IFN-γ + 874 T/A, IFN-γR -611G/A, IL-4 -590C/T, and IL-4Rα Q576R polymorphisms in 126 cITP patients  and 202 healthy control donors. Genotyping was determined by PCR and direct sequencing. The Th1/Th2 ratio was detected in peripheral blood mononuclear cells via flow cytometry.T-helper cell type 1 (Th1) polarization in chronic immune thrombocytopenia (cITP) has been reported at the protein and mRNA levels. We evaluated the impact of Th1/Th2 cytokine and cytokine receptor functional polymorphisms on both susceptibility to, and severity of, cITP. We analysed IL-4Rα 576 non-QQ genotype compared to healthy subjects (P = 0.04). cITP patients with the IFN-γ +874 non-AA genotype (high expression type) showed more severe thrombocytopenia than those with the AA genotype (P < 0.05). cITP patients had a significantly higher Th1/Th2 ratio than control patients (P < 0.01); this ratio was inversely correlated with platelet counts. Furthermore, patients with both IFN-γ +874 non-AA genotype (high expression type) and IFN-γR −611 non-AA genotype (high-function type) had a significantly higher Th1/Th2 ratio (P < 0.05).cITP patients had a higher frequency of the The cytokine polymorphisms affecting Th1/Th2 increase the susceptibility to, and severity of, chronic ITP. Chronic immune thrombocytopenia (cITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia. Several immune mechanisms contribute to the pathogenesis of cITP, including increased platelet destruction in the reticuloendothelial system and impaired platelet production in the bone marrow . Recent Naive CD4+ T cells differentiate into several different Th cells, including Th1, Th2, Th17, and regulatory T (Treg) cells. Th cell functions are determined by their cytokine secretion patterns; Th1 and Th2 cells are defined by their ability to produce interferon-γ (IFN-γ) but not interleukin (IL)-4, and IL-4 but not IFN-γ, respectively –8. Many IFN-γ +874 T/A, IFN-γR -611G/A, IL-4 -590C/T, and IL-4Rα Q576R, on susceptibility to cITP, as well as on its clinical features. Furthermore, we explored the association between the Th1/Th2 ratio and these polymorphisms in both healthy donors and cITP patients.We investigated the existence of a role for Th1/Th2 cytokine and cytokine receptor functional polymorphisms, including 9/L) in the absence of other causes or disorders that may be associated with thrombocytopenia according to the criteria of the ITP International Working Group (IWG) [9/L at presentation of cITP. “Severe bleeding tendency” was defined as gastrointestinal bleeding and cerebral haemorrhage [9/L, and a response was defined as a platelet count between 30 and 100 × 109/L on condition that it was at least double the baseline count. “Loss of R” was defined as a platelet count <30 × 109/L, a less than 2-fold increase in platelet count from baseline, or the presence of bleeding. “Corticosteroid-dependence” was defined as the ongoing need for continuous corticosteroid administration or frequent courses of corticosteroids to maintain a platelet count at or above 30 × 109/L and/or to avoid bleeding [In this study, 126 Japanese cITP patients , as well as 202 race- and sex-matched healthy subjects were examined. cITP was defined as isolated thrombocytopenia (platelet count <100 × 10up (IWG) . cITP waup (IWG) . “Very smorrhage . The resmorrhage . In thesbleeding . “Severebleeding . “Refracbleeding . “SecondIL-4 -590C/T (rs2243250), IL-4Rα Q576R (rs1801275), and IFN-γR -611G/A (rs1327474) genotypes were determined using the polymerase chain reaction restriction fragment length polymorphism method. Genomic DNA was extracted from whole blood using a DNA Isolation kit . The reaction volume was 20 μL, comprising 1 μL of genomic DNA, 0.2 mM of dNTPs, 0.5 U of TaKaRa Ex Taq HS DNA polymerase , and 0.5 μM of each of 2 primers. We used the primers 5'- ACTAGGCCTCACCTGATACG -3' (forward) and 5'- GTTGTAATGCAGTCCTCCTG -3' (reverse) [IL-4), MspI (IL-4Rα), and Hpy188I (IFN-γR)  followed by electrophoresis through a 2.0% agarose gel (for IL-4) or a 6% polyacrylamide gel (for IL-4Rα and IFN-γR).The 76R rs18075, and I611G/A rs27474 genreverse)  for analIFN-γ +874 T/A (rs2430561) genotype was determined using the allele-specific PCR method. Genomic DNA was extracted from whole blood using a DNA Isolation kit (Qiagen). The reaction volume was 20 μL, comprising 1 μL of genomic DNA, 0.2 mM of dNTPs, 0.5 U of TaKaRa Ex Taq HS DNA polymerase , and 0.5 μM of each of 3 primers: 5'-TCA ACA AAG CTG ATA CTC CA-3' (common reverse), 5'-TTC TTA CAA CAC AAA ATC AAA TCT -3' , and 5'-TTC TTA CAA CAC AAA ATC AAA TCA-3'  [The forward) . The ampTo confirm the accuracy of our assays, several PCR products were sequenced and analysed using an ABI Prism Genetic Analyzer .. Whole heparinized blood was added to an equal volume of RPMI 1640 medium  in 15 ml centrifuge tubes. Twenty-five ng/mL of phorbol 12-myristate 13-acetate and 1 μg/mL of ionomycin  were added to all tubes except the resting controls; all tubes were supplemented with 10 μg/mL Brefeldin A (Sigma-Aldrich) except the activation controls. Tubes were incubated at 37 °C in 7% CO2 for four hours. After incubation with FACS lysing solution and FACS permeabilizing solution, cells were stained at 4 °C for 30 min with antihuman CD4-PE-Cy5 , FastImmune™ IFN-γ FITC/IL-4 PE (BD Biosciences) for intracellular cytokine staining and CD69 PE (BD Biosciences) for activation markers. FastImmune™ IgG2a FITC/IgG1 PE isotype control (BD Biosciences) and mouse IgG1 PE control (BD Biosciences) were used as negative controls. Three-color flow cytometric analysis was performed on a FACS Canto flow cytometer (BD Biosciences) using the FACS Diva software (BD Biosciences). Cells were logically gated on CD4 vs. side-scattered light (SSC) and forward-scattered light vs. SSC. Data were analysed using the FACS Diva software and displayed as dot plots of IFN-γ FITC vs. IL-4 PE. IFN-γ+and IL-4− cells were defined as Th1 cells, while IFN-γ− and IL-4+ cells were deemed Th2 cells. Analysis of the stimulation effect was based on the fraction of CD69-positive cells after activation, as evaluated using histograms. In all analyses, CD69 positivity exceeded 95%.We determined the Th1/Th2 ratio using flow cytometry as previously described by Ogawawa et al. [T test. The Th1/Th2 ratio and age were determined using the non-parametric Mann–Whitney U test. P-values were two-tailed, and P-values < 0.05 were considered statistically significant. We also compared the genotype frequencies with those calculated using the Hardy-Weinberg equilibrium theory .The measured continuous data were expressed as mean ± standard deviation. Allele and genotype frequencies were analysed using the chi-square test, and clinical characteristics and treatment response were analysed using the chi-square test and student’s 9/L to 96.0 × 109/L with a median count of 20.0 × 109/L at the initial diagnosis. The patients’ characteristics are shown in Table Of 126 patients, 92 were female (73.0%) and 34 were male (27.0%). Their median age at diagnosis was 47.7 years . The platelet count ranged from 1.0 × 10IFN-γ +874 T/A, IFN-γR -611G/A, IL-4 -590C/T, and IL-4Rα Q576R are shown in Table IL-4Rα 576 QQ genotype compared to healthy controls using a dominant model . However, no significant differences in the genotype frequencies of IFN-γ, IFN-γR, and IL-4 were observed between cITP patients and healthy controls using a dominant model and recessive model. Genotype frequencies of the four polymorphisms were in Hardy-Weinberg equilibrium in both cITP patients and healthy controls. These data show that IL-4Rα Q576R is associated with susceptibility to cITP.The genotype and allele frequencies of IFN-γ +874 non-AA genotype (high expression type) showed a lower minimum platelet count than those with an AA genotype . However, there was no significant association between the other three genotype distributions and their various clinical features. We also explored the association between the four polymorphisms and treatment response (Tables IL-4 -590 cm3 genotype (low expression type) had a higher incidence of second-line treatment than those with non-CC genotypes . No significant difference in treatment response was observed in other genotype distributions. These data suggest that Th1 polarization due to Th1/Th2 gene polymorphisms plays a role in the clinical features of cITP was well as in the response to treatment.We examined the association between the polymorphisms and the clinical characteristics of cITP patients Tables  and 4. ce Tables . cITP paP = 0.002)    Fig. . As the 02) Fig. . The min04) Fig. . These dIFN-γ +874 non-AA genotype (high expression type) had a significantly higher Th1/Th2 ratio compared to those with the IFN-γ +874 AA genotype (low expression type)   had a significantly higher Th1/Th2 ratio compared to those with the IFN-γR −611 AA genotype (low function type)   Fig. . Further01) Fig. . Howeversms Fig. . In contsms Fig. . These dFcgR and HLA were reported to contribute to the pathogenesis of cITP. Thus, cITP is considered to be a consequence of complex immune dysregulation events in conjunction with the presence of genetic risk factors.Although the immune mechanism that initiates cITP has not been identified, several processes of immune dysregulation have been reported . It is wRecent studies using flow cytometry and real-time PCR have revealed a clear Th1-polarized cytokine profile both at the protein and mRNA levels in cITP , 5, 10. Numerous studies have shown that patients with autoimmune disease have polarized Th1 or Th2 responses . IFN-γ iIFN-γ +874 T/A polymorphism and various diseases such as cancer and autoimmune disorders. IFN-γ +874 T/A polymorphism has been reported to affect the production of IFN-γ; the TT genotype has been linked to higher production of IFN-γ compared to the A/A genotype [IFN-γ polymorphisms in cITP patients, they produced inconsistent findings. Pehlivan et al. [IFN-γ +874 TT genotype (high expression type) compared to healthy controls, while Chen et al. reported no significant association between IFN-γ +874 T/A polymorphism and infant ITP in Chinese patients [IFN-γ +874 T/A polymorphism and Japanese patients with cITP. The frequency of IFN-γ +874 T/A polymorphism has been reported to differ by race [IFN-γ +874 T/A polymorphism.Furthermore, recent studies have shown the association between genotype , 28. Altn et al.  reportedpatients . We also by race , 20, whiIFN-γ +874 non-AA genotype (high expression type) had a lower minimum platelet count than those with the AA genotype. Panitsas et al. showed that lower peripheral platelet counts correlated with higher IFN-γ mRNA levels [IFN-γ +874 non-AA genotype may be susceptible to severe cITP. Further studies may be needed to confirm the involvement of the IFN-γ gene in the pathogenesis of cITP.In contrast to cITP susceptibility, our data showed that cITP patients with A levels . Thus, tIL-4 polymorphisms which affect the expression level of IL-4, including IL-4 VNTR intron 3 and IL-4-590C/T. Rosenwasser et al. [IL-4 -590C/T polymorphism, and reported the TT genotype was linked to higher IL-4 levels compared to the C/C genotype. Several IL-4 -590C/T polymorphism studies were reported in various autoimmune diseases, including asthma, rheumatoid arthritis, and multiple sclerosis [IL-4 -590C/T polymorphism and susceptibility to cITP. A similar finding has been reported by Foster et al. [IL-4 -590C/T polymorphism, they produced inconsistent findings of the association between IL-4 VNTR intron 3 and ITP susceptibility. Makhlouf et al. [IL-4 VNTR, however Chen et al. reported showed no significant association between IL-4 VNTR intron 3 and Chinese ITP patients [IL-4 is the Th2 cytokine that is pivotal for the pathogenesis of many autoimmune diseases; it induces the differentiation of Th0 cells to Th2 cells . Th2 celr et al.  analysedclerosis –35. We fr et al. . In contf et al.  reportedpatients , 37.IL-4 -590 CC genotype (low expression type) had a higher number of treatment regimens than those with the non-CC genotype. Thus, the IL-4 -590 CC genotype, which predominantly induced Th1, appears to contribute to poor response to treatment.cITP patients with the IL-4Rα Q576R, which can affect the binding of IL-4 and phosphorylation of intracellular