of the influence of pitch size, the total distance covered, and the distances covered during quick running and high-intensity running were significantly greater in SSGL than in SSGS (p < 0.05). On the other hand, the number of accelerations, decelerations, catching errors, and turnovers were significantly greater in SSGS than in SSGL (p < 0.05). With repeated bouts of SSGs, HR gradually increased and the distance covered gradually decreased, but the number of technical actions and errors remained fairly constant. This study suggests that ultimate Frisbee SSGs provide high-intensity training that stimulates the glycolytic pathway; therefore, manipulating the pitch size is effective to modulate the physical demands of SSG.The present study investigated the physical, physiological, and technical demands on players in ultimate Frisbee SSG  using two pitches of different size (SSG