Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T cells and neurons.We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of γδ T cells towards neurotoxic IL-17+ γδ T cells. These cells feature innate-like characteristics such as localization to non-lymphoid organs, activation in absence of TCR stimulation, and rapid release of inflammatory molecules upon their activation. γδ TCRs recognize ubiquitous protein and non-protein antigens without the requirement of antigen processing and being independent of the presentation of antigen via major histocompatibility (MHC) molecules . γδ T ceet al. described TLR2, TLR3, TLR4, and TLR7 mRNA expression in spleenic γδ T cells [TLRs are pattern recognition receptors (PRRs) that recognize highly conserved PAMPs derived from viruses, bacteria, fungi, and parasites. In mice, twelve functional TLRs have been identified so far . Some TL T cells . A subse T cells . Express T cells –17.et al. showed direct interaction of CD4+ Th17 cells with neurons and axons independently of TCR specificity [γδ T cells were identified as an early source of IL-17, a proinflammatory cytokine, originally described to be produced by CD4+ Th17 T cells, in response to bacterial infection, in autoimmune diseases, and ischemia/reperfusion injury –22. IL-1cificity .in vitro and IL-17+ γδ T cells induced by supernatants derived from microglia activated through TLR2, TLR4, and TLR9 interact with neurons and cause cell contact-dependent neuronal cell death.Here, we systematically examined the neurotoxic capacity of γδ T cells and the role of microglia and TLRs in this context. γδ T cells express TLR2, TLR7, TLR9, and MyD88. However, γδ T cells do not secrete IL-17 in response to TLR ligands without microglia being present. Microglia activated through TLR2, TLR4, TLR7, or TLR9 release soluble factors including IL-1β and IL-23, inducing IL-17+ γδ T cells that display an activated phenotype. Expression of MyD88 is essential for IL-17 induction in γδ T cells by supernatants derived from microglia activated by TLRs. Both IL-17+ γδ T cells polarized by IL-1β and IL-23 C57BL/6J mice were obtained from the FEM, Charité –Universitaetsmedizin, Berlin, Germany. TLR2 knock out (KO), TLR7KO, and MyD88KO mice were generously provided by Dr. S. Akira . All animals were maintained under specific pathogen-free (SPF) conditions according to the guidelines of the committee for animal care. Experimental procedures were approved by the institutional review committee Landesamt für Gesundheit und Soziales, Berlin.Purified microglia were generated from forebrains of 0–3 day-old mice, and purified neurons were generated from mouse embryos at gestational stage 17, as described previously . Murine + T Cell Isolation Kit and magnetic cell separation (MACS) . Purity of isolated γδ T cells was determined by cell surface staining of CD3 and γδ T cell receptor (γδ TCR). Purity obtained usually reached > 90% CD3+γδTCR+ cells.γδ T cells were purified from lymph nodes and spleen of 8–10 week old male C57BL/6J, TLR2KO, TLR7KO, and MyD88KO mice using the mouse TCRγ/δ6/ml naïve γδ T cells were cultured for 3 days in complete RPMI  with IL-1β (10 ng/ml) , IL-23 (10 ng/ml) (R&D Systems), in the absence or presence of anti-CD3 (1μg/ml) and anti-CD28 (10μg/ml) (eBioscience), as described previously [To obtain polarized IL-17+ γδ T cells, 2x10eviously . IL-17 pin vitro toxicity studies, indicated amounts of IL-17 (PeproTech) were added to neuronal cell cultures for indicated durations. LPS (100 ng/ml) was used as an established compound for microglia-mediated neurodegeneration, thereby testing for contamination of cell cultures with microglia. Imiquimod (10 μg/ml) or loxoribine (1mM) served as a positive control for TLR7-mediated effects. For each condition, experiments were performed in duplicates.For 3/96-well in 200 μl DMEM supplemented with 10% heat inactivated FCS, 1% penicillin/streptomycin and left to adhere overnight. After removal of 100 μl of media cells were stimulated with the TLR ligands Pam3CysSK4 (100ng/ml), imiquimod (10μg/ml) , LPS , CpG 1668  for 24 h. Subsequently, conditioned microglial supernatants were transferred to naïve γδ T cells (30x103/96-well in 100 μl complete RPMI), or naïve γδ T cells were co-cultured with stimulated microglia at a 1:1 ratio. After indicated time points cells were collected for flow cytometry and supernatants were recovered for ELISA or multiplex analysis of cytokines, as indicated. TLR stimulation of bone marrow-derived macrophages was carried out likewise. For neutralization of IL-1β and IL-23, conditioned microglial supernatants were pre-incubated for 1 h at 4°C with 10 μg/ml anti-IL-1β (clone B122), anti-IL-23  or respective isotype controls  before supernatants were used for incubation of naïve γδ T cells.Microglia were plated at 30x105/48-well), and polarized IL-17+ γδ T cells including their culture media were added in indicated amounts and cultured for up to 96 h. Addition of complete RPMI served as a control. For co-cultures of neurons and IL-17+ γδ T cells that were induced by supernatants from microglia or BMDMs activated through TLRs, 2x106/ml naïve γδ T cells were cultured for 3 days with conditioned supernatants . Subsequently, γδ T cells and the respective supernatant were transferred to DIV3-neurons  and cells were cultured for 5 days. For ternary co-cultures of γδ T cells, neurons and microglia, 1x104 microglia were added simultaneously with γδ T cells to DIV3-neurons and cells were cultured for 3 days.To generate co-cultures of neurons and polarized IL-17+ γδ T cells, half of the media was removed from DIV3-neurons , washed with phosphate buffered saline (PBS) and were then incubated with the primary antibody: anti-NeuN, anti-neurofilament , anti-Iba1  and anti-CD3 (eBioscience) overnight at 4°C. Subsequently, cell cultures were incubated with the relevant secondary antibody  for 1 h at room temperature. DAPI staining was performed, as previously described . ImmunofViability of neurons was analyzed by quantifying NeuN+ cells in 6 to 8 fields (at 60x magnification) per cover slip, and the mean was calculated. Data are expressed as relative to control (= 100%). All quantifications were performed in a blinded manner. For analysis of apoptotic cells, DAPI+ nuclei were counted in 6 fields (at 100x magnification). After visual verification of apoptotic hallmarks such as shrinkage and fragmentation, amount of apoptotic nuclei were expressed relative to all DAPI+ nuclei in the same high power field as fold change to control.Multiple analyte detection of cytokines and chemokines in supernatants was performed using FlowCytomix (eBioscience).Amounts of IL-17, IL-1β, IL-18, or granzyme B in cell culture supernatants were determined according to the manufacturer’s manual by ELISA .specific antibody-MFIisotype.For cell surface staining, CD3e (clone 145-2C11), γδTCR (clone GL-3), CD25 (clone PC61.5), CD69 (clone H1.2F3) and CD62L (clone Mel-14) antibodies were obtained from eBioscience . TCR Vγ staining was performed with Vγ1.1 (clone 2.11), Vγ2 (clone UC3-10A6), Vγ3 (clone 536) obtained from BioLegend and Vγ5 (clone F2.67), kindly provided by P. Pereira . The nomenclature of TCR Vγ determinants used is that of Garman et al . For intp < 0.05 with * p < 0.05, ** p < 0.01, *** p < 0.001.Statistical differences as indicated in the figure legends were calculated using GraphPad Prism version 5.0a for Mac OS X . Differences were considered statistically significant when CD4+ Th17 cells were described to directly interact antigen independently with neurons and to induce axonal damage and neuronal cell death . To detein vitro.In summary, activated IL-17+ γδ T cells interact with neurons leading to neuronal cell death To examine protein expression of TLR1, TLR2, TLR4, TLR7, TLR9, and MyD88 on naïve γδ T cells flow cytometry was performed using freshly isolated and purified γδ T cells. Whereas pronounced intracellular expression of TLR7, TLR9, and MyD88 was detected, cell surface staining revealed low expression of TLR2 and almost no expression of TLR1 or TLR4 . To deteTaken together, γδ T cells express TLR2, TLR7, TLR9, and the central TLR adapter molecule MyD88.in vitro [Microglia constitutively express mRNA of most of the TLRs identified so far in vitro . Upon TLin vitro , 29, 30.in vitro , we inveIn summary, microglia activated via TLR2, TLR4, TLR7, and TLR9 secrete soluble factors inducing IL-17+ γδ T cells that exhibit an activated phenotype.in vitro. Subsequently, supernatants were collected after 24 h and were used for the incubation of γδ T cells. After 48 h, γδ T cells were analyzed in terms of activation using the markers CD25, CD69, CD62L, and were tested for amounts of IL-17 in the supernatants. In contrast to supernatants derived from microglia activated through TLRs, only supernatants from BMDMs activated by Pam3CysSK4 and LPS, but not by imiquimod or CpG, induced significant up-regulation of CD25 and CD69  were incubated with the TLR ligands Pam3CysSK4, LPS, imiquimod, and CpG and CD69 . Likewisand CD69 . AlthougThese results indicate that activation of γδ T cells through peripheral immune cells differs from activation of γδ T cells through microglia, the major immune cells in the CNS.As outlined above, mere stimulation of γδ T cells through TLRs did not induce IL-17 secretion. Yet, stimulation of microglia through TLRs drove the production of soluble components secreted from microglia, which then induced IL-17 secretion in γδ T cells. This effect may be due to stimulation of TLRs on γδ T cells by TLR ligands still present in the microglial supernatant and to effector molecules secreted by microglia activated through TLRs. Therefore, wild-type microglia were stimulated with Pam3CysSK4 and imiquimod that elicited a strong IL-17 response in γδ T cells see . After 2IL-1β, IL-18, and IL-23 play a key role in the induction of IL-17 in γδ T cells , 23. To To investigate which other cytokines are produced by IL-17+ γδ T cells supernatants from microglia stimulated by TLR ligands were tested by multiplex assay and ELISA. A striking similarity between polarized γδ T cells and γδ T cells induced by microglial supernatants in terms of the cytokine response including IL-13, IL-17, IL-22, and IFN-γ was observed . SupernaTo determine whether IL-1β and IL-23 derived from microglia contribute to the activation of γδ T cells, microglial supernatants were treated with neutralizing antibodies to IL-1β, IL-23, both cytokines, or isotype control antibodies before addition to γδ T cells. Whereas no effect on the expression of CD62L was detected, inhibition of IL-1β or IL-23 led to a reduction of CD25 and CD69 expression on γδ T cells. Simultaneous inhibition of IL-1β and IL-23 had no additive effect and a reduction to baseline levels of γδ T cells incubated with supernatant derived from unstimulated microglia was not achieved . A signiIn summary, these