substrates including signal transducer and activator of transcription 6 (STAT6), has been linked to many autoimmune disorders such as asthma, atopy, and allergy [IL-4Rα 576 non-QQ genotype. To our knowledge, ours is the first report demonstrating that IL-4Rα Q576R influences susceptibility to cITP. Recently Massoud et al. have shown that IL-4Rα Q576R promotes conversion of induced Treg cells toward a Th17 cell fate using Asthma mouse model [IL-4Rα Q576R polymorphism may affect the susceptibility to cITP in our study.IL-4 exerts its biological effects via signalling through its receptor, IL-4R. There are two types of IL-4 receptor complexes; the type I receptor consisting of IL-4Rα and the common gamma chain, and the type II receptor consisting of IL-4Rα and IL-13Rα . IL-4Rα  allergy . Our stuse model . Many inIFN-γ non-AA and IFN-γR non-AA genotypes, which are related to higher IFN-γ expression and higher IFN-γR function, respectively, had a significantly higher Th1/Th2 ratio (P < 0.05). Thus, such genotypes of the IFN-γ pathway components might contribute to a higher Th1/Th2 ratio in cITP patients, and may thus increase the severity of cITP. We also explored the association between these four polymorphisms and Th1/Th2 ratios in healthy control subjects; however, we found no significant association between these polymorphisms and Th1/Th2 ratios.In this study, cITP patients had a higher Th1/Th2 ratio compared to healthy subjects. Among cITP patients, those with a higher Th1/Th2 ratio had a significantly lower platelet count than those with a lower ratio; these results are consistent with previous studies . Our stuIL-4Rα polymorphism is associated with susceptibility to cITP. Moreover, the IFN-γ +874 non-AA genotype is associated with more severe thrombocytopenia and a higher Th1/Th2 ratio in cITP, indicating that the cytokine polymorphisms affecting Th1/Th2 increase the susceptibility to, and severity of, chronic ITP.In conclusion, our study revealed that the


Curcuma longa (Zingiberaceae family), which possess several pharmacological properties. However, in vivo administration exhibited limited applications in cancer therapies.Curcumin is one of the leading compound extracted from the dry powder of 1–10, acetone 11–17 and cyclopentanone 18–24 series. All the curcuminoids were synthesized by the acid or base catalyzed Claisen Schmidt condenstion reactions, in which β-diketone moiety of curcumin was modified with mono-ketone. These curcuminoids 1–24 were screened against HeLa, K562, MCF-7 (an estrogen-dependent) and MDA-MB-231 (an estrogen-independent) cancer cell lines. Among them, acetone series 11–17 were found to be more selective and potential cytotoxic agents. The compound 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Among the cyclohexanone series, the compound 4 exhibited  potential cytotoxicity against four proposed cancer cell lines, respectively. All the curcucminoids were characterized with the detailed 1H NMR, IR, UV–Vis, and mass spectroscopic techniques. The structure of compound 4 was confirmed by using the single X-ray crystallography. Additionally, we are going to report the first time spectral data of -2,6-bis(2-methoxybenzylidene)cyclohexanone (1). Structure–activity relationships revealed that the mono-carbonyl with 2,5-dimethoxy substituted curcuminoids could be an essential for the future drugs against cancer diseases.Twenty-four curcumin derivatives have synthesized, which comprises cyclohexanone 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines.Curcuminoids with diferuloyl(4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound  Curcuma longa  × 100%50 value (concentration of compounds inhibited 50% of cell viability) was determined from the graph of cell viability vs absorbance.IC4 was solved by direct methods and refined by full-matrix least-squares techniques on F2 using SHELXTL software package. Crystallographic data of the reported structures have been deposited at the Cambridge Crystallographic Data Centre with CCDC deposition numbers of 1548735. Copy of available material can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).X-ray analysis for all these samples were performed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ = 0.71073 Å) with φ and ω scans, at room temperature. Data reduction and absorption correction were performed using SAINT and SADABS programs –53. The 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Structure activity relationship revealed that the role of methoxy groups are important. Curcumin derivatives, 4, 5, 9, 14, 11 and 17 exhibited significant cytotoxic activity  cancer cell lines. Curcuminoids with diferuloyl (4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound


One of the racemic title isomers crystallized in a centrosymmetric space group and the other spontaneously resolved into enantiomers. rac-2-(4-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one (C16H14N2O3S) (1) and (2S)-2-(3-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one (C16H14N2O3S) (2) are reported here. While 1 crystallizes in a centrosymmetric space group, the crystal of 2 chosen for data collection has molecules only with (2S) chirality. This is the result of spontaneous resolution during crystallization, as the synthesis produces a racemic mixture. A crystal with (2R) molecules was also found in the same crystallization vial (structure factors available). The six-membered thiazine ring in both 1 and 2 displays an envelope conformation with the S atom forming the flap. The aryl rings in both structures adopt an approximate V shape with angles between their planes of 46.97 (14)° in 1 and 58.37 (10)° in 2. In both structures, the molecules form layers in the ab plane. Within such a layer in 1, one of the O atoms of the nitrophenyl group accepts a C—H⋯O hydrogen bond from the CH group at position 5 of the thiazine ring of a molecule of opposite chirality, forming chains along the a-axis direction. Each of the thiazine rings also participate in C—H⋯O bonds with the same carbon atom as above, resulting in chains along the b-axis direction, albeit of monochiral type. Adjacent layers are consolidated along the c-axis direction by pairs of parallel hydrogen bonds (C—H⋯O type) between the nitrophenyl groups of enantiomers. In 2, the two C—H⋯O hydrogen bonds contribute to chain formation along the b-axis direction. Weak edge-to-face interactions between the aryl groups of neighbouring molecules in 1, and C—H⋯π interactions between a thiazine ring CH group and a phenyl group of a neighboring molecule in 2 are also observed.The crystal structures of isomeric  N-aryl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one scaffold have been shown to have a wide variety of bioactivities, including antifungal -2-(3-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one, 2. We later collected another crystal from the vial and confirmed that it had the (2R) configuration . We also report the racemic (centrosymmetric) structure of the isomeric 2-(4-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one, 1. We have previously reported the crystal structure of rac-2,3-diphenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one  Å, θ = 47.0 (3)°, φ = 339.8 (4)° and in 2: Q = 0.6654 (16) Å, θ = 44.20 (17)°, φ = 353.8 (3)°. The aryl rings in both structures form an approximate V shape with inter-centroid distances of 3.964 (2) and 4.160 (2) Å, and interplanar angles of 46.97 (14) and 58.37 (10)°, in 1 and 2, respectively.Both structures 2 Figs. 1 and 2 ▸ 1, one of the oxygen atoms of the nitrophenyl group accepts a C—H⋯O hydrogen bond from the CH group at position 5 of the thiazine ring of a molecule of opposite chirality. This results in infinite chains of mixed chirality along the a-axis direction. The second oxygen atom of the nitrophenyl group also accepts a hydrogen bond from the thiazine 5-carbon atom, resulting this time in monochiral chains along the b-axis direction. Further, the stacking of layers along the c-axis direction is consolidated by pairs of parallel hydrogen bonds between the nitrophenyl groups of enantiomers. In 2, a monochiral structure, the C—H⋯O hydrogen bonds between the chiral carbon atom and the 4-oxygen atom on the neighboring thiazine ring results in a chain along the b-axis direction. The second hydrogen bond loops back to the second molecule in the reverse direction of the same chain. While weak edge-to-face interactions [Cg⋯Cg distance of 5.340 (3) Å and an interplanar angle of 84.99 (2)°] between the aryl groups of neighboring molecules is observed in 1, in 2, the 6-carbon atom of the thiazine ring interacts with the phenyl group in a C—H⋯π type interaction [C4⋯Cg = 3.581 (2) Å].In both structures, C—H⋯O interactions are observed Tables 1 and 4 ▸,H-1,3-thiazin-4-one . 3-Mercaptopropionic acid  and then 2-methyltetrahydrofuran (2.3 ml) were added and the solution was stirred. Pyridine  and finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  were added. The reaction was stirred at room temperature and followed by TLC. The mixture was poured into a separatory funnel with dichloromethane and distilled water. The layers were separated and the aqueous was then extracted twice with dichloromethane. The organics were combined and washed with saturated sodium bicarbonate and then saturated sodium chloride. The organic was dried over sodium sulfate and concentrated under vacuum to give crude product.General: A two-necked 25 ml round-bottom flask was oven-dried, cooled under NH-1,3-thiazin-4-one (1): the crude product was recrystallized from 2-propanol to give a white powder. Yield: 1.397 g (74%). m.p. 410–412 K. Colorless blocks for data collection were grown by slow evaporation from 2-propanol solution.2-(4-Nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one (2): The crude product was recrystallized from 2-propanol to give a yellow powder. Yield: 1.121 g (59%). m.p. 415 K. Colorless blocks were grown by slow evaporation from ethanol solution; the (2S) and (2R) crystals had identical morphology. The stereochemical configuration of individual crystals was identified by solving the crystal structure. After several were found to be (2S), a crystal was found that was (2R).2-(3-Nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-41 and 2 are summarized in Table 3Uiso(H) = 1.2Ueq(aromatic or methylene C) or 1.5Ueq(methyl C). In structure 2, the absolute configuration for the chiral centres in the molecule was determined as (2S) with a Flack absolute structure parameter of 0.09 (7) for 4055 Friedel pairs.Crystal data, data collection and structure refinement details for both structures 10.1107/S2056989018003444/hb7733sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018003444/hb77331sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989018003444/hb77331sup4.molSupporting information file. DOI: 10.1107/S2056989018003444/hb77332sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989018003444/hb77332sup5.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018003444/hb77331sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018003444/hb77332sup7.cmlSupporting information file. DOI: 1826377, 1826376CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Experimental data indicate that double guests reside within the cavity of Q[8] in both aqueous solution and solid state, generating highly stable homoternary complexes D-NA To the best of our knowledge, however, the detection and recognition of specific enantiomeric amino acids by Q[1H NMR spectroscopy measurements indicate that both D-NA and L-NA form host-guest inclusion complex with Q[8] host. Given that the changes induced by Q[8] host in the 1H NMR spectra of guests D-NA .Their Job’s plots (based on the continuous variation method) clearly show that both UV and fluorescence spectra data of both enantiomers fit well to 1:2 stoichiometry of the host-guest inclusion complexes , inset.  