Vibrio vulnificus (V. vulnificus) is an estuarine bacterium that is capable of causing rapidly fatal infection in humans. Proper polarization and bactericidal activity of macrophages play essential roles in defending against invading pathogens. How macrophages limit V. vulnificus infection remains not well understood. Here we report that tuberous sclerosis complex 1 (TSC1) is crucial for the regulation of V. vulnificus-induced macrophage polarization, bacterial clearance, and cell death. Mice with myeloid-specific deletion of TSC1 exhibit a significant reduction of survival time after V. vulnificus infection. V. vulnificus infection induces both M1 and M2 polarization. However, TSC1 deficient macrophages show enhanced M1 response to V. vulnificus infection. Interestedly, the absence of TSC1 in myeloid cells results in impaired bacterial clearance both in vivo and in vitro after V. vulnificus infection. Inhibition of the mammalian target of rapamycin (mTOR) activity significantly reverses V. vulnificus-induced hypersensitive M1 response and resistant bactericidal activity both in wild-type and TSC1-deficient macrophages. Moreover, V. vulnificus infection causes cell death of macrophages, possibly contributes to defective of bacterial clearance, which also exhibits in a mTORC1-dependent manner. These findings highlight an essential role for the TSC1-mTOR signaling in the regulation of innate immunity against V. vulnificus infection. Vibrio vulnificus (V. vulnificus) is an emerging estuarine bacterium of coastal waters worldwide, such as the United States, Japan, China, South Korea, and Mexico  and M2  polarization. M1 and M2 macrophages are functionally polarized in response to bacterial infection . M1 macrnfection . Additionfection . Rapamycnfection .V. vulnificus-induced the mTORC1 activation and server inflammatory responses in macrophages (V. vulnificus-induced inflammation via mTORC1 activation in macrophages remains mostly unknown. Tuberous sclerosis complex 1 (TSC1) and TSC2 are tumor suppressor genes. TSC1 and TSC2 negatively regulate mTORC1 activity. TSC1 associates with TSC2 to form a heterodimer. TSC1 is also essential for TSC2 activity by the maintenance of TSC2 protein stability  transgenic mice were purchased from The Jackson Laboratory. Tsc1 flox mice were bred with LysozymeCre mice to induce TSC1 deletion in myeloid cells.We purchased the 6- to 8-wk-old C57BL/6J mice from the Chinese Academy of Science Shanghai SLAC Laboratory Animal Center. The Tsc1 flox mice and Vibrio vulnificus CGMCC 1.1758 strain, which was grew at 37°C in brain heart infusion broth (BHI) or on the BHI blood agar plate. The procedures of V. vulnificus that were used in this study were followed from the standard biological hazards at Biosafety Level 2 and the safety procedures of Wenzhou Medical University Laboratory Safety Department.L-929 cells were purchased from the Cell Bank of the Chinese Academy of Science in Shanghai and cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum (Tianhang Bio) and penicillin-streptomycin . The China General Microbiological Culture Collection Center provided the ® Rabbit mAb, phospho-p70 S6 Kinase (Thr389) (D5U1O) Rabbit mAb, and Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb were purchased from Cell Signaling Technology (CST). Phosphatase Inhibitor Cocktail (Millipore) was purchased from Sigma-Aldrich. Mouse TNF-α Elisa kit, Mouse IL-6 Elisa kit, Mouse IL-1β Elisa kit, LEGENDplex™ Mouse Th1/Th2 Panel (8-plex) . LIVE/DEAD Fixable Blue Dead Cell Stain Kit (for UV excitation) was purchased from Invitrogen.Anti-mF4/80-APC, anti-mCD11b-PE, PE Annexin V Apoptosis Detection Kit I andCytofix/Cytoperm™ and perm/wash solutions were purchased from BD Biosciences Pharmingen . Anti-miNOS-PE was purchased from eBioscience. Anti-mCD206-APC was purchased from BioLegend. Hamartin/TSC1 (D43E2) Rabbit mAb, phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XPV. vulnificus infection or PBS treatment were used for RNA sequencing. Briefly, the cells were lysed in TRIzol and subjected to Annoroad Co. for RNA extraction, sequencing, and transcriptome profile analysis. The significantly differentially expressed genes were identified when we compared the normalized reads count between V. vulnificus and PBS groups with p < 0.05 and |Log2FoldChange| > 0.263. The significance of the gene ontology term enrichment was estimated using Fisher’s Exact Test .J774A.1 cells with 6 h 6BMMϕs from both TSC1flox/flox LysM-Cre (TSC1 KO) and C57BL/6J (WT) were equally seeded in six-well plates or 35 mm dishes and cultured at 37°C for overnight. We then added V. vulnificus at the indicated multiplicity of infection (2MOI) to the cells for 2 h and 4 h. The supernatant was collected at the indicated time for cytokine quantification. The infected cells were used for flow cytometry analysis, RT-qPCR analysis, and Western blot analysis.The bone marrow cells were isolated from femurs and tibiae and cultured them with a 10% L-929 cell culture medium, as described previously . Bone maflox/flox LysM-Cre mice with V. vulnificus. For survival experiments, we injected mice with 0.5 × 108 CFU of V. vulnificus suspended in 200 μl of PBS or PBS alone. The mice were observed every 6 h for 48 h, and the mice that survived for 48 h should have been humanely killed. We euthanized and exsanguinated the mice 4 h after infection for blood and tissue collection. The V. vulnificus engulfed by phagocytes consisting of organs could be counted by grinding the organs completely with PBS and detected with BHI blood agar.We used i.p. injection to infect male C57BL/6J and TSC1V. vulnificus for 2 h and 4 h. Total RNA was extracted with TRIzol (Omega Bio-Tek). The reverse transcription was performed with PrimeScript Reverse Transcriptase  according to the manufacturer’s instructions. Real-time qPCR was performed as previously described  with protease and phosphatase inhibitor cocktails (Sigma) for 15 min at 4°C. Protein concentration was determined by Bradford assay. Protein samples were analyzed by SDS–PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat dried milk in TBST  for 1 h, then incubated with indicated primary antibodies overnight on a shaker at 4°C. The appropriate HRP-coupled secondary antibody was then added and was detected through ECL chemiluminescence (BioRad). β-actin was used as a protein loading control.Cell lysates preparation and Western blot analysis were performed by following the previous protocol . BrieflyCytokine concentration was determined using ELISA kits for IL-1β, TNFα, and IL-6 (BioLegend). We purchased a nitric oxide Test Kit from Nanjing Jiancheng Bio to measure nitric oxide production in the supernatant. Briefly, the supernatant from cultured-cells with indicated treatment was collected and centrifuged at 4°C 2,000 g per 15 min, and then was analyzed by following the manufacturer’s instruction.The plasma inflammatory cytokines were detected by flow cytometry with LEGENDplex™ Mouse Th1/Th2 Panel  kit by following our previous protocol . BrieflyV. vulnificus for 4 h and washed with PBS. Supernatant was diluted and plated them on blood BHI plates for 12 h. Then 100 μg/ml gentamicin was added to kill extracellular bacteria. After incubation at 37°C for 30 min, cells were washed twice with PBS and treated with 0.1% Triton-X100. The cell lysis was plated on blood BHI plates for 12 h at 37°C for the bacterial count.Cells were infected with 2 MOI of V. vulnificus at 37°C for 4 h. For the M1/M2 discrimination flow cytometry experiment, cells were stained with CD206-APC and iNOS-PE after fixation and permeabilization by BD Biosciences Cytofix/Cytoperm™ and perm/wash solutions. For the cell apoptosis assay, dying cell was identified by using the BD PE Annexin V Apoptosis Detection Kit I according to the manufacturer’s protocol. Data were collected by using flow cytometry.Cells were infected with 2 MOI of After the spleen, liver, and lung were fixed in 4% paraformaldehyde, we imbedded the fixed organs in paraffin, then cut thin sections, and following standard procedures, stained them with hematoxylin and eosin.t-test. P-values less than 0.05 were considered significant.Data were presented as mean ± SEM and analyzed for statistical differences using the Prism 6.01/GraphPad software. Statistical significance was analyzed using the Student V. vulnificus. To understand how V. vulnificus causes inflammation, cell death, and activation in macrophages, we performed RNA-Seq and transcriptomic analysis in J774A.1 cells before and after V. vulnificus infection. RNA-seq analysis revealed substantial changes in the macrophages transcriptome after 6 h of infection. The KEGG pathway enrichment analysis demonstrated that the top pathways involved in TNF signaling, NOD-like receptor signaling, IL-7 signaling, and cytokine-cytokine receptor signaling pathways were overrepresented in the upregulated genes after 6 h of infection  mRNA expression, suggesting that increased NO production by V. vulnificus was due to the expression of iNOS. Additionally, as shown in V. vulnificus infection induced upregulation of the numbers of iNOS+ M1 macrophages and CD206+ M2 macrophages in WT BMMϕs. Whereas, in contrast to WT BMMϕs, TSC1 KO BMMϕs shown increased M1 macrophages but a reduction of M2 macrophages after V. vulnificus infection. Thus, these results indicated that TSC1 possibly drives V. vulnificus-induced M2 polarization and suppresses M1 polarization. Overall, these results demonstrated that TSC1 negatively regulated V. vulnificus-infected macrophages preferred polarization to M1 instead of M2.The function of macrophages depends on proper activation and polarization into distinct subtypes with individual effector functions, such as the M1 and M2 macrophage subsets . M1 macrvia inhibition of mTORC1 activity  of V. vulnificus pretreated with or without rapamycin. We observed that the upregulation of both protein and mRNA expression of TNF-α, IL-6, and IL-1β in the V. vulnificus-infected WT and TSC1 KO macrophages were significantly inhibited by rapamycin  by the production of various proinflammatory cytokines including TNF-α, IL-6, and IL-1β in the early stage of bacterial infection , Provincial Natural and Science Foundation of Zhejiang (LY13H190007 to D-LX), Wenzhou Municipal Science and Technology Bureau (Y20150113 to D-LX).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The optoelectronic properties of 5,5′‐bichrysenyl and 6,6′‐binaphthotetraphene were compared to their chrysene‐based “monomers”. Oxidative cyclodehydrogenations of bichrysenyl and its higher homologue towards large nanographenes were also investigated.Chrysene and its bisbenzannulated homologue, naphthotetraphene, were synthesized through a PtCl2‐catalyzed cyclization of alkynes followed by the synthesis of the corresponding soluble biaryls. PAHs were further synthesized starting from the biaryls, leading to PAHs with unknown edge‐type structure. Suzuki coupling of 2‐naphthaleneboronic acid with ((2‐bromophenyl)ethynyl)trimethylsilane yielded 1 (93 %),3) was obtained in two steps through desilylation (92 %) and PtII‐mediated cyclization as a colorless solid (81 %). 