results indicate a role for IL-1β and IL-23, but not IL-18, secreted from microglia in the activation of γδ T cells and induction of IL-17 production.To elucidate if IL-17+ γδ T cells induced by supernatants from microglia activated through TLRs can be neurotoxic, γδ T cells were incubated with supernatants of cultured microglia stimulated with Pam3CysSK4 (TLR2), LPS (TLR4), or CpG (TLR9) for 3 days. Supernatants from unstimulated microglia served as a control. Subsequently, γδ T cells were co-cultured with cortical neurons, and neuronal viability was evaluated after another 5 days. As a further negative control, neurons were incubated with the respective TLR ligand or with microglial supernatants only. The TLR7-specific ligand imiquimod, which is known to induce major cell-autonomous neurotoxic effects , 33, wasTo determine whether the presence of microglia has an impact on to the neurotoxic capacity of activated γδ T cells, the experimental set-up with wild-type neurons as described above was repeated, and co-cultures of γδ T cells and neurons were additionally supplemented with microglia . The preTo investigate whether supernatants from BMDMs activated through TLR2, TLR4, or TLR9, which differed from microglial supernatants in terms of activating γδ T cells see , also diin vitro.Taken together, microglia activated through TLR2, TLR4, or TLR9 can contribute to activation of γδ T cells and promote their maturation into neurotoxic effector cells -/- mice exhibit a reduced disease severity and delayed onset of EAE. Further, IL-17 derived from γδ T cells helps to promote IL-17 production in CD4+ T cells [in vitro studies reveal that IL-17 is not neurotoxic by itself, but toxic effects require activated IL-17+ γδ T cells. In line with our results Wang et al. showed that addition of IL-17 to cultured hippocampal neurons under normoxic conditions had no effect on neuronal cell death, whereas oxygen-glucose depriviation stress led to up-regulation of IL-17 receptor (IL-17R) on neurons and neuronal apoptosis after addition of IL-17 [et al. showed that neuronal NF-κB activator 1 (Act1), an intracellular adaptor in IL-17 signaling, is dispensable for Th17-mediated EAE pathogenesis [The current study demonstrates that supernatants from microglia activated through TLRs induce neurotoxic IL-17+ γδ T cells. It is well established that IL-17 induces the expression of pro-inflammatory genes in fibroblasts, endothelial cells and macrophages, permeabilizes the blood brain barrier and mediates recruitment of neutrophils into the CNS , 36, 37. T cells , 38. Acc T cells , 40. Als T cells . Howeverof IL-17 . Similarof IL-17 . On the ogenesis . Thus, Iogenesis .We have detected TLR2, TLR7, TLR9, and MyD88, but hardly any TLR1 or TLR4 on γδ T cells isolated from peripheral lymph nodes and spleen. However, naïve peripheral CD44+ γδ T cells were found to express TLR2, but not TLR4 on protein level . Furtherγδ T cells express IL-1R1 and IL-23R, and IL-1β can synergize with IL-23 to promote IL-17, -22, and IFN-γ release from γδ T cells , 23, 47.Surprisingly, while IL-17 production by γδ T cells was increased by incubation with supernatants derived from TLR-stimulated microglia, the presence of microglia activated through TLR4, TLR7, or TLR9 reduced the production of IL-17 and IFN-γ by γδ T cells. Since activated microglia do not only contribute to neurodegeneration, but can also play a beneficial role in neuroprotection , one canWe found that co-stimulation by secretory molecules and agonists specific for TLR2 and TLR7 was not necessary for IL-17 induction in γδ T cells since γδ T cells from the respective TLR-deficient mice were still able to produce IL-17 upon incubation with microglial supernatants. Still, signaling that leads to the production of IL-17 must involve MyD88, since MyD88KO γδ T cells were not capable of producing IL-17. In general, MyD88 signaling is required for both TLR and IL-1R1 signal transduction , 52. YetInduction of IL-17 in γδ T cells in the presence of other immune cells such as bone marrow-derived dendritic cells has been described before . We showin vitro, and these effects required cell-cell contact between neurons and T cells. In a mouse model of ischemia/reperfusion injury infiltrating macrophages were the main source of IL-23 after ischemia, promoting IL-17 production of γδ T cells. γδ T cells were shown to localize to the infarct boundary zones [in vitro (data not shown). Close contact between γδ T cells and neurons may point to the involvement of other, yet unidentified, cellular mechanisms mediating neurotoxicity. Granzyme B might be one effector molecule mediating neuronal injury as has been described for CD4+ Th17 cells [We observed neurotoxic effects of IL-17+ γδ T cells ry zones , 36. Locry zones . Also, nry zones . However17 cells . However17 cells . FurtherIn summary, soluble factors, including IL-1β and IL-23 secreted from microglia stimulated by TLR-specific ligands, induce MyD88-dependent activation of γδ T cells. Such activated γδ T cells resemble IL-17+ γδ T cells generated by the addition of IL-1β and IL-23. The presence of IL-17+ γδ T cells but not naïve γδ T cells, results in toxic effects towards neurons that require a direct cell-cell contact between neurons and γδ T cells.S1 Fig(A) Microglia were stimulated with Pam3CysSK4 (100 ng/ml) or LPS (100 ng/ml) for 24 h. Unstimulated cells served as a control. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical TLR7KO neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml) or imiquimod (10 μg/ml) alone served as controls. Subsequently, cultures were immunostained with antibodies against CD3, NeuN, and neurofilament. NeuN-positive cells were quantified and expressed as relative neuronal viability. Each condition was performed in duplicate and averaged. Mean ± SEM of 3–5 individual experiments with ANOVA followed by Bonferroni multiple comparison post test, p<0.0001. (B) Primary cortical neurons derived from wild-type (WT) or TLR7KO mice were stimulated with loxoribine (1 mM). Untreated cells served as controls. After 5 days cultures were immunostained with antibodies against NeuN and neurofilament. NeuN-positive cells were quantified and expressed as relative neuronal viability. Each condition was performed in duplicate and averaged. Mean ± SEM of 2–3 individual experiments with ANOVA followed by Bonferroni multiple comparison post test, p<0.0001.(TIF)Click here for additional data file.


Telomere dysfunction is important in carcinogenesis, and recently, stathmin and elongation factor 1α (EF1α) were reported to be up-regulated in telomere dysfunctional mice.WAF1/CIP1 expression were assessed in specimens of hepatitis B virus (HBV)-related multistep hepatocarcinogenesis, including 13 liver cirrhosis specimens, 14 low-grade dysplastic nodules (DN), 17 high-grade DNs, and 14 hepatocellular carcinomas (HCC). Five normal liver specimens were used as controls. TIF were analyzed by telomere fluorescent in situ hybridization (FISH) combined with immunostaining, while the protein expressions of stathmin, EF1α, γ-H2AX, and p21WAF1/CIP1 were detected by immunohistochemistry.In the present study, the expression levels of stathmin and EF1α in relation to telomere length, telomere dysfunction-induced foci (TIF), γ-H2AX, and p21P < 0.05). EF1α histoscores were higher in high-grade DNs than in normal liver and liver cirrhosis . Stathmin mRNA levels and histoscores, as well as EF1α histoscores (but not mRNA levels), were positively correlated with telomere shortening and γ-H2AX labeling index . EF1α histoscores were also positively correlated with TIF (P < 0.001). Significantly greater inactivation of p21WAF1/CIP1 was observed in low-grade DNs, high-grade DNs, and HCC, compared to liver cirrhosis . p21WAF1/CIP1 labeling index was inversely correlated with TIF, stathmin mRNA level, and EF1α histoscore .The expressions of stathmin and EF1α gradually increased as multistep hepatocarcinogenesis progressed, showing the highest levels in HCC. Stathmin mRNA levels were higher in high-grade DNs than normal liver and liver cirrhosis, whereas EF1α mRNA expression did not show such a difference. The protein expressions of stathmin and EF1α were found in DNs of precancerous lesions, whereas they were absent or present at very low levels in normal liver and liver cirrhosis. Stathmin histoscores were higher in high-grade DNs and low-grade DNs than in normal liver (all, WAF1/CIP1 in HBV-related multistep hepatocarcinogenesis. Accordingly, assessment of stathmin and EF1α levels as a reflection of telomere dysfunction may be helpful in evaluating the biological characteristics of precancerous hepatic nodules in hepatitis B viral cirrhotic patients.Stathmin and EF1α are suggested to be closely related to telomere dysfunction, DNA damage, and inactivation of p21 Hepatocellular carcinoma (HCC) is reported as the fifth most common cancer worldwide and the third most common cause of cancer-related deaths . HepatocSTMN1 gene, is a major microtubule depolymerizing protein involved in cell cycle progression [Previously, expression of stathmin and elongation factor 1α (EF1α) was reported to be up-regulated in telomere dysfunctional mice . Stathmigression . Stathmigression . In mitogression . Stathmigression -13. Moregression . Stathmigression ,15. EF1αgression . Overexpgression . A previgression . Additiogression .WAF1/CIP1 expression in human HBV-associated multistep hepatocarcinogenesis.The assessment of telomere dysfunction may be helpful in evaluating the biological characteristics of hepatic nodules in cirrhotic patients. However, systemic assessment of the expression levels of stathmin and EF1α in relation to telomere dysfunction, particularly telomere length and telomerase dysfunction induced foci (TIF), has not been conducted in defined lesions of human multistep hepatocarcinogenesis, including DNs. Accordingly, the present study attempted to evaluate and compare the expression levels of stathmin and EF1α in relation to telomere length, TIF, γ-H2AX, and p21A total of 58 liver specimens from 24 patients were investigated, including 13 cases of liver cirrhosis, 14 cases of low-grade DNs, 17 cases of high-grade DNs, and 14 cases of HCC. The specimens were obtained from 19 men and five women whose ages ranged from 32 to 63 years . They were all serum HBsAg-positive and anti-HCV-negative. The clinicopathological features of the patients are summarized in Table Total RNA was isolated from human fresh frozen liver tissue samples using TRIzol reagent . The transcription thereof into cDNA was performed using a High Capacity RNA-to-cDNA kit . The following TaqMan Gene Expression Assays were purchased from Applied Biosystems: EF1α (Hs00265885_g1), stathmin (Hs01027516_g1), and GAPDH (Hs99999905_m1). All measurements were performed in triplicate. The relative expression levels of target mRNAs were normalized to GAPDH mRNA levels.PCR