To identify site-specific features of amino acid substitutions that confer enhanced H7N9 virulence in humans, we independently generated mammalian-adapted variants of A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9) by serial passaging in Madin-Darby canine kidney (MDCK) cells.Virus was respectively extracted from cell culture supernatant and cells, and was absolutely quantified by using real-time polymerase chain reaction. Viral RNAs were extracted and subjected to sequencing for identifying mutations. Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids, which were used to generate recombinant viruses. The interaction between the recombinant HA and receptors, H7N9-pseudotyped viruses and receptors were detected.Both subtypes displayed high variability in replicative capability and virulence during serial passaging. Analysis of viral genomes revealed multiple amino acid mutations in the hemagglutinin 7 (HA7) , and NA9 (N171S [AH-H7N9] and G335S [AH-H7N9]) proteins in various strains of the corresponding subtypes. Notably, quite a few amino acid substitutions indeed collectively strengthened the interactions between H7N9 strains and sialic acid receptors. Moreover, some of the amino acid substitutions identified were highly and specifically cytopathogenic to MDCK cells.This study demonstrated that AH-H7N9 and SH-H7N9 subtypes can acquire enhanced receptor affinity for sialic receptors through novel amino acid substitutions. Such changes in affinitive interactions are conferred by site-specific mutations of HA7 proteins that affect the virulence and pathology of the virus strain, and/or limited compatibility between the host and the virus strain. Avian and poultry species, particularly wild ducks, swans, gulls, terns and others, are natural hosts for most avian influenza type A virus (IAV). Hemagglutinin (HA) and neuraminidase (NA) are two surface glycoproteins of IAV, which helps the viral genome enter the host cytoplasm and assists in release of newly formed viral progeny from host cells. In current, 18 different subtypes of HA (H1 to H18) and 11 subtypes of NA (N1-N11) have been identified . AccordiHA protein of H7N9 is a dual-specificity ligand for both human- and avian-type receptors. H7N9 virus strains containing HA mutations in the cleavage site potentially show increased pathogenicity and lethality. For instance, HA Q226L –21 and GIn our study, we developed MDCK cell-infection and sequencing assay, aimed to identity novel amino acid substitutions in HA contribute to virulence enhancement through binding with SA receptors in H7N9 virus strains.50) for each virus strain. Madin-Darby canine kidney (MDCK) cells were selected as the infection model for the generation of each virus strain. Briefly, 8 × 105 MDCK cells were seeded in a 25 cm-flask containing DMEM with 10% FBS, 100 μL/mL penicillin and 100 μL/mL streptomycin (pH 7.2–7.4). MDCK cells were cultured at 37 °C in a humid environment containing 5% CO2 until 70% confluence, and washed with TPCK-trypsin (2 μg/mL)-containing DMEM for 3 times. Then, 10 μL of allantoic fluid containing the P0 strain was inoculated and cultured with MDCK cells as described above for 2 h. Afterweards, the medium was discarded, and TPCK-trypsin -containing DMEM was added in cells, which were cultured for another 48 h. Finally, the cytopathic effect (CPE) of the virus was observed under an inverted microscope. Next, the cultures were centrifuged for 10 min and supernatants as well as cells were collected. Absolute quantification was performed for virus in titers in supernatants and cells. Determination of 50% tissue culture infective dose (TCID50) and whole-genome sequencing were performed for supernatants and cell viruses, respectively. During long-term serial passaging in the host, viruses from the supernatants were collected, and inoculation was performed as described above, with a constant inoculation amount of 8 × 104 copies. Routinely, cells were assessed and passaged every 48 h. Viruses in the culture supernatants were passaged repeatedly till the sixth generation in MDCK cells, suggesting the generation of mutant viruses  + distance ratio × log (dilution coefficient).All experiments were strictly performed following the standard procedures of Biological Safety Level 1–3 Laboratory. Two H7N9 virus strains, namely A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9), were provided by the virus resource database, and were isolated from chicken embryos at the Chinese Center for Disease Control and Prevention (China CDC). Following treatment of chick embryos from embryonic (E) day E9 to E10 with these viruses, chick embryos were incubated at 35 °C for 72 h, and placed at 4 °C overnight. The first transfer generations of AH-H7N9 and SH-H7N9 (P0 strain) were collected from allantoic fluid components in chick embryos. Additionally, simultaneous measurement of infectious virus titer was carried out by 50% tissue culture infective dose  was performed with H7 labeled by FAM™ dye on an Applied Biosystems 7500 Fast Dx Real-Time PCR instrument at the conditions of 10 min at 45 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 45 s at 60 °C. Absolute amounts of viral RNA in were determined by the 7500 software v2.0 according to the HA standard curve.Primers for overlapping PCR were designed, and further used for amplification reactions with the Qiagen one step RT-PCR kit. The standard PCR procedure was as follows: 30 min at 50 °C, 15 min at 95 °C, 40 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C and 10 min at 72 °C, respectively. PCR products were collected, and all gene segments from each virus passage replicated in embryonated eggs were amplified by high fidelity PCR using QIAGEN One Step RT-PCR Kit in the supernatants of homogenized cells and the cell supernatants. The expression vector, pGEM-T Easy Vector , was designed for the convenient cloning of the purified PCR-amplified RNAs. Five positive clones for each gene segment from individual virus subtype were randomly selected for sequencing . Assembly and analysis of the sequences was performed with DNASTAR-Lasergene’s SeqMan software and NCBI-Blast.Gene sequences of the HA7 and NA9 proteins isolated from AH-H7N9 or SH-H7N9 were optimized according to host cell characteristics, and sub-cloned into the pCAGGS plasmid (Genewiz). Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids using the Quickchange Lighting Site-Directed Mutagenesis kit. Briefly, primers containing site-specific mutations were synthesized. The standard PCR procedure was as follows: 2 min at 95 °C, 18 cycles of 20 s at 95 °C, 10 s at 60 °C, 8 min at 68 °C, and 10 min at 68 °C. DH5α competent ™ cells were transfected with these PCR products, by adding 8 μL PCR product into 50 μL DH5α cells, keeping the mixture on ice for 30 min, followed by heat-shock at 42 °C for 90 s and immediate placement on ice for 2 min. Transfected cells were added into 400 μL LB media and cultured in a 37 °C shaker for 45–60 min. Then, 50–100 μL cultured cells were transferred into ampicillin (100 μg/mL)-containing LB plates overnight at 37 °C, and single clones were picked for gene sequencing. Colonies with site-specific mutation accuracy were further inoculated after a 1000-fold dilution onto ampicillin-containing LB plates and cultured overnight in a 37 °C shaker. Plasmid recovery was performed using Qiagen Endo free Plasmid Maxi Kit according to the instructions provided by the manufacturer.4 /well) for 2 h at 37 °C in an environment containing 5% CO2. Then, the medium was replaced with 10% FBS supplemented DMEM (2 μg/ml TPCK-trypsin) for another 48 h of culture. Luciferase activity was measured with luciferase assay regents , NA activity was determined with a NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set (Applied Biosystems) and a Luminescence Counter (Promega) according to the manufacturer’s instructions.First, plasmids expressing the HA7 or NA9 proteins were transfected into 293 T cells, respectively. Expression levels of the former protein were detected by Western blot, and the activity of the latter protein was determined with NA Activity Kit. Then, the HA7, NA9 and pNL-4.3-Luc-R-E plasmids were co-transfected into 293 T cells, thereby constructing a pseudo-type influenza virus. Briefly, 293 T cells were inoculated in 6-well plates, allowing rapidly spreading viral infection in cultured cells until 70% confluence. The cells were then washed with PBS, and added to a culture medium with low-serum Opti-MEM (Gibco). Virus packaging was performed with Lipofectamine™ 2000 Transfection Reagent Kit (Life Technologies) according to the manufacturer’s instructions. Briefly, 100 μL solution A was added to 500 ng of each pCAGGS-HA7 and pCAGGS-NA9 plasmids, and 1500 ng of pNL-4.3-Luc-R-E, with 3.5 μL of vectors comprising a strong viral promoter/enhancer sequence. Solution A was rotated