Conversely, much less is known about the roles of PDE2, PDE3 and PDE4 in the regulation of action potential (AP) properties and ICa,L in the sinoatrial node (SAN) and the atrial myocardium, especially in mice. Thus, the purpose of our study was to measure the effects of global PDE inhibition with Isobutyl-1-methylxanthine (IBMX) and selective inhibitors of PDE2, PDE3 and PDE4 on AP properties in isolated mouse SAN and right atrial myocytes. We also measured the effects of these inhibitors on ICa,L in SAN and atrial myocytes in comparison to ventricular myocytes. Our data demonstrate that IBMX markedly increases spontaneous AP frequency in SAN myocytes and AP duration in atrial myocytes. Spontaneous AP firing in SAN myocytes was also increased by the PDE2 inhibitor erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA), the PDE3 inhibitor milrinone (Mil) and the PDE4 inhibitor rolipram (Rol). In contrast, atrial AP duration was increased by EHNA and Rol, but not by Mil. IBMX also potently, and similarly, increased ICa,L in SAN, atrial and ventricular myocytes; however, important differences emerged in terms of which inhibitors could modulate ICa,L in each myocyte type. Consistent with our AP measurements, EHNA, Mil and Rol each increased ICa,L in SAN myocytes. Also, EHNA and Rol, but not Mil, increased atrial ICa,L. In complete contrast, no selective PDE inhibitors increased ICa,L in ventricular myocytes when given alone. Thus, our data show that the effects of selective PDE2, PDE3 and PDE4 inhibitors are distinct in the different regions of the myocardium indicating important differences in how each PDE family constitutively regulates ion channel function in the SAN, atrial and ventricular myocardium.Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotides in the heart. In ventricular myocytes, the L-type Ca Phosphodiesterases (PDEs) are phosphohydrolase enzymes that are responsible for the degradation of the cyclic nucleotides adenosine and guanosine 3′,5′ cyclic monophosphate (cAMP and cGMP) 2+ current  is a critical target of regulation by PDEs in ventricular myocytes. Specifically, it is established that global PDE inhibition with the broad spectrum inhibitor 3-Isobutyl-1-methylxanthine (IBMX) potently increases basal ventricular ICa,L in mice and rats Ca,L; however combined inhibition of PDE3 and PDE4 does increase basal ICa,L and inhibition of PDE2, PDE3 and PDE4 increases basal ICa,L very similarly to IBMX Ca,L have been well characterized in ventricular myocytes much less is known about the role of these PDE families in the sinoatrial node (SAN) and atria, particularly in mice, a very common model organism due to its use in studies incorporating genetic manipulations.Among the PDE families expressed in the heart Ca,L2+ channel, CaV1.2 and CaV1.3, contribute to total ICa,L in SAN myocytes, in contrast to ventricular myocytes which only express CaV1.2 V1.3 channels activate at more negative membrane potentials than CaV1.2 enabling them to contribute prominently to the generation of the DD and thereby to the frequency of spontaneous AP firing and heart rate control. ICa,L is also a critical determinant of AP morphology in working atrial myocytes V1.2 and CaV1.3 both contribute to total ICa,L in atrial myocytes Ca,L, is different in these tissues in comparison to the ventricles.The SAN contains the specialized pacemaker myocytes whose spontaneous activity is responsible for determining heart rate Ca,L in SAN and atrial myocytes in comparison to ventricular myocytes. This work represents the first direct comparison of these inhibitors on ICa,L in these three regions of the myocardium in mice. Our data demonstrate that the patterns of constitutive PDE activity are distinct between SAN, atrial and ventricular myocytes, further adding to the complexity of PDE signaling in the heart.The purpose of this study was to determine the roles of PDE2, PDE3 and PDE4 in regulating AP properties in mouse SAN and atrial myocytes. We also measured the effects of broad spectrum and family specific PDE inhibitors on basal IThis study utilized male wildtype C57Bl/6 mice (Charles River) between the ages of 10–14 weeks. Experimental procedures were in accordance with the regulations of The Canadian Council on Animal Care and were approved by The Dalhousie University Committee on Laboratory Animals. In all experiments, mice were anesthetized by isoflurane inhalation and cervically dislocated before hearts were removed.An expanded Quantitative mRNA expression for PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D in mouse SAN, right atrium and right ventricular free wall was performed using methods we have described previously see also  [20]. ThCa,L was recorded in the whole cell patch-clamp configuration. All recordings were performed at room temperature (22–23°C). The solutions and electrophysiological protocols for these experiments are provided in Mouse SAN, right atrial and right ventricular myocytes were isolated for patch-clamp recordings using procedures we have described previously t-test. P<0.05 was considered significant.All summary data are presented as means ± SEM. Data were analyzed by one or two way ANOVA with Tukey's posthoc analysis or paired Student's Amongst the PDE2, 3 and 4 families, prior studies have shown that the PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D subtypes are expressed in whole ventricular myocardium and/or isolated ventricular myocytes in rodents These data demonstrate that each region of the myocardium has a specific pattern of expression for the PDE2, 3 and 4 isoforms we have measured. Specifically, no differences were observed in the expression of PDE2A in each region of the myocardium; however, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D all displayed significant differences in expression between SAN, right atrium and right ventricle as indicated in P<0.05) spontaneous AP firing in SAN myocytes from 138±10 to 193±14 APs/min with no change in maximum diastolic potential  in DD slope  AP duration at 50, 70 and 90% repolarization  with no change in resting membrane potential  spontaneous AP frequency in SAN myocytes from 137±7 to 160±6 APs/min (P<0.05) DD slope  AP duration in mouse right atrial myocytes  spontaneous SAN myocyte AP frequency from 135±6 to 169±6 APs/min (P<0.05) DD slope from 28.5±3.4 to 49.9±4.5 mV/s (50 from 37±5.4 to 46.2±5.5 ms (50 (8.9±1 vs. 9.4±1), APD70 (16.9±2 vs. 18.2±2.3) and APD90 (45.7±4.2 vs. 46±4.3) were not different  spontaneous AP frequency in SAN myocytes from 130±8 to 170±9 APs/min (P<0.05) DD slope  AP duration in right atrial myocytes  peak ICa,L density from −4.4±0.3 to −11.2±1  ICa,L maximum conductance (Gmax) from 103.9±6.4 to 225.1±5.9 pS/pF and shifted (P<0.05) the V1/2(act) from −28.4±0.4 to −33.3±0.5 mV. There was no change (P = 0.588) in slope factor (6.4±0.6 mV in control vs. 6.3±0.3 mV in IBMX). Similarly, in right atrial myocytes, IBMX increased peak ICa,L density from −3.9±0.2 to −9.7±0.4 pA/pF (P<0.05) ICa,L Gmax from 112.8±8.8 to 195.8±10.4 pS/pF and shifted (P<0.05) the V1/2(act) from −9.1±1.2 to −18.7±1.1 mV.The effects of global PDE inhibition with IBMX (100 µM) on SAN and right atrial myocyte I −11.2±1  in SAN manalysis . IBMX in.4 pA/pF . Steady .4 pA/pF  shows thCa,L in right ventricular myocytes are illustrated in P<0.05) peak ICa,L density from −4.9±0.7 to −11.2±0.9 pA/pF  from 115±15.2 to 197.1±23 pS/pF and V1/2(act) was shifted (P<0.05) from −8.9±0.8 to −15.4±1 mV  on I.9 pA/pF  in ventr5.4±1 mV  in IBMX.Ca,L (P<0.05) peak ICa,L density from −4.7±0.4 to −6.1±0.5 pA/pF (P<0.05) ICa,L Gmax from 106.0±7.7 to 125.5±5.2 pS/pF and shifted the V1/2(act) from −25.1±1.5 to −28.5±1.6 mV (P<0.05) ICa,L density from −3.4±0.2 to −4.9±0.5 pA/pF  from 72.8±2.3 to 99.9±3.2 pS/pF by EHNA and the V1/2(act) was shifted (P<0.05) from −16.0±1.2 to −18.7±1.4 mV (Next we measured the effects of the PDE2 inhibitor EHNA (10 µM) on SAN and right atrial myocyte ICa,L . In SAN .5 pA/pF . Steady 5±1.6 mV . In righ.5 pA/pF . Further7±1.4 mV . These dCa,L (P = 0.92) in ICa,L density upon application of EHNA. Steady state conductance analysis  and V1/2(act) (−13±1 mV in control vs. −14.4±1.2) were not altered (P = 0.85) in the presence of EHNA. Thus, although selective PDE2 inhibition does not affect ventricular myocyte ICa,L, it does increase ICa,L in SAN and atrial myocytes.In contrast to SAN and atrial myocytes, EHNA had no effect on right ventricular myocyte ICa,L . SummaryCa,L  show no analysis  shows thCa,L are illustrated in P<0.05) peak ICa,L density in SAN myocytes from −3.8±0.5 to −6.3±0.2 pA/pF  from 110.7±5.8 to 160.4±3.3 pS/pF and V1/2(act) was shifted (P<0.05) from −16.8±2.3 to −18.4±2.1 mV by Mil  in peak ICa,L density upon application of Mil. Furthermore, ICa,L Gmax (95.1±2.9 pS/pF in control vs. 100.9±2.3) and V1/2(act) (−10.4±1.2 mV in control vs. −9.9±0.9 mV in Mil) were not altered following PDE3 inhibition in atrial myocytes.The effects of the PDE3 inhibitor Mil (10 µM) on SAN and right atrial I.2 pA/pF . Also, IV by Mil . In contV curves  show no Ca,L are illustrated in P = 0.63) on peak ICa,L density  and V1/2(act) (−8.8±0.6 mV in control vs. −9.2±0.4 mV in Mil) were not affected (P = 0.52) by Mil  by Mil . TogetheCa,L are presented in P<0.05) peak ICa,L density from −4.2±0.1 to −8.1±1.1 pA/pF (P<0.05) ICa,L Gmax from 99.2±7.2 to 188.3±2.8 pS/pF and shifted (P<0.05) the V1/2(act) from −18.1±3.1 to −20.2±0.7 mV (P<0.05) peak ICa,L density from −4.5±0.4 to −7.5±0.7 pA/pF (P<0.05) ICa,L Gmax from 130.8±0.7 to 176.9±1.0 pS/pF and shifted (P<0.05) the V1/2(act) from −7.1±0.2 to −12.3±0.2 mV  on SAN and right atrial myocyte I.1 pA/pF . Rol als2±0.7 mV . In righ.7 pA/pF . Further3±0.2 mV . These eCa,L (P = 0.13) in peak ICa,L density following application of Rol. In addition, neither ICa,L Gmax (98.5±1.5 pS/pF in control vs. 95.1±1.9 pS/pF in Rol) or V1/2(act) (−9.1±0.4 mV in control vs. −8.9±0.6) were modulated (P = 0.53) by Rol. Thus, selective PDE4 inhibition elicits increases in ICa,L in SAN and atrial myocytes, but not in ventricular myocytes.In contrast to SAN and atrial myocytes, Rol had no effect on right ventricular myocyte ICa,L . SummaryCa,L in ventricular myocytes is consistent with