was performed in triplicate for each cDNA sample using the ABI PRISM 7300 Sequence Detection System (Applied Biosystems). Dual-labeled FAM probes containing a 5′-fluorescent reporter and a 3′- quencher were used to conduct the PCR experiments.WAF1/CIP1, and γ-H2AX, as previously described [Immunohistochemical staining was performed to detect the expression levels of stathmin, EF1α, p21The staining intensities of stathmin and EF1α were graded on a scale of 0–3 , and the extent of distribution was rated on a scale of 0–4 . Histoscore was defined as the sum of the intensity and distribution scores. For interpretation of the immunohistochemical stain results for γ-H2AX, dark brown stained nuclei were counted as being positive for antibody, and labeling indices were determined as follows: (number of positive hepatocytic nuclei in five randomly selected fields at × 400 magnification)/ × 100%.4  and detected as recommended by the manufacturer. The resulting X-ray film was scanned with a luminescent image analyzer , and the telomere signals in each lane were quantified as a grid object, defined as a single column with 25 rows, using Image Gauge Software 2.54 (Fujifilm).Telomere terminal restriction fragments were measured as previously described . Brieflyin situ hybridization using a telomere-specific peptide nucleic acid probe (Cy3-(CCCTAA)3; Panagene, Daejeon, Korea). Tissue sections were deparaffinized and then rehydrated with graded alcohol. After incubation in 0.2 N HCl for 20 min, slides were boiled in 10 mM citrate buffer, pH 6.0, for 15 min to retrieve antigens. Sections were then treated with protease solution  and fixed. Sections were dehydrated in 100% ethanol for 5 min. After air-drying, slides were applied 10 μL of a telomere peptide nucleic acid probe mixture , denatured at 80°C for 3 min, and hybridized at 30°C for 2 hrs. Slides were then washed sequentially with 0.6X SSC/70% formamide , 2X SSC (2 × 15 min), PBS (1 × 5 min), and PBST (PBS + 0.1% Tween 20:15 min), and then blocked with 5% BSA for 5 min. Immunostaining using primary polyclonal anti-γ-H2AX  and secondary Alexa Fluor 488-conjugated goat anti rabbit antibody  was performed.TIF were detected in representative sections of formalin-fixed paraffin-embedded tissues by staining for γ-H2AX and by telomere http://www.R-project.org), applying the Mann–Whitney test, Spearman’s correlation coefficient, linear model, and log linear model as deemed appropriate. Significance was set at P < 0.05 for all tests.Statistical analysis was conducted using SPSS  and R package software  . Stathmin mRNA levels in HCC were significantly higher than those in normal liver, liver cirrhosis, and low-grade DNs .The mRNA levels of stathmin were 0.3 ± 0.12 (mean ± SD)  in normal liver, 0.5 ± 0.61 (0.1 ~ 2.1) in liver cirrhosis, 0.8 ± 0.57 (0.1 ~ 2.0) in low-grade DNs, 1.5 ± 1.29 (0.1 ~ 4.7) in high-grade DNs, and 2.3 ± 1.95 (0.4 ~ 7.0) in HCC. Stathmin mRNA levels gradually increased as multistep hepatocarcinogenesis progressed from normal liver, low-grade DNs, and high-grade DNs to HCC, which showed the highest level of expression and statistical significance (P for trend < 0.001) .Similarly, the expression of stathmin protein also gradually increased as multistep hepatocarcinogenesis progressed towards HCC  Figure B. Most nP = 0.009), whereas liver cirrhosis and low-grade DNs showed lower levels of EF1α mRNA expression than normal liver (P < 0.05 for both). The mRNA expression of EF1α showed significant increases as multistep hepatocarcinogenesis progressed towards HCC (P for trend = 0.035).The mRNA levels of EF1α were 2.9 ± 0.71 (mean ± SD)  in normal liver, 1.9 ± 0.83 (0.2 ~ 3.2) in liver cirrhosis, 1.6 ± 0.74 (0.3 ~ 3.1) in low-grade DNs, 2.5 ± 1.76 (0.9 ~ 8) in high-grade DNs, and 3.9 ± 3.19 (1.1 ~ 13.2) in HCC . HCC showed the highest EF1α histoscore, which was significantly higher than that for low-grade DNs, liver cirrhosis and normal liver . EF1α histoscores were also significantly greater in high-grade DNs than in liver cirrhosis (P = 0.002) and normal liver (P = 0.013). The histoscores of EF1α and stathmin were shown to be positively correlated , whereas EF1α and stathmin mRNA levels showed no significant correlation .EF1α protein was not expressed in normal liver, but was detected in dysplastic and HCC cells Figure C. The hiP for trend = 0.032); part of this data was previously reported [Telomere length gradually decreased as multistep hepatocarcinogenesis progressed towards HCC, with statistical significance . EF1α histoscore showed a significant negative correlation with telomere length    . γ-H2AX labeling index in HCC was significantly higher than that in normal liver, liver cirrhosis, low-grade DNs, and high-grade DNs . Additionally, γ-H2AX labeling index showed a significant negative correlation with telomere length ; higher expression of γ-H2AX labeling index was found in lesions with shorter telomere lengths   in liver cirrhosis, 2.4 ± 5.67 (0 ~ 21.3) in low-grade DNs, 4.1 ± 10.42 (0 ~ 40.2) in high-grade DNs, and 10.3 ± 17.00 (0 ~ 49.0) in HCC , indicating that an increase in stathmin expression is associated with DNA damage    ; high-grade DNs showed a significantly higher number of TIF, compared to normal liver and liver cirrhosis ; and low-grade DNs showed a significantly higher number of TIF than normal liver (P = 0.048). We further analyzed the relationships between TIF and telomere length, as well as TIF and the protein expression levels of stathmin and EF1α. TIF showed a significant negative correlation with telomere length   .In order to determine the fractions of TIF during human multistep hepatocarcinogenesis, we performed telomere FISH combined with immunostaining for anti-γ-H2AX to detect colocalization of telomeres and γ-H2AX foci Figure A. A grad) Figure B. NormalWAF1/CIP1 protein was investigated in multistep hepatocarcinogenesis; part of the data was previously reported [WAF1/CIP1 labeling indices showed gradual statistically significant decreases as multistep hepatocarcinogenesis progressed towards HCC (P for trend = 0.002)  Figure A.WAF1/CIP1 labeling indices were 10.4 ± 6.09 (mean ± SD)  in normal liver, 24.9 ± 13.55 (1.6 ~ 47.2) in liver cirrhosis, 7.9 ± 2.90 (2.7 ~ 11.2) in low-grade DNs, 11.0 ± 11.11 (0.8 ~ 44.7) in high-grade DNs, and 5.9 ± 8.08 (0.4 ~ 30.2) in HCC. p21WAF1/CIP1 labeling index was highest in liver cirrhosis, which was significantly higher than that in normal liver, low-grade DNs, high-grade DNs, and HCC . The p21WAF1/CIP1 labeling index of HCC was the lowest, and was significantly lower than that in low-grade DNs, high-grade DNs, and liver cirrhosis .The p21WAF1/CIP1 labeling index showed a significant negative correlation with TIF    . EF1α histoscore showed a significant negative correlation with p21WAF1/CIP1labeling index, as higher EF1α histoscores were correlated with lower p21WAF1/CIP1labeling index , whereas EF1α mRNA levels showed no significant correlation with p21WAF1/CIP1labeling index   expression with telomere dysfunction and DNA damage in HBV-related multistep hepatocarcinogenesis. A-B. Scatter plots reveal a correlation between EF1α mRNA expression and telomere terminal restriction fragment (TRF) length (A) and γ-H2AX labeling index (B). C. Correlation between telomere dysfunction induced foci (TIF) and stathmin protein level. D. Scatter plot of a correlation between p21Click here for file


A-C (2–4), together with a new natural product 7,8-dihydro-7R,8S-dihydroxy-3,7-dimethyl-2-benzopyran-6-one (1) were obtained from the endolichenic fungus Myxotrichum sp. by using OMSAC  method. The planar structures of these new compounds were determined by NMR experiment and HRESIMS data, and the absolute configuration of 1 was established by X-ray diffraction, and the stereochemistry of the new compounds 2-4 were determined by same biosynthesis origin, and similar CD spectra with 1. Allelopathic test showed that compound 4 significantly retarded root elongation of Arabidopsis thaliana seed, indicating that this fungus might contribute to the defense of its host lichen. From the view of biosynthetic pathway, all four compounds 1-4 might be originated from Non-Reduced Polyketide synthase (NR-PKS).Three new polyketides myxotritones  Lichens are combinations of a fungus (the mycobiont) and an algal partner (the photobiont or phycobiont). In addition to fungal mycobionts, some nonobligate fungi, such as endolichenic fungi, are also found to live asymptomatically in the bodies  of lichensMyxotrichum spMyxotrichum sp., rice culture was used to activate the potentially silent gene clusters, from which three new polyketides myxotritones A-C (2–4), together with a new natural product 7, 8-dihydro-7R, 8S-dihydroxy-3, 7-dimethyl-2-benzopyran-6-one (1) were obtained. In this report, the structural elucidation, biological evaluation, and possible biosynthetic pathway were present.Analysis of a great number genome sequence from different microbes revealed that many secondary metabolite biosynthetic gene clusters are silent under common cultivation conditions, and their metabolic potentials were underestimated. To activate the cryptic gene cluster to express, different methods were innovated1 is identified to be as 7, 8-dihydro-7R, 8S-dihydroxy-3, 7-dimethyl-2- benzopyran-6-one based on the NMR, MS data and optical rotation [α]D22 = +393.0 , which was known as a synthetic compound but never isolated from a natural specimen61 were confirmed by single-crystal X-ray diffraction analysis with Cu Kα radiation  and negative (247 nm) cotton effects (R)-configuration of chiral center at C-7891112in situ dimolybdenum CD spectra permitted the assignment of absolute configuration as 7R, 8S  was isolated as a yellow powder, [α]D22 = +45.6 . Its molecular formula was determined as C22H22O8 (12 degrees of unsaturation) by TOF-ESI-MS spectral data, which showed a pseudomolecular ion at m/z 437.1209 [M + Na]+ (2 displayed the maximum absorptions at 217 nm (log ε 4.21), 256 nm (log ε 3.86) and 364 nm (log ε 3.97) , two methylenes with one oxygenated, an oxymethine unit, an oxygenated quaternary carbon, 12 olefinic carbons, an ester carbonyl carbon, and a keto carbonyl group, which explained all carbon signals of 2. Analysis of the 1H and 13C-NMR data of 2 revealed the same structural fragment (subunit A) as 1, except the H-5 in 1 was replaced by other moiety in 2, and this conclusion was supported by HMBC correlations  led to construct an isobenzofuran-1(3H)-one fragment (subunit B). The correlations from 10′-CH2- to C-4a, C-5 and C-6 connected the subunit A with subunit B  and negative (259 nm) cotton effects in the CD spectrum  , revealielations . The remubunit B . Considespectrum . Based o3) was obtained as yellow powder, [α]D22 = +6.0 . The molecular formula of 3 was deduced as C23H24O9 on the basis of its TOF-ESI-MS spectrum, in which a pseudomolecular ion was observed at m/z 467.1313 [M + Na]+  was obtained as yellow needle, [α]D22 = +403.3 . Its molecular formula was assigned as C11H12O5 (6 degrees of unsaturation) by TOF-ESI-MS spectral data, which showed a pseudomolecular ion at m/z 225.0760 [M + H]+, 247.0573 [M + Na]+  replaced by a methylol signal (δC 59.9/δH 4.29). This hypothesis was further confirmed by HMBC correlations 4) was ob1–4, all these four compounds might come from same Non-Reduced Polyketide synthase (NR-PKS) origins. The putative biosynthetic pathway was suggested in the From the structural features of compounds A. thaliana as model. The root growth of A. thaliana was inhibited after treatment with the compounds in a dose dependent manner  as references. HRESIMS data were acquired using a LTQ Orbitrap XL Mass Spectrometer .Optical rotations were measured on a Perkin-Elmer 241 Polarimeter  and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were obtained on a Chirascan spectropolarimeter. Myxotrichum sp. was isolated from the lichen Cetraria islandica (L.) Ach. collected from Laojun Mountain, Yunnan Province, People’s Republic of China. The isolate was identified on the basis of the internal transcribed spacer region sequences of the rDNA (Genbank Accession No. HQ324780) (The endolichenic fungus Q324780)  and the 2Cl2–MeOH gradient elution. The fraction (611 mg) eluted with CH2Cl2–MeOH 50:1 was separated by Sephadex LH-20  column chromatography eluting with MeOH to afford 4 subfractions. The resulting subfraction 1 was further purified by semipreparative RP HPLC  to afford 1 , subfraction 4 was purified by RP-HPLC  to afford myxotritone A  and myxotritone B . Fraction (863 mg) eluted with CH2Cl2–MeOH 10:1 were fractionated again by Sephadex LH-20 column chromatography using MeOH as eluent. Purification of the subfraction by RP-HPLC  afforded myxotritone C .The fermented material was extracted with ethyl acetate (6 L for four times). The solution was concentrated to dryness under vacuum to afford a crude extract (22.0 g), which was fractionated by silica gel column chromatography (10 × 100 cm) using CH7R, 8S -7, 8-dihydro-7, 8-dihydroxy-3, 7-dimethyl-2- benzopyran-6-one (1): Brown powder, [α]D22 = +393.0° ; UV(MeOH) λmax (log ε) 349 (4.45) nm; CD (MeOH) 205 (Δε-0.21), 225 (Δε + 0.06), 247 (Δε−0.55), 311 (Δε + 0.34), 362 (Δε + 0.40) nm; IR (KBr) Vmax: 3430, 2981, 2937, 1717, 1645, 1602, 1546, 1457, 1394, 1369, 1326, 1087, 1298, 990 967, 893 cm−1; 1H NMR and 13C NMR, see m/z 209.0813 [M + H]+ .2):Myxotritone A , UV (MeOH) λmax (log ε) 217 (4.21) nm; 256 (3.86) nm; 364 (3.97) nm; CD (MeOH) 230 (Δε + 1.47), 259 (Δε −1.47), 371 (Δε + 0.48) nm; IR (KBr) Vmax: 3436, 2951, 2843, 1732, 1674, 1661, 1599, 1485, 1329, 1279, 1187, 1146, 1098, 1020, 962, 909, 879, 847, 825 cm−1; 1H NMR and 13C NMR, see m/z 415.1394 [M + H]+ , 437.1209 [M + Na] + .3):Myxotritone B ; UV (MeOH) λmax(log ε) 221 (3.91), 256 (3.56) and 370 (3.61) nm; CD (MeOH) 226 (Δε + 0.77), 264 (Δε -0.82), 371 (Δε + 0.44) nm; IR (KBr) Vmax: 3436, 2954, 2844, 1636, 1517, 1457, 1448, 1387, 1350, 1339, 1277, 1187, 1101, 1064, 1032, 1017, 966 cm−1; 1H NMR and 13C NMR, see m/z 467.1313 [M + H]+ .4):Myxotritone C ; UV (MeOH) λmax(log ε) 233 (3.86), 243 (3.83) and 348 (4.46) nm; CD (MeOH) 247 (Δε -1.94), 312 (Δε + 1.28), 366 (Δε + 1.39) nm; IR (KBr) Vmax: 3430, 2913, 2864, 1674, 1653, 1623, 1616, 1545, 1512, 1444, 1398, 1373, 1326, 1270, 1235, 1160, 1123, 1061, 1015, 970, 930, 883, 830 cm−1; 1H NMR and 13C NMR, see m/z 225.0760 [M + H]+ .Arabidopsis thaliana seeds were surface sterilized by 5% sodium hypochlorite for 5 min, followed by washing with sterile distilled water for five times. Compounds were dissolved with DMSO to final concentration of 40 mg/mL. Then 20 μL of them were added to 25 mL 1/2 MS medium supplemented with 0.8% (w/v) agar to get plates with different concentrations of compounds . To eliminate the effect of DMSO on the growth of A. thaliana, plates with 20 μL DMSO were used as blank control. Fifteen seeds were distributed on each Petri dishes described before. Each concentration was conducted in triplicate. The Petri dishes were placed in a growth chamber at 23 ± 1 °C under light for 8 h and darkness for 6 h. The lengths of roots were measured after 9 days. The percentage of growth inhibition of root lengths was calculated as the following equation:where T stands for the average length of treatment (cm) and C stands for the average length of control (cm)14How to cite this article: Yuan, C. et al. Allelopathic Polyketides from an Endolichenic Fungus Myxotrichum SP. by Using OSMAC Strategy. Sci. Rep. 6, 19350; doi: 10.1038/srep19350 (2016).


SYP-F-7919), a fungus obtained from the rhizosphere soil of Panax notoginseng collected from the Yunnan province of China. Their structures were elucidated by extensive NMR analyses, a modified Mosher’s method, chemical derivatization and single crystal X-ray diffraction analysis. Compounds 2–4 exhibited potent cytotoxicity against the U937 and MCF-7 tumour cell lines and showed moderate cytotoxic activity against the SH-SY5Y and SW480 tumour cell lines. The substitution of an acetyloxy or 2-hydroxypropionyloxy group at C-7 significantly increased the cytotoxic activity of the resorcylic acid lactone derivatives. Subsequently, the possible mechanism of compound 2 against MCF-7 cells was preliminarily investigated by in silico analysis and experimental validation, indicating compound 2 may act as a potential MEK/ERK inhibitor. Moreover, proteomics analysis was performed to explore compound 2-regulated concrete mechanism underlying MEK/ERK pathway, which is still need further study in the future. In addition, compounds 2–4 and 7 exhibited a significant inhibitory effect on NO production induced by LPS.Five new 12-membered resorcylic acid lactone derivatives, penicimenolides A-E ( Panax notoginseng is widely distributed in Yunnan, Guangxi and Jiangxi province of China and its roots have been used as a traditional Chinese medicine for the treatment of hemorrhages, blood stasis and improvement of blood circulation and remission painPanax notoginseng have been receiving increasing attention, leading to the various secondary metabolites with antimicrobial2Panax notoginseng, a strain of Penicillium sp. (SYP-F-7919) has drawn our interest because the EtOAc extract of the culture broth exhibited typical resorcylic acid lactones (RALs) UV absorptions (λmax) at 215, 264 and 297 nmLasiodiplodia theobromae7Penicillium sp.Syncephalastrum racemosum10, Pyrenophora teresAcremonium zeae12, lasiodiplodin, was isolated in 197112 compounds had been obtained67891011121314151617181920212223242515121017192012 derivatives, penicimenolides A-E (1–5), a new ring-opened resorcylic acid lactone derivative penicimenolide F (6), and six known biogenetically related derivatives (7–12)  in murine macrophages (RAW 264.7) activated by lipopolysaccharide (LPS). Herein, we report the isolation, structure elucidation, absolute configuration, bioactivities and preliminary mechanism of the compounds obtained from the Penicillium sp. SYP-F-7919.RALs are a class of fungal polyketide derivatives that are produced by a variety of fungal strains, such as s (7–12)  were isoPenicillium sp. was isolated by a combination of column chromatography, including silica gel, ODS, Sephadex LH-20, and reversed phase high performance liquid chromatography (HPLC) to yield twelve resorcylic acid lactone derivatives (1–12).The ethyl acetate extract of the culture broth of the fungus 1) was isolated as colourless needles, c 0.5, MeOH). Its molecular formula was determined to be C16H18O5 by HRESIMS at m/z 291.1231 [M + H]+ . The IR spectrum of 1 revealed the presence of hydroxyl group(s) at 3384 cm−1, carbonyl group(s) at 1708 and 1642 cm−1 and an aromatic ring at 1605 and 1449 cm−1. A comparison of the 1H and 13C NMR spectroscopic data  indicated an E configuration for the double bond. The position of the double bond was confirmed by the 1H-1H COSY correlations of H-6/H-5 and H-7/H-8  (6) was proposed to be an R configuration because of a shared biogenesis. For compound 1, this conclusion was further confirmed by comparing the optical rotation value with 8 (1 was identified to be (3R), (6E)-etheno-9-oxo-de-O-methyllasiodiplodin.Penicimenolide A (pic data  for 1 wi H-7/H-8 . Because (Cu Ka) , the asy2) was obtained as colourless needles, c 0.5, MeOH). The molecular formula C18H22O7 was confirmed by HRESIMS at m/z 351.1435 [M + H]+ . Except for an additional acetyl group, the 1H and 13C NMR data -7-acetoxyl-9-oxo-de-O-methyllasiodiplodin.Penicimenolide B  was obtained as yellow needles, c 0.5, MeOH) and was determined to have a molecular formula of C19H24O8 on the basis of HRESIMS analysis . The 1H and 13C NMR data for 3 . The HMBC correlations from H-7 (δH 5.51), H-2′ (δH 4.21) and H3-3′ (δH 1.35) to C-1′ (δC 175.1), in combination with the 1H-1H COSY correlation between H3-3′ and H-2′, revealed the presence of a 2′-hydroxypropionyloxy fragment attached to C-7 (3. The absolute configuration of C-2′ in 3 was established using the modified Mosher method3 was treated separately with (R)- and (S)-MTPA-Cl to obtained the respective (S)- and (R)-MTPA esters (3a and 3b). The R configuration of C-2′ was determined by the positive ΔδH(S-R) value of H3-3´ and the negative ΔδH(S-R) value of H-7 -7-(2′-hydroxypropionyloxy)-9-oxo-de-O-methyllasiodiplodin.Penicimenolide C (ta for 3  were simd to C-7 . Accordi H-7 see . In addi4) was isolated as a yellowish oil, c 0.5, MeOH). The HRESIMS of 4  showed the same molecular formula as 3. The 1H and 13C NMR spectroscopic data for 4 -7-(2′-hydroxypropionyloxy)-9-oxo-de-O-methyllasiodiplodin.Penicimenolide D  with relative energy from 0-18 kcal/mol used in optimizations at the B3LYP/6-31G(d) level using Gaussian09 package3) with relative energy from 0 to 10 kcal/mol and conformers (28 lowest energy conformers for 4) with relative energy from 0 to 10 kcal/mol were then re-optimized at the B3LYP/6-311+G(d) level. ECD computations for all conformers were carried out at the B3LYP/6-311++G level in the gas phase. Boltzmann statistics were performed for ECD simulations with standard deviation of σ 0.22 eV. The computed ECD of 3 and 4 were looked similar to the experimental values of 3 and 4, respectively (3 and 4 were further confirmed.To compare computed electronic circular dichroism (ECD) with experimental results is a valid method to assign absolute configurations of natural products28293031ectively . Thus, t5) was obtained as a yellowish oil, c 0.25, MeOH). Its molecular formula was confirmed to be C16H20O5, which was deduced by the HRESIMS . The detailed analysis of the 1H and 13C NMR (5 possessed a similar structure to trans-resorcylideδC 201.7) in trans-resorcylide by an oxygenated methine (δC 74.9) in 5, which was further proven by the HMBC correlations from H-7, H-8, and H-10 to C-9 - and (R)-MTPA esters (5a and 5b). The negative ΔδH(S-R) value of