for 1 s, and kept at room temperature for 2–5 min. Then, solution B was prepared by adding 10 μL effective transfection reagent into 100 μL plasmid containing the enhancer sequence, and rotated for 10 s. Next, solutions A and B were mixed and kept for 5–10 min, added to 600 μL of Opti-MEM medium. The mixture was slowly and carefully added into 6- well plates containing cultured 293 T cells, after discarding the medium. After 6 h of incubation, the supernatant was aspirated, fresh Opti-MEM containing TPCK-trypsin (Sigma) at 2 μg/mL was added. Supernatants containing the H7N9-pseudovirus were collected after 48 h of culture, filtered with a 0.45-μm filter (Millipore) and stored at − 80 °C. Since the pNL-4.3-Luc-R-E plasmid contained the luciferase gene as a marker, the activity of the packaged virus was determined with the Firefly Luciferase Reporter Gene Assay kit. Briefly, all H7N9 pseudoviruses were respectively incubated with MDCK cells cultured in serum free DMEM containing TPCK-trypsin (2 μg/mL) in 96 well plates  at 4 °C overnight. The membrane was then incubated with anti-mouse IR dye 800-labeled IgG secondary antibody , measured at a recommended wavelength, and analyzed by the Odyssey software.Cultured cells were washed with PBS twice, lysed with lysis buffer , centrifuged at 15,0002O2 was then added; the chromogenic reaction was terminated by addition of H2SO4. The amounts of the HA protein were measured at 450/650 nm, using a standard curve.HA quantification was first performed following the instructions provided by the manufacturer. Recombinant HAs were coated onto a polystyrene plate at 4 °C overnight. The AH-H7N9 hemagglutinin/HA protein  was used as the standard and serially diluted. The plate was then blocked with 5% bovine serum albumin (BSA) (Sigma) at 37 °C for 2 h. After washing, mouse anti-HA mAbs (1:1000) were added, and the plate was incubated at 37 °C for another hour. The plate was incubated with HRP-labeled secondary anti-mouse IgG (1:5000) (Sigma) at 37 °C for another hour following washing. OPD/HA solid-phase binding assay with competing glycopolymers was performed for determining receptor affinity as described previously. Serially diluted HA proteins (threefold) from 200 μg/mL (3.4 μM) were allowed to incubate overnight at 4 °C in 96-well plates. BSA (5%) was used to block the plates for 2 h at 37 °C. Then,   or   was added to the wells at 200 ng/mL for 1 h at 37 °C, respectively. Horseradish peroxidase (HRP)-conjugated avidin  was added to wells for visual detection of the proteins by a chromogenic reaction. The extent and accuracy of binding of the recombinant HA by a given polysaccharide receptor was determined by absorbance at 450/650 nm. Data were analyzed by the GraphPad Prism 5.0 software.2O2/DAB was added, and the reaction was terminated 15 min later. Virus quantification was carried out by detecting OD at 450/650 nm. Then, p24 protein levels were adjusted to 200 ng/mL after dilution of the virus, which was coated to 96-well plates as described above. The 3′SL and 6′SL were twofold serially diluted from 200 ng/mL, allowing the binding detection of HRP-SA by DAB and OD measurement, as described above for the solid-phase binding assay between HA and glycol- polymers.Interaction detection between recombinant viruses and receptors was performed with the HIV-1 p24 Antigen ELISA kit , by assessing receptor-labeled 3′SL and 6′SL, according to the manufacturer’s instructions. Briefly, p24-antigen containing- and standard samples were added to p24 McAb-coated 96-well plates and incubated at 37 °C for 60 min. Following incubation, the plates were washed, and biotin-labeled p24 polyclonal antibody and HRP-labeled SA were added for further studies. After 5× washing, Hp < 0.05 was considered as statistical significant.The Student’s t-test was utilized to determine the statistical significance of differences between experimental groups. in-vitro Real-Time PCR . Compared with the reference A/Shanghai/2/2013 (H7N9) strain, mutations occurred in the Anhui/1 virus strain at the sites of the M26I, S171N and G335N of NA proteins, A135T in HA, and A373T of NP predicted biological significance. Meanwhile, mutations occurred in Shanghai/02 at the following sites of the T40I, A107T, A135T, T157I, G218E, V223I, and A225K in HA genes, M86V, Y161A in PA, A284T in NP, N72I in NA, and D101N in NS primarily predicted the sites of interest. According to the above the results, the HA and NA gene mutations involved in receptor-binding properties of the virus mainly contributed to virulence alteration during serial passaging. The 3D structures of interactions between the HA protein with amino acid substitution and receptors were analyzed by Flusurver (https://flusurver.bii.a-star.edu.sg/INTERACTIONS/)  HA7A135T-pCAGGS and (SH-H7N9) HA7T157I-pCAGGS displayed relatively higher expression levels compared with average expression levels of (SH-H7N9) HA7T71I-pCAGGS, (SH-H7N9) HA7T71I-V223I-pCAGGS and (SH-H7N9) HA7T71I-T157I-V223I-pCAGGS, as well as (SH-H7N9) HA7T71I-T157I-V223I-T40I-pCAGGS, which showed lowest expression levels. Moreover, the NA variants, pNL-4.3-Luc-R-E vector and blank load transfections showed no detectable HA protein expression  HA7Anhui/1 & Shanghai/2 HA7 = 0.635 ± 0.188 μM to the “avian-type” receptor 3′SL and KD for 6′SLAnhui/1 & Shanghai/2 HA7 = 0.753 ± 0.197 μM to the “human-type” receptor 6′SL). Surprisingly, all HA mutations retained the ability to bind the “avian-type” receptor analog, of which HAA135T had the lowest affinity (KD for 3′SLHA7A135T = 5.553 ± 1.232 μM), taking into account average levels of affinitive interactions of other variants to 3′SL , as well as relatively low affinity for HA7T71I (KD for 6′SL HA7T71I-V223I = 0.388 ± 0.102 μM), in comparison with average affinity for other variants . The results showed that mean values in the AH-H7N9 , SH-H7N9 (HA7: T71I-V223I), SH-H7N9 (HA7: T71I-T157I-V223I), SH-H7N9 (HA7: T71I-T157I-V223I-T40I), and SH-H7N9 (HA7: T71I) virus strains ranged between 6 and 8. Among them, SH-H7N9 (HA7: T71I-V223I) was the only virus strain with a mean value of Log10RLU higher than 7. Moreover, mean values in the AH-H7N9 (HA7: A135T), SH-H7N9 (HA7: T157I), SH-H7N9 wild type, and H5N1 wild type virus strains ranged between 3 and 6. The remaining variants showed no significant differences compared with the negative control , a solid-phase binding assay with competing glycopolymers was performed for measuring receptor affinity Fig. b. We perFurthermore, we also tested the interaction between the major analog of the human receptor (6′SL) and the various H7N9 recombinant strains. In striking contrast, SH-H7N9 (HA7: T157I) became the strain with apparently higher affinitive binding associated with interaction between HA7 and 6′SL, in particular for higher concentrations (> 100 nM) of the ligands. SH-H7N9 (HA7: T71I-V223I) and SH-H7N9 (HA7: T71I-T157I-V223I-T40I) also could be effectively and stably bound. Still, AH-H7N9 , SH-H7N9 (HA7: T71I- T157I-V223I) and H5N1 showed apparently reduced affinity to 6′SL, in particular with the lowest affinity for H5N1, compared with other recombinant strains.In this study, we developed an MDCK cell-infection assay that allowed the screening of unknown amino acid substitutions that potentially have functional alterations, especially in affinitive binding with SA receptors during serial passaging of both AH-H7N9 and SH-H7N9 subtypes. Sequencing confirmed that we indeed generated several new mutations Table , either During serial passaging, the Anhui/1-H7N9 and Shanghai/2-H7N9 strains isolated from sputum of patients with H7N9 virus infection were first cultured in egg embryos equivalent to the second-generation embryonic virus strains (E2), named P0. Then, they were transfected into MDCK (Madin-Darby canine kidney) and A549 (human pulmonary carcinoma) cells, respectively. Indeed, Anhui/1-H7N9 and Shanghai/2-H7N9 strains are transmitted by traces, from poultry to humans (patients infected with H7N9 virus), to egg embryos (similar to poultry), then returned to animals (dogs) and humans (A549), resulting in adaptive gene mutations frequently occurring during growth in diverse species recycled between avians, poultry and humans. Interestingly, analysis of host adaptation and resistance mutations revealed that even different H7N9 subtypes had markedly different surviving curves during serial passaging, when infecting MDCK cells, but not A549 cells. it is well known that the five rounds of H7N9 outbreak occurr from 2013 to 2017  and PB2 (I292V) sites between Anhui/1-H7N9 (p0) and Shanghai/2-H7N9 (p0) strains. P1-P6 gene Flusurver website analysis showed that four of eleven meaningful amino acid mutations were of interest level (1–3), including HA , NA  and NP (373 T) within Anhui/1-H7N9 serial passaging strains. HA (A149T) may create a potential glycosylation site; NP (NP373T) is known to cause antigenic shifts or mild drug resistance. While a preliminary analysis of seventeen sense mutations revealed twelve site-related functions involved in HA , NA (N73I), PA (M86V and Y161A), NP (A284T) and NS1 (D101N) within Shanghai02-H7N9 strains. According to the above results, HA and NA gene mutations involved in receptor-binding properties of the virus mainly contribute to virulence alteration during serial passaging.4. Interestingly, some mutations of the HA 7 protein found above, i.e., T40I, T79I, T157I and V223I, changed into a hydrophobic residue (isoleucine), indicating that they acquire a higher ability to bind the human receptor. The HA T48I mutation removes a potential N-glycosylation site at position 46, which may also affect antigenic and other properties of this strain. In detail, the motif at positions 46–48 changed from NAT (glyco) to NAI (no glyco). On the contrary, HA A143T mutation, which had higher affinity to 6′SL, created a new potential N-glycosylation site at position 141 which may also affect antigenic and other properties of this strain. In detail, the motif at positions 141–143 changed from NGA (no glyco) to NGT (glyco). As observed in resolved structures of proteins from related strains, the HA position equivalent to this mutation is involved in binding of small ligand(s), antibody recognition sites, or viral oligomerization interfaces  and loops 130/150/220 aa) based on H3 numbering. For instance, S128N was detected within the loop (130 aa), which is proximal to the receptor surface (distance ≈ 20 Å) . Unexpec0/220 aa On the other hand, the indispensably important function of NA in H7N9 virus is to promote viral mobility toward/within the infected host through protecting virus particles from entrapment by mucin-rich secretions in the respiratory tract as well as through facilitation of viral egress from infected host cells. Thus, the typical N9 NA protein structure contains conserved residues of active sites that contact neuraminic acid as well as residues that provide basic requirements for catalysis, including calcium ion-binding sites, by interacting with both water molecules and backbone carbonyl oxygen atoms at specific sites (293/297/324/347 aa) based on N2 numbering . In thisApart from mutations occurring in the HA and NA genes, Shanghai/2 passaging strains also had some mutations of interest, including PA , NP A284T) and NS1 (D101N). The SH-NS1 D101N and E208K mutations are not found in sequences used to derive mutation statistics. The NS1 D101N position mutated except for NS1-E208K is involved in binding of small ligand(s) or host protein(s), or viral oligomerization interfaces, as found in resolved structures of proteins from related strains. The PA M86V and PA Y161A mutations have been reported to be involved in viral oligomerization interfaces, as shown in Additional file T and NS1Nevertheless, Shanghai/2-H7N9 strains PB1 , PB2 (I185S), NP (M352I) and NS (E208K), and AH-H7N9 strains PA , PB1 (D76N and V451E), PB2 (D87G) are not found in sequences used to derive mutation statistics, thus requiring further investigation of the underlying mechanisms by reverse genetics in H7N9 viruses.In this study, we identified quite a few human-adapted variants of AH-H7N9 and SH-H7N9 by serial passaging in MDCK cells. Our results revealed that some of these variants displayed higher variability in replicative capability in both subtypes with multiple amino acid mutations in the H7 and N9 proteins, which was further demonstrated by enhanced receptor affinity to 3′SL/6′SL and NA activity, as well as increased virulence in those strains.Additional file 1: Figure 1. Long-term serial passaging of virus strains.Additional file 2: Figure 2. Absolute RNA amounts in viruses from supernatants or cells, quantified by quantitative PCR for the first- or second-generation of viruses in human lung cancer A549 cells..Additional file 3: Figure 3. Illustration of the five rounds of H7N9 outbreak that occurred in China from 2013 to 2017Additional file 4: Figure 4. Crystal structure of representative PA, NP and NA. (A) Shanghai2 PA M86V mutation position (red atoms) on viral chain B (yellow backbone), within 5 A from oligomeric subunit chain A (blue backbone); (B) Shanghai2 PA Y161A mutation position (red atoms) on viral chain C (yellow backbone), within 5 A from oligomeric subunit chain A (blue backbone). (C) Anhui/2 NP A373T mutation position (red atoms) corresponds to position 8 on viral chain F (yellow backbone); (D) Wild Anhui/1 NA-M26I (compared with wild Shanghai2) mutation position (red atoms) corresponds to position 84 on viral chain A (yellow backbone), within 5 A from ligand NAG (pink atoms).


In-cell cytoblot was used to assess levels of proteins; while DNA binding and reporter gene assays were used to measure transcription factor DNA binding and transcriptional activities, respectively. Glucose uptake in adipocytes was evaluated with 2‐deoxy‐2‐[ amino]‐d‐glucose uptake. MTC (5–20 µM) suppressed LPS + IFNγ-induced release of TNFα, IL-6 and IL-1β, as well as NO/iNOS and PGE2/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-IκB and phospho-p65 proteins, accompanied by a reduction in total IκB in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-κB DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5–20 µM) resulted in enhanced DNA binding of Nrf2 and an increase in ARE-luciferase activity. In a macrophage–adipocyte co-culture, the compound reduced the release of TNFα, IL-6, IL-1β, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPKα. Our results suggest that MTC produced anti-inflammatory and antioxidant activities in macrophages. MTC also prevented inflammation in macrophage–adipocyte co-culture. The effect of MTC on glucose uptake in adipocytes is proposed to be linked to activation of AMPK.Methyl 3,4,5-trimethoxycinnamate (MTC) is a bioactive natural phenylpropanoid. We evaluated anti-inflammatory effects of synthetic MTC in RAW264.7 macrophages and RAW264.7–3T3-L1 adipocytes co-culture. Levels of cytokines and chemokines, as well as NO and PGEThe online version of this article (10.1007/s10787-020-00720-8) contains supplementary material, which is available to authorized users. Chronic inflammation still remains a fundamental hallmark of a wide variety of diseases, ranging from arthritis, asthma, inflammatory bowel disease, neurodegenerative disorders, cardiovascular diseases, cancer and metabolic disorders. Consequently, chronic inflammation and inflammatory signalling pathways remain prime targets in identifying new treatments for these inflammatory disorders.Transcription factors such as the nuclear Factor kappa B (NF-κB) and nuclear factor erythroid 2-related factor (Nrf2) play critical roles in inflammation. NF-κB is responsible for the transcriptional control of pro-inflammatory genes, and plays a central role in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmunity, as well as diseases comprising a significant inflammatory component such as cancer and atherosclerosis -induced nitric oxide production in BV-2 microglia  NaHCO3 (twice) and water, dried over anhydrous Na2SO4 and filtered. After removal of ethyl acetate under vacuum, the methyl 3,4,5-trimethoxycinnamate was obtained.MTC was synthesised as earlier described  were cultured in Dulbecco’s modified eagle’s medium (DMEM), supplemented with 2-mM glutamine and 10% foetal bovine serum (FBS). Cell viability experiments were carried out to determine whether incubation of RAW264.7 macrophages with concentrations of MTC used in combination with LPS (1 µg/mL) and IFNγ (10 ng/mL) would produce cytotoxic effects (Supplementary data 1).