5,5′‐Bichrysenyl (5) was obtained from TMS‐protected 1 via Glaser coupling yielding 4 (67 %) followed by a selective twofold cyclization as a pale yellow solid (30 %).4 with trifluoromethanesulfonic acid in DCM2 resulted in a partial rearrangement forming 6,6′‐bichrysenyl , inseparable from 5. The π‐extended naphthotetraphene and 6,6′‐binaphthotetraphene were obtained similarly: Sonogashira coupling of 1,2‐bromoiodonaphthalene (6)7, from which 9 was obtained via Suzuki reaction with anthracene‐2‐boronic acid (8) (54 %). Desilylation to alkyne 10 (67 %) followed by cyclization led to naphthotetraphene (11) as a pale yellow solid in good yields of 80 %. This simple access to 11 significantly improved the hitherto reported synthesis involving the unstable 2‐methyl‐2H‐isoindole.12 could not be obtained in a Glaser coupling reaction directly from the TMS‐protected alkyne 9 even at higher temperatures (up to 100 °C) and reaction times of up to 14 days. Using the more reactive terminal alkyne 10, bialkyne 12 was isolated in yields of 57 % and subsequent PtCl2 induced cyclization led to 6,6′‐binaphthotetraphene (13) as a yellow solid (87 %).Our synthetic access to chrysene and naphthotetraphene (11) also crystallized in this motif , the solubility of 13 nearly doubled (5 mg mL−1 in THF), for which unfortunately only amorphous solids were obtained after various crystallization attempts. Thus, as expected, formal homocoupling resulted in a remarkable solubility increase, even without the introduction of solubility mediating groups, which was rationalized in terms of the twisted nature of the biaryls.Compared to its monomer n‐hexane and in thin films  increased noticeably between chrysene and naphthotetraphene from 11 % to 46 % and was slightly improved in the biaryls .We analyzed the absorption and photoluminescence of the target compounds in s Figure . While t5 and 13 revealed no change of absorption spectra over a period of 5 days under continuous irradiation with a 365 nm hand‐held UV lamp under aerobic conditions in solution , underlining the high photostability of the compounds.Stability measurements involving 5, 11 and 13, spin‐cast from THF, was dramatically red‐shifted compared to that observed in solution: 5,5′‐bichrysenyl (5) displayed a yellow photoluminescence with a broad band around λmax,film=546 nm, bathochromically shifted by 104 nm, and naphthotetraphene (11) and 6,6′‐binaphthotetraphene (13) were yellow‐orange fluorescent in films, with red‐shifted maxima by 149 nm and 121 nm, respectively. Only the thin film emission spectrum of chrysene itself resembled its solution spectrum, although a slight change in the intensity distribution of its vibronic structure was observed. The otherwise red‐shifted emission bands indicated significant π–π interactions of the chromophores of 5, 11 and 13 in thin films. Interestingly, crystalline 5,5′‐bichrysenyl (5)  was blue emissive  compared to that of 3 and 11 , which are in good agreement with the optical gaps determined from their absorption onsets  or by going to terchrysene‐based systems. Additionally, alternative ways of film formation, other than spin‐coating, allowing for more time for the biaryl rotamers to equilibrate and adopt their energetically preferred (twisted) conformation should furnish blue‐emitting thin films for use in OLEDs.Emission of thin films of d naphtho,3‐ctetra5 and 13, may serve as precursors for large, unsubstituted PAHs—this approach could thus complement the hitherto explored one via exhaustive cyclodehydrogenations of polyphenylene dendrimers which are planarized under Scholl conditions.5 and 13 in hand, we further investigated the Scholl cyclodehydrogenations to obtain 14 and 15, respectively, which, as a member of the larger PAHs might be utilized as IR emitters and/or be utilized for stimulated emission.14 was reported so far but separation from a naphthoindenozethrene3SO3H, FeCl3 and DDQ/ScOTf3.[14 as a deep red double [4]helicene (Scheme λabs=495 nm) and emission (λem=516 nm) maxima—14 is yellow fluorescent in n‐hexane. The observed small Stokes shift illustrated the rigidity of the compound. This is the first selective access to 14 in reasonable yields.The synthesis of large PAHs without solubility mediating groups is a challenge for solution‐based chemistry. Soluble biaryls, such as Q/ScOTf3.18 Only e Scheme  with a mc]tetraphene (13) under similar conditions only lead to an insoluble and inseparable mixture of twofold cyclodehydrogenated product of unknown identity and, in traces, to fully oxidized 15 . Higher temperatures and prolonged reaction times up to 7 days did not drive the reaction to completion. While the solubility of 11 as well as that of its monocyclodehydrogenated reaction intermediate is sufficiently high for a complete fusion to yield 14, the limit of this approach is quickly met in terms of solubility of the reaction intermediates when attempting to synthesize 15. As such, cyclodehydrogenation cannot compete with oligophenylene planarization in terms of PAH size,tetraphene, may also be fully dehydrogenated, remains to be investigated.Cyclodehydrogenation of 6,6′‐binaphthotetraphene (11) and 6,6′‐binaphthotetraphene (13) via 6‐endo‐dig cyclizations of 1‐alkynylbiaryls and their Glaser coupled bialkynes. Although biaryl formation does only slightly influence the calculated frontier molecular orbital energy levels, it results in an increased solubility without additional solubility‐mediating groups. The shallow torsional potential, not greatly hindering rotation about the connecting single bond, leads to a pronounced red‐shift in the thin film emission due to partial planarization and intermolecular interactions. In the crystalline state, however, blue emission is retained due to the herringbone motif of the twisted biaryls. Key to soluble, all sp2 hybridized, blue fluorescent derivatives for OLED applications is thus to rigidify the system even further, for example, as in 5,11‐polychrysenylene, which also served as a graphene nanoribbon precursor.4 has the potential to exceed the previously reported oligomers in size due to reduced steric hindrance upon C−C bond formation. Whereas polyphenylene dendrimers are conventionally employed as precursors to large unsubstituted PAHs, as they planarize under Scholl conditions, we explored a new strategy through cyclodehydrogenation of (soluble) biaryl systems in this work, which could furnish nanographenes with hitherto unexplored edge type combinations. These types of sparsely investigated PAHs are of interest for lasing applications11 furnishing 14, but full cyclodehydrogenation of its higher homologue 13 was only observed in trace amounts: Most likely, solubility limits the full conversion of the twofold cyclodehydrogenated intermediate, which will be circumvented by utilizing surface‐assisted fusionIn conclusion, we reported a simple PtCl1996744, 1996745, 1996746, 1996747, 1996748, and 1996749 contains the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition numbers General procedure for the synthesis of acenes: General procedure (GP): In a flame dried Schlenk flask, the starting material (1.00 equiv.) and PtCl2 (0.25 equiv.) were dried for 1 h under vacuum. Dry toluene was added and the reaction was stirred at 80 °C for 3 d. The reaction mixture was allowed to cool to room temperature and purified. The crude product was further purified by flash chromatography  to obtain the compound.5,5′‐Bichrysenyl (5): According to GP, 4  and PtCl2  were dried for 1 h under vacuum. 20 mL of dry toluene (freeze pumped three times) were added and the reaction was stirred at 80 °C for 3 d. The mixture was allowed to cool to room temperature before the solvent was removed under reduced pressure. The crude product was purified by flash chromatography  to obtain 5 as a pale yellow solid . Rf=0.29 , M.p.: >300 °C, 1H NMR : δ=8.98 , 8.94 , 8.28 , 8.16 , 7.98 , 7.80–7.74 , 7.66 , 7.62 , 7.37 , 6.88 ppm , 13C{1H} NMR : δ=141.6, 134.1, 132.3, 131.2, 130.8, 130.6, 129.0, 128.8, 128.3, 127.5, 127.4, 126.5, 125.9, 123.9, 122.2 ppm, IR: ν˜=3054, 3018, 2961, 2922, 2872, 2853, 1592, 857, 797, 757 cm−1, HRMS (MALDI+) m/z: [M+H]+: calc. for [C36H22]+: 454.1720, found 454.1659, correct isotope distribution.Naphthotetraphene (11): According to GP, 10  and PtCl2  were dried for 1 h under vacuum. 50 mL of dry toluene (freeze pumped three times) were added and the reaction was stirred at 80 °C for 3 d. The mixture was allowed to cool to room temperature before the organic phase was washed several times with 1 N aqueous HCl. The precipitated solid was filtered and washed with 1 N HCl, EtOAc, H2O and ethanol. The crude product was purified by flash chromatography  to obtain 11 as a pale yellow solid . Rf=0.25 , M.p.: >300 °C, 1H NMR : δ=9.42 , 8.94 , 8.58 , 8.23–8.21 , 8.12–8.08 , 7.57–7.52 ppm , 13C{1H} NMR : δ=133.4, 133.1, 132.1, 130.4, 129.5, 128.8, 128.6, 127.6, 126.6, 126.5, 123.3, 122.4 ppm, IR: ν˜=3046, 2961, 1467, 1354, 1260, 1088, 1074, 1053, 1017, 1005, 957, 895, 879, 841, 811 cm−1, HRMS (MALDI+) m/z: [M+H]+: calc. for [C26H16]+: 328.1252, found 328.1249, correct isotope distribution.6,6′‐Binaphthotetraphene (13): According to GP, 12  and PtCl2  were dried for 1 h under vacuum. Dry toluene  were added and the reaction was stirred at 80 °C for 3 d. The mixture was allowed to cool to room temperature before the organic phase was washed several times with 1 N HCl solution. The precipitated solid was filtered and washed with 1 N HCl, EtOAc, H2O and ethanol. The crude product was purified by flash chromatography  to obtain 13 as a pale yellow solid . Rf=0.24 , M.p.: >300 °C, 1H NMR : δ=9.66 , 9.18 , 8.77 , 8.51 , 8.35 , 8.32 , 8.27 , 8.22 , 8.04 , 7.79 , 7.58 , 7.52 , 7.19 , 6.92 , 6.69 ppm , 13C{1H} NMR : δ=142.5, 133.7, 133.5, 132.9, 132.0, 131.7, 131.5, 130.1, 130.0, 130.0, 129.8, 129.5, 129.0, 128.7, 128.5, 127.7, 127.6, 127.0, 126.8, 126.5, 126.5, 125.7, 123.6, 122.7 ppm, IR: ν˜=048, 2957, 1672, 1600, 1587, 1474, 1282, 1258, 1071, 1006, 953, 905, 885, 861, 803 cm−1, HRMS (MALDI+) m/z: [M+H]+: calc. for [C52H30]+: 654.2348, found 654.2448, correct isotope distribution.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco ( The ability of plants to dedifferentiate and regenerate cells from differentiated somatic tissues in plants has served as an important tool for producing fully functional plantlets from plant explants or cells. The regenerative capacity of plant cells can be enhanced in vitro in nutrient media supplemented with plant hormones ,2, and iArabidopsis, the genes such as CUP-SHAPED COTYLEDON (CUC), SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and many others are required for shoot apical meristem (SAM) formation during embryogenesis for SAM maintenance during post-embryonic development , and cell density was adjusted to 1 × 106 cells mL−1 using a hemocytometer. 