H-10 and the positive ΔδH(S-R) value of H3-17, H-4, H-5 and H-6 unambiguously confirmed the S configuration of C-9 -(7E)-etheno-9-hydroxy-de-O-methyllasiodiplodin.Penicimenolide E ( 13C NMR  spectros0 to C-9 . To conf C-9 see . Based o6) was isolated as a yellowish oil, c 0.25, MeOH). The HRESIMS exhibited a quasimolecular ion peak at m/z 285.0971 [M + H]+ , indicating a molecular formula of C13H16O7. The 1H NMR spectrum of 6  and 6.19 , two methyl signals at δH 3.68  and 2.45 , four methylene protons at δH 4.47  and 2.06/2.24 , and one methine proton at δH 4.32 . The 13C NMR and DEPT spectra revealed the presence of 13 carbon resonances, including two ester carbonyls , six aromatic carbons, two methyls (one oxygenated), two methylenes (one oxygenated) and one oxygenated methine. The HMBC correlations - and (R)-MTPA esters (ΔδH(S-R)) at H-1´, H-2´ and H3-5´ were analyzed to confirm the absolute configuration of C-3´ as R (see 6 was determined to be (3′R)-3′-hydroxy-4′-oxo-4′-methoxy-orsellinate.Penicimenolide F 8)S, 14R)-13-hydroxydihydroresorcylide (9)S)- methoxyresorcylide (10)R)-methoxyresorcylide (11)12)In addition, six known compounds were isolated and identified as 2–5, 7–12) were evaluated for their cytotoxic activity against six human tumour cell lines, including U937, MCF-7, A549, SH-SY5Y, HepG2 and SW480  of the equalized trajectory was extracted, we found that compound the MEK1 . For ERKectively . Moreove2 system, RMSD of the complex were process about 2ns equilibrium during the whole simulation  fluctuation was an important criterion to gauge whether the protein-ligand system was stable. For MEK1-compound mulation . During d system . Notably protein . The simmulation .In vitro experiments were employed with MCF-7 cells to elucidate the possible mechanism of compound 2, as ERK are often excessively activated in breast cancer2 demonstrated potent cytotoxic effect against MCF-7 cells in a time- and concentration-dependent manner, with an IC50 value of 9.893 μM (24 h)  and 27.53 ± 3.21% (p < 0.01), respectively, when treated with 10 μM and 15 μM of compound 2, indicating that compound 2 increases apoptotic ratio in a dose-dependent manner (M (24 h) . In ordec bodies . These centation . In addit manner .2 induced apoptosis, we investigated the involvements of apoptotic markers in compound 2-treated cells. We found that compound 2 significantly upregulated Bax expression as well as downregulated Bcl-2 expression. And the active form of PARP was also increased during compound 2 treatment , as shown in 2-4 and 7 showed a significant inhibitory effect on NO production, with the IC50 values ranging from 0.7 to 5.8 μM. In addition, compounds 5, 8, 10 and 12 had weak activity.All of the isolated compounds, except for The free radical NO, which is produced by the oxidation of L-arginine catalyzed by a group of nitric oxide synthases (NOSs), can be involved in a series of physiological and pathological processesMelting points were determined with a Tektronix X-4 micromelting-point apparatus . The optical rotations were recorded on a JASCO P-1020 digital polarimeter , and the UV spectra were measured with a Shimadzu UV-2201 UV-Vis recording spectrophotometer . The IR data (KBr disks) were measured by a Bruker IFS-55 spectrometer . The CD spectra were obtained using a Bio-Logic MOS-450 . The 1D and 2D NMR spectra were run on Bruker AVANCE-400 or AVANCE-600 NMR spectrometers . The HRESIMS data were obtained on a Waters Synapt G2 QTOF mass spectrometer . The analytical HPLC was performed using an Agilent 1200  equipped with a DAD detector using an ODS column . Semipreparative HPLC was performed using a Shimadzu LC-6AD  equipped with a UV SPD-20A detector using an ODS column . Column chromatography was carried out using silica gel , ODS , and Sephadex LH-20 . Analytical grade solvents were used for the extraction and chromatographic separation.Panax notoginseng, which was collected from Wenshan, Yunnan province of China in November 2012. The fungus was identified on the basis of the DNA sequence of the internal transcribed spacer (ITSI-5.8S-ITS2 region). The sequence data derived from the fungal strain has been submitted and deposited in GeneBank with the accession number KU380346. BLAST search results revealed that the isolate belongs to the genus Penicillium and had a high sequence identity (99%) to the species Penicillium menonorum. A voucher specimen (No. SYP-F-7919) was deposited at the School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University.The fungal strain used in this work was isolated from the rhizosphere soil of 2O) to obtain conidial suspensions. To inoculate Erlenmeyer flasks (500 mL), 1 mL of conidial suspension was used, each containing 200 mL of the medium . Each flask was incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. Large scale fermentation was carried out in 200 Erlenmeyer flasks (500 mL).The fungal strain was cultured on plates of potato dextrose agar (PDA) at 28 °C for 5 days. The plates were washed with double distilled water (ddH2O stepwise gradient to obtain seven subfractions (F1-F7). Subfraction F5  was further purified using a Sephadex LH-20 column that was eluted with CH2Cl2-MeOH (1:1). Then, it was purified by pHPLC using 55% MeOH to yield compounds 1 , 7  and 8 . Fraction G  was applied to an ODS column that was eluted with a gradient of MeOH-H2O to give seven subfractions (G1-G7). Subfraction G3  was purified by pHPLC (25% MeCN) to afford compounds 6  and 5 . Compounds 2 , 10  and 11  were obtained from subfraction G4  by purification with pHPLC using 55% MeOH. Fraction H (3.0 g) was also obtained from the cyclohexane-EtOAc  eluate on silica gel CC and then was separated into ten subfractions (H1-H10) by ODC CC using MeOH-H2O as the eluent. Subfractions H4  and H5  were purified by pHPLC (43% MeOH and 25% MeCN) to yield compounds 9  and 12 , respectively. Compound 4  was purified from subfraction H6  by pHPLC with 33% MeCN. Compound 3  was obtained from the subfraction H7  using pHPLC (33% MeCN).The fermentation broth (48 L) was centrifuged to separate the supernatant from the mycelia. The supernatant was extracted thoroughly with ethyl acetate (3 × 48 L), and the organic solvent was concentrated under reduced pressure to afford the crude extract (18.6 g), which was subjected to column chromatography over silica gel and eluted with cyclohexane-EtOAc (10:0 to 0:10) and EtOAc-MeOH (10:0 to 0:10) to produce eleven fractions (A-K). Fraction F (2.0 g), which was eluted with cyclohexane-EtOAc , was subjected to an ODS column and a MeOH-H2 (5.0 mg) was dissolved in dry pyridine (1.0 mL), and Ac2O (0.5 mL) was subsequently added. After stirring at 40 °C for 48 h, the mixture was concentrated in vacuo to dryness, and the residue was purified by pHPLC with MeCN-H2O  at a flow rate of 4 mL/min to yield 2a (1.0 mg). Following the above procedure, compound 12 was acetylated to yield 12a.A sample of compound 3 (5.0 mg) and 4% NaOH aqueous solution (5 mL) was stirred at 30–40 °C in a water bath for 3 h. The reaction mixture was neutralized with 10% HCl (4 mol/L) and extracted with EtOAc. After the extract was concentrated in vacuo to dryness, the residue was purified by pHPLC, eluting with MeCN-H2O  at a flow rate of 4 mL/min to obtain the hydrolyzate 3c (1.0 mg). Then, 3c was compared to 12 by HPLC analysis, which showed that the retention time of the two compounds were identical ; UV (MeOH) λmax (log ε) 215 (3.88), 265 (3.54), 302 (3.27) nm; CD  λ (Δε) 206 (-1.21), 217 (0.65), 290 (3.25), 330 (−0.05); IR (KBr) νmax 3384, 2920, 1708, 1642, 1605, 1449, 1384, 1262 cm−1; 1H NMR and 13C NMR data, see m/z 291.1231 .colourless needles; mp 148–150 °C; c 0.5, MeOH); UV (MeOH) λmax (log ε) 214 (4.03), 262 (3.74), 302 (3.47) nm; CD  λ (Δε) 204 (−8.26), 224 (3.84), 260 (5.31), 306 (−1.01); IR (KBr) νmax 3405, 2921, 1722, 1646, 1619, 1449, 1384, 1362, 1263 cm−1; 1H NMR and 13C NMR data, see m/z 351.1435 .colourless needles; mp 128–130 °C; c 0.5, MeOH); UV (MeOH) λmax (log ε) 213 (4.05), 262 (3.80), 301 (3.56) nm; CD  λmax (Δε) 208 (−8.05), 225 (1.51), 261 (5.34), 308 (−0.34); IR (KBr) νmax 3426, 2920, 1727, 1644, 1452, 1384, 1261, 1207 cm−1; 1H NMR and 13C NMR data, see m/z 381.1549 .yellow needles; mp 118–119 °C; c 0.5, MeOH); UV (MeOH) λmax (log ε) 220 (4.31), 264 (4.01), 303 (3.85) nm; CD  λmax (Δε) 209 (−2.91), 223 (0.74), 261 (4.81), 302 (−1.61); IR (KBr) νmax 3417, 2920, 1752, 1645, 1606, 1458, 1384, 1261, 1189; 1H NMR and 13C NMR data, see m/z 381.1548 .yellowish oil; c 0.25, MeOH); UV (MeOH) λmax (log ε) 212 (3.94), 256 (3.27), 289 (3.07) nm; CD  λ (Δε) 204 (4.65), 248 (−3.11), 285 (−0.70); IR (KBr) νmax 3408, 2932, 1690, 1618, 1452, 1385, 1349, 1264 cm−1; 1H NMR and 13C NMR data, see m/z 293.1385 .yellowish oil; c 0.25, MeOH); UV (MeOH) λmax (log ε) 220 (4.16), 264 (3.97), 302 (3.59) nm; CD  λ (Δε) 214 (5.49), 265 (−2.27), 298 (−2.18); IR (KBr) νmax 3429, 2919, 1735, 1648, 1448, 1384, 1263 cm−1; 1H NMR and 13C NMR data, see m/z 285.0971 .yellowish oil; 10 were obtained from CHCl3 containing a small amount of MeOH at room temperature. Crystallography data were obtained on an Agilent Super Nova  single crystal X-ray diffractometer with the Cu Ka (λ = 1.54184 Å). Structure solution and refinement were performed with the SHELXL program package. The crystallography data for 10: C17H22O6; FW = 322.35; monoclinic space group P21, crystal size 0.40 × 0.40 × 0.30 mm3, unit cell dimensions a = 5.05804 (7) Å, b = 11.74745 (14) Å, c = 13.77837 (18) Å, α = γ = 90.00°, β = 100.1922 (13)°, V = 805.778 (18) Å3; Z = 2; T = 150 K; Dcalcd = 1.329 mg/m3; μ = 0.836 mm−1, F (000) = 344, 5093 measured reflections were collected, 3141 independent reflections [Rint = 0.0167]. Completeness to θmax was 98.58%; Final R indices: R1 = 0.0140 and wR2 was 0.1007 [I > 2σ (I)]. The goodness of fit on F2 was 0.971. Flack parameter = 0.08 (10).Colourless lump crystals of 10 were deposited in the Cambridge Crystallographic Data Centre (CCDC 1445050).The crystallography data for 2 humidified incubator at 37 °C. Cell lines in the phase of their growth cycle (6 × 104 cells/mL) were added to each well of 96-well plate (100 μL/well) and cultivated for 24 h. The tested compounds  were serially diluted in DMEM/RPMI-1640 at different concentrations and placed into wells in a total volume of 200 μL. Taxol  was used as a positive control. After 24 h, 20 μL of MTT -2,5-diphenyltetrazolium bromide; 5 mg/ml) was added to each well. After a 4 h incubation, the supernatant was removed from the wells and the formazan crystals were dissolved in DMSO (150 μL). The plates were then shaken for 15 min, and the absorbance was measured at 570 nm using an enzyme immunoassay instrument .The U937 and MCF-7 human cancer cell lines were obtained through commercial purchasing from American Type Cultures Collection . The A549, SH-SY5Y, HepG2 and SW480 human cancer cell lines were purchased from