spectra  gave dou2@Q[8] were grown by slow evaporation of a solution containing the host Q[8] and the guest D-NA under 3.0 M aqueous hydrochloric acid solution in the presence of CdCl2. X-ray structural analysis has established that the D-NA2@Q[8] crystallize in the monoclinic crystal system, space group P21/c. As can be seen in 1H NMR spectroscopy. Obviously, the van der Waals contacts between the naphthyl groups and the inner wall of the Q[8] cavity, together with the electrostatic interactions between the protonated nitrogens in the guests and the carbonyl oxygens at the portals of the Q[8] host, and strong hydrogen-bonding: N(18)–H···O(4) 2.860(5) Å, N(34)–H···O(11) 2.902(3) Å, contribute to the formation of the inclusion complex D-NA2@Q[8]. Furthermore, the π···π interactions between two encapsulated D-NA molecules play a critical role in the formation of this host-guest inclusion complex. Outside of the inclusion complexes, neighboring D-NA molecules contact with each other through not only π···π interaction, but also C–H···π interactions.The synthesis and structure of L-NA2@Q[8]  is probably due to the cooperativity of above mentioned four kinds of weak interactions. On the basis of the corresponding experimental results, we also obtained the association constants of Ka = (6.51 ± 0.19) × 1011 M−2 and (3.17 ± 0.05) × 1011 M−2 for Q[8] with D-NA and L-NA, which are much larger than that of Q[8] with tripeptides reported by Urbach2@Q[8] and L-NA2@Q[8] in aqueous solution.ITC study  on the c2@Q[8] and L-NA2@Q[8]. This study suggests that Q[8] host may be very useful in dimerisating specific amino acids, peptides and proteins with suitable binding groups.In summary, we have investigated the host-guest complexation of Q[8] with two enantiomers D-NA and L-NA in both aqueous solution and solid state by using NMR, UV and fluorescence spectroscopy, MS, isothermal titration calorimetry (ITC), and X-ray crystallography. Driven by the cooperativity of electrostatic interactions, multiple C–H···π interactions, and hydrogen-bondings, both D-NA and L-NA can be encapsulated into the cavity of Q[8] to form stable homoternary complexes D-NA1H NMR spectra were recorded on a Bruker DPX 400 spectrometer in D2O. Absorption spectra of the host-guest complexes were recorded on an Aglient 8453 spectrophotometer at room temperature. Fluorescence spectra of the host-guest complexes were performed with a Varian RF-540 fluorescence spectrophotometer. MALDI-TOF mass spectrometry was recorded on a Bruker BIFLEX III ultra-high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with a-cyano-4-hydroxycinnamic acid as matrix.3-(2-naphthyl)-D-alanine and 3-(2-naphthyl)-L-alanine were obtained from Aldrich and used as supplied without further purification. Q[8] was prepared according to a literature method392@Q[8] and for L-NA2@Q[8] were grown from hydrochloride acid solution by slow evaporation. Diffraction data of both complexes were collected at 273(2) K with a Bruker SMART Apex-II CCD diffractometer using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). Empirical absorption corrections were performed by using the multi-scan program SADABS. Structural solution and full-matrix least-squares refinement based on F2 were performed with the SHELXS-97 and SHELXL-97 program packages, respectively41Single crystals of D-NA2@Q[8]: [(C13H14NO2)2@(C48H48N32O16)]·(CdCl42−)2·(C13H14NO2)2·10(H2O), Mr = 2882.77, monoclinic, space group P21/c, a = 18.875(2) Å, b = 12.5113(16) Å, c = 27.418(3) Å, β = 112.540(6)°, V = 5980.2(12) Å3, Z = 2, Dc = 1.601 g cm−3, F(000) = 2959, GOF = 1.008, R1 = 0.1183 (I > 2σ(I)), wR2 = 0.3541 .Crystal data for D-NA2@Q[8]: [(C13H14NO2)2@(C48H48N32O16)]·(CdCl42−)2·(C13H14NO2)2·10(H2O), Mr = 2882.77, monoclinic, space group P21/c, a = 18.875(2) Å, b = 12.5113(16) Å, c = 27.418(3) Å, β = 112.540(6)°, V = 5980.2(12) Å3,Z = 2, Dc = 1.601 g cm−3, F(000) = 2959, GOF = 1.007, R1 = 0.1164 (I > 2σ(I)), wR2 = 0.3555 .Crystal data for L-NAwww.ccdc.cam.ac.uk/data_request/cif.CCDC 1451630 and 1451631 contain the 2  in 3.0 M HCl (2 ml), Q[8]  was added. The resulting reaction mixture was stirred for 5 min at 50 °C and filtered. Slow solvent evaporation of the filtrate in air over a period of about two weeks provided rhombic colorless crystals of D-NA2@Q[8] with the yield of 1.8 mg (20%).To a solution of D-NA  and CdCl2  in 3.0 M HCl (2 ml), Q[8]  was added. The resulting reaction mixture was stirred for 5 min at 50 °C and filtered. Slow solvent evaporation of the filtrate in air over a period of about three weeks provided rhombic colorless crystals of L-NA2@Q[8] with the yield of 1.7 mg (18%).To a solution of L-NA  and CdClITC data were obtained on a Nano ITC instrument . Titration were performed with Q[8] concentration of approximately 0.1 mM in the sample cell (1.3 mL), and D-NA or L-NA concentration of approximately 2 mM in the syringe (250 μL). The heat of dilution was corrected by injecting the guest solution into deionized water and subtracting these data from those of the host-guest titration. All titrations were repeated three times. Computer simulations (curve fitting) were performed using the Nano ITC analyze software.How to cite this article: Gao, Z.-Z. et al. Host-guest complexation of cucurbit[8]uril with two enantiomers. Sci. Rep.7, 44717; doi: 10.1038/srep44717 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The crystal structures of complexes 1 and 4 were determined by single-crystal X-ray diffraction. In-vitro anti-bacterial activity for the prepared complexes against Gram-positive  and Gram-negative  bacteria and Yeast species (Saccharomyces and Candida) were performed using agar well-diffusion method. Only complex 4 showed reasonable activity against yeast. All compounds showed more anti-bacterial activity against Gram-positive bacteria than Gram-negative.Four novel complexes [Co(HThe online version of this article (doi:10.1186/s13065-017-0268-2) contains supplementary material, which is available to authorized users. The chemical classes of NSAIDs comprise phenylalkanoic acids, anthranilic acids, salicylate derivatives, oxicams, furanones and sulfonamides –24. SuliZ-5-fluor2O)4(sul)2] (1) and [Co(sul)2] (4) are also reported.The synthesis, characterization and anti-bacterial activity of new cobalt(II) sulindac containing complexes with heterocyclic nitrogen based ligands  are described in the present work. The crystal structures of  as also supported by single crystal X-ray determination.Infrared spectral data of KBr pellet of cobalt sulindac complexes c values . In compas(COO−), the symmetric stretching υs(COO−) and the difference between these two values of sulindac group in complexes 1–4 and those of potassium sulindac are shown in Additional file The assignments of IR frequencies for the asymmetric stretching υ2 and 3 have υas(COO−) at 1599, and 1600 cm−1, but υs(COO−) appear at 1390 and 1380 cm−1, so Δυ (COO−) are 219 and 220 cm−1, respectively which is larger than Δυ(COO−)K(sul) = 178 cm−1 and this supports monodentate coordination mode of the carboxylate groups. In addition, complex 3 has an absorption frequency at 3415 cm−1 which may indicate water molecules in the coordination geometry.Complexes 2 two absorption frequencies υas(NH2) at 3374 cm−1 and υs(NH2) at 3268 cm−1 with Δυ(NH2) = 106 cm−1 were observed. These frequencies are assigned to the 1°-NH2 group indicating that the complexation with cobalt is through the pyridine nitrogen atom rather than the NH2 nitrogen atom  (1): 85% (3.81 g) yield; m.p. 201 °C; IR : 3376, 3050, 2911, 2850, 1600, 1563, 1485, 1465, 1416, 1369, 1326,1268, 1217, 1203, 1171, 1133, 1086, 1024, 1008, 967, 918, 891, 891, 868, 805, 776, 717, 672, 659, 572, 473; UV–Vis : 211 (3283), 252 (828), 258 (872), 264 (850), 282 (771), 328 (514); μeff = 2.26 BM.2·7H2O  in 10 ml of methanol, then 2-ampy  dissolved in 15 ml of methanol was added. The mixture was allowed to stir for 24 h, the solvent was evaporated then the residue was dissolved in dichloromethane which was then evaporated and the compound obtained was washed with petroleum ether and dried under vacuum.Sulindac  was allowed to dissolve in a methanolic solution of potassium hydroxide  (40 ml methanol). To this solution was added slowly CoCl[Co(2-ampy)2(Sul)2] (2): 56% (2.50 g) yield; m.p. 180 °C (decomposed); IR : 3374, 3268, 3015, 2914, 2860, 1599, 1515, 1494, 1464, 1424, 1380, 1267, 1195, 1164, 1137, 1086, 1031, 1010, 955, 915, 891, 846, 811, 727, 651, 593, 533, 474, 449; UV–Vis : 207 (1828), 286 (450), 329 (348), 655 (12.7); μeff = 2.41 BM.2·7H2O  in 10 ml of methanol, then 1,10-phenanthroline  dissolved in 15 ml of methanol was added. The mixture was allowed to stir for 24 h, the solvent was evaporated then the residue was dissolved in dichloromethane which was then evaporated and the compound obtained was washed with petroleum ether and dried under vacuum.Sulindac  was allowed to dissolve in a methanolic solution of potassium hydroxide  (40 ml methanol). To this solution was added slowly CoCl[Co(H2O)2(sul)2] (3): 22% (1.0 g) yield; m.p. 140 °C; IR : 3415, 3059, 2911, 2852, 1600, 1515, 1464, 1424, 1380, 1267, 1195, 1164, 1137, 1086, 1010, 956, 915, 891, 846, 811, 727, 651, 593, 533, 474, 441; UV–Vis : 208 (2152), 226 (700), 271 (535), 328 (224), 431 (16.3), 488 (13.2); μeff = 2.4 BM.2·7H2O  in 10 ml of methanol, then 2,9-dimethyl-1,10-phenanthroline  dissolved in 15 ml of methanol was added. The mixture was allowed to stir for 24 h, the solvent was evaporated then the residue was dissolved in dichloromethane which was then evaporated and the compound obtained was washed with petroleum ether and dried. Suitable crystals for X-ray structural analysis were obtained by recrystallization from 1:1 mixture of chloroform/acetonitrile.Sulindac  was allowed to dissolve in a methanolic solution of potassium hydroxide  (40 ml methanol). To this solution was added slowly CoCl[Co(sul)2] (4): 34% (1.54 g) yield; m.p. 150 °C (decomposed); IR : 3040, 2912, 2845, 1599, 1566, 1465, 1441, 1359, 1194, 1157, 1135, 1086, 1031, 954, 916, 891, 855, 812, 761, 728, 644, 533, 474; UV–Vis : 207 (2263), 229 (933), 274 (621), 328 (261), 432 (13.3); μeff = 2.4 BM.−1 region (KBr) on a Perkin Elmer FT-IR spectrometer (2004). UV–Vis spectra were recorded using Hewlett Packard 8453 photo diode array spectrophotometer in the 200–800 nm region using DMSO as solvent. Melting points were determined in capillary tubes with B-545 melt apparatus without any correction. The magnetic susceptibility measurements were determined by Gouy method using mercury cobalt-thiocyanate complex, (HgCo(NSC)4) as standard. Calculation of the effective magnetic moment was obtained by using the following: μeff = 2.83 * (χmT)1/2 .Infrared (IR) spectra were recorded in the 450–4000 cm1 and 4 was carried out at room temperature on a Bruker SMART APEX CCD X-ray diffractometer system (graphite-monochromated Mo Kα radiation λ = 0.71073 Å) by using the SMART software package [1 and 4.X-ray intensity data of complexes  package . The dat package . The str package . H atomsBordetella, E. coli) and gram-positive  and Yeast species (Saccharomyces and Candida) were used in the present work.Agar diffusion method  was usedIn sterile saline single bacterial colonies were dissolved until the suspended cells reached the turbidity of McFarland 0.5 Standard. The bacterial inocula were spread on the surface of the Muller Hinton nutrient agar by means of a sterile cotton swab. Sterile glassy borer were used to make a 6 mm in diameter wells in the agar plate. Samples were dissolved in DMSO in concentration equal to (8 mg/ml), (4 mg/ml) and (2 mg/ml), then 50 μl of the test samples were introduced in the respective wells. DMSO was used as negative control while gentamycin used as positive control. Immediately the plate was incubated at 37 °C for 24 h. The anti-bacterial activity was determined by measuring the diameter inhibition zone of complete growth in millimeter (mm). The averages of two trials determined the results and are stated as average ± standard deviation.Additional file 1: Table S1. Physical properties and yield of Cobalt(II) sulindac compounds.Additional file 2: Table S2. Comparison between some of principle peaks in IR for K(sul) and 1 (cm-1).Additional file 3: Table S3. Summary of principle peaks in IR for complexes 2, 3 and 4 (cm-1).Additional file 4: Table S4. UV-visible spectral data for compounds (1–4).Additional file 5: CCDC 1450310 and CCDC 1450311 contain the supplementary crystallographic data for complexes 1 and 4. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated with this article can be found, in the online version.