200 μL of each sample was immediately incubated in 1.8 mL of PCM at 25 °C in the dark for 3 weeks. After 3 weeks of incubation, micro-calli was transferred to 10 mL of PCM in a 100 mm × 20 mm plastic Petri dish on a gyratory shaker (70 rpm) at 25 °C under a 16 h photoperiod (∼30 µmol m−2 s−1 cool white fluorescent light) during 3–4 weeks.Cotyledons of 11-day-after germination (DAG11) tobacco seedlings were used as explants for protoplast isolation. Protoplasts were isolated as previously described  with sli−1 thiamine, 60 g L−1 myo-inositol, and 3% (w/v) sucrose, 2 mg L−1 NAA, and 0.5 mg L−1 Zeatin; pH 5.8).Cotyledons of DAG7 tomato seedlings were used as explants for protoplast isolation. Cotyledon explants were harvested; each cotyledon explant was cut into 1–2-mm-thick pieces and incubated with 10 mL of an enzyme solution (described above) on a gyratory shaker (40 rpm) at 25 °C for 4.5 h in the dark. Protoplast isolation was carried out in the same manner as described above. After washing, the obtained protoplast pellet was suspended in PCM (MS medium containing 0.4 mg Lw/v) sucrose, 2 mg L−1 BAP, 0.5 mg L−1 NAA, and 0.8% plant agar; pH 5.8] containing 0, 0.1, 0.5, and 1.0 mM of NaB after 5 weeks of incubation at 1-week intervals. Protoplast-derived calli of tobacco were transferred to the CIM and incubated for 2 weeks, after which the callus of 2 mm2 in size was transferred into 0, 0.1, 0.5, and 1.0 mM NaB-containing CIM.To investigate the effect of sodium butyrate (NaB) on callus growth, callus size was measured in the callus induction medium (CIM)  containing 0, 0.1, 0.5, and 1.0 mM of NaBProtoplast-derived calli of tobacco were transferred into the CIM and incubated for 4 weeks; thereafter, the callus of approximately 7 mm−2 s−1 cool white fluorescent light) at 25 °C. Subculture of callus was carried out each month. Callus size was calculated by the program settled in Stereo Microscope (Nikon SMZ1270).The cultures were maintained under a 16 h photoperiod (∼30 µmol mw/v) sucrose, 0.4 mg L−1 thiamine, 100 mg L−1 myo-inositol, 2 mg L−1 NAA, 0.5 mg L−1 Zeatin, and 0.4% Gelite; pH 5.8] for 2–3 weeks, after which the callus of 2.5 mm2 in size was transferred into 0, 0.1, 0.5, and 1.0 mM NaB containing SIM . The callus size was measured during 6 weeks of incubation at 1-week intervals in SIM-1 and SIM-2 containing 0, 0.1, 0.5, and 1.0 mM of NaB. The cultures were maintained under a 16 h photoperiod (∼30 µmol m−2 s−1 cool white florescent light) at 25 °C. Subculture of callus was performed each month. Callus size was calculated by the program settled in Stereo Microscope (Nikon SMZ1270).After 3-4 weeks incubation in PCM, calli of tomato were transferred to the CIM  containing 0, 0.1, 0.5, and 1.0 mM of NaB during 6 weeks of incubation.Cotyledons of DAG7 tomato seedlings were excised and divided into three segments, and cultured proximal segments in the SIM  containing 0, 0.1, 0.5, and 1.0 mM of NaB during 6 weeks of incubation.Cotyledons of DAG11 tobacco seedlings were excised and cultured in the SIM [MS basal salt medium (Duchefa), 3% (−2 s−1 cool white fluorescent light) at 25 °C. The efficiency of adventitious shoots formation was calculated with the ratios from the total numbers of explants and the number of explants showing shoot formation.All cultures were incubated under a 16 h photoperiod (∼30 µmol mhttps://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot) with slight modifications. Histones were extracted by 0.2 N HCl overnight at 4 °C. Supernatant was collected and neutralized with 0.1 volume of 2 N NaOH in the supernatant. Histones were separated by 15% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and detected using a specific antibody against acetylated histone H3  and histone H3 antibody  by Chemiluminescence system Fusion Solo S . The ratio of histone H3 and acetylated histone H3 was calculated using built-in software.The calli cultured in the SIM containing 0, 0.1, 0.5, and 1.0 mM of NaB, respectively. After 1 or 2 weeks of incubation, calli were harvested and finely ground with pestle and bowl. Histone extraction performed using abcam’s protocol . The primer of NbCYCD3-1 was designed to detect four of seven NbCYCD3-1 homologs , and NbWUS gene information was obtained from Mlotshwa et al. (2006) [TM Real-Time PCR Detection System (Bio-Rad). The primers used for PCR were as follows: 5′-AAGCCCCTCTTAACCCAAAG-3′ and 5′-ACCAGAGTCCAACACAATACC-3′ for SlActin4; 5′-CAAGGAGAAGGTGGAAGGATG-3- and 5′-CTGTTGGACTCCCTGGTAATG-3′ for SlCYCD3.1; 5′-TGGAACTTTGGCTATGGAGAAG-3′ and 5′-GGGTAAGTTGCTGGAGAAGTAG-3′ for SlWUS; 5′-CCAAAGGCCAATCGAGAAAAG-3′ and 5′-CAGAGTCCAGCACAATACCAG-3′ for NbActin; 5′-CAACTTGTTGCTGTAGCTTGTC-3′ and 5′-CAAACACATATCTTGGATCCTCAAC-3′ for NbCYCD3-1; 5′-CATCCGCTTCTTCTCATGGT-3′ and 5′-GAGTTGCCACCTGGTGATATT-3′ for NbWUS. SlActin4 (ACT4) and NbActin was used as an internal control.Cotyledon explants of tomato and tobacco or protoplast-derived calli of tobacco were incubated in the SIM containing NaB for 4, 8, and 12 days or 1, 2, and 3 weeks, respectively (as described above). Total RNA was extracted using an Aqueous Kit (Ambion) with a TURBO DNA-Free Kit (Applied Biosystems). cDNA was synthesized using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems). To investigate the transcript levels of . (2006) . The qRTSlCYCD3-1 (Solyc04g078470.2), SlWUSCHEL (Solyc02g083950.2.1), SlActin 4 (Solyc04g011500.2). N. benthamiana gene sequence data from this article can be found in the Sol genomics network and NCBI GenBank under the following accession numbers: NbCYCD3-1 (Niben101Scf05643g01012.1), NbACT (Niben101Scf09133g02006.1), NbWUS .Tomato gene sequence data from this article can be found in the Pyotozome v2.1 under the following accession numbers: p < 0.05.Statistical analyses were carried out using values of experiments performed in triplicate. Data were analyzed using one-way analysis of variance test, followed by Tukey′s multiple comparisons using PRISM Software 8.0 . Differences were considered statistically significant at


Meanwhile, after GA treatment, the morphology of T24 cells were changed significantly. Moreover, GA significantly inhibited T24 cells proliferation and blocked T24 cells cycle in S phase (p < 0.001). GA induced T24 cells apoptosis (p < 0.001), accompanied by reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) depolarization. Western blotting analysis showed that GA significantly increased Cleaved caspase-3, Bax, P53, and Cytochrome C (Cyt-c) proteins expression, and decreased Bcl-2, P-PI3K, P-Akt, P-IκBα, P-IKKα, and P-NF-κB p65 proteins expression in T24 cells (p < 0.05). Real-Time PCR results verified that GA significantly promoted Caspase-3, Bax, P53, and Cyt-c genes expression, and inhibited Bcl-2, PI3K, Akt, and NF-κB p65 genes expression (p < 0.001). However, on the basis of GA (IC50) stimulation, NAC (an oxidative stress inhibitor) pretreatment reversed the apoptotic rate of T24 cells and the expression of Bax, Cleaved caspase-3, P53, Bcl-2 proteins, and the MMP level in T24 cells, as well as the expression of Cyt-c protein in T24 cells mitochondria and cytoplasm. In addition, GA significantly suppressed T24 cells migration and invasion ability with VEGF protein inhibition (p < 0.001). Briefly, GA can inhibit T24 cells proliferation, metastasis and promote apoptosis, and the pro-apoptotic activity is closely associated with mitochondrial dysfunction and PI3K/Akt/NF-κB signaling suppression. Our study will help in finding a safe and effective treatment for bladder cancer.Gallic acid (GA), a hydrolyzable tannin, has a wide range of pharmacological activities. This study revealed that, GA significantly inhibited T24 cells viability in a concentration- and time- dependent manner. The IC Bladder cancer is a common malignant tumor in the urogenital system. It was estimated that 430,000 new cases of bladder cancer were diagnosed worldwide in 2012 . ClinicaRheum rhaponticum L, Punica granatum L, Rubus idaeus L, and so on. At present, more and more scholars have paid close attention to GA, due to its extensive pharmacological activities  and N-acetylcysteine (NAC) were obtained from Sigma (USA). Punicalagin, ellagic acid and punicalin with 99% purity were purchased from Chengdu Institute of biology, Chinese Academy of Sciences . CCK-8 kit was acquired from Dojindo (Japan). The 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit was purchased from Ribobio . Annexin V-FITC apoptosis detection kit and PI/RNase Staining Buffer kit were obtained from BD Biosciences . ROS detection kit was obtained from Jiancheng Bioengineering . Mitochondrial Membrane Potential (MMP) detection kit was obtained from BestBio company . The primary antibodies against Cleaved Caspase-3, Caspase-3, P53, Bcl-2, Bax, Cyt-c, P-Akt, Akt, P-NF-κB p65, NF-κB p65, and P-IκBα were purchased from Cell Signaling Technology . The primary antibodies against PI3K, P-PI3K, P-IKKα, VEGF, COX-IV, and β-Actin were purchased from abcam (UK). The second antibody was obtained from LI-COR (USA). PrimeScipt™ RT reagent Kit with gDNA Eraser and SYBR@Premix Ex Taq™ II were obtained from Takara Bio (Japan). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin were purchased from Gibco . Matrigel was obtained from BD Biosciences .2 incubator . Preparing GA stock solution at a concentration of 500 µg/ml: precisely weighed 2.00 mg GA in a EP tube, added 4 ml FBS free MEM medium, mixed with a vortex to ensure complete dissolution, and then stored at -20°C. The GA stock solution needs to be diluted before use. Specifically, the GA stock solution was taken out and dissolved in the dark at 37°C, and then it was diluted to the appointed concentration with FBS-free MEM medium to obtain the working solution. Attention should be paid here to avoid light in the use of GA.The human bladder cancer T24 cell line was obtained from China Center for Type Culture Collection . T24 cells were cultured in MEM medium (Gibco) with 10% FBS, 100 units/ml penicillin and 100 units/ml streptomycin, then maintained in 37°C with 5% CO50) in each group were calculated.For cell counting kit-8 (CCK-8) assay, T24 cells were firstly plated in a 96-well plate for 24 h incubation. After that, the previous medium was removed, and 100 µl 0, 6.25, 12.5, 25, 50, 75, and 100 µg/ml GA or 5-FU solution were respectively added into each well for 24, 48, and 72 h stimulation. After reaching the appointed time, the previous medium was replaced with 100 µl new MEM medium containing 5% CCK-8. After 1 h incubation at 37°C in the dark, the absorbance at 450 nm was measured by a microplate reader . Finally, the inhibition rate and half inhibition concentration  were added into each well. After 24 h stimulation, the cell morphological changes in each group were observed by an inverted microscope .3 cells/well, and consecutively cultured for one week at 37°C. After the medium was removed, the cells were washed with phosphate buffered saline (PBS) for 3 times. After being fixed with 4% paraformaldehyde for 15 min, the cells were stained with 0.5% crystal violet solution for 15 min. Finally, the cells were washed with PBS and dried in the air, and the number of colonies was counted manually.T24 cells were firstly plated in a 6-well plate and cultured for 48 h, the cells were then treated with different concentrations of GA  for 24 h. Subsequently, the cells in each well were digested, counted, and plated into a new 6-well plate with complete media at a density of 2×102. The cells were then treated with different concentrations of GA  for 24 h. Subsequently, the EdU working solution (20 µM) was added to each well with equal volume and incubated for 2 h. After treatment with 4% paraformaldehyde and PBS containing 0.3% Triton X-100, the cells were stained with the EdU reaction liquid according to the instructions of 5-ethynyl-2-deoxyuridine labeling/detection kit . DAPI was used to label the nuclei for 15 min after PBS washing. Finally, the EdU-positive cells in five randomly