® CL-173™) mouse pre-adipocytes were cultured in DMEM and supplemented with 2-mM glutamine and 10% FBS. All cells were cultured in an incubator at 37 °C and 5% CO2.3T3-L1 (ATCC2 production was measured using PGE2 EIA assay kit (Arbor Assays), according to the manufacturer’s instructions.Cultured RAW264.7 macrophages were treated with MTC (5–20 µM). Thirty minutes later, the cells were stimulated with LPS (1 µg/mL) and IFNγ (10 ng/mL) for a further 24 h. Culture supernatants were analysed for levels of NO using the Griess assay kit (Promega), according to the manufacturer’s instructions. Levels of pro-inflammatory  and anti-inflammatory (IL-10) cytokines in culture supernatants were measured using mouse ELISA kits (Biolegend), according to the manufacturer’s instructions. PGE5 cells per well) in Opti-MEM (Gibco) and incubated at 37 °C for 4 h. Cells were then transfected with either the pNL3.2.NF-κB-RE [NlucP/NF-κB-RE/Hygro] vector  or the pGL4.37[luc2P/ARE/Hygro] vector  using lipofectamine 2000 transfection reagent (Invitrogen) and incubated for a further 4 h. Thereafter, experiments were carried out to evaluate the effects of MTC (5–20 µM) on NF-κB and antioxidant response elements (ARE) luciferase activities, respectively. Luciferase activities were measured using Dual-Glo luciferase assay kit  according to the manufacturer’s instructions.RAW264.7 cells were seeded out in a 24-well plate  for 30 min, followed by stimulation with LPS (1 µg/mL) and IFNγ (10 ng/mL) for a further 24 h in experiments to determine DNA binding of NF-κB. The cells were also treated with MTC (5–20 µM) for 24 h to evaluate effects on binding of Nrf2 to ARE consensus sites. Nuclear extracts were collected and analysed for binding of transcription factors to the DNA using the TransAM NF-κB and Nrf2 transcription factor EMSA kits  according to the manufacturer’s instructions. The TransAM transcription factor assay kits employed a 96-well plate to which oligonucleotides containing the NF-κB consensus site (5′-GGGACTTTCC-3′) or the ARE consensus binding site (5′-GTCACAGTGACTCAGCAGAATCTG-3′) have been immobilised.4 cells/mL). At 70% confluence, cells were treated with MTC (5–20 μM) for 30 min, followed by stimulation with LPS (1 µg/mL) and IFNγ (10 ng/mL) for different incubation periods. Cells were fixed with 8% paraformaldehyde solution (100 μL) for 15 min. and then washed with PBS. The cells were then incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-COX-2 (Abcam), rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 , rabbit anti-phospho-IκB (Santa Cruz Biotechnology), rabbit anti-IκB (Santa Cruz Biotechnology), and rabbit anti-phospho-AMPKα (Santa Cruz Biotechnology). Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100-µL HRP substrate was added to the plate and signal measured at 450 nm with a microplate reader. Readings were normalised with Janus Green stain (Abcam).The in-cell western is now an established method for the rapid quantification of proteins in cells  containing 10% FBS supplemented with 0.5-mm 3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (1 µM) and insulin (200 nM). Adipocyte differentiation was assessed using the oil red O staining quantitative assay kit (Cayman). Culture supernatants were removed from cells, followed by washing with PBS. Thereafter, lipid droplets assay fixative was added to each well and incubated for 15 min at room temperature. Following removal of the fixative, cells were washed and 75-μL oil red O solution was added to each well for 20 min at room temperature. This was followed by removal of the oil red O and thorough washing of the wells with 100-μL wash solution and the addition of 100-μL dye extraction solution and absorbance read on a Tecan F50 microplate at 492 nm. Differentiation was quantitatively confirmed by comparing the absorbance of differentiated and undifferentiated cells (Supplementary data 2).5) were cultured in the lower well; while, 5 × 104 RAW264.7 macrophages were cultured in inserts that constituted the upper chamber. Following the establishment of co-culture, RAW264.7 cells in the upper chamber were treated with MTC (5–20 µM) for 30 min, followed by stimulation with LPS (1 µg/mL) and IFNγ (10 ng/mL) for a further 24 h. Culture supernatants were collected and analysed for levels of NO, TNFα, IL-1β, IL-6 using similar protocols described in experiments on RAW264.7 macrophage mono-culture. Production of monocyte chemoattractant protein-1 (MCP-1/CCL2) was measured using a mouse CCL2 ELISA kit (Invitrogen); while, levels of RANTES in culture supernatants were detected with a mouse CCL5/RANTES ELISA kit (R and D Systems).Following differentiation, 3T3-L1 adipocytes were co-cultured with RAW264.7 macrophages using the Transwell system . In the co-culture, differentiated 3T3-L1 cells (1 × 10d‐glucose (2‐NBDG) (100 µM) in glucose-free DMEM for a further 1 h after treatment. Thereafter, cells were washed with PBS and glucose uptake by fluorescence detection at an excitation/emission wavelength of 485 nm/535 nm.Glucose uptake was determined by incubating differentiated 3T3-L1 adipocytes in the lower chamber with 2‐deoxy‐2‐[ amino]‐t test.Values of all experiments were represented as a mean ± SEM of at least three experiments. Values were compared using one-way ANOVA followed by a post hoc Tukey test. In experiments to confirm differentiation of adipocytes, differences in oil red O staining between undifferentiated and differentiated adipocytes were determined using the Student’s 3) δH 7.59 , 6.73 , 6.33 , 3.87 , 3.79 . 13C NMR  δC 167.5, 153.5, 144.9, 140.1, 129.9, 117.1, 105.2, 61.1, 56.2, 51.8, IR ѵmax  3003, 1697, 2943, 2837, 1632, 1040, 1005, 1582, 1506, 851.Yield 93%; white solid; 1H NMR  and LPS (1 µg/mL) for 24 h. This resulted in ~ 80-fold increase in TNFα secretion into the culture supernatant in comparison with control cells. However, when cells were treated with 5-, 10- and 20-µM MTC, there was ~ 1.8-, 2.1- and 2.5-fold reduction in LPS + IFNγ-induced increase in TNFα production, respectively  reduction in the levels of IL-10 in RAW264.7 cells  increase in IL-10 levels was observed in culture supernatants.In other experiments, LPS + IFNγ stimulation caused a significant (p < 0.05) reduction in NO production. The results show that NO concentrations in cells stimulated with LPS + IFNγ alone was ~ 15.4 µM. This was reduced to ~ 9.1, ~ 9.0, and ~ 7.1 µM in cells pre-treated with 5, 10 and 20 µM of MTC, respectively  released in culture supernatants obtained from LPS + IFNγ-stimulated cells, and observed that pre-treatment with MTC (5–20 µM) resulted in significant  reduction in the expression of phospho-p65 protein at all the concentrations investigated  reduced  reduced by MTC (5–20 µM)  increase in luciferase activity  and concentration-dependent reduction in luciferase activity. Similarly, it was observed that LPS + IFNγ-induced DNA binding of NF-κB was significantly (p < 0.01) reduced in a concentration-dependent fashion  increase in ARE-luciferase activity, when compared with untreated control cells. Experiments to evaluate the effect of MTC on HO-1 protein showed that treatment of RAW264.7 cells with MTC (5–20 µM) caused a significant (p < 0.05) increase in protein levels of HO-1 in the cells  values when the cells were treated with 5-µM MTC. However, binding increased ~ 3.2- and ~ 5.2-fold with 10- and 20-µM concentrations of MTC, respectively  and concentration-dependent reduction in NO production. Similar observations were made following analyses of culture supernatants for levels of pro-inflammatory cytokines TNFα  elevation in the levels of RANTES in the culture supernatants, and these were significantly (p < 0.01) reduced following pre-treatment with MTC (5–20 μM)  and IFNγ (10 ng/mL), there was ~ 23-fold increase in the levels of MCP-1, in comparison with control cells. Supernatants from cells that were pre-treated with 5, 10 and 20 μM of MTC showed ~ 1.30-, ~ 1.42- and ~ 1.71-fold reduction in the concentrations of MCP-1, respectively  increase in 2‐NBDG uptake  for 24 h caused a marked increase in 2‐NBDG uptake by the cells, in comparison with untreated cells Fig. a. Howeveake Fig. a.Fig. 8Tp < 0.001) reduction in phospho-AMPKα protein in adipocytes after 24 h  and IFNγ (10 ng/mL) caused a significant  reversed.In Fig. 2 release through mechanisms involving inhibition of iNOS and COX-2 proteins, respectively. These effects demonstrate anti-inflammatory activity of MTC.Macrophage-mediated chronic inflammation is known to be a major contributor in many diseases, and several strategies are currently being deployed in preventing or blocking inflammation. Secondary metabolites from natural products have been extensively investigated as a potential source of new chemical scaffolds for new anti-inflammatory drugs. In this study, we have shown that a synthetic form of natural 3, 4, 5-trimethoxycinnamic acid, MTC reduced the release of pro-inflammatory cytokines TNFα, IL-6 and IL-1β in LPS + IFNγ-stimulated RAW264.7 cells, while increasing the production of the anti-inflammatory cytokine IL-10. We further observed that MTC treatment resulted in the reduction of both NO and PGEThe release of pro-inflammatory mediators from macrophages has been implicated in many inflammatory and auto-immune diseases. In rheumatoid arthritis, macrophages secrete potent cytokines and chemokines including TNF, IL-1, IL-6, IL-10, IL-12, IL18, IL-15, IL-10, GM-CSF, M-CSF and TGFβ which contribute to the chronic inflammation associated with the disease  and Polygala tenuifolia were reported to inhibit neuroinflammation in LPS-activated BV-2 microglia  phenylpropanoids activate Nrf2 in human keratinocytes. Our studies could not establish a link between the anti-inflammatory activity and activation of Nrf2/ARE mechanisms by MTC, thus warranting further investigations.Investigations by us and others have suggested that the anti-inflammatory action of some compounds could be due, at least in part, to their ability to activate the Nrf2 transcriptions factor Below is the link to the electronic supplementary material.