E configuration with respect to the azo functionality.The organic molecule in the title hydrate shows an  8H8N6S·H2O, the two aromatic groups are close to coplanar with the dihedral angle between the mean planes of the thiazole and pyridine rings being 2.9 (2)°. The organic molecule adopts an E configuration with respect to the double bond of the azo bridge. In the crystal, molecules are linked by (amine)N—H⋯N(pyridine), (amine)N—H⋯O(water) and (water)O—H⋯N(thiazole) hydrogen bonds along with π–π interactions involving pairs of thiazole rings and pairs of pyridine rings. The plane-to-plane distance between two parallel molecules is 3.7856 (4) Å and corresponds to the length of the a axis. In this way, a layer structure parallel to (010) is formed. The layers are linked by weak C—H⋯S hydrogen bonds, eventually resulting in a three-dimensional network.In the title hydrated azo compound, C The amine N5 and N6 atoms are 0.044 (2) and −0.059 (3) Å, respectively, out of this plane. The thiazole ring  makes a dihedral angle of 2.9 (2)° with the pyridine ring . An intramolecular N5—H5A⋯N2 hydrogen bond is observed (Table 1S(6) ring motif.The molecular entities of (I)d Table 1, showingac plane is formed ed Fig. 2. Weak C—et al., 2016E)-2-(pyridin-3-yldiazenyl)thiazole moiety gave no hits. However, structures of substituted thiazolylazo derivatives were found, for example, 5-(diethylamino)-2-(2-thiazolylazo)phenol  with sodium nitrite . The mixture was stirred at a temperature between 268 and 273 K while a solution of 2,6-diaminopyridine  in 40 ml of 4 M hydrochloric acid was added. The reaction mixture was stirred for 1 h and then adjusted to pH 6.0 by 0.001 M sodium hydroxide. The red precipitate formed was filtered through suction and washed with water. Suitable crystals for X-ray analysis were grown by recrystallization using the vapor diffusion technique in a methanol-hexane mixture at 253 K . 1H NMR : δ 6.10 , 7.39 , 7.55 , 7.703 . Mass spec. (ESI) m/z 220.9 (M+), 136.2, 108.3, 81.4. IR–KBr (cm−1): 3335 (w), 3217 (w), 3082 (w), 1660 (s), 1631 (s), 1454 (m), 1292 (s), 1159 (m). Analysis calculated for C8H10N6OS: C, 43.64; H, 3.66; N, 38.16; 14.56. Found: C, 43.80; H, 3.79; N, 38.45; S,14.78.2-Aminothiazole  was dissolved in 6 Uiso(H) = 1.5Ueq(O) and 0.86±0.02 Å with Uiso(H) = 1.2Ueq(N), respectively. The C-bound H atoms were included in calculated positions and treated as riding atoms: C—H = 0.93 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018004693/wm5438sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018004693/wm5438Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018004693/wm5438Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018004693/wm5438Isup4.cmlSupporting information file. DOI: 1831600CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The five-coordinated HgII atoms have distorted square-pyramidal stereochemistry comprising two N-atom donors from bidentate chelate BPMBD ligands and three Cl-atom donors, two bridging and one monodentate. The dihedral angle between the benzene and the pyridine rings in the BPMBD ligand is 7.55 (4)°. In the crystal, the dinuclear molecules are linked by weak C—H⋯Cl hydrogen bonds, forming zigzag ribbons lying parallel to [001]. Also present in the structure are π–π interactions between benzene and pyridine rings [minimum ring-centroid separation = 3.698 (8) Å].The title dinuclear mercury(II) complex, [Hg The Sch2+ ion has a distorted square-pyramidal geometry completed by three Hg—Cl bonds, one monodentate [Hg1—Cl2 = 2.402 (4) Å] and two bridging Hg1—Cl1 [2.459 (3) Å] and Hg1—Cl1i . The environment of a five-coordinated mercuric ion is common among Hg2+ complexes  Å. The observed Hg—Cl and Hg—N bond lengths and bond angles are considered normal for this type of HgII complex  is 3.879 (6) Å, indicating a weak π–π interaction along the c axis  = 3.698 (8) Å -N1,N1-diethyl-N4-(pyridin-2-ylmethylene)benzene-1,4-diamine that has been structurally characterized. A dihalomercury(II) complex has been reported by Baul et al. (2013II atom is coordinated by the bis-chelating N-heterocyclic ligand [(E)-N-(pyridin-2-ylmethylidene)arylamine)], two bridging Cl ligands and one terminal Cl ligand. Similar HgII complexes have also been reported with a slight modification of the ligand -pyridine-κ2N,N′]mercury(II)} aniline-κN]mercury(II)} aniline-κ2N,N′]mercury(II)} -[(pyridin-2-yl-κN)methylidene]benzene-1,4-diamine-κN4} mercury(II)). All of the above compounds show the HgII ion in a distorted square-pyramidal coordination environment formed by the N atoms of the diimine ligand, two bridging Cl atoms and one monodentate Cl atom, as found in the title compound, one of the bridging Hg—Cl bonds being significantly longer than the other.A search of the Cambridge Structure Database -N1,N1-diethyl-N4-[(pyridin-2-yl)methylidene]benzene-1,4-diamine (DPMBD) was prepared by adding portionwise pyridine-2-carbaldehyde  to a methanolic solution (50 ml) of N1,N1-diethyl-p-phenylenediamine . The reaction mixture was stirred for 3 h at room temperature and filtered. The resulting yellow powder was washed with methanol (2 × 3 ml) and hexane (3 × 10 ml). The compound was recrystallized from hot MeOH to give yellow crystals, which were dried in a vacuum desiccator to give the pure product .The iminopyridyl compound  with mercury(II) chloride  in methanol (5 ml), with vigorous stirring for 2 h at room temperature. The red precipitate that formed was filtered off and redissolved in dimethylformamide. Crystals of the red title complex  suitable for X-ray analysis were obtained within 3 d by slow evaporation of the dimethylformamide.Uiso(H) = 1.5Ueq(C) (for methyl H) and Uiso(H) = 1.2Ueq(C) (for other H atoms).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017005874/zs2377sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017005874/zs2377Isup2.hklStructure factors: contains datablock(s) I. DOI: 1531593CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The FeII atom of the [Fe(CN)6]4− anion lies on a site with site symmetry ..2/m, and has an octahedral coordination sphere defined by six cyanido ligands. Both the Etpy+ and the oxonium cations are located on a mirror plane. In the crystal, electron-donor anions of [Fe(CN)6]4− and electron-acceptor cations of Etpy+ are each stacked parallel to the b axis, resulting in a columnar structure with segregated moieties. The crystal packing is stabilized by a three-dimensional O—H⋯N hydrogen-bonding network between the oxonium ions and the cyanide ligands of [Fe(CN)6]4−.The title compound, (C II(CN)6]4− electron-donor species and FeIII electron-acceptor species. Several charge-transfer salts composed of [Fe(CN)6]4− and organic acceptor cations, e.g. 1,1′-dimethyl-4,4′-bipyridinium (methyl viologen) have been reported 6]4− accompanied by monocationic species are rather rare 2[Fe(CN)6] (Etpy+ is 1-ethylpyridinium), (I)The present X-ray crystallographic analysis of the title salt, (Etpy)+ cation and an oxonium ion (both located on a mirror plane), and one quarter of an [Fe(CN)6]4− anion, the FeII atom of which is located on a site with symmetry ..2/m. The FeII atom is coordinated by six cyanido ligands in a slightly distorted octahedral configuration . The bond angle of the ethyl group of the Etpy+ ion [N3—C6—C7 = 110.77 (19) °] is similar to those of Etpy[AlCl4] ] 4− electron-donor anions and the Etpy+ electron-acceptor cations stack separately in columns parallel to the b axis whereby both types of columns are alternately arranged in the a- and c-axis directions.The [Fe(CN)6]4− ions form a three-dimensional O—H⋯N hydrogen-bonding network (Table 1+ cations is enclosed in the hydrogen-bonding cage formed by six [Fe(CN)6]4− ions and six oxonium ions  and the face-to-face distance of the least-square planes (3.731 Å) between two pyridinium rings indicate that π–π interactions are not developed.In the crystal of (I)k Table 1. A pair ns Fig. 3. Two pyr+ cation have been deposited in the Cambridge Structural Database 2[Fe(CN)6] 6] (106 mg) and l-ascorbic acid (60 mg) were dissolved in water (17 ml). The mixture was added to an aqueous solution of 1-ethylpyridinium bromide (177 mg/17 ml). After standing at 277 K for a day, yellow platelet-shaped crystals suitable for X-ray analysis were obtained. Elemental analysis: found: C, 51.52; H, 5.878; N, 24.06%; calculated for C20H26FeN8O2: C, 51.51; H, 5.63; N, 24.03%. Thermogravimetry was measured from 296 to 476 K at a rate of 5 K min−1 under N2 gas flow (100 ml min−1) on a Rigaku TG-DTA Thermo Plus EVO2 TG8121. Found: 7.85% mass loss; calculated: 7.73%. The mass loss of (I)+, remain in the crystal as counter-cations. The IR spectrum of compound (I)−1): 3135–2941 , 2640 , 2075 .Hviz. (0 17 1), (2 16 0) and (5 15 2), were omitted owing to poor agreements between observed and calculated intensities. H atoms of the Etpy+ cation were, at first, located in a difference Fourier map, but finally placed in geometrically calculated positions and refined as riding, with C(methylene)—H = 0.92 Å, C(methyl)—H = 0.98 Å and C(aromatic)—H = 0.95 Å, all with Uiso(H) = 1.5Ueq(C). H atoms of the oxonium ion were located in a difference Fourier map and their positions refined with Uiso(H) = 1.5Ueq(O). The maximum and minimum electron density peaks are located 1.00 Å from atom C1 and 0.71 Å from atom Fe1, respectively.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017000810/wm5355sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017000810/wm5355Isup2.hklStructure factors: contains datablock(s) I. DOI: 1527928CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The Ag+ cation is surrounded by six O atoms from neighboring 1,3-pdta groups and water molecules.In the title complex, the Cr 11H14N2O8)]·3H2O, contains one [Cr]− anion , one Ag+ cation and three water molecules. The Cr3+ ion is coordinated to the four O and two N atoms of the 1,3-pdta ligand, displaying a distorted octahedral geometry. The mean Cr—N and Cr—O bond lengths are 2.0727 (17) and 1.9608 (15) Å, respectively. The conformations of the chelate rings were found to be envelope for the glycinates and twist-boat for the six-membered diamine (T) ring. The Ag+ cation is surrounded by six O atoms from three non-coordinated carbonyl O atoms of neighbouring 1,3-pdta groups and three water molecules. The crystal structure is stabilized by intermolecular hydrogen bonding involving the water O—H group as donor and the carboxyl O atom as acceptor.The asymmetric unit of the title compound, Ag[Cr(C The counter-ion and metal-center oxidation state play a very important role in conformational isomerism. Upon coordination of 1,3-pdta by a metal center, the six-membered T ring can take twist-boat or half-chair conformers 3][Fe]·2H2O ]·3H2O ]·3H2O ]·3H2O ]·2H2O  to 99.41 (6)° and the trans angles are 173.07 (7), 175.01 (6) and 176.04 (7)°. The six-membered propane-1,3-diamine T ring  Å is 0.1119 Å longer than the average Cr—O bond distance. The Cr—N and Cr—O bond distances are in accordance with the values observed in Na[Cr]·3H2O. However, the average Ag—O distance of 2.525 (2) Å is slightly longer than the Na—O distance of 2.437 Å in Na[Cr]·3H2O ]ng Fig. 1 adopts a+ cation is surrounded octahedrally by three water molecules  and three carboxylate O atoms − unit. The crystal structure with an Na+ counter-cation ]·H2O and Ca[Cr]2·4H2O ]− complex with Ag+ cation was found.A search of the Cambridge Structural Database ]·3H2O suitable for X-ray structural analysis. Elemental analysis calculated for Ag[Cr(C11H14N2O8)]·3H2O: C, 25.60; H, 3.91; N, 5.43%; found: C, 25.71; H, 3.23; N, 5.36%. UV–vis data : 201 (vs), 223 (vs), 245 (sh), 385 (s), 506 (s), 700 (w). IR spectrum  : 3447  (ν OH), 3232 (sh), 2977 (vs) and 2941 (s) (ν CH), 1643  (asν COO), 1473 (s), 1428 (m), 1363 (vs) and 1327 (vs) (sν COO), 1271 (sh), 1222 (s), 1144 (s), 1099 (vs), 1061 (m), 1029 (s), 988 (s), 941 (vs), 916 (vs), 897 (m), 853 (vs), 746 (vs), 690 (m), 632 (w), 579 (m), 529 (s), 486 (s), 433 (s).All chemicals were reagent-grade materials and were used without further purification. The UV–Vis absorption spectrum was recorded with a Cary 5000 UV–Vis–NIR Spectrophotometer. The FT–IR spectrum was obtained from a KBr pellet with a JASCO 460 plus series FT–IR spectrometer. Analyses for C, H, N were performed on a Carlo Erba 1108 Elemental Vario EL analyser. The precursor salt, Na[Cr]·3HUiso(H) = 1.2Ueq(C). O-bound H atoms were assigned based on a difference-Fourier map, and were refined with distance restraints of 0.88 (2) Å (using DFIX and DANG commands), and Uiso(H) = 1.2Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018001743/nk2244sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018001743/nk2244Isup2.hklStructure factors: contains datablock(s) I. DOI: 1424813CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