selected fields were viewed by a fluorescence microscopy .T24 cells were firstly plated in a 6-well plate with equal volume and cultured for 48 h at 37°C with 5% COT24 cells were firstly stimulated with an increasing concentration of GA solution  for 24 h. After that, the cells were collected and washed with PBS. 1 ml 70% ethanol was added into each tube and incubated at -20°C overnight for fixing. Subsequently, the fixed T24 cells were collected and washed with 4°C PBS, and 500 µl PI/RNase dye solution was added into each tube and incubated for 15 min at 25°C in the dark. Finally, the fluorescence intensity of each group was detected immediately using the flow cytometry .Briefly, T24 cells were firstly stimulated with an increasing concentration of GA solution . After 24 h treatment, the cells were collected by centrifugation after trypsinization, and washed with cold PBS. Then 500 µl binding buffer solution was added into each tube for resuspension. Finally, 5 µl fluorescein-conjugated Annexin V solution and 5 µl PI solution were added into each tube respectively and incubated for 15 min at 25°C in the dark. Finally, the apoptosis rates of T24 cells were detected using the flow cytometry .T24 cells were firstly stimulated with an increasing concentration of GA solution  for 24 h. After stimulation, T24 cells were digested by trypsin and collected by centrifugation. Then 1 ml DCFH-DA working solution was added to each tube and dyed for 30 min at 25°C in the dark. After incubation, the cells were obtained and washed with PBS to remove the excess DCFH-DA. Finally, the fluorescence intensity was immediately detected by a microplate reader  after adjusting the consistency of cell density.The JC-1 staining method was used to measure the changes in T24 cells MMP level after GA stimulation. Briefly, T24 cells were firstly stimulated with an increasing concentration of GA solution  for 24 h. After stimulation, the cells were collected and washed with PBS. The JC-1 working solution was added to each tube and dyed for 15 min at 25°C in the dark. Then the cells were obtained by centrifugation and resuspended with PBS. Finally, the MMP level changes were detected using the flow cytometry . In addition, the evaluation of MMP level was also performed with a fluorogenic lipophilic cation, according to the manufacturer’s protocol. In cells with hyper-polarized mitochondrial membranes, JC-1 spontaneously forms complexes (J-aggregates) emitting red fluorescence. In cells with depolarized mitochondrial membranes, JC-1 remains in the monomeric form, emitting green fluorescence. The detection of MMP was performed by fluorescent microscopy (200×magnification). All experiments were repeated three times.T24 cells were firstly plated in a 6-well plate for 48 h incubation. After that, the previous medium in each well was removed and replaced with different concentrations of GA solution . After 24 h stimulation, the 6-well plate was moved on the ice and washed by PBS. Then the cells in each well were incubated with 600 µl RIPA lysis buffer solution, and lysed on ice for 20 min, and scraped with a cell scraper, then transferred into an EP tube. The total protein was obtained by collecting supernatant after centrifugation. The BCA method was used to detect the extracted protein concentration, and the sample volume was adjusted according to the protein concentration before electrophoresis. Then the sample was added into each pore respectively and the marker was added at both ends, 10% Tris–Glycine gels were used to separate the protein sample under constant current. After that, the protein gelatin was transferred to PVDF membranes. Five percent BSA in TBST (TBS with 0.1% Tween-20) was used to block the membranes for 1 h at room temperature. Then the membranes were incubated with the specific primary antibodies against Bax, Bcl-2, Caspase-3, Cyt-c, P53, P-Akt, Akt, P-PI3K, PI3K, P-IκBα, P-IKKα, P-NF-κB p65, NF-κB p65, VEGF, COX-IV, and β-Actin at 4°C overnight respectively. The next day, the membranes were washed with TBST for three times, and incubated with the corresponding secondary antibody  for 1 h at room temperature in the dark. The membranes were scanned by Odyssey imaging system (USA). Finally, the relative gray value of the target proteins and internal reference protein were respectively measured by Image J Software.In our experiment, the expression of the target genes was regarded as the mRNA level. The relative expression of Bax, Bcl-2, Cyt-c, Caspase-3, P53, PI3K, Akt, and NF-κB p65 genes in T24 cells after GA stimulation were detected by RT-PCR method. In short, T24 cells were firstly stimulated with an increasing concentration of GA solution respectively. After 24 h stimulation, the cells were collected through trypsinization and transferred to the EP tubes. Trizol was then added into each tube to extract the total RNA according to the manufacturer’s protocol. The ultraviolet spectrophotometer  was used to detect the concentration and purity of the extracted RNA from each group. The reverse transcription was performed using an ABI PCR instrument  with GAPDH as the internal reference. The primer sequence was obtained from Wuhan Google Biotechnology. The design and synthesis of the primer sequences were shown in 2) was measured with ImageJ software.T24 cells from each group were plated in 6-well plates for 48–72 h. When the cell density reached approximately 80%, the monolayer was wounded by scratching with a 200 µl sterile pipette tip lengthwise along the plate surface, and the media was removed. The cells were then washed 2–3 times with PBS and cultured in serum-free media. Images of cell migration were captured at 0, 12, and 24 h by an inverted microscope , and the area of migration , and added 700 µl MEM medium containing 20% FBS on the lower chamber, and placed the 24-well plate in 37°C incubator. After 24 h incubation, the transwell chamber was taken out from the incubator. The non-migrated cells were scraped with a cotton swab. Then the migrated cells were fixed with 95% methanol for 15 min, and then dyed with 0.5% crystal violet solution for 15 min. Finally, the migrated cells were observed and photographed by an inverted microscope . The cells in 5 random fields were counted and the mean was calculated. For transwell invasion assay, the matrigel was firstly diluted in a ratio of 1:8 with FBS-free MEM medium. The transwell upper chambers were then coated with 80 µl diluted matrigel. The subsequent operations were the same as the migration experiments.For transwell migration assay, T24 cells were firstly stimulated with an increasing concentration of GA solution . After 24 h stimulation, the cells were collected by trypsin and suspended in FBS-free MEM medium. After adjusting the density of cells, we added 100 µl 1×10The samples were collected from T24 cells culture supernatant and centrifuged at 4°C, 2,000×g for 10 min. The separated supernatant was used for biochemical analysis. The level of VEGF (cat. no. EK0393) was measured using an ELISA kit purchased from Wuhan Boster Biological Technology, Ltd. . The absorbance at 450 nm was measured on a microplate reader .p values were labeled as follows: *p<0.05, **p<0.01, ***p<0.001.All experiments were repeated at least three times independently. The data were expressed as the mean ± standard deviation (SD). The treated groups were compared by one-way variance (ANOVA) with SPSS 13.0 . The statistically significant p < 0.001). The half inhibitory concentration (IC50) of GA stimulating T24 cells for 24, 48, and 72 h were 21.73, 18.62, and 11.59 µg/ml respectively, while the IC50 of 5-FU were 102.04, 35.63, and 20.01 µg/ml respectively. Compared with the control group, T24 cells viability in GA-treated group was significantly decreased, indicating that GA significantly inhibited T24 cells viability in a time- and concentration-dependent manner (p < 0.01). As GA stimulated T24 cells for 24 h, the inhibition rate of GA with 6.25 and 25 µg/ml was 6.89 and 65.77%, respectively. These results indicated that GA significantly inhibited T24 cells viability. Combining with the results of CCK-8, we determined GA with 6.25, 12.5, and 25 µg/ml as the appropriate action concentration and 24 h as the appropriate action time.In order to find the effective anti-cancer component in pomegranate peel tannins, we firstly compared the effects of each monomer in pomegranate peel tannins on T24 cells viability, and found the inhibition of GA on T24 cells viability was significantly higher than the other three monomers . In the group of GA with 25 µg/ml, the percentage of cells in G1 phase was 57.52%, while that in S phase was 36.49% . The apoptotic rate of T24 cells in the control group was 5.13%, while the apoptotic rate in GA-treated groups with 6.25, 12.5, and 25 µg/ml were 8.64, 18.84, and 36.79% respectively (p < 0.01). In addition, the early apoptotic rate was 3.25% in the control group and 31.17% in GA (25 µg/ml) treated group, indicating that GA mainly caused T24 cells early apoptosis. These results suggested that GA promoted T24 cells apoptosis in a concentration-dependent manner, and mainly induced early apoptosis of T24 cells.In order to explore whether GA could induce T24 cells apoptosis, the FITC/PI staining method was used, and the results were displayed in p < 0.05). These results indicated that GA induced oxidative stress in T24 cells.Current researches have revealed that mitochondrial ROS plays a vital role in cell apoptosis, and the mechanism is involved in many aspects of apoptosis . Since Gp < 0.001). These results indicated that GA caused the decline of MMP level in T24 cells, and implied that GA-induced T24 cells apoptosis might be associated with the mitochondrial dysfunction.An obvious decrease in MMP level is generally regarded as the earliest change in apoptosis. The MMP level changes in T24 cells were expressed as the ratio of red/green fluorescence after stimulating with GA. As shown in p < 0.001). Meanwhile, the expression of P53, Cyt-c, and Cleaved caspase-3 proteins were significantly increased , NAC + GA group . The results showed that, compared with the control group, the apoptotic rate of T24 cells was significantly increased in GA (IC50) group (p < 0.01), while compared with the GA (IC50) group, the apoptotic rate of T24 cells was significantly decreased in GA + NAC group (50) group (p < 0.01), and NAC reversed this effect (50) group (p < 0.01), while compared with the GA (IC50) group, the intensity of green fluorescence in T24 cells was significantly decreased and the intensity of red fluorescence in T24 cells was significantly increased in GA + NAC group (50) group (p < 0.01), while compared with the GA (IC50) group, the expression of Cyt-c protein in T24 cells mitochondria was significantly increased in GA+NAC group (50) group (p < 0.01), while compared with the GA (IC50) group, the expression of Cyt-c protein in T24 cells cytoplasm was significantly decreased in GA + NAC group , GA group . The authors are very grateful to the Central Laboratory of Renmin Hospital of Wuhan University for providing technical assistance, experimental places and equipment facilities.