Qdtotal).To assess the removal performance of low-volume post-hemodiafiltration (HDF) with Japanese hemodiafilters and the removal performance with 20 % reduction in the total dialysate flow rate (Qb) of 250 mL/min and a total volume of substitution fluid (Vs) of 12 L/session(s) for 4 hrs using Fineflux-210Seco (FIX), ABH-21PA (ABH), and NVF-21H (NVF). We assessed removal efficiency of small molecular solutes, low-molecular-weight-proteins and the amount of albumin loss. Study 2: Post-HDF was performed at Vs of 12 L/s under G-1, Qdtotal of 500 and Qb of 250 mL/min; G-2, Qdtotal of 400 and Qb of 250 mL/min; and G-3, Qdtotal of 400 and Qb of 300 mL/min. Removal efficiency was compared and analyzed between these conditions.Subjects were 8 patients undergoing pre-HDF. Study 1: Post-HDF was performed at a blood flow rate  removal rate (RR) (%) were 81.2, 83.1 and 82.8, and the α1-MG RR were 37.4, 40.2 and 38.5, respectively. Study 2: The results in G-1, 2 and 3 are shown in order. The Kt/V and the RR of small solutes, were significantly higher in G-3. The β2-MG RR (%) were 81.2, 80.1 and 81.0, and the α1-MG RR were 37.4, 37.5 and 38.0, respectively.Study 1: The results using FIX, ABH and NVF are shown in order. The Kt/Qb of 250 mL/min with Japanese high-performance hemodiafilters exhibited favorable removal efficiency for all solutes. Even with 20 % reduction in Qdtotal, the removal performance was also favorable.Low-volume post-HDF performed at  Qb) has been reported to improve patient prognosis, and its effect has been reported to be enhanced with increased Vs , creatinine (MW: 113 Da), phosphorus (MW: 30.97 Da), ß2-microglobulin (MG) (MW: 11.8 kDa), and α1-MG (MW: 33 kDa); and the removal rate of prolactin (MW: 22 kDa). To measure the removal amount of each solute and the amount of Alb loss, spent dialysate was pooled at 2 L/h. At the end of a post-HDF session, the pooled fluid was thoroughly stirred, and then samples were collected.To assess removal performance, we measured the standardized dialysis dose . A Study 1 The respective results are presented in the order of FIX, ABH, and NVF. Kt/V values were 1.82 ± 0.29, 1.89 ± 0.32, and 1.83 ± 0.31. There were significant differences between FIX and ABH, and between ABH and NVF in Kt/V. The removal rates of urea (%) were 76.9 ± 4.8, 78.3 ± 4.9, and 77.3 ± 5.1, with a significant difference between ABH and NVF. Removal rates of ß2-MG (%) were 81.2 ± 2.6, 83.1 ± 4.3, and 82.8 ± 3.7. Removal rates of prolactin (%) were 80.2 ± 3.2, 78.0 ± 9.9 and 77.9 ± 7.3, and removal rates of α1-MG (%) were 37.4 ± 3.9, 40.2 ± 8.2 and 38.5 ± 7.0, respectively. Amounts of Alb loss (g/s) were 3.4 ± 0.7, 5.4 ± 2.1, and 5.4 ± 2.8, but no significant difference was observed. Ratios of α1-MG/Alb (mg/g) were 44.4 ± 9.3, 33.0 ± 9.7, and 36.9 ± 10.5. For 1 g of Alb loss, the removal amount of α1-MG was higher for FIX, but it did not significantly differ between the hemodiafilters  were 81.2 ± 2.6, 80.1 ± 4.2, and 81.0 ± 3.6; the rate in G-1 was significantly higher than that in G-2 (p < 0.05). Removal rates of prolactin (%) were 80.2 ± 3.2, 79.6 ± 5.1, and 80.5 ± 5.0. Removal rates of α1-MG (%) were 37.4 ± 3.9, 37.5 ± 6.5, and 38.0 ± 5.8. Amounts of Alb loss (g/s) were 3.4 ± 0.7, 3.8 ± 1.1, and 4.0 ± 0.8, showing no significant difference. Removal amounts of ß2-MG (g) were 0.222 ± 0.03, 0.199 ± 0.03, and 0.198 ± 0.03; the amount was significantly highest in G-1. Removal amounts of α1-MG (g) were 0.146 ± 0.033, 0.163 ± 0.03, and 0.180 ± 0.005; the amount was significantly highest in G-3. In addition, ratios of α1-MG/Alb were 44.4 ± 9.3, 40.2 ± 9.6, and 42.0 ± 8.2 , and the NVF series consists of 3 types of products . The membranes of the FIX-U and NVF-P have the largest pore size among the respective series. From these products, we selected and used the FIX-S and NVF-H, with which post-HDF was assumed to yield adequate removal performance for LMWP and to keep Alb loss within the adequate range. As for ABH-PA, which is the only available type of the product, there was no alternative.Qdtotal of 500 mL/min and Vs of 12 L/s, the net Qd was 450 mL/min. Under this condition, the efficiency in removing small molecular solutes by diffusion was expressed as Kt/V of 1.8 or higher and removal rates of 75 % or higher for urea and 70 % for creatinine, which were favorable. We previously reported that HDF should be set with target removal rates of 80 % for ß2-MG and 35 % for α1-MG when dialysis patients with various complications are treated [2-MG and 35 % or higher for α1-MG.In the present study, because post-HDF was performed at  treated . Post-HDQb to prevent hemoconcentration. For example, in the ESHOL study, Qb and Vs were reported to be 384–392 mL/min and 20.8–21.8 L/s, respectively [Qb are required, because the removal performance of hemodiafilters used for HDF in Europe is unsatisfactory for substances in the LMWP range.The European style of post-HDF requires a high Vs 15–30 L/s) to improve efficiency in removing solutes by convection . High Vs0 L/s to Qb is associated with 2 disadvantages. The first disadvantage is the risk of excessive loss of amino acids. Because amino acids are small molecular solutes, their loss increases with higher Qb. Excessive loss of amino acids obviously has an adverse effect on the nutritional status of patients. The second disadvantage is the increased risk of the development of micro air-bubbles in the blood tube circuit. Stegmayr et al. reported that when Qb is 300 mL/min or higher, micro air-bubbles always develop in the blood tube circuit, enter the body via a dialysis membrane, and adversely affect the body [Qb of 250 mL/min, at which post-HDF was performed in the present study, the risk of the development of micro air-bubbles is low. Based on these 2 disadvantages, the Japanese style of post-HDF, characterized by low-volume of substitution fluid and moderate Qb using Japanese high-performance hemodiafilters, clearly appears to be superior to the European style of post-HDF. Furthermore, the former may also be superior in biocompatibility, because the increase in TMP during post-HDF is mild [The European style of post-HDF at high the body –13. With is mild .V, removal rate of urea, removal rate and amount of β2-MG showed significant differences in several comparisons. However, it is unlikely that these differences could result in a problem in clinical practice. There was no significant difference in the amount of Alb loss. When using FIX, however, even though the serum level of Alb was high, the amount of Alb loss was low and its standard deviation was also small. The ratio of α1-MG/Alb was also at its highest when FIX was used . Because the problem with conventional post-HDF is the difficulty in controlling Alb loss, under the present circumstances, the FIX appears to be the most appropriate hemodiafilter for post-HDF among those 3 types of hemodiafilters.Among the 3 types of hemodiafilters used in the present study, Kt/ed Table . These rQdtotal , the net Qd was 350 mL/min, and the removal efficiency for small molecular solutes was only slightly decreased. Furthermore, because Vs remained the same, it is not surprising that the removal efficiency for LMWP was unchanged. When Qb was 300 mL/min, the removal efficiency for small molecular solutes was significantly improved. However, it should be kept in mind that the loss of amino acids and the risk of development of micro-air-bubbles increase with higher Qb.When post-HDF was performed at Vs of 12 L/s for 4 hrs with 20 % reduction in Qb using Japanese high-performance hemodiafilters can efficiently remove small-, middle-, and large-molecular-weight solutes. In addition, even when Qdtotal is reduced by 20 %, favorable removal performance is achieved for all solutes. Thus, post-HDF will be presumably performed more often, and the European style of high-volume post-HDF is considered unnecessary in Japan.Low-volume post-HDF with moderate