M-KO mice with myeloid-restricted deletion of HO-1 to specifically investigate its influence on the acute inflammatory response to zymosan in vivo. In the mouse air pouch model, we have shown an exacerbated inflammation in HO-1M-KO mice with increased neutrophil infiltration accompanied by high levels of inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, and prostaglandin E2. The expression of the degradative enzyme matrix metalloproteinase-3 (MMP-3) was also enhanced. In addition, we observed higher levels of serum MMP-3 in HO-1M-KO mice compared with control mice, suggesting the presence of systemic inflammation. Altogether, these findings demonstrate that myeloid HO-1 plays an anti-inflammatory role in the acute response to zymosan in vivo and suggest the interest of this target to regulate inflammatory processes.Heme oxygenase-1 (HO-1) is induced by many stimuli to modulate the activation and function of different cell types during innate immune responses. Although HO-1 has shown anti-inflammatory effects in different systems, there are few data on the contribution of myeloid HO-1 and its role in inflammatory processes is not well understood. To address this point, we have used HO-1 Hmox1 in mice increases the severity of many experimental diseases, and the incidence or severity of several human diseases are associated with polymorphisms in the Hmox1 promoter that regulates HO-1 expression [Heme oxygenase (HO) catalyzes the oxidative degradation of heme to carbon monoxide (CO), iron, and biliverdin which is converted to bilirubin by biliverdin reductase . HO-2 ispression , 13.Hmox1−/−) mice exhibit growth retardation, anemia, proteinuria, hepatic and renal iron accumulation, chronic inflammation, and reduced life span [M-KO) do not exhibit any relevant physiologic changes compared with wild-type animals, and unlike HO-1−/− mice, they do not have enlarged spleens or pathological abnormalities [HO-1 knockout  mice were obtained from Alexander Fleming Biomedical Sciences Research Center, Vari, Greece, and crossed with LysMCre (B6.129P2-Lyz2tm1(cre)Ifo/CgnCnrm EM:01145) mice from EMMA (European Mouse Mutant Archive) Repository, Istituto Biologia Cellulare CNR, Monterotondo Scalo, Italy, and MGC Stiftung (Mouse Genetics Cologne (MGC) Foundation, Munich, Germany). Genotyping of mice was performed using the KAPA Mouse Genotyping Kit . Primers used for real-time PCR were as follows: Hmox1 , LysMCre , and LysMWT . For Hmox1, 1 cycle was for 4 min in 94°C; 1 cycle for 45 sg in 52°C; 30 cycles for 45 sg in 93°C, 45 sg in 52°C, and 1 min in 72°C; and 1 cycle for 10 min in 72°C. For Cre, 1 cycle was for 2 min in 95°C; 45 cycles for 30 sg in 95°C, 30 sg in 58°C, 90 sg in 72°C; and 1 cycle for 10 min in 72°C. The products, 545 bp (Hmox1FL), 387 bp (Hmox1WT), 390 bp (LysMCre), and 428 bp (LysMWT) were analyzed with the Agilent Bioanalyzer 2100 . To confirm the suppression of HO-1, we isolated peritoneal macrophages from LysMCre/+Hmox1FL/FL (HO-1M-KO) and Hmox1FL/FL (control) mice and analyzed HO-1 expression by immunocytochemistry and flow cytometry. Macrophages were obtained by peritoneal lavage with saline. HO-1 expression was assessed in adherent cells stimulated with 0.1 μg/ml phorbol 12-myristate 13-acetate  for 18 h at 37°C. Cells were fixed with 2% paraformaldehyde  and permeabilized with 0.1% Triton X-100 for 10 min. After blocking , anti-HO-1 polyclonal antibody (Enzo Life Sciences Inc.) was incubated overnight at 4°C. Unbound antibody was removed with phosphate-buffered saline, and the secondary fluorescent antibody  was incubated for 30 min. Macrophages were observed with a fluorescence microscope . Cells were also analyzed by flow cytometry with the anti-mouse CD16/CD32 antibody for blockade  followed by anti-F4/80-APC, anti-Ly-6G (Gr-1)-PE , and HO-1-FITC mAb (Enzo Life Sciences Inc.) using FACScan and Cell Quest Pro .All studies were performed in accordance with European Union regulations for the handling and use of laboratory animals. All the animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Valencia, Spain. Mice were housed and cared for by the veterinary staff in accredited facilities and were routinely screened for health status. M-KO and control mice between 10 and 12 weeks were used in these experiments. To create the MAP, 10 ml of sterile air was injected subcutaneously into the dorsal area of the mouse (day 0). Three days later, 5 ml of sterile air was injected (day 3), and six days after the initial injection of sterile air (day 6), 1 ml of saline buffer or 1 ml of 1% w/v zymosan (Sigma-Aldrich) in saline buffer was injected into the air pouch [Mice were maintained at 21°C ± 2°C on a 12 h light/dark cycle with feed and water ad libitum. Male HO-1ir pouch . At 18 hβ (IL-1β) and tumor necrosis factor-α (TNFα) were measured by enzyme-linked immunosorbent assay (ELISA)  with a range detection of 15.6–1000 pg/ml and 31.2–2000 pg/ml, respectively. C-X-C motif chemokine ligand 1 (CXCL-1) ELISA was from PromoKine  with a sensitivity of 8.0 pg/ml. MMP-3 levels were measured by ELISA , with sensitivity of 19.0 pg/ml. The eicosanoid prostaglandin E2 (PGE2) was determined by radioimmunoassay [Interleukin-1unoassay .3VO4, 1 μg/ml leupeptin, 0.1 μg/ml aprotinine, and 0.5 mM phenylmethyl sulfonyl fluoride and were sonicated (3 × 10 sec) and centrifuged at 10,000 ×g for 10 min at 4°C. Supernatants were collected and protein concentration was evaluated by the DC protein reagent . Proteins (30 μg) were separated by SDS/PAGE (12.5%) and transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk and incubated with specific polyclonal antibody against HO-1 (1/1000)  or β-actin  for 2 h at room temperature. Finally, membranes were incubated with peroxidase-conjugated polyclonal goat anti-rabbit IgG  and the immunoreactive bands were visualized by enhanced chemiluminescence  using an AutoChemi image analyser . Band intensity was analysed by optical densitometry using ImageJ analysis software . Band densities were corrected for background, and protein levels were normalized against β-actin.Cell pellets from the air pouch exudates were resuspended in a buffer pH 7.46 containing 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol, 5 mM NaF, 1 mM Nat-test was performed. All statistical analyses were performed using GraphPad Prism version 7.00 for Windows . A P value lower than 0.05 was considered to indicate statistical significance.The data were presented as mean ± SD. Normal distribution of data was assessed by the Shapiro-Wilk normality test. Two-way analysis of variance (ANOVA) followed by Sidak's multiple comparisons test, or unpaired Hmox1FL/FL mice were crossed with LysMCre knockin mice for deletion of HO-1 in myeloid cells. The LysMCre transgene mediates the excision of loxP-flanked sequences in these cells [Hmox1FL/FL (control) but not in LysMCre/+Hmox1FL/FL (HO-1M-KO) mice, thus confirming the efficient deletion of myeloid HO-1.se cells . We asseM-KO) was significant for cell migration . M-KO mice. It is interesting to note that inflammatory cell accumulation in air pouch exudate induced by zymosan was significantly higher in HO-1M-KO mice compared with control mice (11.3 ± 3.9 versus 5.1 ± 1.7 × 106 cells/ml). In this experimental model, there is a time-dependent increase in HO-1 expression in the cells migrating to the air pouch exudate [M-KO mice . Zymosan elicited a higher IL-1β level in HO-1M-KO mice (1582.0 ± 40.3 pg/ml) compared with control mice (1185.0 ± 191.1 pg/ml). Similarly, TNFα levels in the presence of zymosan were enhanced in HO-1M-KO mice (521.1 ± 175.0 pg/ml) with respect to control animals (290.8 ± 49.02 pg/ml). In addition, the interaction between treatment and genotype was significant for the production of the chemokine CXCL-1 . The levels of CXCL-1 in inflammatory exudates were increased by zymosan administration in both control and HO-1M-KO mice . M-KO mice. The production of PGE2 in HO-1M-KO mice (109.0 ± 16.1 ng/ml) was significantly higher than that of control animals (42.7 ± 21.9 ng/ml).There were no significant differences in levels of proinflammatory mediators between control and HO-1 Figures . Our res-KO mice  althoughF = 16.75, P = 0.0006 and F = 58.93, P < 0.0001, resp.). MMP-3 levels in the air pouch exudate were enhanced by the inflammatory response to zymosan (M-KO mice (153.5 ± 54.8 ng/ml) compared with controls (61.3 ± 28.6 ng/ml). We also assessed MMP-3 levels in serum as a marker of systemic inflammation. As shown in M-KO mice (50.1 ± 9.9 versus 11.6 ± 5.4 ng/ml). Zymosan injection did not increase serum MMP-3 in the control group (10.6 ± 2.5 ng/ml) but exerted a dramatic effect in HO-1M-KO mice (151.2 ± 32.5 ng/ml).There was a significant interaction between treatment and genotype for MMP-3 levels in air pouch exudate and serum  is able to cleave and activate other MMPs such as collagenases [α and IL-1β. In addition, MMPs such as MMP-3 can induce macrophage secretion of TNFα leading to cyclooxygenase-2 induction and PGE2 production which in turn increases MMP expression. This nexus between MMPs and prostanoids may be relevant in the pathogenesis of chronic inflammatory diseases and cancer [Proinflammatory cytokines and elevated levels of PGEagenases  and the d cancer .in vivo, which may contribute to the inflammatory amplification process involving cytokines, eicosanoids, and proteases which results in tissue damage.There are a number of ways in which MMP-3 participates in tissue destruction. Studies in MMP-3 knockout mice have shown the key role of this MMP in cartilage lesion and the contribution of synovial macrophages to MMP-mediated damage . We haveM-KO mice showed high levels of serum MMP-3 even in the absence of zymosan treatment, which indicates the presence of a systemic inflammatory response in these animals. In fact, serum MMP-3 levels are related to inflammation and tissue damage in conditions such as rheumatoid arthritis, where synovial macrophages are crucial in early MMP activity and serum MMP-3 may be a biomarker of disease activity and predictor of joint destruction [Interestingly, HO-1truction , 35. Thein vivo. These data point to new directions in therapeutic interventions that may potentially limit the deleterious effects of excessive inflammation.In conclusion, this study has revealed that myeloid HO-1 mediates protection against the inflammatory response to zymosan


Distribution and association between environmental and clinical isolates of Cryptococcus neoformans in Bogotá-Colombia, 2012-2015Norida Vélez, Patricia Escandón/+Grupo de Microbiología, Instituto Nacional de Salud, Bogotá, ColombiaFollowing authors’ decision, the article “Distribution and association between environmental and clinical isolates of Cryptococcus neoformans in Bogotá-Colombia, 2012-2015” published in Memorias do Instituto do Oswaldo Cruz 111(10): 642-648, 2016, DOI: 10.1590/0074-02760160201, authored by N Vélez & P Escandón was retracted due to an authorship conflict.