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hemerocallis plants are important vegetables with nutritional and health value, especially in eastern Asia, where they have been used as medicines to cure disease such as depression and inflammation for thousands of years. The present study concerns the determination of flavonoids and phenolic acids, as well as antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase and antimicrobial properties of taxa cultivated in Poland. For chemical composition estimation, LC-ESI-MS/MS analysis and spectrophotometric assays were performed. The results show the presence of sixteen compounds in all analyzed species. Among the investigated cultivars, it was found that H. “Chicago Apache” and H. fulva var. kwanso have the highest total phenolic acid and flavonoid content. The most abundant compounds in all analyzed extracts were chlorogenic acid (209.8 to 1010.0 µg/g of DE) and quercetin-3-O-rutinoside (114.7 to 1049.7 µg/g of DE). The studied extracts exhibited moderate to high skin-related activities. These properties were correlated with a high concentration of polyphenols. The present study demonstrated that Hemerocallis cultivars contain significant amounts of phenolic compounds with good skin-related activities and could be interesting as novel sources of bioactive agents for the pharmaceutical, food and cosmetic industries. Plant extracts are a valued source of biologically active compounds. Numerous scientific studies confirm the antibacterial, antiviral, antifungal and anti-inflammatory effects of plant raw extracts . CosmetiHemerocallis (daylily) genus belongs to the Asphodelaceae family and Hemerocallidoideae subfamily -Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), Tricine  were obtained from Sigma-Aldrich . Phosphate-buffered saline (PBS) was purchased from Gibco . Reference substances were from ChromaDex . Acetonitrile, formic acid and water for LC analysis were from Merck . All others chemicals were of analytical grade and were obtained from Polish Chemical Reagent Company .Ascorbic acid, collagenase from Hemerocallis cultivars  were gathered in the Maria Curie-Skłodowska University (UMCS) Botanical Garden in Lublin (Poland), at altitude of 181.2 m a.s.l.  in August 2017. All details about these species, such as inventoty numbers and origin, were described in our previous research -Leu-Gly-Pro-Ala (FALGPA) as a substrate, and activity was measured according to Mandrone et al. . Collage50 values of the Hemerocallis extracts based on concentration–inhibition curves.All results were expressed as the ICStaphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25992). Clinical strains  isolated from infected wounds were obtained and stored since August 2016 in the strain bank at the Department of Biochemistry and Biotechnology, Medical University of Lublin, Lublin, Poland. Stock cultures were held at −70 °C. Tested bacterial strain, before the experiments, was passaged onto fresh Mueller–Hinton agar (M-H)  at 37 °C for 24 h. Next, inocula were made with fresh microbial cultures in sterile 0.9% NaCl to 0.5 McFarland turbidity standard. The antibacterial properties of extracts against bacteria was estimated by disk diffusion method, measuring the zones of inhibition. (Kirby-Bauer Disk Diffusion Susceptibility Test Protocol) on Petri plates with solid medium (M-H agar). Appropriate strain cultures were separately spread over the agar surface using a cotton swab. Next, tested liquid samples (100 μg) were placed using sterile disc . After 18 h of incubation at 37 °C, zones of microbial growth produced around the studied extracts were estimated and recorded as the diameters of inhibition [mm]. All analyses were carried out in triplicate. The results are expressed as mean ± RSD.Zones of bacterial growth inhibition, caused by obtained extracts, were estimated for the reference microorganisms from the American Type Culture Collection (ATCC): Gram-positive bacteria , using built-in “prcomp” function.All results were stated as means ± standard deviation (SD) of three independent tests. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis of significance of differences between means. Hemerocallis cultivars, H. “Chicago Apache” (H5) and H. fulva var. kwanso (H1) had the highest phenolic content , followed by H. “Jaskółka” (H7) (45.5 ± 0.0 mg GAE/g DE), H. “Danuta” (H6) (35.3 ± 0.5 mg GAE/g DE), H. “Aten” (H2) (28.0 ± 0.1 mg GAE/g DE), H. “Rebel Cause” (H8) (22.1 ± 0.4 mg GAE/g DE), H. “Bożena” (H3) (16.1 ± 0.5 mg GAE/g DE), and H. “Catherine Woodbuery” (H4) (9.0 ± 0.4 mg GAE/g DE). The values obtained in our study are slightly higher than those of Lin and co-authors (2011) for various extracts of the flowers of H. fulva (from 25.3 ± 2.7 to 34.6 ± 2.7 mg GAE/g of extract) and lower than those of H. fulva leaves  }-β-D-galactopyranoside, chrysoeriol 7-O-}-β-D-galactopyranoside, were isolated from the aqueous-ethanolic extract of H. fulva flowers [According to the literature reports, entified . In studisolated . Moreove flowers .Hemerocallis species are rich in terms of phenolics.Therefore, our results are in agreement with previous investigations and it can be seen that extracts, especially aqueous-ethanolic, of Hemerocallis cultivars. In the study, we examined in vitro antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase and antibacterial properties of eight Hemerocallis cultivars collected in Poland.Skin aging is a natural, unavoidable, complex process, progressing over the years. Basic life functions disappear gradually, creating an incorrect response to external factors. Various intrinsic and extrinsic factors are responsible for this process, including hormonal, genetic, metabolic changes, as well as exposure to environmental stress . As seveHemerocallis cultivars exhibited moderate to high scavenging capacity in a concentration-dependent manner. For comparison, the radical scavenging activity of ascorbic acid was tested in the same conditions. The higher DPPH scavenging activity was showed for the extract of H. “Chicago Apache” (H5) (IC50 = 0.9 ± 0.2 mg/mL) followed by H. fulva var. kwanso (H1) (IC50 = 1.1 ± 0.1 mg/mL). However, the IC50 value for Hemerocallis extracts was from two- to even forty-times higher (H. “Catherine Woodbuery”—IC50 = 19.4 ± 0.1 mg/mL) than the for ascorbic acid (IC50 = 0.5 ± 0.0 mg/mL)  .Hemerocallis cv. Stella de Oro flowers were tested for their antioxidant activity. The flavonol 3-O-glycoside from this species demonstrated low antioxidant activities at 10 µM [The results of other published reports can be tough to compare due to the other conditions of experiments used. Nevertheless, several phenolic compounds isolated from at 10 µM . The scaat 10 µM . The autIt is well known that polyphenols can reduce oxidative stress by several mechanisms that depend on their chemical structure. One of these mechanisms is the chelation of metal ions, such as iron, which plays a key role in the production of harmful oxygen species . Under n2+ complex formation. The extracts from H. fulva var. kwanso (H1), H. “Chicago Apache” (H5), H. “Danuta” (H6) and H. “Jaskółka” (H7) were the most active ones interfering with the formation of iron and ferrozine complexes, which suggests their high chelating capacity and ability to capture iron ions before ferrozine. These samples had IC50 values comparable (9.2 ± 0.1 µg/mL—H7 and 9.6 ± 0.1 µg/mL—H6) and higher (1.9 ± 0.3 µg/mL—H5 and 6.1 ± 0.6 µg/mL—H1) than Na2EDTA*2H2O (IC50 = 9.8 ± 0.0 µg/mL) used as positive control. Slightly lower activity was noted for H. “Bożena” (H3) with IC50 = 12.9 ± 0.1 µg/mL. The lowest chelating activity was found for extracts from H. “Catherine Woodbuery” (H4) and H. “Rebel Cause” (H8) (The chelating capacity was based on measuring the percentage of inhibition of ferrozine-Fese” (H8) .Hemerocallis cultivars may be responsible for their strong antioxidant effects [High levels of chlorogenic acid and rutin in the extracts of  effects , which wHemerocallis cultivars was measured using C. histolyticum collagenase. The results are presented in Collagen is the predominant tissue constituent of normal human dermis which is mainly responsible for structural stability. The reduction of collagen is started by collagenases that split interstitial collagens . The inhH. “Chicago Apache” (H5) showed the highest anti-collagenase activity with IC50 value of 38.5 ± 0.2 μg/mL. H. fulva var. kwanso (H1) and H. “Jaskółka” (H7) also showed high activity with IC50 values of 40.3 ± 0.4 μg/mL and 41.2 ± 0.3 μg/mL, respectively. These results were comparable to those achieved for EGCG, used as the positive control (IC50 = 34.9 ± 0.4 μg/mL). The lowest chelating activity was found for extracts from H. “Catherine Woodbuery” (H4) and H. “Rebel Cause” (H8) with IC50 = 83.8 ± 0.2 and 78.8 ± 0.4 μg/mL, respectively.The extract of Hemerocallis extracts, have demonstrated a high collagenase inhibitory effect which was shown by different reports [In many studies, it has been proved that polyphenols are mostly compounds that are responsible for collagenase inhibition ,55. More reports ,56.In normal adult skin, the elastin dominates, representing over 90% of the total content of a developed elastic fiber, and it is a protein responsible for the elasticity . Because50 = 62.4 ± 0.1 μg/mL. As in the case of collagenase inhibition, the extract from H. “Chicago Apache” (H5) shows the highest anti-elastase activity with the IC50 value of 45.5 ± 0.1 μg/mL. The inhibition of elastase activity of H. fulva var. kwanso (H1) and H. “Jaskółka” (H7) was also high . All the results of anti-elastase activity are presented in The analysis was achieved with using a N-Succinyl-Ala-Ala-Ala-p-nitroanilide (substrate molecule) and elastase from porcine pancreas (enzyme). The extracts were tested in various concentrations (6.25–100 μg/mL). EGCG was used as the positive control and it showed ICAs with anti-collagenase activity, it has also been demonstrated that polyphenols are significant elastase inhibitors .Tyrosinase is a copper-containing enzyme widespread in nature, that is, a key enzyme in the biosynthesis of melanin, biopolymer responsible for the color of skin and hair, and also protect skin from ultraviolet . Though Hemerocallis species and kojic acid used as positive control were tested in various concentrations (6.25–100 μg/mL). The extract from H. “Chicago Apache” (H5) was the most effective amongst all samples with IC50 = 16.6 ± 0.1 μg/mL. Other extracts showed slightly weaker activity with IC50 values from 20.6 ± 0.1 (H1) to 79.3 ± 0.2 μg/mL  and Gram-positive  bacterial species was examined using a plate antimicrobial test. The obtained effects are presented in Hemerocallis “Chicago Apache” (H5) and H. “Jaskółka”(H7) showed the highest activity among the eight tested samples. The zones of reference S. aureus and S. epidermidis growth inhibition caused by Hemerocallis “Chicago Apache” (H5) were respectively 27.3 ± 3.1 mm and 19.5 ± 2.3 mm. Importantly, the growth of the tested clinical strains of S. aureus and S. epidermidis was also inhibited in highest amount by H5, creating zones of 19.5 ± 2.3 mm and 16.5 ± 2.1 mm, respectively. Inhibiting the growth of clinical strains is important when samples with potential therapeutic properties are tested. However, the tested samples showed no inhibition zones against Gram-negative E. coli strains. Therefore, Hemerocallis samples demonstrated narrow spectrum, limited only to Gram-positive bacteria, which