previous studies in mice and rats, which have shown that selective inhibitors must be given in combination to augment basal ICa,LCa,L , increased ICa,L Gmax  and shifted the V1/2(act)  compared to control. Thus, our data illustrating the effects of global and specific PDE inhibitors on ventricular ICa,L fully agree with previous studies, which supports the conclusion that specific PDE inhibitors have distinct effects in different regions of the myocardium. These ventricular ICa,L data also confirm that the PDE inhibitors we have used are selective at the doses we have studied. We also measured the effects of combined PDE3/4 inhibition on AP firing and ICa,L in SAN and right atrial myocytes. We observed that Mil + Rol augments AP firing and ICa,L in SAN and atrial myocytes similarly to IBMX (data not shown).The observation that selective PDE inhibitors, when given alone, have no effect on basal Ia,LCa,L . SummaryCa,L in mouse SAN and atrial myocytes. We also measured the effects of these inhibitors on mouse ventricular ICa,L in order to make direct comparisons between the three myocyte types. This is the first direct comparison of these effects in the mouse myocardium. The main finding of these experiments is that PDE effects on AP firing and basal ICa,L are distinct in the different regions of the myocardium.PDEs are critical regulators of cAMP and cGMP levels in cardiomyocytes, representing the primary enzymes responsible for cyclic nucleotide degradation, and acting as the only counter balance to the mechanisms responsible for cyclic nucleotide production (adenylyl and guanylyl cyclases) 50. Similarly IBMX markedly increased AP duration at 50, 70 and 90% repolarization in mouse atrial myocytes. These data show that there is a high level of constitutive PDE activity that regulates AP properties in the SAN and atrial myocardium; however, subsequent experiments with specific PDE inhibitors revealed important differences between the two cell types. Specifically, in mouse SAN myocytes, EHNA, Mil and Rol each increased spontaneous AP frequency on their own, indicating that PDE2, PDE3 and PDE4 all contribute to the regulation of AP properties in basal conditions. In contrast, in mouse atrial myocytes, only EHNA and Rol were able to increase AP duration whereas Mil had no effect, indicating that constitutive PDE2 and PDE4, but not PDE3 activity, regulates atrial AP properties.Our AP data clearly demonstrate that global PDE inhibition with IBMX potently increases spontaneous AP frequency in mouse SAN myocytes in association with increases in DD slope and APDCa,L in SAN and atrial myocytes. Specifically, IBMX increased basal ICa,L by approximately 185% in SAN myocytes and 140% in atrial myocytes. Consistent with our AP measurements, SAN myocyte ICa,L was increased by each of the selective inhibitors EHNA (∼31%), Mil (∼66%) and Rol (∼93%). In atrial myocytes only EHNA (∼38%) and Rol (∼72%) increased basal ICa,L. Mil had no significant effect on right atrial ICa,L once again confirming a key difference between SAN and atrial myocytes in terms of which specific PDE inhibitors are able to modulate basal AP properties and ICa,L in mice.Similar trends were also observed for ICa,L in mouse right ventricular myocytes and observed increases of approximately 130%. In agreement with prior studies Ca,L in ventricular myocytes. Only when Mil and Rol were applied together was ventricular ICa,L increased as also noted previously Ca,L in all regions of the myocardium, the ability of selective PDE inhibitors  to modulate basal ICa,L is strikingly different in SAN, atrial and ventricular myocytes.For further comparison, we measured the effects of IBMX on basal ICa,L in the different regions of the myocardium are not strictly related to differences in expression. For example, PDE2A is similarly expressed in each region of the myocardium yet PDE2 inhibition with EHNA augments ICa,L in SAN and atrial myocytes, but not ventricular myocytes. Also, PDE3A  contributes prominently to the DD in SAN myocytes and is well known to be directly modulated by cAMP f in mouse SAN myocytes f in rabbit SAN myocytes 2+ release from the SR and a subsequent activation of the Na+-Ca2+ exchanger (INCX) 2+ release mechanism is also cAMP sensitive and has been shown to be constitutively regulated by PDE3 + currents, such as IKs, which can be modulated by IBMX and Mil in guinea pig SAN myocytes Ks, but it has been shown to express IKr channels that are thought to be cAMP sensitive 2+ current  Ca,T, including in SAN myocytes, is not modulated by cAMP Ca,T is affected by PDE inhibitors.Our experiments have focused on the PDE2, 3 and 4 families and on the role of IIt is also possible that other PDE families contribute to AP and ionic current regulation in SAN and atrial myocytes. For example, PDE1 and PDE5 have been shown to be expressed in the heart and to play functional roles in some settings Ca,L in SAN, right atrial and right ventricular myocytes. Although IBMX potently increased ICa,L in all cell types our data demonstrate that each region of the myocardium displays a unique pattern of response to family specific PDE inhibitors. SAN myocytes were responsive to each specific inhibitor tested, while atrial myocytes were responsive to PDE2 and PDE4, but not PDE3 inhibitors. Ventricular myocytes, on the other hand, do not respond to any family specific PDE inhibitors when given alone. These differences may be important when considering cyclic nucleotide regulation of ion channels in different regions of the heart. It is now known that PDE signaling in the heart is complex, based on the hypothesis that PDEs can be compartmentalized to distinct subcellular regions. Our data show that another aspect of the complexity of PDE regulation in the heart is related to regional differences in the roles of specific PDE families on ion channel regulation in the SAN compared to the working  myocardium.In summary, we have characterized the effects of inhibitors of PDE2, PDE3 and PDE4 on AP properties in mouse SAN and atrial myocytes and directly compared the effects of these inhibitors on basal IFigure S12+ current in right ventricular myocytes.Effects of IBMX on L-type CaA. Representative ICa,L recordings (at 0 mV from −40 mV) in right ventricular myocytes in control conditions, in the presence of IBMX (100 µM), and after IBMX washout. B. Summary I–V relationships for the effects of IBMX on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of IBMX on right ventricular myocytes. Summary data are means ± SEM; n = 6 ventricular myocytes; *P<0.05 vs. control by paired Student's t-test.(TIF)Click here for additional data file.Figure S22+ current in right ventricular myocytes.Effects of PDE2 inhibition with EHNA on L-type CaA. Representative ICa,L recordings (at 0 mV from −40 mV) in right ventricular myocytes in control conditions, in the presence of EHNA (10 µM), and after EHNA washout. B. Summary I–V relationships for the effects of EHNA on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of EHNA on right ventricular myocytes. Summary data are means ± SEM; n = 10 ventricular myocytes; EHNA had no effect on right ventricular ICa,L (paired Student's t-test).(TIF)Click here for additional data file.Figure S32+ current in right ventricular myocytes.Effects of PDE3 inhibition with milrinone on L-type CaA. Representative ICa,L recordings (at 0 mV from −40 mV) in right ventricular myocytes in control conditions, in the presence of Mil (10 µM), and after Mil washout. B. Summary I–V relationships for the effects of Mil on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Mil on right ventricular myocytes. Summary data are means ± SEM; n = 5 ventricular myocytes; Mil had no effect on right ventricular ICa,L (paired Student's t-test).(TIF)Click here for additional data file.Figure S42+ current in right ventricular myocytes.Effects of PDE4 inhibition with rolipram on L-type CaA. Representative ICa,L recordings (at 0 mV from −40 mV) in right ventricular myocytes in control conditions, in the presence of Rol (10 µM), and after Rol washout. B. Summary I–V relationships for the effects of Rol on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Rol on right ventricular myocytes. Summary data are means ± SEM; n = 5 ventricular myocytes; Rol had no effect on right ventricular ICa,L (paired Student's t-test).(TIF)Click here for additional data file.Figure S52+ current in right ventricular myocytes.Effects of PDE3 and PDE4 inhibition with milrinone and rolipram on L-type CaA. Representative ICa,L recordings (at 0 mV from −40 mV) in right ventricular myocytes in control conditions, in the presence of Mil + Rol (10 µM each), and after drug washout. B. Summary I–V relationships for the effects of Mil + Rol on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Mil + Rol on right ventricular myocytes. Summary data are means ± SEM; n = 8 ventricular myocytes; *P<0.05 vs. control by paired Student's t-test.(TIF)Click here for additional data file.Appendix S1Supplemental materials and methods.(PDF)Click here for additional data file.Table S1Effects of IBMX on spontaneous action potential parameters in isolated mouse SAN myocytes.(PDF)Click here for additional data file.Table S2Effects of IBMX on stimulated action potential parameters in isolated mouse right atrial myocytes.(PDF)Click here for additional data file.Table S3Effects of EHNA on spontaneous action potential parameters in isolated mouse SAN myocytes.(PDF)Click here for additional data file.Table S4Effects of EHNA on stimulated action potential parameters in isolated mouse right atrial myocytes.(PDF)Click here for additional data file.Table S5Effects of milrinone on spontaneous action potential parameters in isolated mouse SAN myocytes.(PDF)Click here for additional data file.Table S6Effects of milrinone on stimulated action potential parameters in isolated mouse right atrial myocytes.(PDF)Click here for additional data file.Table S7Effects of rolipram on spontaneous action potential parameters in isolated mouse SAN myocytes.(PDF)Click here for additional data file.Table S8Effects of rolipram on stimulated action potential parameters in isolated mouse right atrial myocytes.(PDF)Click here for additional data file.


Their antitumor effects on BEL-7402, HUH-7, and HepG2 cells were studied by MTT assay. The antitumor biological mechanism of these two complexes was studied in BEL-7402 cells by cell cycle analysis, Hoechst 33342 staining, Annexin V-FITC/PI assay, and detection of mitochondrial membrane potential (ΔΨm). The results showed that the growth of cancer cells was inhibited significantly, and complexes 1 and 2 mainly caused in BEL-7402 cells G0/G1 cell cycle arrest and induced apoptosis. Both 1 and 2 decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Complex 2 showed greater antitumor efficiency than that of complex 1.Two oxovanadium(IV) complexes of [VO(msatsc)(phen)], ( Schiff bases are an important class of ligands because such ligands and their transition metal complexes have a variety of applications including biological, clinical, and analytical applications . The devin vitro). Much effort has been done for vanadyl species coordinated to organic ligands on the research of their mimetic effects in hopes of developing vanadodrugs  and (2)  (2 have been synthesized and characterized. Their antitumor effects on BEL-7402 human liver (Bel7402), HUH-7, HepG2 cells were studied by MTT assay. The reported compounds may be an addition of new class of compounds as the metal-based drugs.Because V(IV) complexes have no charge, they are perceived to be candidates for easy bioabsorption. On the other hand, vanadyl(IV) complexes incorporating thiosemicarbazones have been studied extensively for their insulin-like effects which result in the inhibition of glycerol release and enhancement of glucose uptake by rat adipocytes and have been used in the treatment of tuberculosis –20. In vrbazone)  2 have b2 (acac = acetylacetonate) and 1,10-phenanthroline (phen) were commercially available and used as received. DMSO, CHCl3 were purchased from Aldrich (USA). Other chemicals and reagents of analytical grade were obtained commercially without further purification unless specifically mentioned.VO(acac)BEL-7402, HUH-7, and HepG2 cell lines were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences . RPMI 1640 medium was purchased from Hyclone , Trypsin, fetal calf serum and Annexin V-FITC/PI apoptosis detection Kit were purchased from GIBCO Company (USA). Hoechst 33342 staining solution was purchased from Beyotime Institute of Biotechnology (China). MTT and Rhodamine123 were purchased from Sigma Company (USA).1H NMR spectra were recorded on a Varian-500 spectrometer. All chemical shifts are given relative to tetramethylsilane (TMS). Infrared spectra were recorded on a Bomen FTIR model MB102 instrument using KBr pellets. UV-Vis spectra were recorded on a Schimadzu UV-3101 PC spectrophotometer at room temperature. Emission spectra were recorded on a Perkin-Elmer Lambda 55 spectrofluorophotometer. Magnetic susceptibility measurements were recorded on a MPMSXL-7 , at room temperature. Solutions of compounds were freshly prepared 2 h prior to biochemical evaluation. Microanalysis  was carried out with a Perkin-Elmer 240Q elemental analyzer. Electrospray mass spectra (ES-MS) were recorded on a LCQ system  using methanol as mobile phase. Cell viability assay was carried out with a microplate reader . Cell cycle analysis, Annexin V-FITC/PI assay of apoptotic cells and detection of mitochondrial membrane potential were recorded on a FACScan flow cytometer . Fluorescence microscopy of Apoptosis assays was carried out with the fluorescence microscope .9H11N3O2S: C, 48.01; H, 4.89; N, 18.67; S, 14.22%; Found: C, 47.89; H, 4.69; N, 18.53; S, 14.11%. 1H NMR : 11.34 , 9.88 , 7.21 , 6.86 , 6.81 , 6.79 , 3.53 . ES-MS (CH3OH): m/z 226.0 ([M+H]+). UV λmax, nm  in DMSO: 340 (26310). 4-chlorosatsc: White solid. Yield: 83%. Anal. Calcd. for C8H8N3OSCl: C, 42.01; H, 3.49; N, 18.38; S, 14.00%; Found: C, 41.85; H, 3.24; N, 18.13; S, 13.92%. 1H NMR : 11.54 , 9.97 , 8.16 , 7.75  6.86 ,. ES-MS (CH3OH): m/z 230.5 (M+). UV λmax, nm  in DMSO: 346 (18655). Methoxylsalicylaldehyde thiosemicarbazone (msatsc) and 4-chlorosalicylaldehyde thiosemicarbazone (4-chlorosatsc) were prepared with a method similar to that described earlier –21. An e2O (phen·H2O)  in absolute alcohol (20 cm3) were heated at 72°C under argon for 30 min. After dissolution, VO(acac)2  was added and the brown red precipitate was formed immediately. This suspension was kept stirring under reflux for about 3.5 h and then the coloured solid formed was filtered off from the hot solution, washed with mixed solvents of ethanol and ether for three times and dried under vacuum. Yield: 90%. Anal. Calcd for C21H17N5O3SV: C, 53.63; H, 3.62; N, 14.89; S, 6.81%. Found: C, 53.54; H, 3.56; N, 14.63; S, 6.64%. 1H NMR : 9.11 , 8.51 , 7.97 , 7.78 , 7.22 , 6.86 , 6.82 , 6.77 , 3.65 . ES-MS (CH3OH): m/z 470.9 ([M+H]+). UV λmax, nm  in DMSO: 265 (41770), 340 (15255), 383 (6525), 760 (20), 797 (20). Magnetic moment: μeff: 1.70 BM. Molar conductance ΩM (Ω−1 cm2 mol−1): 9.65.A mixture of methoxylsalicylaldehyde thiosemicarbazone (msatsc)  and 1,10-phennanthroline·H1. Yield: 89%. Anal. Calcd for C20H14N5O2ClSV: C, 50.59; H, 2.95; N, 14.76; S, 6.75% Found: C, 50.45; H, 2.68; N, 14.53 S, 6.44%. 1H NMR : 9.11 , 8.51 , 8.01 , 7.79 , 7.78 . ES-MS (CH3OH): m/z 475.5 (M+). UV λmax, nm  in DMSO: 264 (41015), 347 (10975), 400 (7815), 786 (20). Magnetic moment: μeff: 1.73 BM. Molar conductance ΩM (Ω−1 cm2 mol−1): 10.56. This complex was synthesized with the same method described for 2/95% air. The medium was changed every two days and the cells were subcultured every three days.The cells were routinely cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum. The culture was maintained at 37°C with a gas mixture of 5% CO4 cells/mL, 100 μL aliquots were added to each well of 96-well flat-bottomed microtiter plates, followed by addition of 100 μL of the complexes 1 and 2. Three replicate wells were used for each data point in the experiments. After incubation for the indicated intervals, 20 μL of MTT (5 mg/mL in PBS) solution was added to each well and plates were then incubated for 4 h at 37°C. The medium with MTT was removed from the wells. Intracellular formazan crystals were dissolved by adding 150 μL of DMSO to each well, and the plates were shaken for 10 min. The absorbance was read at 490 nm with a microplate reader. Percentage of survival was calculated as a fraction of the negative control (medium only). The half-maximal inhibitory concentration (IC50) was obtained from the dose-response curve with an original 6.0 software. Cell viability was determined using the MTT assay. Briefly, the cells were collected and resuspended in RPMI1640 medium at 4 × 101 and 2, harvested by centrifugation at 1000 ×g for 5 min, and then washed with ice-cold PBS. The collected cells were fixed overnight with cold 70% ethanol, and then stained with PI solution consisting of 50 μg/mL PI, 10 μg/mL RNase. After 10 min incubation at room temperature in the dark, fluorescence-activated cells were sorted in a FACScan flow cytometer using CellQuest 3.0.1 software. Analysis of the cell cycle of control and treated cancer cells was determined. Using standard methods, the DNA of cells was stained with PI, and the proportion of non-apoptotic cells in different phases of the cell cycle was recorded. The cancer cells were treated with complexes 1 and 2 for 48 h, BEL-7402 cells were washed twice with PBS, then stained with 10 μg/mL Hoechst 33342 staining solution at 37°C for 30 min according to the manufacturer's instructions. Finally, the cells were observed under the fluorescence microscope.This method was modified from a previous report , 22. Bri1 and 2 for 48 h were determined by flow cytometry using a commercially available Annexin V-FITC/PI apoptosis detection Kit. After treatment, cells were harvested and washed twice in ice-cold PBS and resuspended in 500 μL of binding buffer at 1–5 × 105 cells/mL. The samples were incubated with 5 μL of Annexin V-FITC and 5 μL propidium iodide in the dark for 15 min at room temperature. Finally, samples were analyzed by flow cytometry and evaluated based on the percentage of cells for Annexin V positive.BEL-7402 cells treated with complexes μM) is the positive control. Treated with complexes 1 and 2 for 48 h, BEL-7402 cells were incubated with Rhodamine123 (2 μM/mL) at 37°C for 30 min, and washed with PBS. The cell pellets were collected by centrifugation  and resuspended in 500 μL PBS. Green fluorescence intensities of Rhodamine 123 in cells were analyzed by FL-1 channel of the flow cytometer.In this study, ΔΨm was measured by using Rhodamine123, and 5-Fluorouracil . Significance was accepted at P value lower than 0.05. Data were expressed as the mean ± SD from these independent experiments. Statistic analysis was performed using the SPSS 13.0 for Windows. Comparisons between two groups were performed by unpaired Oxovanadium(IV) complexes of formulation [VO(msatsc)(phen)] 1  and its novel derivative [VO(4-chlorosatsc)(phen)]  2 are prepared in high yield from a general synthetic procedure in which vanadylacetylacetonate is reacted with the ligands in ethanol .−1 and/or 829 cm−1 corresponding to the C=S vibration. A strong band is observed in the free ligands at 1614 cm−1 or 1603 cm−1, characteristic of the imine (C=N) , 27−1 an1H NMR spectra of complexes 1 and 2 are in excellent agreement with the proposed structures. In the 1H NMR spectra of the two complexes, the chemical shifts of the phenolic hydroxy protons of 11.34 ppm and 11.54 ppm, and amino protons linking directly to the imine groups of 9.88 ppm and 9.97 ppm for complexes 1 and 2, respectively, in comparison with the metal-free ligands are unobserved. These facts also affirm that the free ligands are coordinated to metal. The π-π* transitions of aromatic rings of phenanthroline  and (2)  have been synthesized and characterized. Their antitumor effects on BEL-7402, HUH-7, and HepG2 cells were studied by MTT assay. In vitro experimental results show that both 1 and 2 possess significant antiproliferative effects. The results also showed that they can cause G0/G1 phase arrested of the cell cycle and decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Both complexes exhibit significant induced apoptosis in BEL-7402 cells and displayed typical morphological apoptotic characteristics, and complex 2 showed greater antitumor efficiency than that of complex 1. These results may provide further evidence to exploit the potential medicine compounds from the metal complexes.In summary, two oxovanadium(IV) complexes of [VO(satsc)(phen)] (


In this study, we investigated the potential role of astaxanthin, an antioxidant marine carotenoid, in re-establishing SHP-1 negative regulation on pro-inflammatory cytokines secretion in U-937 cell line stimulated with oxidative stimulus. ELISA measurement suggested that ASTA treatment (10 µM) reduced pro-inflammatory cytokines secretion  induced through H2O2, (100 µM). Furthermore, this property is elicited by restoration of basal SHP-1 protein expression level and reduced NF-κB (p65) nuclear expression, as showed by western blotting experiments.It has been suggested that oxidative stress activates various intracellular signaling pathways leading to secretion of a variety of pro-inflammatory cytokines and chemokines. SHP-1 is a protein tyrosine phosphatase (PTP) which acts as a negative regulator of immune cytokine signaling. However, intracellular hydrogen peroxide (H Nuclear extracts were prepared according to Osborn et al. [2, 0.2% NP-40 and 0.5 mmol/L PMSF) and placed on ice for 10 min to allow lysis. Nuclei were pelleted by centrifugation at 3500 g for 10 min at 4 °C, resuspended in 1 mL of Buffer B  and placed on a rocking platform for 30 min at 4 °C. The nuclear lysates were then clarified by centrifugation at 14,000 g for 20 min at 4 °C and the supernatants (nuclear extracts) collected. The protein concentrations of the extracts were determined using the Bradford method . For Western blot analysis, 50 µg of protein per lane was separated on a 4–12% NuPAGE gradient gel (Gibco Invitrogen), electro-transferred on to a nitrocellulose membrane and blocked with 10% skimmed milk in PBS containing 0.1% Tween-20. Blots were probed and incubated overnight at 4 °C with the rabbit polyclonal IgG anti-SHP-1, the rabbit polyclonal IgG anti-pNF-κB and NF-κB (p65) all at 0.2 µg/mL in Tris-buffered saline (TBS)/0.1% Tween-20. A rabbit antihuman monoclonal antibody recognizing the human β-actin was used as control in all experiments. Blots were then washed and incubated for 1 h with goat antirabbit–horseradish peroxidase  diluted 1:10,000 in TBS/0.1% Tween-20. Immunoblot signals were developed using the Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology). The blot images were analyzed with a gel analysis software package . Data are expressed as mean ± SD intensity of optical density.U937 cells were washed once in cold phosphate-buffered saline  and harvested by gentle scraping, and used to prepare total protein or nuclear extracts. Total protein extracts were prepared by treating cells with lysis buffer [50 mmol/L Tris–HCl pH 7.5, 0.4% Nonidet P-40 (NP-40), 120 mmol/L NaCl, 1.5 mmol/L MgCln et al. . Cells wp < 0.05) was considered statistically significant. The comparison H2O2 vs. H2O2 + ASTA and H2O2 + ASTA vs. H2O2 + ASTA + SS was performed using post hoc test with the alpha level at 0.05.The results are expressed as mean ± SD. Statistical analysis was performed using analysis of variance (ANOVA). The probability of null hypothesis of <5% .It is well recognized that persistent inflammation contributes to the pathogenesis of many diseases, including cancer, heart disease and atherosclerosis ,26,27. T


The model was confirmed with elevated portal pressures and increased serum CD163 levels. The inducible nitric oxide synthase (iNOS) or heme oxygenase-1 (HO-1) in portal triads was assessed. The isolated portal perfused rat liver (IPPRL) was performed at d0, d28, d56, and d84 in the progression of chronic hepatitis. After constricting with phenylephrine, the portal veins were relaxed with Sal B. The EC50 of Sal B for relaxing portal veins was−2.04 × 10−9, 7.28 × 10−11, 1.52 × 10−11, and 8.44 × 10−11 mol/L at d0, d28, d56, and d84, respectively. More macrophages infiltrated in portal triads and expressed more iNOS or HO-1 as PH advanced. The areas under the curve (AUCs) of Sal B for reducing PH were positively correlated with the levels of iNOS or HO-1 in portal triads, and so did with serum CD163 levels. Sal B reduces PH in IPPRL with chronic hepatitis, via promoting portal relaxation due to macrophage-originated NO or CO in portal triads, partly at least.This study is aimed to investigate the effects of Sal B on portal hypertension (PH). PH with chronic hepatitis was induced by carbon tetrachloride (CCl Portal hypertension (PH) is a common complication in the patients with advanced chronic hepatitis . The incSalvia miltiorrhiza (Danshen), which is a traditional Chinese medicine widely used for cardiovascular diseases . Ra. Ra4 in ectively , 21; olieciously . The exuanalized , 21. TheThe blood samples were centrifuged at 1200 g for 10 min at 4°C, and serum was stored at −80°C until the assays. Serum CD163 levels was measured using enzyme-linked immunosorbent assay kits  following the manufacturer's guidelines. μm thickness and then stained with Masson's trichrome (Masson) . The average of ten random fields generated a single data for statistic analysis [For immunohistochemical analysis, sections were incubated with rabbit polyclonal antibody against iNOS  or HO-1 . Staining was visualized using avidin-biotin peroxidase immunostaining kit with diaminobenzidine . The mean optical density (OD), positive staining area , respectively [−10, 2.64 × 10−10, 5.82 × 10−10, and 8.24 × 10−10 mol/L, respectively, to constrict portal veins [−13–10−7 mol/L) was added into the recirculating perfusate. Dose-response curves were obtained from the Sal B concentrations and the changed percentage of the perfused pressure from the baseline of phenylephrine constriction.The isolated portal perfusion system was performed with controlled velocity as described previously , 21. At t-test or Mann-Whitney. Significant differences were established at the 0.05 level. The equation, the EC50 with its 95% confidence intervals of Sal B, and the area under the curve (AUC) of Sal B were analyzed using GraphPad Prism 4 (GraphPad Software). The EC50 of Sal B (y) was regressed with the durations  and serum CD163 levels (x) in the progression of chronic hepatitis, and the AUC of Sal B (y) was regressed with the amounts of existed iNOS or HO-1 from immunohistochemical staining and the serum CD163 levels (x).All data are expressed as mean ± S.E.M. Comparisons between groups were performed using Student's d0 to d84 (P < 0.01) as the progression of chronic hepatitis  as the procession of chronic hepatitis  as the procession of chronic hepatitis , d56 (1.5-fold), and d84 (3-fold) compared with that at d0, respectively, (P < 0.01); these were decreased at d56 and increased at d84 compared with that at d28, respectively (P < 0.01); so did increased that atd84 compared with that at d56 (P < 0.01).The iNOS-IHC OD per volume  in the pd0 , d56 (2-fold), and d84 (3-fold) compared with that at d0, respectively (P < 0.01); these at d56 and d84 significantly increased compared with that at d28, respectively (P < 0.01); so did that at d84 compared with that at d56 (P < 0.01).The total HO-1-IHC OD per volume  in the rd0, Sal B constricted portal veins of normal rats (y = 0.5290 + 2.2160/[1 + 10x); the EC50 with its 95% confidence intervals was 2.04 × 10−9 (1.02 × 10−10–4.10 × 10−8) mol/L. At d28 (d56 (d84 (y = −0.0563 + 0.0520/[1 + 10x), y = −0.0672 + 0.0585/[1 + 10x), and y = −0.1203 + 0.0918/[1 + 10x), respectively; the EC50 with their 95% confidence intervals were 7.28 × 10−11(1.23 × 10−11–4.30 × 10−10) mol/L, 1.52 × 10−11(3.90 × 10−12–5.90 × 10−11) mol/L, and 8.44 × 10−11(1.21 × 10−11–1.97 × 10−10) mol/L, respectively.At mal rats , the equ At d28 , d56  from the EC50 (y × 10−11 mol/L) of Sal B to the durations (x = d × 24 + 11.47 (hours); d = 0, 28, 56, and 84 days) of chronic hepatitis progression. So did the equation was y = 201.9300x − 3993  from CD163 levels to the durations.The liner regressive equation was y = 0.3587x − 8.0364  from the AUCs of Sal B to the iNOS-OD/V (%) in portal triads at d0, d28, d56, and d84 in the progression of CCl4-induced chronic hepatitis.The liner regressive equation was y = 0.4120x − 9.3727  from the AUCs of Sal B to the HO-1-OD/V (%) in portal triads at d0, d28, d56, and d84 in the progression of CCl4-induced chronic hepatitis. The liner regressive equation was y = 0.8531x + 26.2360  from the AUCs of Sal B to the serum CD163 levels at d0, d28, d56, and d84 in the progression of CCl4-induced chronic hepatitis. It was y = 22.8210x + 19.3530  from the AUCs to the serum CD163 levels at d28, d56, and d84 in the progression of CCl4-induced chronic hepatitis.The liner regressive equation was 4-induced chronic hepatitis. Its mechanisms are related to the inhibition of oxidative stress from macrophages and the increase of NO bioavailability or CO potency in portal triads. Sal B is the most active antioxidant extracted from Danshen and has obvious effects for liver fibrosis, chronic hepatitis, or PH in clinic [It was demonstrated in the present study that Sal B relaxed portal veins in IPPRLs of CCl− to further reduce NO bioavailability and aggregate PH [− generation [It has been reported that the portal resistance is mainly located at the terminal portal venules (TPV) in portal triads . The actegate PH . The HO-egate PH . The locneration . Therefoneration  for PH [neration , 21, 25,neration , and to neration , especianeration . We hypo4-induced chronic hepatitis and was confirmed with increased portal pressure and pathological changes, such as the peritoneal exudation, enlarged hepatic indexes, serum hepatic biomarker levels, collagen deposition, and pseudolobule formation [50 of Sal B relaxation was positively correlated with the duration of CCl4-induced chronic hepatitis, indicating the action of Sal B which was pathological dependent. Our results demonstrated that increased iNOS or HO-1 levels in the macrophages infiltrated in portal triads are involved in the mechanism of Sal B relaxation. The existed levels of iNOS or HO-1 in lobules disappeared gradually, these in portal triads strengthened continuously along with the progression of CCl4-induced chronic hepatitis, especially in the infiltrated macrophages. We also reported here that iNOS and HO-1 levels in portal triads are correlated positively with the AUCs of Sal B for reducing PH.In present study, PH model in rat was replicated by CClormation , 2. The ormation . We foun− generation [− formation [− generation from NO [2+ influx [The IPPRL was used in this study to evaluate the effect of Sal B on PH. The hepatic artery was ligated to ensure that the portal resistance originated mainly from the smooth muscle cells in terminal portal venule (TPV) and the sphincter-like endothelia at hepatic sinusoid inlets . In PH rneration , rescuedneration , and conneration –11, 23. neration . (2) CO neration  and elevneration , 17. Espormation . (3) EcS from NO . Then th from NO , Sal B a from NO ; Sal B s from NO . On huma+ influx . It's a + influx . Further+ influx .Sal B for reducing PH might be used to explain the actions of medical plants in Chinese prescription for the ascitic patients with chronic hepatitis. It is an interesting clue to discover more effective candidates depending on the macrophage iNOS or HO-1 in portal triads, at least partly. Consequently, Sal B or its derivative might be exploited as a candidate to increase NO bioavailability or CO potency, especially from free radical damages in inflammatory diseases.


O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.(-)-Epigallocatechin 3- Ex=280 nm, catechin  s: ε=Absorbance/lightpath (cm)/concentration (M).Absorbance of EGCG was measured at 10 µM with Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.) at room temperature and standardized for 1 cm lightpath according to the Nanodrop’s manual. Extinction (Fluorescence of EGCG was measured at 10 µM with Hitachi F-7000 spectrofluorometer (Hitachi High-Technologies Co.) in a 1 cm quartz cell thermostated at 20°C. The slit width was 5 nm, PMT voltage 700 V, scan speed 240 nm/min.The absorbance and fluorescence spectra were exported to and plotted with Origin 9 software (OriginLab Co).2 makes pH uncertain that may affect fluorescence and EGCG stability. We started initial measurements in DMSO, an aprotic solvent and included AN since DMSO has a cut-off at 265 nm (determined as absorbance of 1.00 in a 1 cm cell vs. water) rendering measurements of absorbance at shorter wavelengths impossible. Spectral measurements in DMSO were done in 100% DMSO, while solutions in AN and AB contained 0.1% DMSO. Therefore, 0.1% DMSO was added to the AN and AB blanks. It is noteworthy that even 0.1% DMSO can present a problem for UV absorbance and fluorescence measurements because the cut-off of 0.1% DMSO in EtOH is 229 nm, and in AB is 223 nm (not shown).EGCG was prepared as a 30 mM stock in DMSO, aliquoted with an Eppendorf Repeater® plus at 2 µL to avoid decomposition due to the freeze-thaw cycling and stored at -20°C. Before experiments, 4 µL of DMSO were added to a vial to bring EGCG concentration to 10 mM before 1/1000 dilution to the experimental concentration of 10 µM. Spectral characteristics were measured at 10 µM in AN, an aprotic solvent, and in AB containing KCl (150 mM), HEPES (10 mM) intended to mimic the aqueous cellular environment and at pH=7.0. We chose to measure fluorescence in a pH-buffered AB at a neutral pH=7.0 rather than distilled deionized water because ambient COAll chemicals were purchased from Sigma-Aldrich Co. The purity and structure of EGCG were confirmed by NMR and LCMS. 18.2 MΩ water  was used for all experiments.max=275 nm was 0.151±0.004 (n=4), 0.175±0.017 (n=4), and at UVmax=277 nm it was 0.138±0.014 (n=4) in three independent experiments  at UVmax=275 nm. When excited at UVmax=275 nm, the intensity of fluorescence was 1,258 arbitrary units (au) at Emmax=365 nm (emission scan)  . An addi= 389 au .max=275 nm, 0.100±0.005 (n=4) at UVmax=270 nm, and 0.087±0.007 (n=4) at UVmax=276 nm in three independent experiments  in AB at UVeriments . When exx=388 nm . The 3D x=389 nm . Interes 18.5 au .max=280 nm was 0.092±0.008 (n=6), 0.121±0.009 (n=4), and at UVmax=279 nm it was 0.095±0.008 (n=4) in three independent experiments .Absorbance of 10 µM EGCG in DMSO at UVeriments . When exx=353 nm ; Table 1Ex~235-237 nm due to the cut-off effects of DMSO (an aprotic solvent) to contrast with protic solvents such as AB and EtOH, made us use AN, a non-hydrogen bonding polar solvent with a cut-off of 190 nm similar to water (191 nm). Absorbance of 10 µM EGCG in AN at UVmax=231 nm was 1.712±0.004 (n=4)  , Table 1x=275 nm , and twoA- νF)*10-3 (cm-1) was plotted against the orientation polarizability Δf for the different solvents from A- νF)*10-3 (cm-1) = 104/Ex(nm) - 104/Emmax(nm) (2=0.95 (The Stokes shift (νmmax(nm) . The larmax at 396 nm in AB, ~344 nm in AN, and 373 nm in EtOH  and 388 nm (Emmax in AB) because orientation polarizability ∆f of AN (0.304) is between those of EtOH (0.298) and AB (0.320)   2=0.95 (and DMSO  points trtain pH  is diffiWe demonstrated that EGCG is a fluorescent molecule and, importantly, its fluorescence is significantly dependent on the polarity of solvent. Interaction of EGCG with a binding pocket of a protein is likely to transfer EGCG from aqueous environment to one with different polarity that is expected to significantly change fluorescence intensity and shift emission maxima. We suggest that both changes in fluorescence intensity and fluorescence emission shifts can be used to study interaction of EGCG with HSP90 or other proteins. In addition, high EGCG fluorescence is useful for studies of binding to proteins with fluorescence anisotropy approach .


CA arrested the growth of dermatitis-inducing Gram-positive and Gram-negative microorganisms such Propionibacterium acnes, Pseudomonas aeruginosa, and Staphylococcus aureus. CA also blocked the nuclear translocation of nuclear factor (NF)-κB and its upstream signaling including Syk/Src, phosphoinositide 3-kinase (PI3K), Akt, inhibitor of κBα (IκBα) kinase (IKK), and IκBα for NF-κB activation. Kinase assays revealed that Syk could be direct enzymatic target of CA in its anti-inflammatory action. Therefore, our data strongly suggest the potential of CA as an anti-inflammatory drug against skin inflammatory responses with Src/NF-κB inhibitory properties.Carnosic acid (CA) is a diterpene compound exhibiting antioxidative, anticancer, anti-angiogenic, anti-inflammatory, anti-metabolic disorder, and hepatoprotective and neuroprotective activities. In this study, the effect of CA on various skin inflammatory responses and its inhibitory mechanism were examined. CA strongly suppressed the production of IL-6, IL-8, and MCP-1 from keratinocyte HaCaT cells stimulated with sodium lauryl sulfate (SLS) and retinoic acid (RA). In addition, CA blocked the release of nitric oxide (NO), tumor necrosis factor (TNF)- Propionibacterium acnes, Pseudomonas aeruginosa, and Staphylococcus aureus) and fungi , exposure to irritating chemicals such as sodium lauryl sulfate (SLS) or retinoic acid (RA), and various allergens, such as 2,4-dinitrophenol (DNP), are regarded as major inflammatory signaling inducers causing skin rash [α cytokines, IL-8 and monocyte chemotactic protein (MCP)-1 chemokines, and nitric oxide (NO) and prostaglandin E2 (PGE2) mediators, are produced [κB and activator protein (AP)-1 transcription factors [α for TNF-α secretion, inducible NO synthase (iNOS) for NO release, and cyclooxygenase (COX)-2 for prostaglandin E2 (PGE2) production [Skin inflammation (dermatitis) includes many inflammatory symptoms occurring in the skin layer in the form of a rash . Infectikin rash , 6. In aproduced , 8. For produced , 10 or i factors , 12, traoduction –16. Rosmarinus officinalis L. [Carnosic acid CA; , isolatenalis L. , is a prnalis L. . This conalis L. –21. How nalis L. –24.Although polyphenolic compounds including CA are currently being investigated for industrial applications, their mass production by chemical syntheses and their photostability remain critical factors for consideration. CA is chemically stable and CA production and yield have been greatly improved by current synthetic methods , 26. In E. coli 0111:B4) were purchased from Sigma Chemical Co. . Piceatannol (picea) and PP2 were obtained from Calbiochem . Luciferase constructs containing promoters sensitive to NF-κB, CREB, and AP-1 were gifts from Profs. Chung Hae Young  and Man Hee Rhee . Enzyme immunoassay (EIA) kits and enzyme-linked immunosorbent assay (ELISA) kits for determining PGE2, IL-6, IL-8, MCP-1, and TNF-α were purchased from Amersham . Fetal bovine serum and RPMI1640 were obtained from Gibco . The murine macrophage cell line, RAW264.7, the human keratinocyte cell line, HaCaT, the rat basophilic leukemia mast cell line, RBL-2H3, and the human embryonic kidney cell line, HEK293, were purchased from the ATCC . All other chemicals were of analytical grade and were obtained from Sigma. Phosphospecific or total antibodies to p65, p50, Src, Syk, PDK1, p85, Akt, IκBα, lamin A/C, and β-actin were obtained from Cell Signaling .CA (95% purity), phenoxyethanol, octanediol, methylparaben, magnotics, ampicillin, 3--2,5-diphenyltetrazolium bromide (MTT), peptidoglycan (PGN), and lipopolysaccharide , the proportion of dead cells was less than 1% as measured by Trypan blue dye exclusion.RAW264.7, HaCaT, RBL-2H3, and HEK293 cells were cultured in DMEM or RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum , glutamine, and antibiotics (penicillin and streptomycin) at 37°C under 5% CO6 cells/mL) for 18 h, CA (0 to 20 μg/mL) was added to the cells and incubated for 24 h. The cytotoxic effect of CA was then evaluated by a conventional MTT assay, as reported previously [μL MTT solution  was added to each well, and the cells were continuously cultured until termination of the experiment. The incubation was halted by the addition of 15% sodium dodecyl sulfate (SDS) into each well, solubilizing the formazan [570–630) was measured using a SpectraMax 250 microplate reader.After preincubation of RAW264.7 and HaCaT cells  for 18 h, cells were pretreated with CA (0 to 20 μg/mL) for 30 min and further incubated with LPS (1 μg/mL), pam3CSK (10 μg/mL), or PGN (10 μg/mL) for 6 (TNF-α) or 24  h. The inhibitory effect of CA on the production of NO, PGE2, IL-6, IL-8, MCP-1, and TNF-α was determined by analyzing NO, PGE2, IL-6, IL-8, MCP-1, and TNF-α levels with Griess reagent and enzyme-linked immunosorbent assay (ELISA) kits as described previously [After preincubation of RAW264.7 and HaCaT cells (1 × 10eviously . 2 atmosphere. RBL-2H3 cells (2 × 105 cells/well) pretreated with CA were sensitized with 1 μg/mL anti-DNP IgE for 1 h at 37°C. IgE-sensitized cells were washed with PBS and stimulated with 4 μg/mL 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) in PBS at 37°C for 30 min. Degranulation was assessed by measuring β-hexosaminidase release. Briefly, 40 μL supernatant and 100 μL 2 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide  were added to each well of a 96-well plate, and color was developed for 30 min at 37°C. The enzymatic reaction was terminated by adding 200 μL 0.2 M glycine-NaOH, pH 10.7. The absorbance at 405 nm was measured using a SpectraMax 250 microplate reader.RBL-2H3 cells were grown in DMEM supplemented with 10% FBS in a 5% COTo determine cytokine mRNA expression levels, total RNA was isolated from LPS-treated RAW264.7 cells using TRIzol Reagent (Gibco BRL) according to the manufacturer's instructions. Total RNA was stored at −70°C until use. Semiquantitative RT reactions were conducted as reported previously , 33. The6 cells/mL) were transfected with 1 μg plasmid containing NF-κB-Luc or AP-1-Luc along with β-galactosidase using the calcium phosphate method in a 12-well plate according to the manufacturer's protocol [HEK293 cells (1 × 10protocol . The celprotocol .6 cells/mL) were washed three times in cold PBS with 1 mM sodium orthovanadate and lysed by a sonicator in lysis buffer  for 30 min with rotation at 4°C. The lysates were clarified by centrifugation at 16,000 ×g for 10 min at 4°C and stored at −20°C until needed.RAW264.7 cells , 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 20 μg/mL aprotinin, and 100 μM 1,4-dithiothreitol (DTT) on ice for 4 min. Cell lysates were then centrifuged at 19,326 ×g for 1 min in a microcentrifuge. In the second step, the nuclear fraction pellet was washed once in washing buffer, which was the same as the lysis buffer without Nonidet P-40. In the final step, nuclei were treated with an extraction buffer containing 500 mM KCl, 10% glycerol, and the other reagents in the lysis buffer. The nuclei/extraction buffer mixture was frozen at −80°C and then thawed on ice and centrifuged at 19,326 ×g for 5 min. The supernatant was collected as a nuclear extract. Soluble cell lysates were immunoblotted, and protein levels were visualized as previously reported [μg) from RAW264.7 cells (1 × 107 cells/mL) treated with or without LPS (1 μg/mL) for 2.5 min were precleared with 10 μL protein A-coupled Sepharose beads (50% v/v)  for 1 h at 4°C. Pre-cleared samples were incubated with 5 μL anti-JAK2 antibody overnight at 4°C. Immune complexes were mixed with 10 μL protein A-coupled Sepharose beads (50% v.v.) and rotated for 3 h at 4°C.Nuclear lysates were prepared in a three-step procedure . After treported . For immμL, purified enzymes (1–5 mU) were incubated with the reaction buffer. The reaction was initiated by the addition of MgATP. After incubation for 40 min at room temperature, the reaction was stopped by the addition of 5 mL 3% phosphoric acid. Ten microliters of the reaction mixture was then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.For evaluating the inhibition of Src and Syk kinase activity using purified enzymes, the kinase profiler service from Millipore  was used. In a final reaction volume of 25 μg/mL) of the fourteen synthesized compounds was determined under conditions described in the literature [Propionibacterium acnes (ATCC 6919), Staphylococcus aureus (ATCC 33592), Escherichia coli (ATCC 25922), Candida albicans (ATCC 10231), and Aspergillus niger (ATCC 9142).The minimum inhibitory concentration  of two independent experiments. Other data are representative of three different experiments with similar results. For statistical comparisons, results were analyzed using analysis of variance/Scheffe's posthoc test and the Kruskal-Wallis/Mann-Whitney test. All P values < 0.05 were considered statistically significant. All statistical tests were conducted using SPSS .Data Figures  are exprCA is a multipotential diterpene displaying antioxidative, anticancer, antiangiogenic, anti-inflammatory, antimetabolic disorder, photoprotective, hepatoprotective, and neuroprotective activities –21. Althμg/mL (μg/mL) remarkably reduced the production of these molecules to basal levels , but not the recently developed antibiotic herb, Magnotics, which is known to inhibit pimples [P. aeruginosa, E. coli, S. aureus, C. albicans, and A. niger with MIC values ranging from 125 to 2,000 μg/mL, even though the activity was not as powerful as that of several antiseptics such as phenoxyethanol, octanediol, and methylparaben    stronglyat 5 min . Consistat 5 min , suggestpression  and in Teinase-9 . μg/mL) only directly and partially blocked the kinase activity of Syk but not Src (2 exhibited higher level (80 to 50%) at even 5 and 10 μg/mL , Sorbus commixta, and Sanguisorba officinalis were shown to target these enzymes in their anti-inflammatory actions [It is known that the phosphorylation of Src and Syk, initially activated protein tyrosine kinases by inflammatory signaling , 51, is  not Src . When th Figures  is consi Figures , indicat Figures  could afel 80 to % at evenreported . The crureported , 53. Fur actions –56. Therα, and PGE2 managed by macrophages and keratinocytes. Specifically, CA was clearly diminished the activation of NF-κB through the inhibition of its upstream signaling cascades composed of Syk, Src, PI3K, PDK1, Akt, IKK and IκBα (κB inhibitory drug available for skin inflammatory symptoms induced by various irritants, microorganism-derived immunogens, and allergens. To investigate this possibility, the in vivo efficacy using skin inflammatory models will be tested in the future. Furthermore, since two major pathways, JAK2/STAT-1 and TBK1/IRF-3 [In summary, we have found that CA strongly blocks several skin inflammatory responses such as the production of TNF-and IκBα . ConsideK1/IRF-3 , 58, areK1/IRF-3  which isK1/IRF-3 . Since t


The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose.Azohydromonas lata, Bacillus megaterium, Bacillus cereus, Burkholderia cepacia, Pseudomonas olevorans, Pseudomonas pseudoflava and Ralstonia eutropha, against seven fermentation inhibitors produced by the saccharification of lignocellulose: acetic acid, levulinic acid, coumaric acid, ferulic acid, syringaldehyde, furfural, and hyroxymethyfurfural. There was significant variation in the sensitivity of these microbes to representative phenolics ranging from 0.25-1.5 g/L coumaric and ferulic acid and between 0.5-6.0 g/L syringaldehyde. Inhibition ranged from 0.37-4 g/L and 0.75-6 g/L with acetic acid and levulinic acid, respectively. B. cepacia and P. pseudoflava were selected for further analysis of polyhydroxyalkanoate production.We examined the sensitivity of seven polyhydroxyalkanoate producing bacteria: B. cepacia demonstrated the greatest inhibitor tolerance. Similarly, of two organisms examined for polyhydroxybutyrate production, B. cepacia was notably more efficient when fermenting pentose substrates.We find significant differences in sensitivity to the fermentation inhibitors tested and find these variations to be over a relevant concentration range given the concentrations of inhibitors typically found in lignocellulosic hydrolysates. Of the seven bacteria tested,  Numerous bacteria have been shown to synthesize polyhydroxyalkanoates (PHAs) as intracellular carbon storage granules in response to nutrient limitations . PHAs arSaccharification of lignocellulosic material from the aforementioned potential reservoir of inexpensive carbon is complicated by the concomitant generation of a number of fermentation inhibitors ,5. TheseB. cepacia and P. pseudoflava, selected for their known broad monosaccharaide utilization capabilities, and assess their PHA production efficiency using authentic lignocellulosic-derived sugars arabinose, xylose, glucose and mannose as fermentation substrates.Microorganisms vary considerably in their tolerance of, and their ability to catabolize, these inhibitors. A number of bacteria have been identified that are able to metabolize both phenolic compounds and furans and these traits may be useful in overcoming the barriers presented by these compounds in PHA bioproduction ,7. In thWe find significant variation in both the tolerance of these microbes to the panel of fermentation inhibitors and in PHA production. While fermentation inhibitors can be removed by a variety of means including liming, membrane filtration, and adsorption to charcoal these methods add costs that undermine the effort to recover inexpensive carbon sources. Characterization of inhibitor tolerance and PHA production will allow a more informed basis for microbe selection and strain improvement for PHA production from lignocellulosic material, affording a biological approach to overcoming fermentation inhibitors.A. lata), 4 g/L (P. pseudoflava) and 6 g/L for all other organisms tested  for furfural was 1.5 g/L  to 1.5 g/L (B. cepacia) (Table P. pseudoflava) to 6.0 g/L (B. cepacia and P. olevorans).Coumaryl and coniferyl-derived phenolic representatives were the most toxic on a w/v basis. Coumaric acid and ferulic acid MIC values ranged from 0.25 g/L (P. pseudoflava) to 4 g/L B. cepacia) and levulinic acid MIC values ranging from 0.75 g/L (P. pseudoflava) to 4.0 g/L (B. cepacia)  Table .B. cepacia exhibited the greatest inhibitor tolerance of the organisms tested. With the exception of A. lata sensitivity to furfural, P. pseudoflava was consistently the most sensitive organism in these assays. These data indicate that B. cepacia represents a promising candidate for lignocellulosic bioconversion when considering tolerance to fermentation inhibitors.Within each inhibitor class, B. cepacia and P. pseudoflava, we examined the PHB production potential of these two bacteria in chemically defined media supplemented with 10 g/L of glucose, mannose, arabinose or xylose. Both organisms achieved higher dry cell weight (DCW) on hexose substrates glucose and mannose compared to pentose substrates arabinose and xylose  supplemented with 1 g/L yeast extract, and 15 g/L agar as appropriate.600 nm was observed. Briefly, 80 g/L stocks of the indicated inhibitors were prepared in DMSO and sterilized by 0.2 μm filtration. Serial dilutions were performed in 96 well plates containing a 1:50 dilution of overnight cultures of the indicated bacteria suspended in 1/2x NBY media using initial inhibitor concentrations of 6 g/L and 4 g/L generating parallel overlapping serial dilution profiles. Plates were incubated at 30°C and 85% relative humidity and analyzed at 24 hours.Minimal inhibitory concentrations (MICs) were determined by standard methods . MICs we2HPO4. 7H2O, 1.5 g KH2PO4, 60 μl 10% (w/v) ferric ammonium citrate, 1.5 g (NH4)2SO4, 0.5 g CaCl2. 2H2O, 1 g MgSO4. 7H2O and 1 ml trace element solution. The trace element solution contained (per liter): 300 mg H3BO, 200 mg CoCl2. 6H2O, 100 mg ZnSO4. 7H2O, 30 mg MnCl2. 4H2O, 30 mg NaMoO4. 2H2O, 20 mg NiCl2. 6H2O and 10 mg CuSO4. 5H2O. Filter sterilized (0.2 μm) carbon sources were added as indicated. Culture volumes were 200 ml in 2 L Erlenmeyer flasks and agitated at 200 rpm on 1 inch orbits at 30°C. Samples for analysis were collected at 12, 24, 48, 72 and 96 hours (Figure Shake flask cultures for PHA production were performed essentially as described . Minimal-1 for 20 min at 16°C and dried overnight at 100°C. Samples were weighed and methyl ester derivatization was performed essentially as described [2SO4 were added to samples followed by 3 ml CHCl3 and incubated at 100°C for 4 hr. 1.5 ml dH20 was added after which the samples were vortexed vigorously. The chloroform layer was recovered and 2 μl was injected using a HP5890A equipped with a 7683 auto sampler, split-splitless capillary column injection port, a Flame Ionization Detector and a 35 m Agilent DB-Wax capillary column. The temperature of the injector was 230°C and the detector temperature 275°C. The oven program used was 80°C for 4 minutes, then ramping 8°C/min to 160°C, for 6 minutes for a total run time of 20 minutes. Helium was used as the carrier at 20 ml/min.10 ml samples of PHA-producing shake flask cultures were collected at the indicated times, pelleted by centrifugation at 5 kgescribed . 3 ml ofThe authors declare they have no competing interests. This work was performed at and financially supported by the USDA – Forest Service, Forest Products Laboratory, Madison WI.DD performed sample preparation and GC analysis. BI provided experimental concepts and edited the manuscript. CC designed the experiments, performed MIC assays and fermentations, and wrote the manuscript. All authors read and approved the final manuscript.


Eriosema robustum, a plant used for the treatment of coughs and skin diseases.The aim of this study was to evaluate the antimicrobial activity and the cytotoxicity of the ethanol crude extract, fractions and isolated compounds from the twigs of 1) and B (2), orostachyscerebroside A (3), stigmasterol (4), 1-O-heptatriacontanoyl glycerol (5), eicosanoic acid (6), 3-O-β-D-glucopyranoside of sitosterol (7) and 6-prenylpinocembrin (8), from E. robustum. A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against fungi and bacteria, and the 3--2,5-diphenyltetrazolium bromide reduction assay was used to evaluate the cytotoxicity.Column chromatographic and spectroscopic techniques were used to isolate and identify eight compounds, robusflavones A (Aspergillus fumigatus and Cryptoccocus neoformans (MIC 0.08 mg/ml), whilst the crude extract and fraction A had moderate activity against A. fumigatus and Candida albicans (MIC 0.16 mg/ml). Fraction A however had excellent activity against Staphylococcus aureus (MIC 0.02 mg/ml), Enterococcus faecalis and Escherichia coli (MIC 0.04 mg/ml). The crude extract had significant activity against S. aureus, E. faecalis and E. coli. Fraction B had good activity against E. faecalis and E. coli (MIC 0.08 mg/ml). All the isolated compounds had a relatively weak antimicrobial activity. An MIC of 65 μg/ml was obtained with robusflavones A (1) and B (2) against C. albicans and A. fumigatus, orostachyscerebroside A (3) against A. fumigatus, and robusflavone B (2) against C. neoformans. Compound 8 had the best activity against bacteria (average MIC 55 μg/ml). The 3 fractions and isolated compounds had LC50 values between 13.20 to > 100 μg/ml against Vero cells yielding selectivity indices between 0.01 and 1.58.Fraction B had significant antimicrobial activity against E. robustum to treat infections. The crude extract had a good activity and low preparation cost, and may be useful in topical applications to combat microbial infections.The isolated compounds generally had a much lower activity than expected based on the activity of the fractions from which they were isolated. This may be the result of synergism between different compounds in the complex extracts or fractions. The results support the traditional use of  The need for new, effective and affordable drugs to treat microbial diseases in the developing world is one of the issues facing global health today. Available drugs for the treatment of these diseases are limited by factors ranging from microbe resistance to safety, compliance and cost. The use of medicinal plants for curative purposes is as old as mankind and can be traced from the beginning of civilisation. The interaction between man and his environment is such that his food, shelter, the diseases that afflict him and the cure of such diseases are all within the environment. Thus, man uses his environment and the resources of nature to combat diseases that afflict him. Consequently over many centuries, people of various countries of the world have embarked on what is now known as traditional medicine . It is eEriosema robustum, a member of Fabaceae family, is a perennial non-climbing shrub with yellow flowers native to Burundi, Ethiopia, Kenya, Rwanda, Tanzania, Uganda, Democratic Republic of Congo and Cameroon , B [ and C [ and EtOAc - MeOH ]. Fraction C did not have as many active antimicrobial constituents separated by TLC in bioautography as fractions A and B, and was not further investigated.The dried and powdered twigs of 25-26 and F27-30 eluted with n-hexane: acetone (17:3) gave compounds 2 (9 mg), 4 (8 mg) and 6 (12 mg). Subfractions F20-26 eluted with n-hexane: acetone  and F27-35 eluted with n-hexane: acetone  were similarly subjected to further silica gel column chromatography eluted with n-hexane, ethyl acetate, methanol in gradient polarity followed by preparative TLC and Sephadex LH-20 to yield 1 (4 mg), 3 (10 mg), 5 (7 mg) and 7 (9 mg).Fraction B was subjected to purification applying silica gel column chromatography eluted with n-hexane, acetone and methanol in order of increasing polarity to yield 50 fractions of 150 ml each which were combined in subfractions after monitoring with Co-TLC. Subfractions F4 (3 mg) and 8 (17 mg).Fraction A was subjected to Sephadex LH-20 to remove chlorophyll and the eluate was concentrated and fractionated using similar silica gel column techniques as described above for fraction B to give mainly Orostachyscerebroside A (3): colourless amorphous; 1H NMR : δ 4.38 , 4.58 , 4.08 , 3.76 , 3.70 , 1.86-2.02 , 5.63 , 1.23-1.33 , 1.40-1.65 , 1.24-1.31 , 1.50 , 0.84 , 3.80 , 1.26 , 2.02 , 1.02 , 1.02-1.20 , 1.20-1.33 , 4.12 , 3.32 , 4.96 , 3.60 , 3.03 , 3.38 , 3.65 , 7.55 ; 13C NMR  δ: 69.4 (C-1), 50.3 (C-2), 70.9 (C-3), 71.3 (C-4), 34.8 (C-5), 129.8 (C-6), 130.1 (C-7), 27.4 (C-8/C-11′), 32.2 (C-9), 130.3 (C-10), 130.7 (C-11), 31.8 (C-12), 29.0-30.9 (C-13 to C-22), 22.6 (C-23/C-19′), 14.4 (Me-24/Me-20′), 174.2 (C-1′), 71.3 (C-2′), 34.8 (C-3′), 24.4 (C-4′/C-8′), 27.1 (C-5′), 130.1 (C-6′/C-10′), 129.8 (C-7′/C-9′), 29.0-30.9 (C-12′ to C-18′), 103.9 (C-1″), 73.9 (C-2″), 76.9 (C-3″), 70.4 (C-4″), 77.3 (C-5″), 61.4 (C-6″); MALDI-FT-MS m/z: 866.6720 , MALDI-FT-MS2 (CID at 45 eV) (rel. int.) m/z: 704.6171 +, 686.6065 +, 500.3176 +.IR spectra were recorded on a Bruker Alpha FT-IR spectrometer . UV spectra were recorded on Thermo Electron Helios spectrophotometer .1H- and 13C-NMR spectra were recorded with a Bruker spectrometer at 500 MHz and a Varian spectrometer at 400 MHz. Chemical shifts (δ) were quoted in parts per million (ppm) from the internal standard tetramethylsilane (TMS). Deuterated solvents as dimethyl sulfoxide , and chloroform  were used for the NMR experiments. Mass spectrometry was performed on a Thermo Scientific LTQ-Orbitrap instrument coupled to a MALDI (matrix-assisted laser desorption ionization) or liquid chromatography electrospray ionization source. Column chromatography was performed on MN silica gel 60 (0.063-0.2 mm/70–230) mesh. Preparative TLC was performed using high-purity grade silica gel powder  Sigma-Aldrich, Germany. Pre-coated plates of TLC silica gel 60 F254  were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 30% H2SO4 followed by heating up to 110°C.et al. (2005) was used for fungi [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212), two Gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922); and three clinical isolates of the pathogenic fungi Candida albicans, Cryptococcus neoformans and, Aspergillus niger along with Candida albicans (ATCC 10231). Some fungal strains used were cultured from clinical cases of fungal infectious diseases in animals (before treatment) in the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science. C. albicans was isolated from a Gouldian finch, C. neoformans from a cheetah, while A. fumigatus was isolated from a chicken which suffered from a systemic mycosis.The two-fold serial microdilution method described by Eloff 1998) was used to determine the minimum inhibitory concentration (MIC) values for the extract, fractions and isolated compounds against bacteria  and a mo was used12 cfu/ml, S. aureus; 1.5 × 1010 cfu/ml, E. faecalis; 5.2 × 1013 cfu/ml, P. aeruginosa and 3.0 × 1011 cfu/ml, E. coli, obtained by adding 99 ml of MHB to 1 ml of each microorganism from the freshly prepared overnight bacterial culture in Mueller Hinton broth [p-iodonitrotetrazolium violet (INT)  was added to each well to indicate microbial growth. The microtitre plates were further incubated at 37°C for 2 h and the minimal inhibitory concentration (MIC) was determined as the lowest concentration inhibiting microbial growth, indicated by a decrease in the intensity of the red colour of the formazan [In the antibacterial tests, 100 μl of the (10 mg/ml) crude extract and fractions or (1 mg/ml) compounds in duplicate dissolved in dimethyl sulfoxide (DMSO) were serially diluted two-fold with sterile distilled water in 96-well microtitre plates and 100 μl bacterial culture in Mueller Hinton broth (MHB)  was added to each well. DMSO was used to dissolve the compounds because acetone used in the original method  did not C. albicans and A. fumigatus, and after 24 and 36 h incubation for C. neoformans as the lowest concentration of the sample that inhibited fungal growth. The colourless salt of tetrazolium acts as an electron acceptor and is reduced to a red coloured formazan product by biologically active organisms [For the antifungal assay, 100 μl of the crude extract, fractions and compounds dissolved in DMSO at the same concentrations, as described above for the bacteria, were two-fold serially diluted with distilled water in 96-well microtitre plates, and 100 μl of fungal culture in Potato Dextrose broth  was added to each well . Amphoterganisms ,12. The g for 5 min, and resuspended in growth medium to 5 × 104 cells/ml. The growth medium used was Minimal Essential Medium  supplemented with 0.1% gentamicin (Virbac) and 5% foetal calf serum (Sigma). A total of 200 μl of the cell suspension is pipetted into each well of columns 2 to 11 of a sterile 96-well microtitre plate. A 200 μl aliquot of growth medium was added to wells of columns 1 and 12 to minimize the “edge effect” and maintain humidity. The plates were incubated for 24 h at 37°C in a 5% CO2 incubator, until the cells were in the exponential phase of growth. The MEM was aspirated from the cells, and replaced with 200 μl of extract, fractions and compounds at differing concentrations . The cells were disturbed as little as possible during the aspiration of medium and addition of test samples. Each dilution was tested in quadruplicate. The microtitre plates were incubated at 37°C in a 5% CO2 incubator for 48 h with test compounds, fractions or extract. Untreated cells and positive control  were included. After incubation, cells were washed with 150 μl phosphate buffered saline (PBS) and fresh MEM (200 μl) was added to each well together with 30 μl MTT . The plates were then incubated for a further 4 h at 37°C. After incubation with MTT the medium in each well was carefully removed, without disturbing the MTT crystals in the wells. The MTT formazan crystals were dissolved by adding 50 μl DMSO to each well and the plates gently shaken until the MTT solution was dissolved. The amount of MTT reduction was measured immediately by detecting absorbance in a microplate reader  at a wavelength of 570 nm and a reference wavelength of 630 nm. The wells in column 1, containing medium and MTT but no cells, were used to blank the plate reader. The LC50 values were calculated as the concentration of test sample resulting in a 50% reduction of absorbance compared to untreated cells.The MTT -2,5-diphenyltetrazolium bromide) reduction assay is one of the most frequently used methods for measuring cell proliferation and cytotoxicity. The intensity of colour  of the MTT formazan produced by living, metabolically active cells is proportional to the number of live cells present. MTT is a yellow water-soluble tetrazolium dye that is reduced by live, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. The viable cell growth after incubation with extract, fractions and isolated compounds was determined using the tetrazolium-based colorimetric MTT assay described by Mosmann (1983) . In shorEriosema robustum was subjected to repeated silica gel column chromatography followed by preparative TLC and Sephadex LH-20 to afford eight compounds including robusflavones A  and B  [3) [4, white powder, m/z 412, C29H48O) [O-heptatriacontanoyl glycerol  [6, white powder, m/z 312, C20H40O2) [O-β-D-glucopyranoside of sitosterol  [8, yellow, m/z C20H20O4) [3), from the genus Eriosema. Orostachyscerebroside A (3) was found for the first time in the aerial parts of Orostachys japonicas in 2012 [The EtOH crude extract from the twigs of C29H48O) , 1-O-hep35H60O6)  and 6-pr20H20O4)   were determined and the results are presented in Tables Aspergillus fumigatus and Cryptoccocus neoformans with an MIC of 0.08 mg/ml, while the crude extract and fraction A had moderate activity against Aspergillus fumigatus and Candida albicans (ATCC) with an MIC value of 0.16 mg/ml in all cases. Fraction B had the highest antifungal activity among the tested fractions and extract with an average MIC of 0.61 mg/ml followed by fraction A (average MIC of 0.65 mg/ml) and the crude extract (average MIC of 0.88 mg/ml).The antimicrobial activity and cytotoxicity of the crude extract, fractions from the column chromatography of the extract and isolated compounds –8 were d1) and B (2) against C. albicans (ATCC) and A. fumigatus, compounds 4 and 7 against C. albicans (ATCC), as well as compound 6 against A. fumigatus and C.neoformans, orostachyscerebroside A (3) against A. fumigatus, and robusflavone B (2) against C. neoformans. Compounds 5 and 7 had weak antifungal activity against C. neoformans (MIC > 250 μg/ml in both cases). The animal pathogenic isolate C. albicans was more resistant to the tested samples with MIC values between 0.63 to 1.25 mg/ml for the extract and fractions and ranging from 125 to 250 μg/ml for compounds, respectively. Nevertheless, robusflavone B (2) had the best antifungal activity (average MIC of 88 μg/ml), followed by compounds 3 and 6 (average MIC of 110 μg/ml), compound 7 (average MIC of 157 μg/ml) and robusflavone A (1) (average MIC of 165 μg/ml).The most active isolated compounds had moderate antifungal activity with the MIC value of 65 μg/ml. These included robusflavones A  and had moderate activity against P. aeruginosa . Fraction A had excellent activity against S. aureus (MIC of 0.02 mg/ml), E. faecalis and E. coli (MIC of 0.04 mg/ml) and reasonable activity against P. aeruginosa . The Gram-negative P. aeruginosa and the Gram-positive S. aureus were more resistant to fraction B with MIC values of 0.31 and 0.16 mg/ml, respectively. However, fraction B had significant activity against E. faecalis and E. coli with an MIC value of 0.08 mg/ml. Compound 8 had moderate activity against S. aureus  and had reasonable activity against other bacteria with an MIC value of 63 μg/ml. Other constituents had also reasonable antibacterial activity against P. aeruginosa with the MIC value of 63 μg/ml as well as robusflavone B (2) and compound 6 against E. faecalis (MIC of 63 μg/ml). Amongst all the isolated compounds, 6-prenylpinocembrin (8) had the highest antibacterial activity with an average MIC of 55.1 μg/ml and average total activity of 20 ml/mg followed by robusflavone B (2), and compounds 5 and 6 , as well as robusflavone A (1) with an average MIC of 203.3 μg/ml and average total activity of 7 ml/mg.The antibacterial activity of the crude extract, fractions and isolated compounds were determined against four bacteria with MIC values ranging from 0.02 to 0.31 mg/ml and from 31.3 to 250 μg/ml, respectively  and B (2) characterized earlier as two new antioxidant flavones [3 and 5) had similar antibacterial activity (both with average MIC of 109.5 μg/ml) but had slightly different antifungal activity with average MIC values of 220 and 173 μg/ml, respectively.The crude extract and fractions were found to be more active than the isolated compounds against the bacteria and the fungi. The antibacterial activity of fraction A is noteworthy against the bacteria with an average MIC value of 100 μg/ml ,23 and a1–3, 5, 6 and 8) are reported here for the first time.From the results, it is evident that the isolated compounds did not have good activity against the tested microorganisms as observed for extract and fractions. This supports the conclusion of Lewis and Ausubel (2006) that plants contain many compounds with low antimicrobial activity and use other mechanisms to combat microbial growth . In someE. robostum were evaluated for their cytotoxicity against monkey kidney Vero cells in vitro using the MTT -2,5-diphenyl-2H-tetrazolium hydrobromide) assay and the results are presented in Table 6) that was not tested, compounds 3 and 7 had LC50 value greater than 100 μg/ml whilst the crude extract, fractions and others isolated compounds  had cytotoxic activity against the Vero cells with LC50 values ranging from 13.20 to 91.52 μg/ml. Our samples, and especially the tested compounds, could be considered less toxic than the positive control doxorubicin (LC50 = 3.32 μM) and by taking into consideration the criterion of the American National Cancer Institute (NCI) regarding the cytotoxicity of pure compounds (LC50 < 4 μg/ml) [50 values of 13.20, 30.42 and 21.87 μg/ml for compounds 1, 2 and 8 with the same basic skeleton (flavonoid) were lower than those of others compounds and more cell lines should be tested in a comprehensive screen to confirm their probable safety in terms of cytotoxicity. The selectivity index (SI) values of the tested samples ranged from 0.01 to 1.58 and could be considered as poor when taking in consideration that the ratio for a good therapeutic index for a remedy or drug should be ≥ 10 [1, 2, 3, 5 and 8 from E. robustum against Vero cells.The crude extract, fractions and isolated compounds from the twigs of 4 μg/ml) . However be ≥ 10 . This isA. fumigatus, fraction A against C. albicans and A. fumigatus, and fraction B against C. albicans, A. fumigatus and C. neoformans. It is noteworthy that the crude extract had a fungicidal activity against the two Candida and the Cryptococcus species, but the fractions had mainly fungistatic activity. This again points to the probability of synergistic activities.Fraction B had good antifungal activity with a total activity of 3018 ml/g for the fraction. One should keep in mind that the 5 g of extract separated by column chromatography was obtained from 87 g of the original dried plant material. In comparing the activity of different plants the concept of total activity was developed [With the high total activity of the crude extract and the ease of preparation, there is a reasonable possibility that the crude extract can be used as topical treatment for fungal and especially bacterial infections. Manipulation of the extract by for example solvent-solvent fractionation may lead to extracts with a higher activity and possibly a lower cytotoxicity .E. robustum to treat infections. The crude extract had good activity with low toxicity to animal cells, and may be useful in topical applications to combat microbial infections.The results obtained support the traditional use of The authors declare that they have no competing interests.MDA carried out the phytochemical and antimicrobial studies, and wrote the manuscript; LJM carried out the cytotoxicity and participated in the manuscript writing; SG and RM assisted with the structure elucidation of some compounds; PT, MS and JNE supervised the work. All the authors read and approved the final manuscript after revision by JNE.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/289/prepub


The human body produces small amounts of H2S and uses it as a signaling molecule. The cocktail method was used to evaluate the influence of H2S on the activities of CYP450 in rats, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: bupropion, metroprolol, midazolam, omeprazole and tolbutamide, respectively. The rats were randomly divided into two groups, control group and H2S group. The H2S group rats were given 5 mg/kg NaHS by oral administration once a day for seven days. The mixture of five probes was given to rats through oral administration and the blood samples were obtained at a series of time-points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. In comparing the H2S group with the control group, there was a statistically pharmacokinetics difference for midazolam and tolbutamide; the area under the plasma concentration-time curve (AUC) was decreased for midazolam (p < 0.05) and increased for tolbutamide (p < 0.05); while there was no statistical pharmacokinetics difference for bupropion, metroprolol and omeprazole. H2S could not influence the activities of CYP2B6, CYP2D6 and CYP2C19 in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in rats.Hydrogen sulfide (H It is or system . H2S haside (CO) ,6. It iside (CO)  and is aide (CO) . H2S is  disease ,9.in vivo after administering H2S. CYP450 are the main metabolic enzymes at the in vivo level; therefore, to investigate the effects of H2S on the CYP450 activity is very important for further chemotherapy. Cytochrome P450 enzymes comprise a superfamily of hemoproteins, and three families  are mainly involved in the metabolism of drugs in both humans and rats [It may cause a change in the activity of metabolic enzymes and rats . They arand rats . More thand rats .In order to assess various individual CYP450 activities, probe drugs have been widely used in many clinical investigations in the field of drug metabolism and pharmacogenetics –15. ProbIn this paper, the cocktail probe drugs approach is used to evaluate the induction or inhibition effects of H2S on the activities of rats CYP450 isoforms such as CYP2B6, CYP2D6, CYP3A4, CYP2C19 and CYP2C9 in rats, which are reflected by the changes of pharmacokinetic parameters of five specific probe drugs , then provide guidance for rational clinical oral administration after administration of NaHS.2.2.1.y = 0.0017x + 0.0381 (r = 0.9982) for bupropion, y = 0.0035x + 0.092 (0.9962) for metroprolol, y = 0.0037x + 0.1538 (r = 0.9979) for midazolam, y = 0.0006x + 0.0321 (r = 0.9961) for omeprazole, y = 0.0008x − 0.0037 (r = 0.9991) for tolbutamide. Each probe drug peak area ratio with concentration has a good linear relationship. The lower limit of quantification (LLOQ) for each probe drug in rat plasma was 10 ng/mL.Calibration curves for five probe drugs were generated by linear regression of peak area ratios against concentrations, respectively. The calibration plot of the probe drugs in the range of 10–2000 ng/mL were, The relative standard deviation (RSD%) of the five probe drugs in low, medium and high three concentrations were less than 15%. The intra-day and inter-day relative error (RE%) ranged from −15% to 13%. The results demonstrate that the values were within the acceptable range and the method was accurate and precise. The extraction recoveries were ranged from 80% to 95%. The results of matrix effect, the percent nominal concentration were more than 85% or less than 110%.2.2.vs. time profiles of twelve rats are presented in The main pharmacokinetic parameters after oral administration of bupropion, metroprolol, midazolam, omeprazole and tolbutamide from non-compartment model analysis are summarized in 2S group with the control group, the pharmacokinetic parameters of bupropion had changed, t1/2 from the 1.9 increased to 2.3 h, but there no statistical significance (p > 0.05), AUC(0–t) from the 532.7 reduced to 393.0 ng/mL h with no significant difference (p > 0.05); CL from 13.7 reduced to 21.7 L/h/kg, but there was no significant difference (p > 0.05); Cmax varied from 279.2 to 308.4 ng/mL, but there was no significant difference (p > 0.05). Compared to the control group, the H2S group, AUC(0–t) reduced, CL increased, however, there was no significant difference for these pharmacokinetic parameters (p > 0.05), which indicates that the H2S will not induce or inhibit the activity of CYP2B6 enzyme.As could be seen from 2S group, but there was no significant difference (p > 0.05), which shows that the H2S would not induce or inhibit the activity of CYP2D6 enzyme.Similar results were found in metoprolol, the pharmacokinetic parameters of metoprolol had changed between control group and H2S group with the control group, the pharmacokinetic parameters of midazolam had changed, AUC(0–t) from the 509.2 reduced to 210.0 ng/mL h, there was significant difference (p < 0.05); CL from 18.6 increased to 44.6 L/h/kg, there was significant difference (p < 0.05); Cmax varied from 330.9 to 152.9 ng/mL, there was no significant difference (p > 0.05). Compared to the control group, the H2S group, AUC(0–t) reduced (p < 0.05), CL increased (p < 0.05), Cmax becomes lower, which indicates that the H2S could induce the activity of CYP3A4 enzyme.Compared H2S group, and there was no significant difference for these pharmacokinetic parameters (p > 0.05), which indicates that the H2S would not induce or inhibit the activity of CYP2C19 enzyme.The pharmacokinetic parameters of omeprazole had almost not changed between control group and H2S group with the control group, the pharmacokinetic parameters of tolbutamide had changed, CL from the 0.11 h reduced to 0.08 h, and there was statistical significance (p < 0.05), AUC(0–t) from the 6799.3 increased to 10,429.3 ng/mL h with significant difference (p < 0.05); Cmax varied from 484.7 to 640.4 ng/mL, and there no statistical significance (p > 0.05). Compared to the control group, H2S group, AUC(0–t) increased (p < 0.05), CL reduced (p < 0.05), Cmax became higher, showing that the H2S would inhibit the activity of CYP2C9 enzyme.Compared HAUC of bupropion and omeprazole H2S group is the almost same as the control group, the AUC and Cmax tolbutamide in H2S group is higher than the control group, the AUC and Cmax of midazolam in H2S group is lower than the control group, this result is consistent with As could be seen from + and HS−in vivo, it will form H2S when HS− combine with H+in vivo. 5 mg/kg NaHS given to the rats, the level of H2S in exposed individuals may be about 89.2 μmol/kg H2S. H2S could not influence the activities of CYP450 isoforms CYP2B6, CYP2D6, CYP2C19 in vivo in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in vivo in rats. It has certain guiding significance to the clinical treatment, when the drugs metabolism through CYP2C9 orally combined with NaHS, we must pay attention to dosage, appropriate reduce the dose, the plasma drug concentration may be too high and poisoning. When the drugs metabolism through CYP3A4 orally combined with NaHS, should increase the dose, so as not to hinder the effect of drugs. However, the human case is not the same as rat, the result is only for clinical reference.NaHS can be decomposed into Na3.3.1.Bupropion, metroprolol, midazolam, omeprazole and tolbutamide  and the internal standard carbamazepine (IS) were purchased from Sigma-Aldrich Company . HPLC grade acetonitrile and methanol were purchased from Merck Company . Ultra-pure water (resistance >18 mΩ) prepared by a Millipore Milli-Q purification system .3.2.Male Sprague-Dawley rats (250 ± 20 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. . The animal license number was SCXK  2012–0005. All twenty rats were housed at Wenzhou Medical University Laboratory Animal Research Center . Animals were housed under controlled conditions (22 °C) with a natural light-dark cycle. All experimental procedures were conducted according to the Institutional Animal Care guidelines  and approved ethically by the Administration Committee of Experimental Animals, Laboratory Animal Center of Wenzhou Medical University.3.3.All analysis was performed with a 1200 Series liquid chromatograph  equipped with a quaternary pump, a degasser, an autosampler, a thermostatted column compartment, and a Bruker Esquire HCT mass spectrometer  equipped with an electrospray ion source and controlled by ChemStation software (Version B.01.03 (204), Agilent Technologies, Waldbronn, Germany).Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C18 column at 30 °C. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: 0–4.0 min (10%–80% B), 4.0–8.0 min (80%–80% B), 8.0–9.0 min (80%–10% B), 9.0–13.0 min (10%–10% B). The flow rate was 0.4 mL/min.m/z 240 for bupropion, m/z 268 for metroprolol, m/z 326 for midazolam, m/z 198 for omeprazole, and m/z 237 for IS) in positive ion electrospray ionization interface. Drying gas flow was set to 7 L/min and temperature to 350 °C. Nebuliser pressure and capillary voltage of the system were adjusted to 25 psi and 3500 V.The quantification was performed by the peak-area method. The determination of target ions were performed in selective ion monitoring mode . Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations . The analytical standards and QC samples were stored at −20 °C.3.5.2S group (n = 6). Control group were give saline by oral administration once a day for 7 days; while H2S group were give NaHS (5 mg/kg) by oral administration once a day for 7 days. After complete the modeling, the H2S and control group were given the mixtured five probe drugs by oral administration, the dose of bupropion, metroprolol, midazolam, omeprazole and tolbutamide were 8, 8, 8, 8 and 0.8 mg/kg, respectively.Twelve male Sprague-Dawley rats (250 ± 20 g) were randomly divided to control group and HBlood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 5, 15, 30 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48 h after oral administration of five probe drugs. The samples were immediately centrifuged at 8000 r/min for 5 min, and 100 μL plasma was obtained for each sample.The plasma samples were extracted and measured by LC-MS. In a 1.5 mL centrifuge tube, an aliquot of 10 μL of the internal standard working solution (2.0 μg/mL) was added to 0.1 mL of collected plasma sample followed by the addition of 0.2 mL of acetonitrile. After the tube was vortex-mixed for 1.0 min, the sample was centrifuged at 15,000 r/min for 10 min. The supernatant (2 μL) was injected into the LC-MS system for analysis.2S group and control group with the t-test inspection were analyzed by SPSS 16.0 statistical software . A p < 0.05 was considered as statistically significant.Plasma probe drugs concentration versus time data for each rat was analyzed by DAS software . The pharmacokinetic parameters of probe drugs of the H4.2S could not influence the activities of CYP450 isoforms CYP2B6, CYP2D6, CYP2C19 in vivo in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in vivo in rats. This has certain guiding significance to clinical treatment. In the case of about 16% of common drugs’ metabolism through CYP2C9, when these drugs are orally combined with NaHS, we must pay close attention to dosage and appropriately reduce the dose of the drug, otherwise, the plasma drug concentration may be too high and have a poisoning effect. Furthermore, in the case of about 50% of prescription drugs metabolism through CYP2C9, when orally combined with NaHS, the dose must be increased, so as not to hinder the effect of the drug. However, it is noted that the human case differs from rat, therefore, the result is only provided for clinical reference.H