We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO−). In this specific case of P-transfer from PPi(III) to 5′-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO−. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5′-AMP, [P(III)P(V)-5′-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H In addition, we ouline how divalent metal ions influence this process, in a similar manner to that observed with PPi(III-V) formation , the closely related condensed P-material, pyrophosphite, PPi(III) ; δ -10.5 . 3‘/2‘-P(III)-5’AMP: δ = 6.1 ; δ 2.3 . 2′/3′-P(III)-5’AMP: δ = 4.9 , δ 2.3 . Full measured compositional data for PPi(III), Pi(III), 5′-AMP, PPi(III-V)-5′-AMP, 2′-P(III)-5′-AMP and 3′-P(III)-5′-AMP are collected in accompanying spread-sheet file and were assigned by 31P-NMR spectroscopy and by comparison to previously reported data  was phosphonylated in the presence of Na2-PPi(III), , 5‘-AMP  and MgCl2 , was adjusted to pH 7 by slow addition of solid NaOH. This solution was allowed to stand for 24 hrs at ambient temperature before analysis by 31P-NMR spectroscopy which reveals the following speciation by integration of peaks: unreacted 5’AMP: 21.5 %; 3′/2′-P(III)-5’AMP: 11.0 % & P(III)P(V)-5‘-AMP: 67.4%  and unreacted PPi(III) 82 %). This solution was transferred to a micro-distillation apparatus connected to a Schlenk line and the D2O removed under reduced pressure. Subsequently, the apparatus was filled with dry dinitrogen gas and H218O (1.0 g) added to the residues by syringe. After standing at room temperature for 6 days hydrolysis was found to be complete with the bulk PPi(III) hydrolyzing to Pi(III) and the P(III)P(V)-5′-AMP hydrolyzing back to 5′-AMP. The 3′/2′-P(III)-5’AMP products are relatively more resistant to hydrolysis. Additionally some hydrolysis of the phosphate ester linkage within 5′-AMP generated some Pi(V) which has reacted with PPi(III) to afford PPi(III-V). Excess H218O was removed under reduced pressure and replaced with D2O (1 mL) and the pH adjusted to 12 via slow addition of solid NaOH. Analysis of the Pi(III) resonances by 31P-NMR shows that 18O has been incorporated and with a Δδ value of 20 ppb which is consistent with isotopic incorporation.An aqueous (1 mL) solution containing sodium pyrophosphite, Na2-PPi(III) (1.5 M) at ambient temperature (ca. 20 °C) using a procedure modified from Yamamoto . The major product under these conditions is P-phosphonylated 5'-AMP [PPi(III-V)-5'-AMP; Fig. In our previously published work on the P-transfer abilities of pyrophosphite, PPi(III) , CaCl2 (0.1 M), KCl (0.1 M), G1 (1.0 M), G2 (1.0 M) (see attached spreadsheet of raw and processed data: 5’AMP KK45–52 Collated Results.xlsx) from which MgCl2 appears to have the most significant effect, affording ca. 2.5 times the amount of PPi(III-V)-5′-AMP after 1 day at 20 °C  as a co-factor with MgCl2 (0.1 M) further accelerates PPi(III-V)-5′-AMP formation to >3 times after 1 day  and G2 (1.0 M) respectively. Both have distinct but significantly more modest acceleratory effects on PPi(III-V)-5′-AMP formation than when combined with MgCl2. It can also be seen distinctly from Fig. 2 and MgCl2/G2 lead to a decrease in PPi(III-V)-5′-AMP concentration over time when compared to the growth of product over an 8 h period  which is subject to both general acid and general base catalysis.It is noted that PPi(III-V)-5′-AMP formation is accelerated in the presence of certain additives: MgCl2+ acts as a Lewis acid to bring together 5′-AMP and PPi(III) at a common reaction center. Preliminary molecular modeling of such a composite at the PM3 level using the Spartan 1.0.3 (http://www.wavefun.com/products/spartan.html) package, locates a reasonable transition state and returns an activation energy of ca. 16.6 kcalmol−1  to afford acylphosphonates, the key intermediate in PPi(III)-mediated G1 coupling. As both 5′-AMP  or the Pi(V) moiety of 5′-AMP. If the 18O was found to be more concentrated in the Pi(V) moiety of 5′-AMP, it would suggest that this Pi(V) moiety had been activated towards hydroysls by having been phosphonylated. If however, the isotopic enrichment was found solely in the Pi(III) groups, then the logical conclusion is phosphonylation of PPi(III-V)-5′-AMP does not activate the Pi(V). Our working model mechanism for isotopic enrichments follows the sequence: (i) PPi(III) is hydrolysed by H218O to Pi(III) with 18O incorporated; (ii) this heavy Pi(III) can then undergo degenerative Pi(III)-exchange via nucleophilic attack at PPi(III) to generate a heavy PPi(III); (iii) nucleophilic attack of heavy Pi(III) now on heavy PPi(III) has a 50 % chance of generating Pi(III) with double 18O. The 31P-NMR spectroscopy  signals above clearly display the 16O-isotopomer as dominant but also two further sets of isotopomers with separations, Δδ of 20 and 40 ppb, indicative of incorporation of one and two 18O-atoms respectively. The three isotopomers are present in the ratio 71.6:13.7:1.0. Incorporation of 18O-isotope is also seen in newly formed PPi(III-V) which we envisage to originate from reaction between PPi(III) and Pi(V) which accrues from the hydrolysis of 5′-AMP when adjusted to pH 12.As illustrated in Fig. opy Fig.  shows on18O incorporation  of what appears to be triplets of doublets (td). We believe that the difference between the sets of triplets of 70 ppb is too large to be explained as isotopomers of 5‘-AMP (which would indicate incorporation of three 18O-atoms) but that the larger td-pattern is due to unreacted 5’-AMP together with the 5′-Pi(V) nucleus of either 3′/2′-P(III)-5’AMP isomers and that the smaller td-pattern is the remaining 3′/2′-P(III)-5’AMP isomer  in PPi(III-V) have isotopomers but the peak due to Pi(V) does not appear to show any ion Fig. b. Close sis Fig. c revealsmer Fig. c.2+ and Ca2+ and organic co-factors containing acyl-functionalities, scenarios reminiscent of those employed within contemporary biochemistry. The major product of this P-transfer process is the functionalized isohypophosphate, PPi(III-V)-5‘-AMP. In attempting to ascertain whether this condensed P-compound could be considered as an activated form of 5’-AMP, isotopic exchange studies reveal that hydrolysis of PPi(III-V)-5′-AMP via H218O takes place preferentially at the Pi(III) rather than Pi(V) terminus, arguing for a greater inherent reactivity at Pi(III). Further studies are continuing to better place some of the above chemistry within putative Hadean geological environments.Whilst we recognize that many of the concentration ranges and chemical environments used in this study likely map only poorly to early earth geological scenarios Sleep , much is


Crystal structures of two 4-substituted derivatives of 2-acetylphenol are discussed comparatively with reference to modes of hydrogen bonding. 10H8O2, (I), and C13H14O3, (II), the 2-acetyl-4-ethynylphenol unit displays a planar geometry, which is stabilized by an intramolecular O—H⋯O hydrogen bond. The crystal structure of (I) is constructed of infinite strands, along [101], of C—H⋯O=C hydrogen-bonded molecules, which in turn are linked by C—H⋯π interactions. In the crystal of (II), which crystallized with three independent molecules per asymmetric unit, the non-polar parts of the molecules form hydrophobic layered domains, parallel to (10-1), which are separated by the polar groups. While the 2-acetylphenol part of the molecules are involved in O—H⋯O=C hydrogen bonding, the ternary OH groups creates a cyclic pattern of O—H⋯O hydrogen bonds.In the title compounds, C As a result of the presence of an intramolecular O–H⋯O hydrogen bond, the molecule has an almost planar geometry with largest atomic distances from the mean plane being −0.034 (1) Å for atom C5 and 0.069 (1) Å for atom O1. Because of substituent effects, the bond distances within the aromatic ring of the molecule deviate significantly from those observed in the polymorphous structures of ethynylbenzene  and 83.9 (2)° (gauche) for molecules 1 and 3 and 173.0 (2)° (anti) for molecule 2 . The ethynyl segment of the molecules also deviates from linearity, possibly because of packing forces and intermolecular interactions.The crystal structures of the title compounds (I) 2 Fig. 1b. The ed(H⋯O) 2.28 Å]  2.72 Å; Table 1et al., 2009Infinite strands of C—H⋯O hydrogen-bonded molecules , which create a cyclic hydrogen-bond motif of graph set A as donors and the acyl oxygen atoms O2A and O2B as acceptors , forming layers parallel to ) Table 2. Furtheres Fig. 3.et al., 2016p-substituted 2-acetylphenols excluding their co-crystals and complexes yielded 23 hits, only two of them containing the 4-ethynyl-2-acetylphenol element, namely 1,1′-diethanone and 1,1′-diethanone [CSD refcodes: TEVLAJ and TEVLEN; Hübscher et al., 2013p-position of the phenolic OH as in 4-cyano-2-acetophenol [LIWFUT; Filarowski et al., 2007et al., 2003et al., 2004via C—Harene⋯O=C bonding. Inter-strand association is accomplished by π–π stacking forces. In these structures, the p-substituents are excluded from intermolecular interactions. In the latter compound, the chlorine atom acts as a bifurcated acceptor for C—H⋯Cl bonding  and cyclohexane, respectively, upon slow evaporation of the solvents at room temperature.Compounds (I)Uiso(H) = 1.5Ueq(C-methyl and O) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016013451/su5317sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989016013451/su5317Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016013451/su5317IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016013451/su5317Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016013451/su5317IIsup5.cmlSupporting information file. DOI: 1500395, 1500394CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here, we also report on an esterification process of TCEP, which takes place in the presence of alcohols, leading to a platinum complex coordinated to an ester tmTCEP (2-methoxycarbonylethyl phosphine) ligand. The stability in solution of the three compounds and their interaction with biological models such as DNA  and proteins (lysozyme and RNase) have also been studied.Three platinum complexes with  Metallodrugs such as organometallic AuIII/RuII/OsII/RhIII/IrIII complexes have emerged as a new trend in non-conventional metallodrugs due to their potential activation by the redox balance in cancer cells and/or their protein interaction  (1) complex 2Cl2]; only one signal in the 31P-NMR and 195Pt-NMR spectra at 4.87 and at −4420.5 ppm, respectively  configuration with only one active ν(Pt–Cl) stretching band in the IR spectrum at 325 cm−1 expected for a D2 h symmetry group. 31P-NMR and 195Pt-NMR signals at 9.25 and at −4357.5 ppm ·1,25H2O) and its structure had been solved ) described in the literature /[compound]), in the absence and in the presence of compounds 1, 2 and 3 are plotted in A classical intercalator molecule like ethidium bromide causes a significant increase in the viscosity of the DNA solution due to the separation of base pairs at the interaction site and an increase in overall double helix length, whereas a groove binder agent like Hoechst 33258 does not appreciably lengthen the DNA helix and, therefore, causes less-pronounced changes or no changes at all in the viscosity of DNA solutions. A covalent binder case, for example cisplatin, causes a decrease in the relative viscosity of the DNA solution +: m/z 731.A 4 ml aliquot of a degassed water solution containing 37.2 mg of K3.4.2.2PtCl4 in methanol under an argon atmosphere. The mixture was stirred at room temperature for 2 days. The final white solid was filtered and washed with water and methanol. 850.52 g mol−1. Yield: 73.9%. 1H-NMR  δ 2.44 , δ 2.76 , δ 3.30 ; 13C-NMR  δ 20.81 (C1). δ 29.90 (C2). δ 52.68 (C4). δ 174.0 (C3); 31P-NMR  δ 4.87 ppm . 