may be beneficial in the prevention and fight against classic infections caused by Gram-positive strains.The antibacterial activity of the Hemerocallis cultivars are the first results published to date. As mentioned above, the strongest effects were found in extracts from Hemerocallis “Chicago Apache” (H5) and H. “Jaskółka”(H7). This may be ascribed possibly to an action of chlorogenic acid and rutin detected in high amounts in these extracts, which are known to cause damage to bacterial cell membranes, causing effluence of cellular matters, leading to the death of bacteria [The antibacterial effects of bacteria ,61,62.50). Then, the lower value denotes the higher activity. To perform a holistic view of the results, we used a chemometric multivariate approach. It allows us to examine some independent trends in changes among the investigated properties. During the introductory analysis of the correlations between investigated properties , one canTherefore, we also performed scaled Principal Component Analysis of the whole data matrix, containing 24 rows (three samples for each eight species) and eight columns . This approach allows us to examine intercorrelations between the properties, graph them in one two-dimensional graph and place the investigated samples in this reduced space. From a mathematical point of view, this is equivalent to the projection of multivariate space to the bivariate plane, where this plane is located in the best possible way . Of course, the visualized information depends on the data structure, so if the dataset is not intercorrelated , this method would not help us to understand the internal structure, as there is no optimal position on this plane.The dataset was found to be easy to compress and 81.8% of the overall variance was located in the first PC. Of the variance, 11.6% was located in the second one, so the PC1-PC2 plot explains 93.3% of the overall variability inside this dataset. Therefore, almost all information can be presented in a two-dimensional projection and only several percentages of information are lost inside the orthogonal complement of this plane. It was also found that interesting information was located inside the PC3, explaining 5.1% of variance. No explainable trends were found in the further PCs.H. “Catherine Woodbuery” (H4) and H. “Rebel Cause” (H8), whereas the lowest for H. fulva var. kwanso (H1) and H. “Chicago Apache” (H5). It should be emphasized that species are spread uniformly according to the PC1 values, so this trend should be perceived as continuous and fluent. It can be concluded that flavonoids and phenols are responsible for all investigated biological activities, as the activities are expressed as IC50, and thus are negatively correlated with the content of particular compounds.The first main trend in this dataset, placed in PC1  is conneH. “Aten” (H2), H. “Bożena” (H3), H. “Catherine Woodbuery” (H4) and H. “Rebel Cause” (H8) species. H. fulva var. kwanso (H1), H. “Chicago Apache” (H5), H. “Danuta” (H6) and H. "Jaskółka” (H7) have the second PC value close to zero. This trend is connected with increasing DPPH with a simultaneous decreasing of TPCA and slight decrease of ELA  or opposite change . This trend can be interpreted as antioxidant activity, orthogonal to all other activities and negatively correlated with TPCA. As DPPH is measured as the concentration, the dependence is opposite, and it proves that TPCA is mainly responsible for antioxidant activity.On the contrary, the second PC represents mainly the difference between The third PC  presentsDue to the fact that natural ingredients in cosmetics are no longer just a trend, but a standard in creating new care products, there is a constant search for new plant raw materials. Plant extracts could contain specific secondary metabolites that provide a very wide range of possibilities in use.Hemerocallis cultivars may have a value in the production of cosmetics. We found that aqueous-ethanol extracts of the flowering aerial parts of eight daylilies were rich in phenolics. Among the investigated cultivars, it was found that H. “Chicago Apache” and H. fulva var. kwanso have the highest total phenolic acids and flavonoids content. The most abundant compounds in all analyzed extracts were chlorogenic acid and quercetin-3-O-rutinoside. The studied extracts exhibited moderate to high skin-related activities. These properties were correlated with a high concentration of polyphenols. The present study demonstrated that Hemerocallis cultivars contain significant amounts of phenolic compounds with good skin-related activities, and could be interesting as novel sources of bioactive agents for the pharmaceutical, food and cosmetic industries.The results of this study suggest that the


This study compared steady-state pharmacokinetics of IND, GLY and MF between Japanese and Caucasian male subjects after multiple inhalations of IND/GLY/MF o.d.A once-daily (o.d.) fixed-dose combination of indacaterol acetate (IND), glycopyrronium bromide (GLY), and mometasone furoate (MF) delivered via the BreezhalerThis was a single-center, open-label, 2-treatment crossover study with a 21-day washout period. Japanese and Caucasian subjects received IND/GLY/MF 150/50/80 μg  or 150/50/160 μg o.d. (ICS high-dose) for 14 days in each period. Pharmacokinetics were characterized up to 24 h post-dose on Days 1 and 14.max for IND, GLY and MF at the high ICS dose on Day 14 were 1.31  1.38  and 1.07 , respectively. Geometric mean ratios (Japanese/Caucasian) [90% CI] for AUC0–24h on Day 14 for IND, GLY and MF at the high ICS dose were 1.17 , 1.05  and 1.15  respectively. Similar trends were noted for all components for the medium ICS dose treatment. IND/GLY/MF was safe and well tolerated; no AEs suspected to be study drug-related were observed.In total, 16 Japanese  and 17 Caucasian subjects  were randomized. Geometric mean ratios (Japanese/Caucasian) [90% confidence interval (CI)] for CPharmacokinetics of IND, GLY and MF (high and medium dose) when delivered as a fixed-dose combination were comparable between Japanese and Caucasian subjects. The IND/GLY/MF combination at the administrated doses was safe and well tolerated in both ethnic groups.Japan Registry of Clinical Trial: jRCT2031200227, retrospectively registered on 04, December, 2020. Asthma is a chronic inflammatory disorder associated with airway hyper-responsiveness, airway inflammation, and airway structural remodeling that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning . As of 2® device (IND/GLY/MF) is a fixed-dose combination (FDC) of IND, a LABA, GLY, a LAMA, and MF, an ICS, in development for o.d.-maintenance treatment of asthma GINA (Step ≥ 4). Each of these three mono-components, IND ). All subjects provided written informed consent prior to any assessments being performed. As per the key inclusion criteria, participants were healthy male subjects, aged 20–45 (inclusive) years of age, a BMI of 18–30 kg/mFor the PK assessments of IND, GLY and MF, blood samples were collected pre-dose, and at 5 min (IND and GLY only), 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h after dosing on Days 1 and 14. Assessment windows were: 30 min from study drug administration for pre-dose samples on Day 1, − 10 min from study drug administration for pre-dose samples on Day 14, ± 3 min for 5 min, 15 min post-dose samples, ± 5 min for 30 min, 1 h, 2 h and 3 h post-dose samples, ± 10 min for 6 h, 12 h and 24 h post-dose samples.18 column using acidified water and acetonitrile mobile phase at a flow rate of 0.750 mL/min within 3.30 min. The lower limit of quantification (LLOQ) of IND was 5.00 pg/mL. For GLY determination, sample processing was performed by means of solid phase extraction using a volume of 0.2 mL plasma. Separation was obtained within 2.5 min on an ACQUITY UPLC BEH C18 analytical column at a flow rate of 0.750 mL/min with binary mobile phases made of water and acetonitrile both acidified 0.1% formic acid. The LLOQ of the method was 1.00 pg/mL.All blood samples were taken by either direct venipuncture or an indwelling catheter inserted in a forearm vein. At specified time points, 4 mL blood was collected in lithium heparin tubes for determination of IND and GLY, and 6 mL blood was collected in K2-EDTA tubes for determination of MF. The plasma samples of each compound were assayed using three separate validated liquid chromatography tandem mass spectrometry methods. Briefly, for IND determination, 0.2 mL plasma was processed using solid phase extraction and then separated on an ACQUITY UPLC BEH C18 column at a flow rate of 1.00 mL/min within 3.51 min, using a binary mobile phase made of 0.05% ammonia in water and acetonitrile. The LLOQ of the method was 0.250 pg/mL. Additional analytical parameters and performances of the three methods used for the determination of the three compounds are summarized in Additional file 0–24 h, Cmax and Tmax) were determined by non-compartmental analysis using WinNonlin Phoenix .For the MF determination, the method consisted of liquid–liquid extraction using 0.8 mL of plasma, and the separation of the extracted sample was achieved on an ACQUITY UPLC BEH CSafety was monitored in terms of adverse events (AEs) and serious adverse events (SAEs). There was regular monitoring of hematology, blood chemistry, urinalysis, and ECGs, and regular assessments of vital signs, physical condition, and bodyweight.0–24 h and Cmax) for IND, GLY and MF on Day 1 and Day 14 were analyzed by day using a linear mixed-effects model. The model included ethnic group, sequence, period, treatment and the interaction between ethnic group and treatment as fixed effects. Subject and matched pairs were considered as random effects. All matched pairs with evaluable PK parameters for at least one subject were included in the analysis. The above analysis was also repeated by including baseline age and bodyweight as covariates into the model.All subjects who received at least one dose of study medication were included in the safety analysis set. The PK analysis set included all subjects with at least one available valid PK concentration measurement, who received any study drug and had no protocol deviations that were expected to impact the PK analysis. Log-transformed primary PK parameters  were randomized to one of the two IND/GLY/MF treatment sequences. Twenty nine out of 33 subjects (14 Japanese and 15 Caucasian subjects) completed the study while four subjects (2 Japanese and 2 Caucasian subjects) discontinued the study. Three subjects (one Japanese and two Caucasian subjects) receiving IND/GLY/MF 150/50/80 μg discontinued the study due to the withdrawal of consent by subject in treatment period 1. One Japanese subject receiving IND/GLY/MF 150/50/80 μg was discontinued from the study by the Investigator due to AEs of gastroenteritis and headache, in treatment period 2.All the subjects were men; the baseline characteristics are presented in Table max of IND was 0.25 h, on both Day 1 and Day 14. The accumulation ratios (Racc for AUC0–24 h and Cmax) after o.d. dosing for 14 days were 3.09–3.32 and 1.57–1.76, respectively in Japanese subjects, and were 3.30–3.31 and 1.74–1.75 in Caucasian subjects (Table max on Day 14 in IND/GLY/MF high- and medium-dose treatment groups were 1.31 (90% CI 1.13–1.51) and 1.30 (90% CI 