Life Science College Cell Resource Center of Chinese Academy of Sciences . The MCF-7, A549, SH-SY5Y and HepG2 cells were cultured in DMEM medium with supplement of 10% fetal bovine serum . The U937 and SW480 cells were cultured in RPMI-1640 medium with supplement of 10% fetal bovine serum . All these cells were cultivated in a 5% COhttp://sea.bkslab.org/)Target proteins of the small molecule compounds, were obtained from the similarity ensemble approach (SEA) (2 with MEK1 and ERK1. MD simulations were carried out by GROMACS package (version 5.1.2)+ and Cl−, so as to mimic a physiology concentration NaCl (0.15 M). Prior to operate the MD simulation, energy minimization was carried out to clear bad contacts. Then, 100ps NVT and 100ps NPT ensembles with simultaneous protein-ligand position restraints were also carried out to equilibrate the system. Consequently, 3ns MD production simulation was performed with a 2fs time step under constant temperature (300 K) and pressure (1 atm).To further verify the binding model of compound 5 cells/mL treated or untreated with compound 2 and cultured for 24 h. The subcellular structure alterations of apoptosis were observed under fluorescence inversion microscope.MCF-7 cells were seeded in each well of 6-well culture plates at a density of 2.5 × 102 and cultured for 24 h. The collected cells were then fixed with 500 μL PBS. The apoptotic ratio was measured by flow cytometry  after employing Annexin-V-FLUOS Staining Kit .MCF-7 cells were seeded into 6-well culture plates with different concentrations of compound 2. All the cells were collected and were lysed by lysis buffer at 4 oC for 1 h. After 12, 000 rpm centrifugation for 15 min, the protein level of the supernatant was quantified by Bio-Rad DC protein assay . Equal amounts of the total protein were separated by 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Subsequently, membranes were blocked with 5% nonfat dried milk. Proteins were detected using primary antibodies , followed by HRP-conjugated secondary antibody and visualized by employing ECL as the HRP substrate.MCF-7 cells were cultured in 6-well culture plates for 24 h. Then cells were treated with different concentrations of compound 2-treated MCF-7 cells. In simple terms, cells were dissolved in lysis buffer containing protease inhibitor (Sigma). Centrifuging the lysate for 1 h at 15 °C to get the supernatant, and stored it at −80 °C for further use. Protein quantitation was performed using Bio-Rad DC protein assay Kit (Bio-Rad). iTRAQ labeling was carried out using iTRAQ Reagent 4-Plex kit (AB SCIEX) based on the manufacturer’s protocol with minor modifications. The whole cell lysates were labeled with iTRAQ labeling reagent 114 and 115 for the pair of control MCF-7 and compound 2-treated MCF-7, respectively. After 2D LC analysis and tandem mass spectrometry analysis, protein identification and relative iTRAQ quantification were performed with ProteinPilot™ Software 4.2 (AB SCIEX) using the Paragon™ algorithm for the peptide identification, which was further processed by Pro GroupTM algorithm where isoform-specific quantification was adopted to trace the differences between expressions of various isoforms. Results with iTRAQ ratio cutoff values of 1.2 and 0.8 for fold-change and number cutoff values of 3 for quantifiable peptides for in protein abundance were accepted. Moreover, the results of 114 and 115 were adopted when their expression level were changed at the same trend.The iTRAQ and MS/MS analysis were performed in compound To build the MEK1/ERK1 kinase protein–protein interaction (PPI) network, we collected diverse sets of biological evidence from seven online databases. Different PPIs were collected from the Database of Interacting Proteins (DIP)http://david.abcc.ncifcrf.gov/). The functional categories used were GO term related to Biological Process (BP). And, the apoptosis-related proteins were detected and used as the background set.Differentially expressed proteins were assigned to 16 functional synaptic protein groups. Enrichment was only considered relevant when over represented functional groups contained at least 4 proteins. In addition, functional enrichment was determined using the DAVID functional annotation tool  using a previously reported method475 cells/mL in RPMI 1640 medium. After incubation for 1 h, the tested samples at various concentrations and LPS  were simultaneously added to the wells. After being incubated at 37 °C for 24 h, the culture supernatant (100 μL/well) was transferred to the enzyme-labelled plates and then mixed with an equal volume of Griess reagent. After 10 min at room temperature, the absorbance of the reaction products was measured at 540 nm. The cell culture without the tested compounds was used as the LPS group, the cell culture sample without the tested compounds and LPS was used as control group, and hydrocortisone was used as a positive control. All tests were performed in quadruplicate. The concentration of NO2− was calculated using a working curve of 1, 5, 10, 50 μM NaNO2 solutions, and the inhibitory rate on NO production was calculated by the level of NO2−. Cytotoxicity was measured using a 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay after 24 h of incubation with the tested compounds.Mouse monocyte-macrophages (RAW 264.7) were maintained in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) and heat-inactivated fetal bovine serum (10%) at 37 °C in a 5% COHow to cite this article: An, Y.-N. et al. Penicimenolides A-F, Resorcylic Acid Lactones from Penicillium sp., isolated from the Rhizosphere Soil of Panax notoginseng. Sci. Rep.6, 27396; doi: 10.1038/srep27396 (2016).


Their optical, electrochemical and morphological properties have been evaluated and compared to those of the non-rotaxane counterpart 3. The influence of TM-βCD or TM-γCD encapsulation on the thermal stability, solubility in common organic solvents, film forming ability was also investigated. Polyrotaxane 3·TM-βCD exhibits a hypsochromic shift, while 3·TM-γCD displays a bathochromic with respect to the non-rotaxane 3 counterpart. For the diluted CHCl3 solutions the fluorescence lifetimes of all compounds follow a mono-exponential decay with a time constant of ≈0.6 ns. At higher concentration the fluorescence decay remains mono-exponential for 3·TM-βCD and polymers 3, with a lifetime τ = 0.7 ns and 0.8 ns, whereas the 3·TM-γCD polyrotaxane shows a bi-exponential decay consisting of a main component  with a relatively short decay constant of τ1 = 0.7 ns and a minor component with a longer lifetime of τ2 = 5.4 ns (2%). The electrochemical band gap (ΔEg) of 3·TM-βCD polyrotaxane is smaller than that of 3·TM-γCD and 3, respectively. The lower ΔEg value for 3·TM-βCD suggests that the encapsulation has a greater effect on the reduction process, which affects the LUMO energy level value. Based on AFM analysis, 3·TM-βCD and 3·TM-γCD polyrotaxane compounds exhibit a granular morphology with lower dispersity and smaller roughness exponent of the film surfaces in comparison with those of the neat copolymer 3.Two alternating polyfluorene polyrotaxanes ( O-methyl-βCD (TM-βCD) or 2,3,6-tri-O-methyl-γCD (TM-γCD) have been synthesized in the course of our investigations.Over the last decades, conjugated polymers (CPs) have been actively investigated as an alternative to conventional inorganic materials in many electronic applications due to their low cost and easy processability –6. Among20alt-2,7-fluorene/TM-βCD)] (3·TM-βCD) and poly (3·TM-γCD) polyrotaxanes have been synthesized. Thus, 3·TM-βCD and 3·TM-γCD have been obtained through the Suzuki cross-coupling reaction of 2,7-dibromofluorene (1) encapsulated into TM-βCD or TM-γCD cavities (1·TM-βCD and 1·TM-γCD) with 9,9-dioctylfluorene-2,7-diboronic acid bis ester (2), as bulky stopper units , β- and γCD, bromobenzene (Br–Ph), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and quinine sulfate dehydrate in 0.5 M sulfuric acid were purchased from (Sigma-Aldrich) and used as received. TBAClO4 for electrochemical analysis (99.0%) (Fluka) was used without further purification. Acetonitrile (ACN) (Fischer), DCM, CHCl3, toluene and all other solvents were purchased from commercial sources  and used without further purification.1H NMR spectra have been recorded on a Bruker Avance DRX 400 MHz instrument equipped with a 5 mm QNP direct detection probe and z-gradients. Spectra have been recorded in CDCl3 at room temperature. The chemical shifts are reported as δ values (ppm) relative to the residual peak of the solvent. The FTIR (KBr pellets) spectra were obtained on a Bruker Vertex 70 spectrophotometer. The molecular weights of copolymers were determined by GPC in THF by using a Water Associates 440 instrument and polystyrene (Pst) calibrating standards. DSC was performed with a Mettler Toledo DSC-12E calorimeter with two repeated heating–cooling cycles at a heating rate of 5 °C·min−1 under N2 atmosphere. TGA analysis was performed under constant nitrogen flow (20 mL·min−1) with a heating rate of 10 °C·min−1 using a Mettler Toledo TGA/SDTA 851e balance. UV–vis and fluorescence spectra in CHCl3 solutions were performed using 3, 3·TM-βCD and 3·TM-γCD with the same concentration (either 10−1 mg∙mL−1 or 10−3 mg∙mL−1) of the 3 cores without macrocyclic molecules. Time-resolved photoluminescence (PL) measurements were performed with a time-correlated single photon counting (TCSPC) spectrometer previously reported [reported . The PLQ4 solution (0.1 M) in anhydrous ACN was used as the supporting electrolyte. The set-up was introduced into a glove box and controlled by AUTOLAB PGSTAT 101 (Ecochemie) using NOVA software. The pseudo-reference was calibrated with a 10−3 M of Fc solution in ACN. The polymer samples were drop-casted onto the working electrode from a concentrated DCM solution and studied in the interval −2.5 and +2.0 V vs Ag wire. Cathodic and anodic scans were performed independently.CVs were carried out in a three-electrode cell in which Pt (1 mm diameter) was used as a working electrode, a Pt wire as counter-electrode and an Ag wire as pseudo-reference electrode. A TBAClO3 solution at 3000 rpm for 60 s on a WS-400B-6NPP-Lite Single Wafer Spin Processor . Scan areas of 3 × 3 μm2, were analyzed with a resolution of 512 × 512 pixels. Advancing and receding contact angle measurements were performed by using the drop shape analysis profile device equipped with a tiltable plane . Ultrapure water  or a diiodomethane drop was first deposited on the sample using a variable volume micropipette. The drop volume was set to 15 µL for water and 10 µL for diiodomethane. In order to perform dynamic contact angle measurements, the sample surface sustaining the drop was tilted at a constant speed (1 deg·s−1) and the images of the drop simultaneously recorded. The advancing contact angle was measured at the front edge of the drop, just before the triple