195Pt-NMR  δ −4433.08 ppm . Anal. (%) calcd. for C24H42Cl2O12P2Pt. C: 33.89, H: 4.98, N: 0. Found. C: 33.83, H: 4.82, N: 0. ESI-MS [M–Cl]+: m/z 815.14.TCEP (49.8 mg) previously dissolved in 2 ml of MeOH was added to a suspension of 36.5 mg of K3.4.3.Cis-[Pt(TCEP)2Cl2], 2 (20 mg) was heated in 5 ml of D2O, at 100°C for 20 min. Lowering the temperature of the final solution to room temperature gave the full precipitation of a yellow solid. The solid was isolated by filtration and washed with cold water and dried in a drying oven. 766.36 g mol−1. Yield: 92%. 1H-NMR  δ 2.35 , δ 2.83  12.3 ppm ; 13C-NMR  δ 16.2 (C1), δ 28.5 (C2), δ 172.7 (C3); 31P-NMR  δ 9.25; 195Pt-NMR  δ −4357.51. Anal. (%) calcd. for C18H30Cl2O12P2Pt. C: 28.21, H: 3.95, N: 0. Found. C: 28.63, H: 3.78, N: 0. ESI-MS [M+Na]+ : m/z 788.99.3.5.2 and 3, they were initially dissolved in DMSO in a 5 mM concentration. For all experiments, the desired concentration of complexes was achieved by dilution of the stock DMSO solution with aqueous buffer. Cisplatin stock solutions were prepared in 5 mM NaClO4 and diluted in Milli-Q water. All the solutions and buffers were previously tempered to 37°C. Afterwards, the freshly prepared complex solutions were mixed in a thermoshaker at 37°C with Milli-Q water (for stability K) /[CT-DNA]). Complexes 2 and 3 were initially dissolved in DMSO : water at a 5 mM concentration. The dilution was performed using Tris–HCl buffer to a final volume of 2 ml containing a maximum amount of 1.0% of DMSO.The viscosity experiments were carried out in automated AND viscometer model SV-1A, at constant temperature of 25.0 ± 0.1°C using a water bath with thermostat control. The concentration of CT-DNA was maintained constant .3.8.−1 stock) in 10 mM Tris–HCl (pH 7.6) and 1 mM EDTA were incubated with the platinum compounds at several ri values (0.01–0.5) using the corresponding amount of platinum from either a 5 or 50 µM stock solution. (Stock solutions were prepared in water and used fresh.) The samples were incubated at 37°C for 24 h, after which time 2 µl of a loading dye containing 50% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol was added. The whole of the sample (20 µl) was loaded in the wells of a 0.8% agarose gel. Electrophoresis was carried out for a period of 2.5 h at approximately 50 V. After electrophoresis, the gel was immersed in 800 ml of Millipore water containing 64 µl of a 10 mg ml−1 stock solution of ethidium bromide for 30 min to stain the DNA.The DNA-binding studies were performed in a total volume of 20 µl. The DNA aliquots containing 8 ml of DNA-pBR322  with the same results. The experimental time-dependent spectral profile was analysed as a pseudo first-order reaction by plotting the variation of the absorbance as a function of time .To investigate the interaction of cisplatin and complexes 4.cis configuration for complex 2 and trans for complex 3. A comparative study of the supramolecular interactions in the solid state has been performed. The cis complex 1 affords slow isomerization to complex 3 in water solution, while complexes 2 and 3 showed good stability in water solution and Tris–HCl buffer. The reactivity of these two stable compounds is higher when they interact with model proteins than with DNA. The complexes do not react when they face the secondary DNA structure model (pBR322), and with CT-DNA they act very differently compared with cisplatin.We report the synthesis of three platinum complexes using TCEP and tmTCEP (where esterification and coordination take place in the same step). The crystal structures demonstrated


The title compounds were synthesized from the commercially available dye quinizarin. In both compounds, the anthraquinone frameworks are close to planarity but there is a large difference in the conformation of the propyloxy group. 17H14O4, (I), and its acetyl derivative, 4-acetyloxy-4-propyloxy-9,10-anthraquinone, C19H16O5, (II), were synthesized from the commercially available dye quinizarin. In both compounds, the anthraquinone frameworks are close to planarity. There is a large difference in the conformation of the propyloxy group; the molecule of (I) adopts a gauche conformation [O—C—C—C = −64.4 (2)°], although the molecule of (II) takes a trans-planar conformation (zigzag) [O—C—C—C = 176.1 (3)°]. In the molecule of (I), there is an intramolecular O—H⋯O hydrogen bond. In both crystals, the molecules are linked by C—H ⋯O hydrogen bonds. A difference in the molecular arrangements of (I) and (II) is found along the stacking directions.1-Hydroxy-4-propyloxy-9,10-anthraquinone, C As a part of a project on the study of the substitution effects of the anthraquinone ring on optical properties in solution as well as in the solid state, we have been synthesizing new anthraquinone derivatives. Recently, we found that the recrystallization of 1,4-dipropyloxy-9,10-anthaquinone from hexane solution afforded two polymorphs, red prisms and yellow needles, whose crystal structures were different from each other (Kitamura gauche conformation [O2—C15—C16—C17 torsion angle = 64.4 (2)°], and in compound (II)trans-planar (zigzag) conformation [O2—C17—C18—C19 = 176.1 (3)°]. In (I)S(6) ring motif if Fig. 1. In compi, C15—H15A⋯O1ii . As shown in Figs. 7The crystal packing structures of the title compounds, (I)s Figs. 5 ▸ ▸. Aet al., 2016et al., 2015bet al., 2016et al., 2015aet al., 2014et al., 2012aet al., 2012bet al., 2012bA literature search found no reports of crystal structures of 1-hydroxy-4-propyloxy-9,10-anthraquinone (I)The title compounds, (I)Compound (I) A mixture of quinizarin , 1-bromopropane , K2CO3  in DMF (5 mL) was stirred at 353 K for 3 h under N2. After cooling to room temperature, water (60 mL) was added to the reaction mixture. The brown solid that precipitated was filtered off. The resulting solid was solubilized with CH2Cl2. The organic layer was washed with 1 M NaOH to remove the unreacted quinizarin, then washed sequentially with brine, dried over Na2SO4, and evaporated under reduced pressure. The residual brown solid was purified by chromatography on silica gel with an eluent of CH2Cl2. The title compound (I)1H NMR : δ 1.14 , 1.91–2.00 , 4.11 , 7.28–7.32 , 7.39–7.41 , 7.73–7.82 , 8.27–8.31 , 13.03 . Crystals suitable for X-ray diffraction were grown by slow evaporation of an AcOEt–hexane (>v:v = 1:10) solution.Compound (II) A mixture of compound (I)2CO3  in acetic anhydride (5 mL) was stirred at 383 K for 3 h under air. After cooling to room temperature, water (50 mL) was added into the resulting mixture, then the mixture was stirred for 20 min at room temperature. The mixture was extracted with CH2Cl2. The organic layer was washed with 10% NaHCO3 solution and then brine, and dried over Na2SO4, and evaporated under reduced pressure. The residual yellow solid was purified by recrystallization from a hexane–toluene (>v:v = 3:1) solution to provide title compound (II)1H NMR : δ 1.146 , 1.93–2.02 , 2.48 , 4.13 , 7.32–7.36 , 7.68–7.76 , 8.12–8.22 . Crystals suitable for X-ray diffraction were grown by slow evaporation of a hexane-toluene (>v:v = 18:1) solution.Uiso(H) = 1.5Ueq(C) for CH3 and 1.2Ueq(C) for CH2 and aromatic C—H.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017015973/nr2068sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017015973/nr2068Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017015973/nr2068IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017015973/nr2068Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017015973/nr2068IIsup5.cmlSupporting information file. DOI: 1583677, 1583676CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The IC50 values of the water extracts of its four herbs were as follows: Coptidis Rhizoma, 96.1 μg/ml; Phellodendri Chinensis Cortex, 108.6 μg/ml; Scutellariae Radix, 303.5 μg/ml; Gardeniae Fructus, 285.0 μg/ml. Thirteen compounds found in the plasma profile of HLJDT were also identified as potent NA-1 inhibitors, which included jatrorrhizine, palmatine, epiberberine, geniposide, oroxylin A, berberine, coptisine, baicalein, wogonoside, phellodendrine, wogonin, oroxylin A-7-O-glucuronide and baicalin . Their inhibitory activities were consistent with molecular docking analysis when considering crystallographic water molecules in the ligand-binding pocket of NA-1. Our current findings suggested that HLJDT can be used as a complementary medicine for H1N1 infection and its potent active compounds can be developed as NA-1 inhibitors.Huang-Lian-Jie-Du-Tang (HLJDT), a traditional formula with four TCM herbs, has been used for hundred years for different diseases. The current study aimed to assess the inhibitory activity of HLJDT against H1N1 neuraminidase (NA-1), and identify potent NA-1 inhibitors from its plasma profile. The  Several crystal structures of NA are obtained, and these structures facilitate structure-based drug discovery of NA inhibitors1. Two commercial drugs zanamivir (Relenza) and oseltamivir (Tamiflu), as derivatives of sialic acid, have been developed through this process. However, the supply of these drugs is limited. It is not possible to prescribe these drugs in the countryside of China when patients get influenza virus infection without serious symptoms. Therefore, it is necessary to discover new drug candidates for treating H1N1 infection. Currently, natural products  are considerable resources for the discovery of NA inhibitors.Highly infectious influenza A virus is pandemics and recurrent annual epidemics, and causes severe respiratory illness and death, especially in the elderly, children, and weakness. Neuraminidase (NA), a surface glycoprotein antigen, is one of biomarkers for subtype classification of influenza A virus. NA facilitates the release of influenza A virus via hydrolyzing glycosidic linkages of terminal sialic acid residues, which is critical to infection progression in the host. Current treatment strategy for influenza virus infection is to inhibit NA function5, inflammation6, cardiovascular diseases7, and Alzheimer’s disease8. After its oral administration, the major chemical components found in rat plasma have been identified, which include alkaloids  and flavones 9.Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese herbal formula used for hundred years, which consists of four herbs such as Coptidis Rhizoma , Scutellariae Radix , Phellodendri Chinensis Cortex  and Gardeniae Fructus  at the weight ratio of 3:2:2:3. It has been clinically used for treating sepsis10, berberine11 and coptisine12, have been identified as effective inhibitors of various NA subtypes. Other major components, detectable in the plasma profile of HLJDT, are supposed to be a potential resource for discovering NA inhibitors due to their similar structures. The aim of our current study was to evaluate the inhibitory activity of the water extracts of HLJDT and its four herbs on NA-1, and identify potent NA-1 inhibitors from its plasma profile , HLJDT showed potent inhibition activity on NA-1.When the substrate (MUNANA) concentration was 20 μM, the ICely Fig. . When co13. Secondary Lineweaver-Burk plot for Ki (obtained by the slopes of the regression lines in the Primary Lineweaver-Burk plot versus HLJDT’s concentrations) in Fig. Ki value of HLJDT on NA-1 activity was 55.6 μg/ml.For graphical inspection on the inhibition type of HLJDT, Primary Lineweaver-Burk plot  and Dixon plot  were firstly applied. As shown in Fig. This is the first time to report NA-1 inhibition activity of HLJDT. Since HLJDT is a clinically used formula, it is very safe for TCM practitioners to prescribe it for patients with influenza-like symptom before prescribing western drugs like Tamiflu.50 and IC10 values of each herb on NA activity were as follows: Phellodendri Chinensis Cortex (HB), IC50 = 108.6 ± 8.6 μg/ml and IC10 = 8.4 ± 3.3 μg/ml; Coptidis Rhizoma (HL), IC50 = 96.1 ± 7.6 μg/ml and IC10 = 9.9 ± 1.1 μg/ml; Scutellariae Radix (HQ), IC50 = 303.5 ± 21.9 μg/ml and IC10 = 28.0 ± 8.7 μg/ml; Gardeniae Fructus (ZZ), IC50 = 285.0 ± 16.6 μg/ml and IC10 = 37.4 ± 2.7 μg/ml. According to One-way ANOVA with Tukey’s multiple comparisons post-test, there were no significant difference among the IC50 values of HB, HL and HLJDT (P > 0.05). Meanwhile, the IC50 value of HLJDT was significantly lower than the one of HQ and ZZ (P < 0.01). These suggested that HB and HL were the major ingredients of HLJDT responsible for NA-1 inhibition, and HQ and ZZ were also involved in NA-1 inhibition. Since the HLJDT formula was designed according to the TCM theory, its safety has been observed in its clinical application for hundreds of years. Its composition should not be changed in its application.In Fig. 50 values as follows: palmatine (17.8 ± 0.8 μg/ml) ~ jatrorrhizine (22.7 ± 0.2 μg/ml) > epiberberine (33.6 ± 0.3 μg/ml) ~ geniposide (34.0 ± 1.8 μg/ml) > oroxylin A (57.3 ± 6.3 μg/ml) > berberine (78.6 ± 5.5 μg/ml) > coptisine (104.6 ± 18.8 μg/ml) > baicalein (159.5 ± 13.4 μg/ml) > wogonoside (186.4 ± 5.6 μg/ml) > phellodendrine (205.1 ± 18.9 μg/ml) > wogonin (246.1 ± 10.3 μg/ml) > oroxylin A-7-O-glucuronide (286.1 ± 44.2 μg/ml) > peramivir (478.8 ± 15.6 μg/ml) > baicalin (735.2 ± 59.1 μg/ml). The IC10 values of alkaloids including epiberberine, jatrorrhizine, berberine, palmatine, coptisine, and geniposide were about 1.9~20 μg/ml . As shown in the Fig. 20. As shown in Table Current approach using Autodock Vina provided maximum 9 possible binding conformations of each compound in the ligand-binding pocket with different binding free energies, but this method ignored the existence of water molecules by removing water molecules at the beginning of docking procedures. The favorable conformations of zanamivir, jatrorrhizine and phellodendrine were observed with respective lowest binding free energies of −8.1, −6.8 and −7.4 kal/mol, and did not overlap the poses of crystallographic water molecules in the ligand-binding pocket of NA-1 Table . However1. As such, the volume of NA-1 co-complexed with zanamivir (PDB ID 3B7E) used in the current study was smaller than the one of its native structure (PDB ID 3BEQ). This is may be the reason that there is no proper conformations of MUNANA and baicalin with bigger volumes in the ligand-binding pocket of NA-1 (PDB ID 3B7E).Except baicalin and MUNANA, all other tested compounds bound to the ligand-binding pocket with lower binding free energies and appropriate distance to the corresponding crystallographic waters when compared to the one of sialic acid. As reported before, the 150 cavity, as part of the ligand-binding pocket, is closed when the inhibitor bound to the ligand-binding pocket of NA-11. As illustrated in Fig. 1, it directly interacted via Hydrogen-bonding with amino acid residues including Arg118, Asp151, Arg152, Trp178, Glu227, Glu276, Arg292, and Arg371, and through hydrophobic contacts with Glu119, Arg224, Ser246, Glu277, Asn294, and Tyr406, which contributed to the inhibition of zanamivir in the catalytic site. Similarly, inhibitors from the plasma profile of HLJDT bound to the catalytic site by interacting with these amino acid residues via H-bonding and/or hydrophobic contacts. These interactions took responsibility for the inhibition of these compounds on NA-1-mediated 4-MU formation.As mentioned above, the binding conformations of active compounds with lower binding free energy and proper distances (more than 1 Å) to the water molecules in the ligand-binding cavities of NA-1 were selected, and simulated by both LigPlot + and PyMOL. Among these residues, eight charged and polar residues  directly interacted with the substrate in the ligand-binding cavities  of NA-112. Ser246 formed H-bondings with 1,3-dioxolane at C-2, 3 of coptisine and epiberberine, hydroxyl group at C-2 of phellodendrine, respectively. Hydrogen bonds of geniposide were formed through hydroxymethyl group at C-7 with Arg292, Arg371 and Tyr406, 7-(hydroxymethyl)-methyl ester with Ser246, 4-carboxylic acid with Asn294, and glucosyl group with Glu227, Trp178 and Asp151.Among these compounds, jatrorrhizine and palmatine interacted with amino acid residues in the catalytic site of NA-1 only through hydrophobic contacts. Besides hydrophobic contacts, other inhibitors tested in this study strongly interacted amino acid residues through hydrogen bonds. Hydroxyl groups at C-5, C-6 and C-7 of baicalein interacted with Glu227, Glu277 and Tyr406 via H-bonding. Hydroxyl group at C-7 of oroxylin A generated H-bonding with Arg118 and Arg371, while oroxylin A 7-O-β-D-glucuronide generated H-bondings via hydroxyl group at C-5 with Asn294, as well as its glucuronide with Asn221, Gly244, and Ser246. Wogonin formed H-bondings through ketone group at C-3 with Arg292 and Tyr406, hydroxyl group at C-5 with Arg292, and hydroxyl group at C-7 with Ser246. Likewise, wogonoside developed H-bondings through ketone group at C-3 with Asn294, and glucuronide with Asp151, Arg156, Trp178, Glu227, and Glu277. Methoxyl group at C-9 and 1,3-dioxolane at C-2, 3 of berberine interacted via H-bondings with Asn294 and Ser179, respectively, which were different from those of bacterial NA50 values) of tested compounds on NA-1, structure-activity relationships of flavones and isoquinoline alkaloids tested in the current study were explored and summarized as follows.According to the inhibition activities (IC50: 57.3 ± 6.3 μg/ml) and oroxylin A 7-O-β-D-glucuronide (IC50: 286.1 ± 44.2 μg/ml), containing a methoxyl group at C-6, showed stronger inhibitory activity than baicalein (IC50: 159.5 ± 13.4 μg/ml) and baicalin (IC50: 735.2 ± 59.1 μg/ml), respectively. The comparison of inhibitory effects on NA-1 between oroxylin A (IC50: 57.3 ± 6.3 μg/ml) and wogonin (IC50: 246.1 ± 10.3 μg/ml) indicated that compounds substituted with methoxyl group at C-6 instead of C-8 may increase NA-1 inhibition. Additionally, structures of these flavones with a glucuronic acid moiety at C-7 showed lower activity against NA-1. For example, NA-1 inhibition of baicalin (IC50: 735.2 ± 59.1 μg/ml) and oroxylin A 7-O-β-D-glucuronide (IC50: 286.1 ± 44.2 μg/ml) were lower than those of baicalein (IC50: 159.5 ± 13.4 μg/ml) and oroxylin A (IC50: 57.3 ± 6.3 μg/ml), respectively. However, wogonoside (IC50: 186.4 ± 5.6 μg/ml) with a substitution of glucuronic acid group at C-7 showed a better NA-1 inhibitory effect than wogonin (IC50: 246.1 ± 10.3 μg/ml), which remains to be further explored.Among flavones, structures with a methoxyl group instead of hydroxyl group at C-6 could increase NA-1 inhibition. For instance, oroxylin A (IC50: 78.6 ± 5.5 μg/ml), epiberberine (IC50: 33.6 ± 0.3 μg/ml) and palmatine (IC50: 17.8 ± 0.8 μg/ml) were all better than that of coptisine (IC50: 104.6 ± 18.8 μg/ml). In addition, compound substituted with hydroxyl group at C-3  instead of methoxyl group at C-3  could not increase NA-1 inhibition. Finally, compounds with saturated aliphatic hydrocarbon groups at C-8 and C-13 and hydroxyl group at C-11 showed lower activity against NA-1. For example, phellodendrine had lower inhibitory effect than palmatine (IC50: 17.8 ± 0.8 μg/ml) and jatrorrhizine (IC50: 22.7 ± 0.2 μg/ml).Among isoquinoline alkaloids, compounds substituted with free methoxyl group(s) instead of 1,3-dioxolane at C-2, 3 and C-9, 10 could increase NA-1 inhibition. For instance, NA-1 inhibition of berberine .The LDin vitro inhibition activities of 13 compounds found in the plasma profile of HLJDT should be responsible for the possible therapeutic effect of HLJDT due to the correlation of their Cmax and NA-1 inhibition activity. Besides, it is the first time to report the inhibition effects of epiberberine, oroxylin A, oroxylin A 7-O-β-D-glucuronide, and phellodendrine on NA-1 in vitro and in silico.This is the first time to report the NA-1 inhibition activity of HLJDT as a ready-to-use potent agent for anti-H1N1 infection, and it is suggested that it is valuable for TCM practitioners to use HLJDT and evaluate its efficacy in patients with influenza-like symptoms when they can not be diagnosed at the beginning. The All authentic standards (purity ≥ 98%) were purchased from ChromaBio Biotechnology . The screening kit for H1N1 neuraminidase inhibitors was provided by Beyotime Institute of Biotechnology . All other unspecified chemicals and reagents were supplied by Sigma .Scutellariae Radix (Lot No. 160562), Coptidis Rhizoma (Lot No. 160374), Phellodendri Chinensis Cortex (Lot No. 160528) and Gardeniae Fructus (Lot No. 160605) were purchased from Yan-He-Ling Pharmaceutical Company . Raw herbs were authenticated by the supplier through thin layer chromatography and HPLC with authentic compounds according to Chinese Pharmacopoeia version 2015. Their voucher specimens were deposited at the Military Institute of Chinese Medicine, 302 Military Hospital, Beijing, China.Raw herbs for the formula  were extracted by boiling with water (300 mL) for 60 min. After filtered, the residue was extracted again in the same way for another hour. The supernatant was mixed and subjected to freeze-dry. The dried extract was kept in the desiccator before use. The dried yield of HLJDT extract was ~31%. Each herb with respective amount in the formula was individually prepared by the same extraction method with 300 mL of water, and their extraction yields were as follows: Scutellariae Radix, ~55%; Coptidis Rhizoma, ~22.6%; Phellodendri Chinensis Cortex, ~24.6%; Gardeniae Fructus, ~35.4%.The inhibition study was performed with modification according to the manufacture’s instructions. Briefly, the inhibitor or extracts/compounds tested was mixed with recombinant NA-1, and then the substrate 2-O-4-Methylumbelliferyl-4,7,8,9-tetra-O-acetyl-N-acetyl-alpha-D-neuraminic acid methyl ester  was added for 1-hour enzymatic reaction. After that, 200 μL of stopping solution was added for the termination of reaction. The formed metabolite 4-methylumbelliferone (4-MU) was measured by BioTek Synergy H1 florescent plate reader  with excitation wavelength at 355 nm and emission wavelength at 460 nm. DMSO (0.1%) served as vehicle control. The inhibition rate was calculated as follows.Ki) values and inhibition types were measured by using three concentrations  of substrates and four concentrations of HLJDT . Other procedures were performed as mentioned above.For enzymatic reaction, the inhibition constant  were obtained from the Protein Data Bank1. The docking parameters were set with default values. The sizes of grid boxes were set as 20 Å × 20 Å × 20 Å for encompassing the ligand-binding pocket. Zanamivir was used as positive control.Molecular docking analysis was preliminarily used to screen compounds from the plasma profile of HLJDT by using the software AutoDock Vina v.1.0.2http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/)22 and PyMOL Molecular Graphics System v.1.3 , respectively.The binding modes of active compounds with lower binding free energy and proper distances (more than 1 Å) to water molecules in the ligand-binding cavity of NA were chosen for further analysis of docking conformation. The 2D and 3D simulation results were illustrated by LigPlot + v.1.4.5 . The inhibition constants (Ki) and modes of different inhibitors to neuraminidase-1 were measured by graphical inspection from different plots  according to the previous report24. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons post-test. A p value less than 0.05 was considered statistically significant.Data were expressed as mean ± standard error of Mean (SEM). IC