1.13–1.49), respectively. The geometric mean AUC0–24 h on Day 14 in IND/GLY/MF high- and medium-dose treatment groups were 1.17 (90% CI 1.01–1.35) and 1.21 (90% CI 1.05–1.40), respectively. The geometric mean Cmax and AUC0–24 h values were slightly higher up to 1.31-fold for Cmax and 1.21-fold for AUC0–24 h in Japanese subjects than in Caucasians. Similar trends were observed for single doses of IND/GLY/MF medium and high dose on Day 1 (Table max on Day 14 in IND/GLY/MF medium- and high-dose treatment groups for IND were 1.20 (1.04–1.40) and 1.22 (1.05–1.42), respectively. The geometric mean ratios (90% CI) (Japanese/Caucasian) for AUC0–24 h on Day 14 in IND/GLY/MF medium- and high-dose groups were 1.17 (1.00–1.36) and 1.12 (0.96–1.31), respectively.Overlapping mean plasma concentration–time profiles of IND were observed between treatment groups (IND/GLY/MF medium and high-dose) in both Japanese and Caucasian subjects. The mean plasma concentrations were slightly higher in Japanese subjects than in Caucasian subjects Fig. a. Followmax of GLY was 0.0833 h both on Day 1 and on Day 14. The accumulation ratios (Racc for AUC and Cmax) after o.d. dosing for 14 days were 2.74–2.86 (AUC0–24 h) and 1.69–1.80 (Cmax) in Japanese subjects, and were 2.83–2.84 (AUC0–24 h) and 1.68–1.90 (Cmax) in Caucasian subjects (Table max on Day 14 in IND/GLY/MF high- and IND/GLY/MF medium-dose treatment groups were 1.38 (90% CI 1.13–1.69) and 1.54 (90% CI 1.27–1.87), respectively. The geometric mean ratios (90% CI) (Japanese/Caucasian) for AUC0–24 h on Day 14 in IND/GLY/MF high- and IND/GLY/MF medium-dose treatment groups were 1.05 (90% CI 0.92–1.20) and 1.06 (90% CI 0.93–1.21), respectively. The geometric mean Cmax was up to 1.54-fold higher in Japanese than Caucasians subjects, while the geometric mean AUC0–24 h was comparable. A similar trend was observed following a single dose of IND/GLY/MF medium and high dose on Day 1 (Table max on Day 14 in IND/GLY/MF medium- and high-dose treatment groups for GLY were 1.33 (90% CI 1.09–1.61) and 1.20 (90% CI 0.98–1.46), respectively. The mean ratios (90% CI) (Japanese/Caucasian) for AUC0–24 h on Day 14 in IND/GLY/MF medium- and high-dose groups were 0.95 (90% CI 0.84–1.09) and 0.95 (90% CI 0.83–1.08), respectively.Comparable mean GLY concentration time profiles were observed between treatment groups (IND/GLY/MF medium and high dose) in both Japanese and Caucasian subjects. The mean concentrations over 24 h were slightly higher in Japanese subjects than in Caucasian subjects   in both Japanese and Caucasian subjects  after o.d. dosing of IND/GLY/MF medium dose for 14 days were 1.37 (AUC0–24 h) and 1.35 (Cmax) in Japanese subjects, and were 1.33 (AUC0–24 h) and 1.28 (Cmax) in Caucasian subjects. The accumulation ratios (Racc for AUC0–24 h and Cmax) after o.d. dosing of IND/GLY/MF high dose for 14 days were 1.50 (AUC0–24 h) and 1.38 (Cmax) in Japanese subjects, and were 1.40 (AUC0–24 h) and 1.31 (Cmax) in Caucasian subjects (Table max on Day 14 in IND/GLY/MF medium- and IND/GLY/MF high-dose treatment groups were 1.15 (90% CI 1.04–1.26) and 1.07 (90% CI 0.97–1.18), respectively. The geometric mean ratios of AUC0–24 h on Day 14 in IND/GLY/MF high- and IND/GLY/MF medium-dose treatment groups were 1.15 (90% CI 1.05–1.27) and 1.23 (90% CI 1.12–1.34), respectively. A similar trend was observed following a single dose of IND/GLY/MF medium dose and IND/GLY/MF high dose on Day 1 (Table max on Day 14 in IND/GLY/MF medium- and high-dose treatment groups for MF were 1.10 (90% CI 0.99–1.23) and 1.03 (90% CI 0.92–1.15), respectively. The geometric mean ratios (90% CI) (Japanese/Caucasian) for AUC0–24 h on Day 14 in IND/GLY/MF medium and high dose were 1.16  and 1.09 (90% CI 0.99–1.20), respectively.After single and multiple doses of IND/GLY/MF (either medium or high-dose) in Japanese and Caucasian subjects, the median time to reach Tmax and AUC0–24 h on Day 14 were 1.91 (1.79–2.02) and 2.00 (1.92–2.09), respectively. In Caucasian subjects, the geometric mean ratios (90%CI) for Cmax and AUC0–24 h on Day 14 were 2.04 (1.93–2.16) and 2.13 (2.04–2.21), respectively  (90%CI) of CIND/GLY/MF was well tolerated and had a similar safety profile at both doses in Japanese and Caucasian subjects. No AEs reported during the study was considered to be related to the study drug by the Investigator and all AEs were resolved by the end of the study. There were no deaths or SAEs reported in the study. One Japanese subject receiving IND/GLY/MF medium-dose reported two AEs (gastroenteritis and headache) that led to discontinuation of study treatment.We report the results of a Phase I study providing pharmacokinetic data for the LABA/LAMA/ICS combination of IND/GLY/MF in Japanese and Caucasian subjects. This multiple-dose study assessed the PK of IND, GLY, MF on Day 1 and Day 14 following IND/GLY/MF o.d. dosing for 14 days. The study included only healthy male subjects in accordance with local clinical practice and regulatory considerations for Phase I healthy volunteer studies in Japan. Healthy subjects were selected instead of asthma patients to minimize impact of confounding variables . This was considered the most sensitive approach to investigate the effect of ethnicity on PK of the components of IND/GLY/MF.max for the 3 analytes  and treatments (150/50/80 μg or 150/50/160 μg) were explored as part of data analysis. There was no correlation between the exposure of the different analytes as evidenced by the lack of any systematic trends for higher exposure for all analytes in an individual subject. These data confirmed generally consistent use of inhalation technique across subjects. Similar accumulation was noted in an individual subject for a particular analyte in both treatments; however, the extent of accumulation was different for different analytes. This observation is consistent with the slightly different mean accumulation ratios for AUC and Ces Table . The exp0–24 h on Day 14, across both dose groups were in the range of 1.17 to 1.21 for IND, 1.05 to 1.06 for GLY, and 1.15 to 1.23 for MF. Mean bodyweight of Caucasian subjects was slightly higher (approximately 10%) than that of Japanese subjects. This was considered one of the factors that contributed to the slightly higher exposures to all three analytes in Japanese subjects. Based on the results of exploratory statistical analysis including age and bodyweight as covariates, the geometric mean ratios (Japanese/Caucasian) of steady-state AUC0–24 h on Day 14, across both dose groups were in the range of 1.12 to 1.17 for IND, 0.948 to 0.954 for GLY and 1.09 to 1.16 for MF. Overall, the mean total systemic exposure  in Japanese healthy subjects increased by 23% at a maximum as compared to Caucasian subjects, across all analytes.The geometric mean ratios (Japanese/Caucasian) of steady-state AUCmax on Day 14, across both dose groups were in the range of 1.30 to 1.31 for IND, 1.38 to 1.54 for GLY, and 1.07 to 1.15 for MF. Based on the results of exploratory statistical analysis including age and bodyweight as covariates, the geometric mean ratios (Japanese/Caucasian) of steady-state Cmax on Day 14, across both dose groups were in the range of 1.20 to 1.22 for IND, 1.20 to 1.33 for GLY and 1.03 to 1.16 for MF. Interpretation of Cmax data is challenging for inhaled drugs with rapid absorption in the lungs. For example, GLY reaches peak plasma levels at 5 min post-dose, at the first sampling point post-inhalation. This reflects the instantaneous absorption of GLY in the lungs. Due to the rapid absorption, small differences in the actual sampling time can have a large impact on the apparent Cmax. The planned assessment window for the 5 min post-dose sampling time point was ± 3 min. While there were no deviations from the sampling time noted during the study, a narrower assessment window would have potentially led to lesser variability in MF Cmax than that observed in the current study  of steady-state C® device was evaluated in Japanese and Caucasian subjects [The results of this study are consistent with the finding for IND and MF exposure reported in a previous ethnic sensitivity study where the fixed-dose combination of IND and MF (QMF149 150/80 μg or 150/320 μg) administered via the Breezhalersubjects . IND andsubjects  whereas Single and multiple doses of IND/GLY/MF medium and high dose were safe and well tolerated in Japanese and Caucasian healthy subjects. Drug-related AEs or SAEs were not reported in the study.max for inhaled drugs and the available safety data at doses higher than proposed doses of IND [Overall, no clinically relevant ethnic effects on the systemic exposure to IND, GLY and MF was observed after multiple-dose administration of IND/GLY/MF in healthy subjects. The observed differences were small in magnitude and were not considered clinically relevant in view of the observed exposure variability, challenges with characterizing Cs of IND , GLY [26s of IND  as part s of IND , while ts of IND . Overalls of IND , 30, GLYs of IND  or the Is of IND , 33 in Cs of IND  or a muls of IND . Overall® inhalation device, the Cmax and AUC values were about two to threefold higher for Japanese patients with asthma compared with Caucasian patients with asthma [max between ethnicities.This study was conducted as a parallel group evaluation in Japanese and Caucasian subjects in a single center with demographically matched subjects from both ethnic groups. Assessment of PK in both ethnic groups at the same site within the same study avoids inter-study or inter-center variability, which may contribute to an apparent difference in PK of inhaled compounds due to potential site-specific differences in inhalation technique used by study volunteers. In prior studies for MF delivered with the Twisthalerh asthma . This dimax and AUC0–24 h of GLY on Day 14 following administration of GLY alone in asthma patients (Caucasian) were 166 pg/mL and 464 h × pg/mL, respectively. Mean Cmax and AUC0–24 h of IND on Day 14 following administration of IND alone or as part of the IND/MF fixed-dose combination were 307–317 pg/mL and 1910–1950 h × pg/mL, respectively, in healthy subjects. However, acknowledging limitations of cross-study comparisons, overall the observed exposure levels are consistent with those observed in previous PK studies.The mean total and peak exposures of IND and GLY observed in this study were higher than noted in previous studies . Mean Cmmax for IND, GLY and MF across both dose groups was 15 min, 5 min and 1–2 h, respectively. Slightly higher exposure of each individual drug in Japanese subjects as compared with Caucasian subjects was observed following single or multiple doses of IND/GLY/MF medium or high dose but is not considered clinically relevant. For MF, Cmax and AUC0–24 h increased in a dose-proportional manner between IND/GLY/MF medium and high dose on both Day 1 and Day 14 for both ethnicities. Single and multiple doses of IND/GLY/MF medium and high dose were safe and well tolerated in Japanese and Caucasian healthy subjects.IND, GLY and MF were systemically available shortly after the inhalation of single or multiple doses of IND/GLY/MF at the medium and high ICS dose strengths in both Japanese and Caucasian subjects. Median plasma TAdditional file 1. Analytical parameters and performances of the three methods used for IND, GLY and MF.