Their structures were determined by combination of NMR, ECD spectroscopy, and X-ray diffraction analysis. Compounds 1 and 6–8 were tested to exhibit potential antitumor properties, of which 6 and 7 inhibited cell growth through inducing apoptosis and cell cycle arrest. In addition, these compounds could induce autophagy and PINK1/Parkin-mediated mitophagy in cancer cell lines, as well as suppress lung cancer A549 cells metastasis in vitro.A series of dearomatized isoprenylated acylphloroglucinols derivatives, hyperhenols A–E (The online version of this article (10.1007/s13659-019-00229-w) contains supplementary material, which is available to authorized users. Hypericum and Garcinia in the family Guttiferae . Silica gel , and MCI gel  were used for column chromatography. Fractions were monitored by TLC , and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shmadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. All 1D and 2D NMR spectra were recorded on Bruker DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts  in September 2018. The plant was identified by ZHANG Yong-Zeng. A voucher specimen (No. 2018H01) was deposited in Kunming Institute of Botany.The plants of 3 to afford a fraction (695.2 g). This fraction was separated over a MCI-gel column (MeOH-H2O from 7:3 to 10:0) to produce five fractions (Fr. A–E). Fr. A (262.3 g) was chromatographed on a silica gel column, eluted with petroleum ether-acetone (100:1 to 0:1), to yield five fractions (Fr. A1–A5). Fr. A2 (37.7 g) was separated over a RP-18 silica column (MeOH–H2O from 85:15 to 100:0) and obtained eleven fractions (Fr. A2-1–A2-11). Fr. A2–5 was purified by preparative TLC and semipreparative HPLC to afford 9 (12.3 mg), 10 (11.5 mg) and 2 (10.8 mg). Fr. B (100 g) was chromatographed on a silica gel column, eluted with petroleum ether-ethyl acetate (50:1 to 0:1) to yield ten fractions (Fr. B1–B10). Fr. B3 (11.0 g) was purified by chromatograph on a silica gel column and preparative HPLC  to afford 11 (25.9 mg) and 12 (4.7 mg). Fr. B4 (755.9 mg) and B6 (1.2 g) were further purified by prearative HPLC  to afford 1 (15.1 mg), 3 (13.3 mg), 6 (26.5 mg) and 7 (12.0 mg). Fr. B2 (18.0 g) was separated over a RP-18 silica column (MeOH–H2O from 85:15 to 100:0), and obtained ten fractions (Fr. B2-1–B2-10) Compounds 4 (7.8 mg), 5 (1.3 mg) and 8 (3.2 mg) were obtained from Fr. B2-2 by preparative HPLC and semipreparative HPLC.The sample (20.0 kg) was extracted with MeOH at room temperature and filtered, and the solvent was evaporated in vacuo. The crude extract was subjected to silica gel column chromatography eluted with CHClα] + 250.8 ; UV (MeOH) λmax (log ε) 202 (4.14), 225 (4.10), 346 (4.02) nm; IR (KBr) νmax 3417, 2968, 2932, 1636, 1520, 1460, 1337, 1304, 1233 cm−1; 1H and 13C NMR data, see Tables m/z 443 [M − H]−; HRESIMS m/z 443.2803 [M − H]− .Yellow oil; +; HRESIMS m/z [M + H]+; 519.3106 .Yellow oil;  + 199.0 ; UV (MeOH) λmax (log ε) 202 (4.08), 228 (3.98), 239 (3.97), 281 (3.83), 326 (3.91) nm; IR (KBr) νmax 3431, 2970, 2932, 2878, 1651, 1522, 1470, 1437 cm−1; 1H and 13C NMR data, see Tables m/z 429 [M − H]−; HRESIMS m/z 429.2653 [M − H]−; .Yellow oil; [α] + 5.3 ; UV (MeOH) λmax (log ε) 320 (2.14), 275 (2.30), 241 (2.42), 197 (2.50), 310 (2.17), 269 (2.30), 215 (2.34) nm; IR (KBr) νmax 3422, 2970, 2935, 2876, 1657, 1618, 1530, 1462, 1379 cm−1; 1H and 13C NMR data, see Tables m/z 427 [M + H]+; HRESIMS m/z 427.2855 [M + H]+ .Yellow oil; +; HRESIMS m/z 501.3008 [M + H]+ .Yellow oil; 45.0 c 0.2, MeOH; , MeOH; α26H38O5, M = 430.56, a = 22.6496(4) Å, b = 9.4550(2) Å, c = 23.8898(4) Å, α = 90°, β = 94.6410(10)°, γ = 90°, V = 5099.27(16) Å3, T = 100(2) K, space group P21, Z = 8, μ (CuKα) = 0.609 mm−1, 56,500 reflections measured, 17,671 independent reflections (Rint = 0.0269). The final R1 values were 0.0363 (I > 2σ(I)). The final wR(F2) values were 0.0966 (I > 2σ(I)). The final R1 values were 0.0366 . The final wR(F2) values were 0.0971 . The goodness of fit on F2 was 1.056. Flack parameter = 0.02(2). Crystallographic data for the structure of 8 have been deposited in the Cambridge Crystallographic Data Centre .C2 at 37 °C.HeLa cells, GFP-LC3 HeLa cells, YFP-Parkin HeLa cells and A549 cells were maintained in DMEM  supplemented with 10% fetal bovine serum, FBS  and 100 U/mL penicillin–streptomycin  in a humidified atmosphere containing 5% COμL of complete medium, attached overnight, and then treated with a series of concentrations of compound for 72 h. At the end of the incubation period, 10 μL MTT solution was added into each well of a 96-well plate for 4 h at 37℃. After the medium was removed, 100 μL DMSO was added to dissolve the purple crystals. After shaking for 5 min, the optical densities at 490 nm were measured using a Microplate Reader.The cells were seeded in a 96-well tissue culture plate at a predetermined density in 100 6 and 7 for 48 h. We then removed the media from the dish and added staining solution containing MitoTracker red (100 nM) incubation 30 min at 37 °C. The cells were fixed with 4% PFA in PBS for 15 min and observed using a fluorescence microscope.HeLa cells were seeded on coverslips and treated with compounds 7 and 8 for 48 h. Subsequently, the cells were harvested, washed with PBS and fixed with 70% alcohol at 4 °C overnight. Then cells were washed with PBS and stained with 20 μg/mL PI/RNase staining buffer for 30 min and analyzed using FACSCalibur flow cytometer .HeLa cells were treated with various concentrations of The GFP-LC3 or YFP-Parkin HeLa cells were treated with compounds for the indicated time point, and then the cells were fixed with 4% PFA in PBS for 15 min at room temperature. The cells were observed under a fluorescence microscope .μL pipette tips and loose cells were washed away. The cell migration was observed and imaged under an IX83 microscope for each condition and timepoint . .Wound healing was used to evaluate cell motility as our previous study . BrieflyμM. For the migration assay, 4.5 × 104 A549 cells resuspended in 100 μL serum-free medium were seeded in the upper chamber with serum-containing medium in the lower chamber of 24-well transwell plates . After 24 h, the experiment was terminated by wiping the cells from the wells with a cotton swab and fixed and stained with 0.05% crystal violet for 20 min, scored under a light microscope in five random fields.Cell migration assay were performed as described previously . In brieCells were harvested and lysed in a lysis buffer , proteins were separated on SDS polyacrylamide gels and transferred to PVDF membranes . The membranes were blocked with 5% nonfat milk, and immunoblotted with primary antibodies at 4 °C overnight. After washed three times with TBST, membranes were incubated for 1 h with appropriate secondary antibodies at room temperature. The follow antibodies were used in our experiments: Caspase-3 , Cleaved-caspase-3 , Caspase-9 , PARP , LC3 , P62 , PINK1 , Tim23 , Tom20 (sc-17764), E-cadherin , Vimentin , pAKT , AKT , Cofilin  and GAPDH . GAPDH was used as the loading control. Membranes were visualized with Image Quant LAS 4000 .Supplementary file1 (DOC 6653 kb)Below is the link to the electronic supplementary material.


The crystal and molecular structures of 2-chloro-3-nitro-5-(trifluoromethyl)benzoic acid and 2-chloro-3-nitro-5-(trifluoromethyl)benzamide, two precursors for the synthesis of 8-nitro-1,3-benzothiazin-4-ones, a promising class of new antituberculosis drug candidates, are described. 8H3ClF3NO4 (1), and 2-chloro-3-nitro-5-(trifluoromethyl)benzamide, C8H4ClF3N2O3 (2), are reported. In 1 and 2, the respective carboxy, carboxamide and the nitro groups are significantly twisted out of the plane of the benzene ring. In 1, the nitro group is oriented almost perpendicular to the benzene ring plane. In the crystal, 1 and 2 form O—H⋯O and N—H⋯O hydrogen-bonded dimers, respectively, which in 2 extend into primary amide tapes along the [101] direction. The trifluoromethyl group in 2 exhibits rotational disorder with an occupancy ratio of 0.876 (3):0.124 (3).8-Nitro-1,3-benzothiazin-4-ones are a promising class of new antitubercular agents, two candidates of which, namely BTZ043 and PBTZ169 (INN: macozinone), have reached clinical trials. The crystal and molecular structures of two synthetic precursors, 2-chloro-3-nitro-5-(trifluoromethyl)benzoic acid, C Starting from 2 benzonitrile upon reaction with nitrating acid mixture. Treatment of 1 with thionyl chloride affords the corresponding acid chloride, which is reacted with concentrated ammonia solution to give amide 2 in good yield . In 1, the plane defined by the carboxy group non-hydrogen atoms  is twisted out of the mean plane of the benzene ring (C1–C6) by 22.9 (1)°. Remarkably, the plane defined by the nitro group  is oriented nearly perpendicular to the mean plane of the benzene ring with a tilt angle of 85.38 (7)°.Fig. 22 crystallizes with two molecules in the asymmetric unit (Z′ = 2), one of which exhibits partial rotational disorder of the trifluoromethyl group. With respect to the mean plane of benzene ring (C1–C6), the plane defined by the non-hydrogen atoms of the amide group  is inclined at 49.0 (2) and 43.4 (2)° in molecule 1 and 2, respectively. The tilt angle between the plane of the nitro group  and the benzene ring mean plane is 46.1 (1)° in molecule 1 and 46.7 (1)° in molecule 2, which is significantly smaller than in 1.Compound 1H NMR spectrum of 2 in DMSO-d6 at room temperature shows two distinct broad singlets for the amide hydrogen atoms (see supporting information), indicating restricted rotation about the C—N bond due to partial double-bond character , two characteristic N—H stretching bands at 3356 and 3178 cm−1 are present .The supramolecular structures of 1 Table 1 and the 2 Table 2 are with1 and 2, respectively, the solid-state supramolecular structures of both compounds feature a number of possible weak interactions  and the nitro group appears to accept a donating bifurcated weak C—H⋯O hydrogen bond from the C6—H6 moiety . The latter interaction links the molecules into chains in the [110] direction and may be discussed in connection with the remarkable twist of the nitro group out of the plane of the benzene ring. A packing index for 1 of 74.3%, as calculated with PLATON . Based on the corresponding C—F⋯F angles of 152.1 (1)° at F1 and 168.6 (1)° at F3, these contacts may be classified as type I fluorine–fluorine interactions . The electron-withdrawing effect exerted by the ring substituents