line starts moving. The angle was obtained using the tangent of the drop profile at the triple line. For each sample, contact angles were measured on four samples and three drops per sample. The reported contact angle values correspond to the average of all measurements with an error bar corresponding to the standard deviation.The surface profiles of copolymers films were evaluated by AFM measurements. AFM were performed in the tapping mode, using a Solver PRO-M scanning probe microscope  with commercially available NSG10 cantilever. Films were prepared onto mica substrates by spin-coating from CHClSynthesis of 2,3,6-tri-O-methyl-CD (TM-βCD) and 2,3,6-tri-O-methyl-CD (TM-γCD): TM-βCD and TM-γCD as macrocyclic molecules were synthesized according to previously reported procedure [rocedure –48.Synthesis of 1·TM-βCD: To prepare 1·TM-βCD inclusion complex, 0.572 g (0.4 mmol) of TM-βCD were dissolved in water (5.0 mL) and 0.067 g (0.2 mmol) of 2 were added. The mixture was stirred at room temperature under nitrogen atmosphere for 48 h to give a turbid dispersion. The water was removed by lyophilization and the complex, as a white powder was used for the preparation of 1·TM-βCD. The synthesis of the inclusion complex 1·TM-γCD was performed under similar experimental conditions as those used for the preparation of the 1·TM-βCD inclusion complex, except  of TM-γCD was used instead of TM-βCD.Synthesis of 3·TM-βCD and 3·TM-γCD polyrotaxane copolymers: 1·TM-βCD  and 2  were dissolved into 6 mL of toluene in a flask under argon (Ar) protection. The mixture was flushed with Ar several times, and then 1.5 mL of a 3 M solution of sodium carbonate (Na2CO3) and 18.2 mg of (Ph3P)4Pd(0), as catalyst dissolved in 4 mL of degassed toluene were added into the flask. The solution was flushed with Ar again for another three times, and the reaction mixture was protected against light. The oil bath was heated to 90 °C, and the reaction mixture was stirred for 72 h. Then, an excess of 0.005 g (0.01 mmol) of monomer 2 dissolved in 3 mL of toluene was added and the reaction was continued for 12 h. Finally, 1.0 μL of Br-Ph was added as end-capper of the copolymer chain and the reaction was continued overnight. After cooling, the mixture was poured into water and extracted with toluene. The organic extracts were washed with water and dried over magnesium sulfate (MgSO4). The toluene solution was concentrated by rotary evaporation and precipitated in CH3OH. The solid was filtered, dried and purified by Soxhlet extraction with methanol and acetone in succession to remove the oligomers. The polymer was further purified by reprecipitation from concentrated CHCl3 solution with methanol, collected by centrifugation and vacuum dried at 60 °C to afford 3·TM-βCD  as a yellow-brownish solid. 1H NMR  7.92–7.55 , 6.85–6.81 , 5.13 H), 4.11–4.09 , 3.88–3.80 H, C(6)H), 3.65 -CH3), 3.62–3.58 H, C(6)H), 3.54–3.49 H, O(2’)-CH3), 3.39 -CH3), 3.19 H), 2.10–1.93 , 1.26–1.11 , 0.83–0.71 ; FTIR : 3433, 2927, 2853, 1724, 1614, 1459, 1410, 1357, 1159, 1091, 1042, 968, 875, 813 cm−1; GPC : Mn = 24300 g·mol−1, Mw/Mn = 1.94.3·TM-γCD was synthesized by similar experimental conditions as described for 3·TM-βCD, except that TM-γCD was used instead of TM-βCD. 3·TM-γCD polyrotaxane was also obtained as a yellow-brownish solid in a 24.7% yield. 1H NMR  7.92–7.38 , 6.91 , 5.26–5.02 H), 4.1–3.25 H, O-CH3), 2.11 , 1.11 , 0.81 ; 13C NMR  151.79–140.10 , 132.18–120.21 , 98.09 (C1), 82.11 , 71.04 , 61.07–59.02 , 55.41 (C g), 40.49 (C h), 37.18 (Cg’), 31.79 (Ci), 30.05–29.20 (Cj–m), 22.59 (Cn), 14.02 (Co); FTIR : 3416, 3058, 2923, 2850, 1634, 1610, 1457, 1405, 1373, 1291, 1095, 888, 810, cm−1; GPC : Mn = 20100 g·mol−1, Mw/Mn = 2.24.Synthesis of the non-rotaxane 3 copolymer: The non-rotaxane copolymer 3 was synthesized under similar experimental conditions as those described for 3·TM-βCD or 3·TM-γCD polyrotaxanes, except that free monomer 1 was used instead of 1·TM-βCD or 1·TM-γCD. The crude polymer 3 was collected by filtration and then extracted with a Soxhlet extractor using methanol and acetone. Further the solid was redissolved in CHCl3, precipitated with methanol, collected by filtration and vacuum dried at 50 °C. The copolymer was obtained as an orange solid in a yield of 47.8%. 1H NMR  7.98–7.39 , 6.93–6.87 , 4.14–4.06 , 2.16 , 1.16 , 0.86 ; 13C NMR  151.77–140.05 , 128.79–120.24 , 55.37 (Cg), 40.47 (Ch), 37.18 (Cg’), 31.79 (Ci), 30.04–29.21 (Cj–m), 22.60 (Cn), 14.06 (Co); FTIR : 3438, 3024, 2954, 2922, 2850, 1605, 1457, 1405, 1378, 1261,1196, 1092, 1023, 810 cm−1; GPC : Mn = 27900 g·mol−1, Mw/Mn = 1.83.File 11H NMR and 13C NMR spectra of the investigated copolymers.Characterization data of the compounds: The stability constant, FTIR,


Polygonum multiflorum Thunb.  are commonly used in anti-aging medicine. Few reports have combined chemical profiles with bioactivity to evaluate the quality of the processed HSW. This study aims to integrate chemometric fingerprints of antioxidant activities and high-performance liquid chromatography–diode array detection–chemiluminescence (HPLC–DAD–CL) to assess the quality of processed HSW.The processed roots of An online HPLC–DAD–CL based on the three reactive oxygen species (ROS), superoxide anion, hydrogen peroxide, and peroxynitriteanion, was developed to screen the potential anti-aging constituents for a comprehensive quality evaluation of processed HSW. Additionally, antioxidant-activity-integrated fingerprints were constructed and hierarchical cluster analysis and principal component analysis were used to evaluate the variations among 14 batches of processed HSW samples purchased from drug stores in different habitats.trans-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside, 2,3,5, 4′-tetrahydroxy-stilbene-2-O-β-d--glucoside, torachrysone-8-O-glucoside, and emodin-8-O-β-d-glucoside exerted relatively large influences on the differences between processed HSW samples.Fourteen batches of processed HSW samples were highly similar and classified into two clusters using hierarchical cluster analysis. Twelve active compounds exhibited antioxidant activity on the ROS with different degrees of sensitivity that constituted specific fingerprints. Among them, protocatechuic acid, catechin, 2O2, O2•−, and ONOO−scavenging experiments.Our study established the antioxidative activity-integrated fingerprint for processed HSW and achieved a screening of the potential anti-aging constituents using the online HPLC–DAD–CL method with HThe online version of this article (doi:10.1186/s13020-016-0087-8) contains supplementary material, which is available to authorized users. Polygonum multiflorum Thunb.   Fig.  are ofteproducts  and exhiproducts  and antiproducts .Fig. 1PhHSW contains numerous phenols, anthraquinones, and stilbene glycosides , 9. Most2O2), 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), superoxide anion (O2•−), and 2, 2-diphenyl-1-picrylhydrazyl, to screen bioactive constituents and evaluate the antioxidant activity of CM [The fingerprint technique is the predominant tool to evaluate the quality of CM. However, the chromatographic fingerprint only contains the chemical message and is insufficient to demonstrate the overall quality of material , 16. Thety of CM –17. Howe2•−, H2O2, and peroxynitriteanion (ONOO−), are involved in the aging process and cause oxidative damage [Aging is a complex physiological process and the oxidative stress theory of aging has gained considerable support .Numerouse damage –22. Antie damage , 23, but2•−, H2O2, and ONOO−).This study aims to investigate the antioxidant profile of processed HSW by HPLC–DAD–CL combined with chemometrics to rapidly screen potential anti-aging constituents of processed HSW by scavenging three reactive species (O2O2 in water), sodium nitrite (NaNO2), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), and hydrochloric acid (HCl) were all purchased from Nanjing Chemical Regent Corporation . Pyrogallol was obtained from Zunyi Second Chemical Corporation . Ethylenediaminetetraacetic acid was supplied by Shanghai Chemical Reagent Corporation . Sodium hydroxide (NaOH) and manganese dioxide (MnO2) were purchased from Xilong Chemical Corporation . The reagents used were all of analytical grade. The purified water used was prepared by a Millipore water purification system .HPLC grade acetonitrile was obtained from Tedia . Luminol , hydrogen peroxide solution , Shanxi , Hebei , Guizhou , Yunnan , Anhui , Guangdong , Hubei , Sichuan , Sichuan , Henan , Guangxi , Guizhou , and Hunan . All samples were authenticated by Professor Bo-Yang Yu based on their morphological features according to the Chinese Pharmacopoeia . Their v2CO3 and 0.1 M NaHCO3 solution. A 1.8 × 10−2 M stock solution of luminol was prepared in a 0.1 M Na2CO3 solution and stored in a refrigerator for at least 3 days before dilution. A 1.0 × 10−2 M stock solution of pyrogallol was prepared in a 0.1 mM HCl solution and then stored in a dark bottle at 4 °C. Reagent solutions for the determination of H2O2, and O2•− scavenging activity were prepared according to previous work conducted by our research group [−6 M luminol solution (Solution I) was prepared with 0.1 M carbonate buffer solution  and a 4.97 × 10−4 M peroxynitrite solution (Solution II) was prepared with a 0.1 M NaOH solution on an ice bath.Carbonate buffers  were prepared by mixing appropriate volumes of 0.1 M Nach group . Peroxynch group  and we uch group . Reagent18 column  with a flow rate of 1.0 mL/min. The column temperature was set at 30 °C. The mobile phase was composed of A (acetonitrile) and B . The gradient elution program was as follows: 0–6 min, isocratic gradient 6 %A; 6–10 min, linear gradient 6 %–10 %A; 10–30 min, linear gradient 10 %–20 %A; 30–70 min, linear gradient 20 %–50 %A; 70–75 min, linear gradient 50 %–60 %A; 75–85 min, isocratic gradient 60 %A.The HPLC system used was a Shimadzu LC-2010C HT system consisting of a quaternary pump, an autosampler , a thermostated column compartment , and a DAD . HPLC separation was achieved with a Venusil MP C2O2, pyrogallol, and peroxynitrite were transported by peristaltic pumps at the same flow rate of 1.1 mL/min each. CL reagents were mixed with sample solution eluted from the HPLC detector then passed through the CL detector; the CL emission was detected by the BPCL system  was used to monitor the CL emission. The CL detector was equipped with a flat glass coil (80 µL) as detection cell and a photomultiplier operated at −800 V. The HPLC–DAD–CL detection system was interconnected with PEEK tubes . CL reagents, luminal solutions, H0 is the baseline intensity of CL (without sample) and CL1 is the inhibited CL intensity of every compound in the extracts.An Agilent 1100LC/MSD Trap XCT ESI  was used to obtain information about the structure of the constituents in processed HSW. The mobile phase for HPLC–MS was the same as described for the HPLC