Anomalocalyx uleanus (Pax & K. Hoffm.) Ducke (Euphorbiaceae) is a singular species in the genus and is restricted and exclusive to the Brazilian Amazon. A phytochemical study of A. uleanus leaves was performed, yielding the isolation of five major compounds: catechin/epicatechin, afzelin, quercetin 3-O-α-L-rhamnopyranoside, and astilbin. The phytochemical compositions of the methanolic extracts of leaves, roots, bark, and stem bark were determined using a dereplication approach. Forty-six compounds were annotated from the liquid chromatography-mass spectrometry (LC-MS/MS) data, while four lipids were identified using gas chromatography-mass spectrometry (GC-MS). In total, fifty compounds were detected, and they belonged to the primary metabolism and several classes of natural products such as flavonoids, flavonoids O-glycosides, flavonoids C-glycosides, biflavonoids, procyanidin, triterpene, triterpenes esterified with phenylpropanoids, phenylpropanoid derivatives, flavonolignans, coumarins, quinic acid derivatives, and benzoic acid derivatives. This is the first report on the phytochemical data of the genus Anomalocalyx, and the results of this study will contribute to the chemosystematic knowledge of the Euphorbiaceae family and justify the need for investigation of the pharmacological potential of the species A. uleanus. The Euphorbiaceae family comprises 219 genera and 6300 species, which are distributed in tropical and subtropical regions, and many of them are endemic to Brazil ,5.Senefelderopsis  . 1H-NMR : δ 6.84 , δ 6.76 , δ 6.72 , δ 5.85 , 5.92 , δ 4.56 , δ 3.97 , δ 2.84 , δ 2.50 . These data are consistent with the catechin structure reported in the literature [Brownish yellow amorphous powder (1.4 mg). QTOF-MS/MS spectrum (negative ionization mode) displayed a molecular ion peak at terature .m/z 289.0720 [M − H]− . 1H-NMR : δ 5.94  and 5.91 , δ 6.97 , δ 6.76 , δ 6.80 , δ 4.82 , δ 4.18 , δ 2.86 , 2.73 . These data are consistent with the epicatechin structure reported in the literature [Brownish yellow amorphous powder (1.1 mg). QTOF-MS/MS spectrum (negative ionization mode) displayed a precursor ion peak at terature . m/z 431.0977 [M − H]− . 1H-NMR : δ 7.77 , δ 6.38 , δ 6.21 , δ 6.94 , δ 5.38 , δ 4.22 , δ 3.71 , δ 3.48 , δ 3.33 , δ 0.92 . These data are consistent with the afzelin structure reported in the literature [Yellow powder (2.8 mg). QTOF-MS/MS spectrum (negative ionization mode) displayed a precursor ion peak at m/z 447.0926 [M − H]− . 1H-NMR : δ 7.34 , δ 7.31 , δ 6.91 , 6.37 , δ 6.20 , δ 5.35 , δ 4.22 , δ 3.42 , δ 3.75 , 3.33 , δ 0.94 . These data are consistent with the quercetin structure reported in the literature [Yellow powder (1.1 mg). QTOF-MS/MS spectrum (negative ionization mode) displayed a precursor ion peak at terature .m/z 449.1083 [M − H]− . 1H-NMR : δ 6.96 , δ 6.82 , δ 5.92 , δ 5.90 , δ 5.07 , δ 4.58 , δ 4.25 , δ 4.04 , δ 3.66 , 3.54 , δ 3.33 , δ 1.19 . These data are consistent with the astilbin structure reported in the literature [Yellowish-green amorphous powder (7.4 mg). QTOF-MS/MS spectrum (negative ionization mode) displayed a molecular ion peak at terature .1 and MS2 analyses were performed on an Agilent 6545 Q-TOF mass spectrometer  equipped with an electrospray ionization (ESI) source. MassHunter® workstation software (version B.08.00) was used for data acquisition and processing. The samples were analyzed in triplicate of authentic biological replicates. The chromatographic separation was carried out on an Agilent Zorbax SB-C18 column (3.0 × 50 mm) and water–acetonitrile with 0.1% formic acid (LC-MS grade). The mobile phase flowed at a rate of 0.3 mL/min after injecting 5 µL of the analytical solutions. The mass data were acquired with a positive (+) and negative (−) ion ESI source in the TOF-MS mode for the molecular ions and in the auto-MS/MS mode for the fragment ions using collision energy  obtained in the TOF-MS mode were identified. MSn energy . The ope example . The acq2 spectra and peak alignment was performed using the MS-Dial software . Then, the processed, deconvolved, and aligned MS2 spectra were extracted and submitted for processing in the MS-FINDER software in order to achieve a greater number of databases aimed at the annotation of the secondary metabolites Aiming to process at MS-DIAL, MS1 at ±0.02 Da and MS2 at ±0.06 Da mass tolerance, minimum peak height of 1 × 104, MS/MS amplitude abundance cut-off at 30, retention time tolerance of ±0.5 min, and MS1 tolerance of ±0.02 Da were used to align the chromatogram. Thus, the processed data were exported to the .mgf format postprocessing and subjected to a feature-based molecular networking (FBMN) analysis at the global natural product social molecular networking (GNPS) platform (https://gnps.ucsd.edu/). The confidence annotated metabolites are presented in this site: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=deca49b0f49d4af087be4e852e9a4b91). The MN was processed using Cytoscape version 3.8.0 software (https://cytoscape.org/) to construct the molecular network. Parent ions for all the extracts were different. Besides, 5 fragment ions were used to construct the molecular network, and 4 fragment ions were used for library annotation.The molecular networking visualization (MN) was constructed using each MS2 spectrum as a node, and the connection between nodes were made by the mass difference from the precursor ion (edges); then, each individual molecular networking has a similar fragmentation profile with a minimum fragment-ion characteristic. Further, Trichilia catigua, brand “Chá do Brasil”®) was used to prepare the extracts following the protocol described by Beltrame et al. (2006) [A. uleanus extract and catuaba standard extract containing cinchonain Ib were subjected to the same chromatographic conditions, ionization energy, and collision energy. The chromatographic separation was performed on a reverse column using the Agilent 1200 system. The column temperature and injection volume were 33 °C and 3 µL, respectively. The proportion of solvent B (acetonitrile) was linearly varied as follows: 0–4 min, 5–15%; 4–15 min, 15–60%; 15–24 min, 60–100%; 24–27 min, 100% (isocratic elution); 27–27.50 min, 100–5%; and 27.50–30 min, 5% (isocratic elution). The ESI source conditions were supported by the mass ranges 200–1500 Da to MS1 and 70–1500 Da to MS2 data. The other parameters were as follows: capillary voltage, 2.5 kV; nozzle voltage, 0 (zero); fragmentor voltage, 125 V; skimmer voltage, 65 V; octupole RF peak, 750 V; gas temperature, 300 °C; gas flow, 12 L/min; nebulizer pressure, 35 psi; sheath gas temp, 350 °C; and sheath gas flow, 10 L/min. MS2 spectra acquisition was performed under the following collision energies: 100–300 Da (30–35 eV), 300–500 Da (35–40 eV), 500–700 Da (40–45 eV), 70–1000 Da (45–50 eV), and 100–1500 Da (50–60 eV).A commercial sample of catuaba tea . This derivatized form of sample 9D was analyzed on a GC-MS chromatograph , using a DB-5 MS column (30 m × 0.25 mm × 0.25 µm). The chromatography was conducted in the split injection mode, with a column oven temperature of 100 °C and injection temperature of 225 °C. Column flow rate was 1 mL/min, equilibration time was 3.0 min, and helium was used as the carrier gas. The results were compared with the NIST library database, and it was possible to identify four esters (47–50) [Sample 9D (58 mg), obtained from the hexane fraction of  (47–50) .A. uleanus was investigated using both phytochemical and dereplication approaches. Five major compounds were isolated from the leaf extracts and identified using NMR and MS: catechin/epicatechin (5a/5b), afzelin (12), quercetin 3-O-α-L-rhamnopyranoside (15), and astilbin (16). All the compounds were used as reference standards  and were important in confirming the accuracy of the MS/MS data in the dereplication study. The chemical composition of Anomalocalyx. It was the first time that the chemistry of the genus was described in the literature. Dereplication was the main investigative process adopted to avoid the re-isolation of the compounds already described, revealing the metabolic pathways present in A. uleanus. The use of the molecular networking approach is extremely important and powerful as a strategy for analyzing clusters of the main compounds. It was possible to establish a structural relationship between these known substances and other structures present in the cluster by analyzing the fragmentation pattern. Forty-six compounds were annotated from the LC-MS/MS data and four from GC-MS, totaling fifty compounds. These compounds belong to the primary metabolism and several classes of natural products, such as flavonoids, flavonoids O-glycosides, flavonoids C-glycosides, biflavonoids, procyanidin, triterpene, triterpenes esterified with phenylpropanoids, phenylpropanoid derivatives, flavonolignans, coumarins, quinic acid derivatives, and benzoic acid derivatives. UPLC-Q-TOF-MS/MS and GC-MS analyses provided abundant information for the genus 33). This compound was obtained from Trichilia catigua bark extract, and it was used as a standard to confirm its presence and derivatives in A. uleanus barks by comparing MS spectral data. This is the first time that cinchonain Ib (33) and derivatives were detected in a species of the Euphorbiaceae family.One of the compounds annotated in the dereplication study was flavonolignan cinchonain Ib (Anomalocalyx will contribute greatly to the chemosystemic and chemophenetic knowledge of the genus and suggest the need for its pharmacological potential, which has never been investigated, to be investigated.All the information obtained about the metabolism of the genus