in both 1 and 2 should activate the C—H moieties for weak hydrogen bonding benzene (CSD refcode: JIHNOG) and 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene, also known as chloralin (JIHNUM) phenyl](piperidin-1-yl)methanone (MUPZAB), a structurally characterized side product in benzothiazinone synthesis, the twist angle is 38.1 (2)° . Abbreviations: s = singlet, d = doublet, q = quartet. IR spectra were measured on a Bruker ALPHA Platinum ATR–FT–IR spectrometer. Mass spectra were recorded on a Thermo Fisher Q ExactiveTM Plus Orbitrap mass spectrometer for 1 and on an Advion expressionS compact mass spectrometer for 2, using methanol as solvent.2-Chloro-3-nitro-5-(trifluoromethyl)benzoic acid (1): Compound 1 was synthesized from 2-chloro-5-(trifluoromethyl)benzonitrile (Lundbeck) using a literature method : δ = 8.70 , 8.40  ppm; 13C{1H} NMR  δ = 164.3, 149.7, 135.7, 129.7 , 128.8 , 127.2, 124.0 , 122.3  ppm; IR(ATR): ν~ = 3097 (C—H stretch), 2865 (O—H stretch), 1702 (C=O stretch), 1542, 1323 (NO2 stretch), 1117 (C—F stretch) cm−1; MS (ESI−) m/z [M − COOH]− calculated for C7H2ClF3NO2− 224.0, found 224.0, [M − H]− calculated for C8H2ClF3NO4− 268.0, found 268.0.2-Chloro-3-nitro-5-(trifluoromethyl)benzamide (2): Compound 2 was prepared from 1 following a published procedure : δ = 8.60 , 8.22 , 8.18 , 8.03  ppm; 13C{1H} NMR  δ = 165.1, 148.9, 140.8, 128.8 , 128.2 , 125.8, 122.42 , 122.4  ppm; IR(ATR): ν~ = 3356, 3178 (N—H stretch), 3096 (C—H stretch), 1659 (C=O stretch), 1629 (N—H bend), 1537, 1317 (NO2 stretch), 1116 (C—F stretch) cm−1; MS (APCI+) m/z [M + H]+ calculated for C8H5ClF3N2O3+ 269.0, found 268.9.1 in methanol/water and of 2 in ethanol.Crystals suitable for single-crystal X-ray diffraction were obtained by slow evaporation at room temperature of 1 were measured at the P11 beamline at PETRA III at DESY :0.124 (3). Carbon-bound H atoms were placed in geometrically calculated positions with C—H = 0.95 Å, and refined with the appropriate riding model and Uiso(H) = 1.2 Ueq(C). The carboxy hydrogen atom in 1 was located in a difference-Fourier map and refined freely. The amide H atoms in 2 were also located in difference-Fourier maps and refined semi-freely with the N—H distances restrained to a target value of 0.88 (2) Å and with Uiso(H) = 1.2 Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021000517/tx2035sup1.cifCrystal structure: contains datablock(s) global, 1, 2. DOI: 10.1107/S2056989021000517/tx20351sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989021000517/tx20352sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989021000517/tx2035sup4.pdfAdditional structure pictures, 1H and 13C NMR spectra, IR spectra and mass spectra. DOI: Click here for additional data file.10.1107/S2056989021000517/tx20351sup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021000517/tx20351sup6.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021000517/tx20352sup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021000517/tx20352sup8.cdxSupporting information file. DOI: 2055889, 2055890CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R,3S)-camphoric acid and o-phenylenediamine.The chiral title compound, which can be used for producing active pharmaceutical ingredients for treatment of type 2 pancreatic diabetes and other pathologies dependent on insulin resistance, was prepared from -camphoric acid and o-phenylenediamine. It crystallized from an ethanol solution in the chiral monoclinic 1P2 space group. The five-membered ring adopts a twisted conformation with the methyl-substituted C atoms displaced by −0.273 (5) and 0.407 (5) Å from the mean plane through the other three atoms. In the crystal, molecules are linked by O—H⋯N hydrogen bonds, forming chains along the a-axis direction. Hirshfeld surface analysis and two-dimensional fingerprint plots were used to analyze the intermolecular contacts present in the crystal.The chiral title compound, C According to the forecasts of the World Health Organization, the number of patients with diabetes will double and reach 300 million people by 2025 -3-(1H-benzo[d]imidazol-2-yl)-1,2,2-trimethylcyclopentane-1-carboxylic acid, 4, exhibits pronounced antidiabetic activity and, in particular, antihyperglycemic effect, which reduces insulin resistance and restores the physiological function of pancreatic β-cells -3-(1H-benzo[d] imidazol-2-yl)-1,2,2-trimethylcyclopentane-1-carboxylic acid 4, the enantiomerically pure -camphoric acid 1 was used.Racemic and enantiomeric crystals are known to possess different activities, which is very important in the pharmaceutical industry. We have found that the disadvantage of the (±) and (-) forms of compound  al. 2009 is theirR,5S)-1,8,8-trimethyl-3-oxabicyclo[3.2.1]octane-2,4-dione [d-(+)-camphoric anhydride] 2 was obtained by refluxing a mixture of -camphoric acid and acetic anhydride for 2 h -3-(1H-benzo[d]imidazol-2-yl)-1,2,2- trimethylcyclopentane-1-carboxylic acid 4 was carried out according to Fig. 2via cyclocondensation of d-(+)-camphoric anhydride 2 with o-phenylenediamine 3 in a mixture of toluene and DMF (383 K) by refluxing for several hours  hydrogen bond additionally stabilizes such a location of the saturated ring. The methyl group on the C11 atom is located in the axial position [C9—C10—C11—C15 = 87.5 (6)°]. The carboxyl group has an equatorial orientation and is almost coplanar to the endocyclic C10–C11 bond . This position is stabilized by the formation of weak intramolecular C10—H10A⋯O1 and C15—H15B⋯O2 hydrogen bonds between the vicinal and geminal substituents . The presence of geminal substituents on neighboring atoms of the pentane ring leads to significant steric repulsion  plane as a result of the strong N2—H⋯O1 and O2—H⋯N1 and weak C14—H14C⋯C2(π) intermolecular hydrogen bonds . The neighboring layers are not bound any specific interactions . It is interesting to note that the molecules are linked by hydrogen bonds that use the O—H⋯N heterosynthon instead of the carboxylic acid dimer homosynthon. Despite the presence of an aromatic ring in the molecule, no stacking interactions are observed in the crystal of 4. Instead of π– π interactions, C—H⋯π interactions are formed (Table 2b).In the crystal, molecules of s Table 2a,b. Thens Fig. 4a. It isd Table 2b.Crystal Explorer 17.5  to 1.406 (blue) a.u. The red spots indicate regions of donor–acceptor interactions or short contacts. There are three red spots in the dnorm surface for 4 , C⋯H/H⋯C , O⋯H/H⋯O  and N⋯H/H⋯N  contacts. The H⋯H interactions appear in the middle of the plot scattered over a large area, while the C⋯H/H⋯C contacts are represented by the ‘wings’ of the plot. O⋯H/H⋯O interactions appear as inner spikes and the N⋯H/H⋯N contacts, corresponding to the O—H⋯N interaction, are represented by a pair of sharp outer spikes, which indicate they are the strongest interactions in the crystal of 4.All of the intermolecular interactions of the title compound are shown in the two-dimensional fingerprint plot presented in Fig. 6et al., 2016viz. -(+)-cis-1,3-bis(benzothiazol-2′-yl)-1,2,2-trimethylcyclopentane  Å and −0.273 (5) Å in the title compound].A search of the Cambridge Structural Database -3-(1H-Benzo[d]imidazol-2-yl)-1,2,2-trimethylcyclopentane-1-carboxylic acid, 4d-(+)-camphoric anhydride 2 , o-phenylenediamine 3 , toluene (11.46 L) and dimethylformamide (0.91 L) were charged. Under stirring, the reaction mixture was heated to boiling (383 K). The mixture was refluxed and the released water was collected in the Dean–Stark receiver. When the removal of water had finished, the reaction mixture was cooled to room temperature. The precipitate that formed was filtered in vacuo using a Nutsche filter. The precipitate was thoroughly squeezed, washed twice with toluene (1.4 L) and re-squeezed. Then the precipitate was washed on the filter with 70% water–ethanol (3.7 L), heated to a temperature of 348±5 K. Finally, the precipitate of the product 4 was thoroughly squeezed and dried at 343 K for 4 h, yielding 2.41 kg (73.2%) of a white crystal-like powder that is practically insoluble in water, soluble in 96% alcohol, m.p. 527–528 K. UV (ethanol) λmax (∊): 204 nm (48960), 245 nm (6800), 275 nm (9160), 281 nm (9320); IR (KBr): ν (cm−1) 3450 (O—H), 3286 (N—H), 2970, 2935, 2887 (C—H), 1673 (C=O), 1529, 1456, 1436, 1373, 1279, 1358, 1167, 1124, 1057, 740; 1H NMR  δ 12.22 , 12.10 , 7.52 , 7.44 , 7.10 , 3.41–3.31 , 2.64–2.54 , 2.43–2.33 , 2.05–1.95 , 1.55–1.45 , 1.25 , 1.14 , 0.61 ; LC/MS m/z (%): 273.2 [MH]+ (100); found, %: C 70.88; H 7.83; N 10.55. C16H20N2O2. Calculated, %: C 70.56; H 7.40; N 10.29.In a glass reactor equipped with a Dean–Stark receiver, Further crystallization by slow evaporation of an ethanol solution was carried out to provide single block-like colorless crystals Fig. 7 suitableUiso(H) = 1.5Ueq(C-methyl and O-hydroxyl) and 1.2Ueq(C) for other H atoms. The Flack parameter cannot be determined reliably, because there is no X-ray anomalous scattering because of the absence of heavy atoms in the molecule.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020010439/dj2007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020010439/dj2007Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020010439/dj2007Isup3.cmlSupporting information file. DOI: 2019647CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Although the title compounds crystallize in different crystal systems [triclinic for (I) and monoclinic for (II)], both asymmetric units consist of one-half of the complex molecule, i.e. one metal(II) cation, one PM-TMA ligand, and one N-bound thiocyanate anion. In both complexes, the metal(II) cation is located on a centre of inversion and adopts a distorted octahedral coordination environment defined by four N atoms from two symmetry-related PM-TMA ligands in the equatorial plane and two N atoms from two symmetry-related NCS− anions in a trans axial arrangement. The trimethylbenzene and pyridine rings of the PM-TMA ligand are oriented at dihedral angles of 74.18 (7) and 77.70 (12)° for (I) and (II), respectively. The subtle change in size of the central metal cations leads to a different crystal packing arrangement for (I) and (II) that is dominated by weak C—H⋯S, C—H⋯π, and π–π interactions. Hirshfeld surface analysis and two-dimensional fingerprint plots were used to quantify these intermolecular contacts, and indicate that the most significant contacts in packing are H⋯H [48.1% for (I) and 54.9% for (II)], followed by H⋯C/C⋯H [24.1% for (I) and 15.7% for (II)], and H⋯S/S⋯H [21.1% for (I) and 21.1% for (II)].Two new mononuclear metal complexes involving the bidentate Schiff base ligand 2,4,6-trimethyl- They have been employed extensively with neutral N-donor co-ligands in the development of novel functional coordination materials, particularly in the field of molecular-based magnets methylidene]aniline (C15H16N2 or PM-TMA). Reaction of the PM-TMA ligand and M(NCS)2 precursors  in methanolic solutions resulted in the formation of discrete mononuclear complexes with the formula [M(NCS)2(PM-TMA)2], M = Mn (I), Ni (II). Their molecular and crystal structures as well as Hirshfeld surface analysis are reported.In this work, we combined 2-pyridinecarboxaldehyde and 2,4,6-trimethylaniline to synthesize a new bidentate Schiff base ligand with two potential I) and (II) are shown in Fig. 1PI) and P21/n for (II)], in both cases the asymmetric unit consist of one-half of the molecule, i.e. one metal(II) cation, one PM-TMA ligand and one thiocyanate anion. Each metal(II) cation is located on a centre of inversion and adopts a distorted octahedral coordination environment with four N atoms from two symmetry-related PM-TMA ligands in the equatorial plane and two N atoms from symmetry-related NCS− anions in a trans axial arrangement. The M—N bond lengths [2.174 (2) to 2.312 (2) Å for (I) and 2.027 (3) to 2.184 (2) Å for (II)] and N—M—N bond angles [74.27 (6) to 180° for (I) and 78.4 (1) to 180° for (II)] for both complexes are all in the normal range for similar Schiff base complexes with MnII  is somewhat more bent than the corresponding Ni1—N1—C1 bond angle [176.7 (3)°] in (II). The PM-TMA ligands are not co-planar, with the trimethylbenzene ring oriented to the pyridine ring at a dihedral angle of 74.18 (7) and 77.70 (12)° for (I) and (II), respectively. An overlay of the complex molecules of (I) and (II) is illustrated in Fig. 3The molecular structures of (I) and (II) is dominated by weak C—H⋯S, C—H⋯π, and π–π interactions. In the crystal structure of (I), pairs of weak C—H⋯S hydrogen bonds along with the C—H⋯π interactions involving the methyl H atoms (H14A and H14B) and the trimethylbenzene ring or the midpoint of thiocyanate C=N group, Table 1a axis, Fig. 4II), adjacent molecules are linked together into a sheet extending parallel to (101), Fig. 6II) also features weak π–π stacking interactions arising from the pyridine rings and the trimethylbenzene rings . There is an additional C—H⋯π interaction between the methyl H atom and the midpoint of the thiocyanate C=N group  and (II) were quantified by Hirshfeld surface analysis  and 54.9% for (II)], which represent van der Waals interactions. Minor contributions are due to H⋯C/C⋯H [24.1% for (I) and 15.7% for (II)] and H⋯S/S⋯H (21.1% for (I) and 21.1% for (II)) contacts, associated with weak C—H⋯π and C—H⋯S interactions, respectively. These contributions are characterized as bright-red spots on the Hirshfeld surface mapped over dnorm and are observed as two sharp peaks in the two-dimensional plots. The C⋯C contacts associated with aromatic π—π stacking contribute only with a small percentage in (I) (2.6%) and about twice the amount in (II) (5.5%). H⋯N/N⋯H contacts in both cases are negligible.The intermolecular interactions between the molecules in the crystal structures of methylidene]aniline. A general search for thiocyanato coordination compounds involving transition metals and N-[(pyridin-2-yl)methylidene]aniline as the main skeleton resulted in 122 structures with different substituents on the benzyl rings. In these structures, the two bidentate Schiff base ligands and the two thiocyanate anions are octahedrally arranged around the central metal cations in a cis-conformation. There is only one complex with a trans-conformation and the same skeleton as in the title complexes, viz. trans-[Cd(NCS)2(C14H14N2)2]  and (II).A search of the Cambridge Structural Database methylidene]aniline (C15H16N2 or PM-TMA) was synthesized according to a literature method 2·6H2O  in methanol (5 ml) was added KNCS , and the solution was stirred at room temperature for 30 min and then filtered to remove a white precipitate of KClO4. The solution was then carefully layered on the methanol solution of PM-TMA. After slow diffusion at room temperature for 3 d, yellow block-shaped crystals of (I) were obtained in 88% yield (44.7 mg) based on the manganese(II) source. Complex (II) was prepared following the procedure described above for (I), except that Ni(ClO4)2·6H2O  and KNCS  were used. Yellow block-shaped crystal of (II) were obtained in 82% yield (47.7 mg) based on the nickel(II) source.All reagents were of analytical grade and were used as received without further purification. The bidentate Schiff base ligand, 2,4,6-trimethyl-Uiso(H) = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020000870/wm5536sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989020000870/wm55361sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989020000870/wm55361sup4.cdxSupporting information file. DOI: 10.1107/S2056989020000870/wm55362sup3.hklStructure factors: contains datablock(s) 2. DOI: 1979499, 1979498CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures were elucidated by thoroughly analyzing of NMR and HRMS data, and single-crystal X-ray diffraction of 1. The oxetane ring moiety in 1 was rare in limonoids and other natural products. Compound 1 showed nitric oxide (NO) inhibitory effect with an IC50 38.45 ± 0.41 µM in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.Four new limonoids, toonayunnanaes F − I (The online version of this article (10.1007/s13659-020-00274-w) contains supplementary material, which is available to authorized users. Toona, belonging to the family Meliaceae, contains 15 species mainly distributed in the regions of tropical Asia and Africa, of which four species and six varieties grow in China + . The 1H and 13C NMR data of 1 , an α,β-unsaturated ketone , and five singlet methyl groups . Those observations together with 26 carbon resonances and 11 indices of hydrogen deficiency, suggested that 1 might be a ring-intact limonoid with an α,β-unsaturated carbonyl moiety in A ring +  and its 13C NMR data. Apart from the signals for an acetoxy substituent, the remaining 26 carbon resonances observed in the 1D NMR spectra indicated that 2 might also be a ring-intact limonoid + . The 1H and 13C NMR spectroscopic data showed many similarities to those of the known 6α-acetoxyazadirone D24 + 7.6 ; UV (MeOH) λmax (log ε) 218 (5.30) nm; IR (KBr) νmax 3553, 2986, 2931, 2859, 1669, 1455, 1387, 1158, 1025 cm−1; 1H and 13C NMR data, see Table m/z 409.2376 [M + H]+ .Toonayunnanae F : colorle2): white amorphous powder; [α]D24 + 9.2 ; UV (MeOH) λmax (log ε) 208 (3.95) nm; IR (KBr) νmax 3409, 2964, 1724, 1384, 1247, 1028 cm−1; 1H and 13C NMR data, see Table m/z 493.2551 [M + Na]+ .Toonayunnanae G : white amorphous powder; [α]D24 + 7.6 ; UV (MeOH) λmax (log ε) 208 (3.55) nm; IR (KBr) νmax 3421, 2977, 1732, 1664, 1249, 1027 cm−1; 1H and 13C NMR data, see Table m/z 469.2577 [M + H]+ .Toonayunnanae H : white amorphous powder; [α]D24 − 30.2 ; UV (MeOH) λmax (log ε) 208 (4.02) nm; IR (KBr) νmax 3466, 2954, 1747, 1717, 1226, 1026 cm−1; 1H and 13C NMR data, see Table m/z 550.2631 [M + NH4]+ .Toonayunnanae I  were evaluated for their inhibitory effects on NO production in LPS-activated RAW 264.7 macrophages as described in the literature [6 cells/mL) were seeded in 96-well plates and treated with different concentrations of tested compounds for 1 h, and 1.0 μg/mL LPS solution was subsequently added to stimulate the cells for 18 h. NO level was evaluated by measuring the standard of accumulated nitrite in cell supernatants with the reagent of Griess. N-Monomethyl-L-arginine Monoacetate (L-NMMA) was used as a positive control (IC50 = 42.38 ± 0.72 μM).The new compounds (terature . BrieflySupplementary file1 (PDF 1690 kb)Below is the link to the electronic supplementary material.