analysis conditions but the composition of the B pump substituted 0.1 % aqueous phosphoric acid for 0.1 % aqueous formic acid. The ESI–MS spectra were acquired in negative ionization mode. Capillary voltage was 3300 V. Drying gas temperature was set at 350 °C with a flow rate of 9.0 L/min and nebulizing pressure was set at 275.8 kPa. Data were processed using the 6300 Series HPLC/MSD Trap and Data Analysis 3.4 .The characteristic constituent in processed HSW samples was selected as the reference peak to calculate relative retention time (RRT) and relative peak area (RPA). The relative standard deviations (RSDs) of RRT and RPA for each common peak were calculated to estimate precision, repeatability, and stability. The method precision was evaluated using intraday and interday variation tests. The intraday variation test was evaluated using five replicate injections of the same sample and the interday variation test was evaluated over 3 days with five replicate injections each day. Method repeatability was analyzed for the six replicate samples. The stability of the sample solution was evaluated at different time points in one day .We used a similarity evaluation system for chromatographic fingerprints of CM (Version 2004 A) to evaluate the similarity of the samples. Six samples from different places were prepared. The similarities between the chromatograms of the samples from different origins were examined by comparing the samples with the reference fingerprint that was generated by the median value of all the chromatograms.Hierarchical cluster analysis (HCA) was performed by SPSS statistics software  was used to sort the samples into different groups. The distance between the samples was used to assess the similarities among processed HSW samples. Samples with high similarity were clustered into homogenous groups. Principal components analysis (PCA) is an effective method to determine the main factors in large amounts of data by feature extraction and dimensionality reduction. PCA analysis of the fingerprint data of processed HSW was performed using SIMCA-P 12.0 software .P < 0.05 and values of P < 0.01, P < 0.001, respectively.Data were expressed as the mean ± standard deviation (SD). The statistical significance of differences between means was established by One-way ANOVA with Turkey post hoc tests. Significant differences and highly significant differences were classed as values of Trans-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside (peak 9) was selected as the reference peak. The RSDs of RRT and RPA for the precision of each common peak ranged from 0.51 to 2.33 % (n = 6) in the intraday variation test, respectively, and ranged from 0.83 to 3.97 % (n = 5) in the interday variation test, respectively. The RSDs for the repeatability of RRT and RPA of each common peak ranged from 0.9 to 4.1 % (n = 5), respectively. The RSDs of the stability test were below 2.8 % (n = 6)  and the scavengingrate (%) (x) for resveratrol scavenging O2•−, H2O2, and ONOO− in different concentrations were obtained. The equations for resveratrol scavenging the H2O2, O2•−, and ONOO− were, respectively, as follows: Ln(y) = 1E − 05x3 − 0.0015x2 + 0.1083x − 6.775, Ln(y) = 2E − 05x3 − 0.0032x2 + 0.2001x − 6.0478 , Ln(y) = 1E − 05x3 − 0.0022x2 + 0.1373x − 6.3504. If resveratrol (1 µg) was a potency unit, the relative total activities should be the sums of peak potency. The relative total activities of peaks in 14 batches of processed HSW were calculated according to Eq. (P < 0.001) and the antioxidative activities of samples S2 and S12 exhibited insignificant differences (P = 1). The S2 and S12 samples had strong antioxidative activities, particularly in scavenging O2•− and H2O2. The qualities and antioxidative effects of S7 in scavenging O2•−, H2O2 and ONOO− were significantly different from S1, although the two samples were from Guangdong province . The same was true for samples S4 and S13. Compared with S4, the antioxidative effects of S13 were obviously weak in scavenging O2•−, H2O2 and ONOO− (P < 0.001). The detailed comparison data was shown in Additional file The standard potency equation of Ln, trans-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside (peak 9), and 2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d--glucoside (peak 11), were sensitive in scavenging three reactive species, and trans-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside exhibited significant antioxidant activity because of its high content in processed HSW. Gallic acid (peak 1), protocatechuic acid (peak 3), catechin (peak 5), and torachrysone-8-O-glucoside (peak 12) exhibited scavenging activity on H2O2, O2•−, or ONOO− , and emodin (peak 16) demonstrated higher selectivity in scavenging ONOO− than on other two reactive species, especially emodin. These constituents had different scavenging capacities on different ROS and could affect different aspects of the aging process, such as directly scavenging free radicals, decreasing the oxidation of nucleic acid and nitration of protein tyrosine residues, and inhibiting apoptosis. In addition, peak 4 had no antioxidant activity, and peaks 17 and 18 showed certain antioxidant activity in the ROS scavenging test despite their weak ultraviolet absorption or unavailable MS messages. The relationship between constituents of processed HSW and the corresponding free radicals are summarized in Fig. In the reactive species scavenging test, stilbene glucosides, including trans(cis)-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside, torachrysone-8-O-glucoside, emodin-8-O-β-d-glucoside, and emodinall exhibit pharmacological effects on the age-related pathological process [trans(cis)-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside, torachrysone-8-O-glucoside, and emodin-8-O-β-d-glucoside all significantly promote hair growth [cis-2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d-glucoside, torachrysone-8-O-glucoside, and 2,3,5,4′-tetrahydroxy-stilbene-2-O-β-d--glucoside were screened and proposed as chemical markers for the quality control of processed HSW. To the best of our knowledge, this is a novel research finding.Gallic acid, protocatechuic acid, epicatechin, catechin,  process –35. In pThe relationship among samples was ascertained by comparing the similarity of the samples chromatographic fingerprint series. The relationship between sets of chromatographic fingerprints was analyzed by comparing the similarity between the objects and the reference fingerprints. The similarity values of 14 batches of samples (S01–S14) were 0.989, 0.990, 0.990, 0.989, 0.982, 0.995, 0.995, 0.986, 0.970, 0.870, 0.905, 0.989, 0.945, and 0.996 (n = 6), respectively. Overall, the processed HSW samples were very similar. Compared with other samples, the similarity indexes of samples S10 and S11 were relatively lower. The different habitats and processing technology could have caused variation in the content or chemical compositions of processed HSW .Peak 1, peak 3, peak 5, peak 6, peak 8, peak 9, peak 11, peak 12, peak 13, and peak 16 were the antioxidants in all processed HSW samples. Peak 17 and peak 18 were not examined for weak chemical messages. HCA analysis, was performed by the SPSS statistics software. The samples of processed HSW could be classified into two clusters despite high similarity: samples S10, S11, S5, S9, S6, S8, S14, S7, S13, and S3 were in Cluster 1; samples S2, S12, S1, and S4 were in Cluster 2. The HCA results were similar to the HPLC similarity index results -glucoside, torachrysone-8-O-glucoside, and emodin-8-O-β-d-glucoside had relatively large influences on the difference between processed HSW samples -glucoside appeared to be chemical markers for the quality control of processed HSW.The PCA analysis produced a classification of samples similar to that produced by the HCA results Fig. a. S1, S3les Fig. b. In add2O2, O2•−, and ONOO−scavenging experiments.Our study established the antioxidative activity-integrated fingerprint for processed HSW and achieved the potential anti-aging constituents using the online HPLC–DAD–CL method with H


Each of the bridges within the chain, which extends parallel to the a axis, is centrosymmetric, with Na⋯Na separations of 3.4807 (16) and 3.5109 (16) Å. In the crystal, there are three water molecules of solvation and these, as well as the coordinating water molecules and the amino group of the 4-amino-3,5,6-trichloropicolinate anion, are involved in extensive inter-species hydrogen-bonding interactions with carboxyl and water O atoms, as well as the pyridine N atom. Among these associations is a centrosymmetric cyclic tetrawater R44(8) motif, resulting in an overall three-dimensional structure.In the structure of the title complex, {[Na(H N,O chelating ligand with MnII 6]2+ cation. Although the structure of picloram has not been reported, that of the guanidinium salt is known 3]+·C6H2Cl3N2O2−·3H2O}n, and the structure is reported herein.4-Amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram) is a commercial herbicide  and 3.5109 (16) Å, respectively; for symmetry codes, see Table 1W) in a non-bridging mode , which is similar to that in the anhydrous guanidinium picloramate salt (73.3°) , 3.080 (2) Å].The structure of the title salt also contains non-coordinating picloramate anions and three water molecules of solvation  Å; symmetry code (xi): x, y − 1, z].In the crystal there are numerous inter-species water O—H⋯Os Table 2, includire Fig. 2. Cyclic 2-aqua)-bridged Na2(H2O)2 units in the coordination polymeric cationic chains in the title structure have precedents in a large number of reported crystal structures. However, with few exceptions, these are based on NaO6 polyhedra, with octahedral or distorted octahedral stereochemistry, having two non-bridging water molecules [Na2(H2O)82+], compared to one non-bridging water molecule in the NaO5 coordination [Na2(H2O)62+] of the title complex. The [Na2(H2O)82+] dications may be discrete, such as found in the anionic aryltelluronic anhydride salt 164+] tetra-cations as found in the dianionic biphenyl-4,4′-diphosphonate salt 8]n, e.g. in the monoanionic salt of the anti-allergic drug tranilast acrolyl]amino}benzoic acid; Geng et al., 2013e.g. the CuII complex with pyrophosphate, [Cu(H2O)(phen)(P2O7)]2−  [Ru2(hedp)2X]4− (H2O)2]−  ethanol–water. Room temperature evaporation of the solution to dryness gave minor colourless crystal blocks of the title complex from which a specimen was cleaved for the X-ray analysis.The title compound was synthesized by briefly heating together 0.5 mmol of 4-amino-3,5,6-trichloropicolinic acid (picloram) with excess NaHCOUiso(H) = 1.2Ueq(N) or 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015012633/wm5173sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015012633/wm5173Isup2.hklStructure factors: contains datablock(s) I. DOI: 1409779CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: