Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units.The dominant, cell surface lipophosphoglycan (LPG) of −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs  and NF-kB activation were evaluated.Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB.Macrophages stimulated with These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions. Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous (CL) and visceral leishmaniasis (VL), respectively Man(α1-3)Man(α1-4)-GlcN(α-1) heptasaccharide; (iii) a portion of disaccharide repeats of the Gal(β1-4)Man(α1)PO4 units and (iv) a terminal neutral oligosaccharide (“cap”)  substitutions to the proximal mannose, respectively [L. braziliensis GIPLs compared to L. infantum. However, the role of LPGs from those species in this process has not been investigated.Structurally, LPG has four distinct domains: (i) a well conserved GPI anchor composed of 1- (“cap”) . In L. iresidues . These s residue   of Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte (BH), Minas Gerais (MG), Brazil (Protocol P-0297-06). Knock-out mice handling protocol was approved by the National Commission of Biosafety (CTNBio) (protocol #01200.006193/2001-16).L. braziliensis (MHOM/BR/1975/M2903) and L. infantum (MHOM/BR/1970/BH46) were used. Promastigotes were cultured in M199 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin 100 units/ml, streptomycin 50 μg/ml, 12.5 mM glutamine, 0.1 M adenine, 0.0005% hemin, and 40 mM Hepes, pH 7.4 at 26°C [World Health Organization Reference strains of  at 26°C  until la2O/ethanol/diethylether/pyridine/NH4OH; 15:15:5:1:0.017) after a sequential organic solvent extraction [2 evaporation, resuspendend in 2 ml of 0.1 N acetic acid/0.1 M NaCl, and applied onto a column with 2 ml of phenyl-Sepharose, equilibrated in the same buffer. The column was washed with 6 ml of 0.1 N acetic acid/0.1 M NaCl, then 1 ml of 0.1 N acetic acid and finally 1 ml of endotoxin free water. The LPG was eluted with 4 ml of solvent E then dried under N2 evaporation. LPG concentration was determined as described elsewhere [in vitro macrophage cultures, LPG was diluted in fresh RPMI. All solutions were prepared in sterile, LPS-free distilled water .For optimal LPG extraction, late log phase cells were harvested and washed twice with PBS prior to extraction of LPG. The LPG extraction was performed as described elsewhere with solvent E (Htraction . For purlsewhere . Prior t−/− and TLR4−/− knockouts) by peritoneal washing with ice cold serum-free RPMI and enriched by plastic adherence for 1 h at 37°C/5% CO2. Cells (3 × 105 cells/well) were washed with fresh RPMI then cultured in RPMI, 2 mM glutamine, 50 U/ml of penicillin and 50 μg/mL streptomycin supplemented with 10% FBS in 96-well culture plates (37°C/5% CO2). Cells were primed with gamma interferon (IFN-γ) (3 IU/mL) [Leishmania parasites (10:1), LPG (10 μg/mL) or lipopolysaccharide (LPS) (100 ng/mL).Thioglycollate-elicited peritoneal macrophages were extracted from BALB/c, C57BL/6 and C57BL/6 (TLR23 IU/mL)  for 18 h2, and antibiotics. All of the cell lines are derived from clone 3E10, that has been stably transfected with a reporter construct containing the structural gene for CD25 under the control of the human E-selectin promoter. This promoter contains a NF-kB binding site; CD25 surface expression is completely dependent upon NF-kB translocation to the cell nucleus [The CHO reporter cell lines . LPG (10 μg/mL) and live stationary promastigotes (10:1 ratio) were added and incubated for 48 h, LPS (100 ng/mL) was added as a positive control. For negative controls fresh medium was added. Supernatants were collected and cytokines  were determined using the BD CBA Mouse Cytokine assay kits according to the manufacturer’s specifications . Flow cytometry measurements were performed on a FACS Calibur flow cytometer . Cell-Quest™ software package provided by the manufacturer was used for data acquisition and the FlowJo software 7.6.4  was used for data analysis. A total of 1,800 events were acquired for each preparation [For CBA multiplex cytokine detection, cells were plated, primed as described above and incubated for 18 h  was added to the medium and incubated for another 24 h at 37°C/5% CO2. Supernatants were collected and nitrite concentrations determined by Griess reaction. Results shown are the mean of two experiments in triplicate [For the inhibition assay, purified cells were primed for 6 h with 3 IU/ml of IFN-γ prior to stimulation with LPG (10 μg/ml). Cells were incubated for 18 h at 37°C/5% COiplicate .5 cells/well in 24-well tissue culture dishes. The following day, either molecule or bacteria , positive control of TLR4; or LPG (0.2 μg or 0.02 μg/well) from L. braziliensis or from L. infantum was added as indicated, for 18 h. The cells were harvested with trypsin-EDTA, washed with medium and with PBS. Subsequently, the cells were counted and 1×105 were cells stained with PE-labeled anti-CD25  1:200 in PBS, on ice, in the dark, for 30 min. After labeling, the cells were washed twice with same buffer, resuspended in 1 mM sodium azide in PBS, and examined by flow cytometry  for the expression of surface CD25 as described [In order to evaluate the activation of NFκB by LPG, CHO reporter cells were plated at a density of 1×10escribed . Analyse6/well) for 18 h prior to assay. The cells were washed with warm RPMI and incubated with LPG from both species for different times  or with medium (negative control) or LPS (100 ng/ml) as positive control. Cells were then washed with ice-cold PBS and lysed in lysis buffer , 50 mM sodium fluoride, 150 mM NaCl, 5 mM ethylenediamine tetraacetic acid (EDTA), 10% glycerol (v/v), 0.5 mM dithiothreitol (DTT) and protease inhibitor cocktail from Sigma®). Cells were harvested with a plastic scraper and centrifuged at 13,000 × g . Supernatants were transferred to new tubes and stored at −20°C until used for immunoblotting. Cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and blocked (5% milk in TBS-0.1% Tween 20) for 1 h. Primary Abs  were incubated for 16 h at 4°C. Membranes were washed (3 × 10 min) with TBS-0.1% Tween 20 and incubated 1 h with anti-mouse IgG conjugated with peroxidase . The reaction was visualized using luminol [Thioglycollate-elicited peritoneal macrophages were plated as above on 6 well tissue culture plates  from L. braziliensis and L. infantum. Nitrite and cytokine concentrations determined on the supernatants after 48 h  suggesting the involvement of TLR4 in this activation. No significant difference of NO production was noticed in macrophages from TLR2−/− or TLR4−/− mice stimulated with live promastigotes when compared to wild type C57BL/6 (P < 0.05)  Figure A. There L. braziliensis LPG stimulated cells (P <0.05). In all experiments, live parasites from both species induced cytokine production close to background levels ; on the other hand, a significant production of TNF-α, IL-1β and IL-6 was observed  critical glycosyltransferases are down regulated in the metacyclic phase [Leishmania are consistent with the reports from many Old World Leishmania species and strains which showed that LPG with its varied structural polymorphisms induced different levels of NO and TNF-α in murine macrophages [Because most  biology . The intsferases ,24. For ic phase . Such inic phase . In thisrophages ,21,34.Leishmania GPI-anchored glycoconjugates was also observed elsewhere, where mouse peritoneal macrophages failed to produce IL-12 when co-incubated with L. braziliensis or L. infantum GIPLs and when also stimulated with IFN-γ or LPS [L. major LPG [L. braziliensis and L. infantum GIPLs [No IFN-gamma, IL-10 or IL-12 production was observed by cells stimulated with LPGs from both New World species. Similar observations can be made in the human visceral leishmaniasis where immune suppression and a mixed Th1/Th2 profile modulate most of the immune response . The lacγ or LPS . It is aajor LPG  and L. bum GIPLs .in vivo experiments demonstrated the importance of TLR3, TLR4 and TLR9 in different Leishmania species [L. donovani LPG in IFN-γ primed macrophages [In vivo, it was shown that TLR4 deficient mice are more susceptible to Leishmania infection, failing to efficiently resolve the lesions [L. braziliensis-infected dendritic cells [in vitro with macrophages and CHO cells that TLR2 and, to a lesser extent, TLR4 were recognized by LPGs from both species clearly suggesting their participation in the LPG signalling. The inability of L. infantum LPG to activate NF-kB and ERK could be suggested as an evasion mechanism compared to L. braziliensis LPG.In the present work we also evaluated the role of TLRs on the recognition and signaling of LPG. TLR2 was first incriminated as the LPG receptor in macrophages and NK cells ,42. Addi species . By usinrophages . In vivo lesions  while TLic cells . Here, wL. infantum LPG was not able to activate ERK1/2. In contrast, L. braziliensis LPG strongly activated MAPK activity after 15 min. Interestingly, p38 and JNK activation exhibited a gradual and transient profile in L. infantum and L. braziliensis, respectively  , LPG frotivation . Feng ettivation  reportedL. braziliensis and L. infantum LPG stimulated macrophages. The LPG from these species have a limited proinflammatory potential since they fail to activate important proinflammatory cytokines such as IL-12 and only activate low amounts of IL-1β while inducing activation of early inflammatory cytokines such as TNF-α and IL-6. Additionally, as seen in Figure Leishmania.The data presented here demonstrated a differential production of NO, cytokines and MAPK activation profile by L. infantum and L. braziliensis, causative agents of visceral and tegumentary forms, respectively [Together with the complex interface of interaction between parasite and host, glycoconjugate interspecies polymorphisms, not only in the LPG, but also in GIPLs, gp63 and other GPI-anchored molecules, are important for differential regulation of initial events of the immune response as well as establishment of infection. Our results with New World species are consistent with this issue of the importance of LPG polymorphisms; structural variations in LPG resulted in differential activation of macrophages . Those polymorphisms could result in different clinical outcomes, such as those shown by ectively .The authors declare that they have no competing interests.Conceived and designed the experiments: ICI, RRA, RPS, MNM, MAC, SJT; performed the experiments: ICI, RRA, NLM, MAC; analyzed the data: ICI, RRA, MAC, RPPS; helped to draft the manuscript: ICI, RRA, MNM, MAC, NLM, SJT, RPS. All authors read and approved the final manuscript.


Sulfitobacter and highly similar to KMM 3554T. A series of experiments demonstrated that this strain M44 had many distinctive characteristics: its cells were gram-negative and mesophilic; its colonies were slightly yellowish, round, convex, and smooth; and it could grow at 10–28°C, pH 6.0–10.0, and in the presence of 0–12.5% (w/v) NaCl; the optimum growth conditions were 25°C and pH 7.0, and the optimum Na+ concentration was 2.5%. In addition, strain M44 contained 18 : 1 ω7c, 11 methyl 18 : 1 ω7c and 16 : 0 fatty acids as major fatty acids, and the genomic DNA G+C content was 58.04 mol%. According to our results of the secondary metabolites, six cyclodipeptides were isolated from the strain M44, which were Cyclo , Cyclo , Cyclo (Phe-Leu), Cyclo (Leu-Ile), Cyclo (Phe-Ile), and Cyclo (Trp-Pro). It is the first study of secondary metabolites isolated from this genus.A marine bacterium M44 was separated from 30 m deep seawater in the East China Sea (26° 28.3′ N 122° 29.0′ E) in 2006. 16S rDNA gene sequence comparison showed that the strain M44 was a member of the genus  As marine bacteria live in hypothermic, hyperbaric, and oligotrophic environments that are significantly different from those of terrestrial ones, it is reasonable to suppose that they should have particular physiological and biochemical traits and metabolic pathways. In recent years, there has been more interest in isolation and identification of marine bacteria. Natural products of marine bacteria have been recognized as an important source of novel and biologically active substances . Sulfitobacter was first discovered by Sorokin  was also investigated. General physiological tests were performed using conventional methods. Biochemical traits were determined using API kits . The ability to oxidize sulfite was tested by the method of Pukall et al. [; Olympusμm × 0.03 μm). The GC parameters were as follows: carrier gas, ultrahigh-purity hydrogen; carrier gas flow, 0.4 mL·min−1; injection volume, 2 μL; column split ratio, 100 : 1; column temperature, 170–260°C at 5°C min−1, 260–310°C at 40°C min−1 and keep 1.5 min ; injection port temperature, 250°C; detector temperature, 310°C.Fatty acids were determined in cells grown on Zobell 2216E agar plates at 28°C for 2-3 days. Fatty acid methyl esters were obtained from a freeze-dried biomass (approx. 10 mg) by saponification, methylation, and extraction using the method of Svetashev et al. . The fatAccording to the method described by Rainey et al. , the genThe almost complete 16S rDNA gene sequence of strain M44 was submitted to GenBank to search for similar sequences by using the BLAST algorithm. A phylogenetic tree was constructed by using Kimura's two-parameter and pairwise-deletion model analysis in the program MEGA version 3.0 . The resThe G+C content of the DNA was determined by using the method of Mesbah et al. . DNA of The bacterium was grown on Zobell 2216E agar medium and incubated at 25°C for a day. A loopful of bacterium was inoculated into a 500 mL Erlenmeyer flask containing 150 mL of marine Zobell 2216E broth and incubated on a rotatory shaker at 130 rpm, 25°C, for 7 days.3OH to get five fractions, one of which was subsequently rechromatographed on C18 reversed-phase column with a gradient of water to methanol. The fractions obtained were further purified by reversed-phase high-performance liquid chromatography (RP-HPLC)  using CH3CN-H2O isocratic elution. 1H and 13C NMR spectra were recorded at 600 and 300 MHz, respectively, on a Bruker AMX-600 spectrometer. Mass spectra were recorded on a Fisons TRIO 2000 spectrometer.The entire culture broth (60 L) was centrifugated at 4000 rpm for 5 min, and the supernatant extracted 3 times with an equal volume of ethyl acetate. The upper layer of liquid was evaporated in vacuum at 30°C to yield 5 g of the crude extract, which was subjected to Sephadex LH-20 gel column and eluted with CHμm in diameter, and did not form endospores.The colonies were slightly yellowish, regularly round, convex and smooth, and about 0.8–1.0 mm in diameter after incubation for 48 h on marine agar. No diffusible pigment was produced in the medium. Cells were gram-negative, chemoorganotroph with respiratory metabolism, mesophilic rod-shaped and single, about 0.6–0.8 2S production, lysine decarboxylase, and ornithine decarboxylase reactions were negative, while catalase, gelatin liquefaction, production of arginine dihydrolase, tryptophane desaminase, Voges-Proskauer reaction, and citric acid reactions were positive. Alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase were present, while cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase were absent in assays with the API ZYM system. D-sucrose was utilized as the sole carbon source in assays with the API 50CH system. Acid was weakly produced from mannitol. The rest substrates were not utilized as sole carbon sources.The growth condition was determined at 10–28°C, pH 6.0–10.0, and the NaCl concentration was 0–12.5% (w/v), in which the optimum growth condition was 25°C, pH 7.0, and at a 2.5% NaCl concentration. The strain did not oxidize thiosulfate or elemental sulfur but oxidized sulfite. Oxidase, nitrate, indole, urease, Hω7c (67.01%), 11 methyl 18 : 1 ω7c (11.76%), 16 : 0 (8.40%), 10 : 0 3OH (5.42%), and 12 : 1 3OH (4.78%). Minor components included 12 : 0 3OH, 17 : 1 ω8c, 17 : 0, and 18 : 0 isomers. The main cellular fatty acids of the strain were 18 : 1 Sulfitobacter and formed a distinct cluster with KMM 3554T  in the neighbour-joining tree.Phylogenetic analysis  based onThe G+C content of DNA was determined to be 58.04 mol% for strain M44.1H-NMR, 13C-NMR, ESI-MS), six extracellular cyclodipeptide constituents, which were Cyclo  [Sulfitobacter sp. M44  , Cyclo  , Cyclo  , Cyclo  , Cyclo  , and CycVal-Leu) , have be sp. M44 .Sulfitobacter dubius and should be classified to the genus of Sulfitobacter, inwhich many different characteristics can be determined. The fatty acid profile of M44 and related strains gave patterns in which 18 : 1 ω7c ranging from 59.9% to 79.1% predominated, but different compositions could be distinguished on the basis of the remaining fatty acids. Firstly, although all these five strains could produce 11 methyl 18 : 1 ω7c, 16 : 0 and 3-OH 10 : 0 fatty acids, there were some differences in the distribution of them. For unsaturated fatty acid, M44 produced much more 11 methyl 18 : 1 ω7c than other related strains. For straight-chain 16 : 0 fatty acid, M44 showed a certain difference when compared with Sulfitobacter dubius ATCC BAA-320T. The percentage of 16 : 0 fatty acid produced by M44 and Sulfitobacter dubius ATCC BAA-320T was 8.4% and 17.8%, respectively. In addition, M44, Sulfitobacter dubius ATCC BAA-320T, and Sulfitobacter delicatus ATCC BAA-321T produced 3-OH 12 : 1 fatty acid, whereas Sulfitobacter pontiacus DSM 10014T and Sulfitobacter donghicola DSW 25T did not. The details of comparison of the fatty acid compositions were listed in Based on the phenotypic properties and the results of physiological study and molecular identification, strain M44 has a great similarity to Sulfitobacter, we cannot confirm the strain M44 belongs to a new Sulfitobacter species based on the current phenotype and physiological study.The results of physiological and other characteristics of M44 are described in Sulfitobacter widely exists in coastal and open ocean environments. Several bioactivities associated with Sulfitobacter have been reported, including organic sulfur cycling in the ocean [Sulfitobacter may play important roles in marine ecosystems. However, there has been no report on the elucidation of secondary metabolites of Sulfitobacter. Six cyclodipeptides were isolated from M44 according to our work. It is the first report on the secondary metabolites of this genus. A strain-termed Oceanibulbus indolifex, located in the same phylogenetic branch, has been reported to produce cyclodipeptides as well, but structurally different from M44 [According to recent studies, he ocean , producthe ocean , host chhe ocean , and marhe ocean . Consequfrom M44 . Publishfrom M44 . In addifrom M44 . Recentlfrom M44 , which aSulfitobacter strain M44 isolated from the East China Sea, which has some similarities and some differences to the known Sulfitobacter strains. And for the first time, we isolated and identified six cyclodipeptides from this genus. The further study should be focused on the molecular mechanism of cyclodipeptides, which may reveal why cyclodipeptides existed in microorganism widely.Generally, our work describes a


Salvia officinalis L. (Lamiaceae) is a Mediterranean species, naturalized in many countries. In Jordan, it is used in traditional medicine as antiseptic, antiscabies, antisyphilitic, and anti-inflammatory, being frequently used against skin diseases. This study aimed the assessment of the antifungal and anti-inflammatory potential of its essential oils, and their cytotoxicity on macrophages and keratinocytes. The oils were investigated by gas chromatography and gas chromatography-mass spectrometry and the antifungal activity was evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of cell viability was made by the 3--2,5-diphenyltetrazolium bromide assay and the in vitro anti-inflammatory potential was evaluated by measuring nitric oxide production using lipopolysaccharide-stimulated mouse macrophages. The main compounds of S. officinalis oils were 1,8-cineole (39.5–50.3%) and camphor (8.8–25.0%). The oils revealed antifungal activity against dermatophyte strains and significantly inhibited NO production stimulated by LPS in macrophages, without affecting cell viability, in concentrations up to 0.64 μL/mL. This is the first report addressing the in vitro anti-inflammatory potential of S. officinalis oil. These findings demonstrated that bioactive concentrations of S. officinalis oils do not affect mammalian macrophages and keratinocytes viability making them suitable to be incorporated in skin care formulations for cosmetic and pharmaceutical purposes. Salvia L. (Lamiaceae) comprises about 900 species, spread throughout the world, some of which with great economic value since they are used as spices and flavoring agents by perfumery and cosmetic industries [The genus dustries , 2.Salvia sp. has also been used for a long time in folk medicine as medication against fever, rheumatism, perspiration, sexual debility, and in the treatment of chronic bronchitis, as well as mental and nervous diseases [diseases .Salvia officinalis L.), trilobed sage , and Spanish sage  [The most well-known species are common sage (ia Vahl) . S. officinalis  is a perennial, evergreen subshrub, with woody stems, grayish leaves, and blue to purplish flowers. It is native to the Mediterranean region, being currently cultivated in various countries [Salvia deriving from the Latin verb salvare = to save or to cure, and officinalis also means medicinal. ountries , 5. Sageα- and β-thujone, 1,8-cineole, camphor), diterpenes  triterpenes (oleanoic and ursolic acids), and phenolic compounds like rosmarinic acid [Sage is largely used as a savory food flavoring either as dried leaves or essential oil . Sage lenic acid –9.Essential oil of sage and their preparations are externally used for inflammations and infections of the mucous membranes of throat and mouth . Internally, the essential oil is used for dyspeptic symptoms and excessive perspiration .S. officinalis produces monoterpenes with a broad range of carbon skeletons, including acyclic, monocyclic, and bicyclic compounds. Three distinct monoterpene synthases are responsible for the first steps in the formation of the most characteristic monoterpenes of S. officinalis essential oil. The (+)-sabinene synthase catalyzes the production of sabinene, which undergoes further rearrangements leading to the two major monoterpenes, α- and β-thujone. The 1,8-cineole synthase produces in one-step 1,8-cineole. Finally, (+)-bornyl diphosphate synthase produces bornyl diphosphate, which is subsequently hydrolyzed to borneol and then oxidized to camphor [ camphor –15.Salvia, the major monoterpenes 1,8-cineole, camphor, and the two thujones show pronounced dynamics during a vegetative cycle that has been confirmed under different geographical conditions [α- and β-thujone increase gradually during the vegetative period. Environmental conditions such as temperature, day length, and light influence quantitative compositions of essential oils . These cnditions –20. Acconditions , 1,8-cinThe antibacterial properties of sage oils have been attributed to the presence of 1,8-cineole, thujone, and camphor . HoweverS. officinalis as antiseptic, antiscabies, antisyphilitic, and anti-inflammatory, being frequently used against skin and eye diseases and also in pleurisy [Common sage is well known in Palestitian traditional medicine by possessing antimicrobial properties . In 2008pleurisy , 25. In pleurisy . S. officinalis, in the present work we aim to characterize the chemical composition of the essential oil obtained from the aerial parts of S. officinalis collectedin different regions of Jordan, as well as to evaluate the antifungal and the anti-inflammatory activities of the oil at concentrations safe to mammalian cells. Thus, taking into account these traditional uses of S. officinalis were collected during April, in six localities of Jordan: S1-Shoubak (South Jordan); S2:Ma'an (South Jordan); S3:Tafillah (South Jordan); S4:Jerash (North Jordan); S5:Ajloun (North Jordan); and S6:Amman (Middle Jordan). Voucher specimens were deposited at the Herbarium of Shoubak University College-Al-Balqa Applied University, Jordan with the following references: Abu-Darwish 1_2010 (S1); Abu-Darwish 4_2010 (S2); Abu-Darwish 7_2010 (S3); Abu-Darwish 10_2010 (S4); Abu-Darwish 13_2010 (S5); and Abu-Darwish 16_2010 (S6).Aerial parts of All the six samples were used for essential oil isolation and the oils were characterized. Only the oils chemically more distinct (S1 and S2) were chosen for biological assays.The essential oil of the six samples was isolated by hydrodistillation for 3 h using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia .μm) and SupelcoWax-10 . The oven temperature was programmed from 70°C to 220°C at 3°C/min and then held isothermal (15 min); injector temperature: 250°C (injection mode: split 1/40); detectors temperature: 250°C; carrier gas: helium, adjusted to a linear velocity of 30 Cm·s−1.Analytical GC was carried out in a Hewlett-Packard 6890  chromatograph with a HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detectors (FIDs). A graphpak divider  was used for simultaneous sampling to two Supelco  fused silica capillary columns with different stationary phases: SPB-1 , interfaced with an Hewlett-Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00. GC parameters as described above; interface temperature: 250°C; MS source temperature: 230°C; MS quadrupole temperature: 150°C; ionization energy: 70 eV; ionization current: 60 μA; scan range: 35–350 units; scans·s−1: 4.51.GC-MS was carried out in a Hewlett-Packard 6890 gas chromatograph fitted with a HP1 fused silica column  and Ma'an, South Jordan (S2), was evaluated against yeasts and filamentous fungi strains (Aspergillus spp. and dermatophytes).The antifungal activity of the oils obtained from the aerial parts of (1) Yeasts. Three American Type Culture Collection (ATCC) type strains ; one CECT (Colección Española de Cultivos Tipo) type strain (Cryptococcus neoformans CECT 1078); and two clinical strains are isolated from recurrent cases of vulvovaginal candidiasis (Candida guillermondii MAT23 and C. krusei H9).(2) Aspergillus Species. Two ATCC type strains (Aspergillus niger ATCC 16404 and A. fumigatus ATCC 46645) and one clinical strain are isolated from bronchial secretions (A. flavus F44).(3) Dermatophytes. Three dermatophyte clinical strains are isolated from nails and skin  and four CECT (Colección Española de Cultivos Tipo) type strains .Candida parapsilosis ATCC 90018 was used as control.The fungal isolates were identified by standard microbiology methods and stored on Sabouraud broth with glycerol at –70°C. Prior to antifungal susceptibility testing, each isolate was inoculated on Sabouraud agar to ensure optimal growth characteristics and purity.All chemicals were supplied by Sigma Aldrich, except fluconazole, supplied by Pfizer, UK under the name Diflucan.Broth macrodilution methods based on the Clinical and Laboratory Standards Institute (CLSI) reference protocols M27-A3  and M38-μL/mL. Final concentration of DMSO never exceeded 2%. Recent cultures of each strain were used to prepare the cell suspension adjusted to 1-2 × 103 cells per mL for yeasts and 1-2 × 104 cells per mL for filamentous fungi. The concentration of cells was confirmed by viable count on Sabouraud agar. The test tubes were incubated aerobically at 35°C for 48 h/72 h (Candida spp. and Aspergillus spp./Cryptococcus neoformans) and at 30°C for 7 days (dermatophytes) and MICs were determined. To evaluate MLCs, aliquots (20 μL) of broth were taken from each negative tube after MIC reading and cultured in Sabouraud dextrose agar plates. Plates were then incubated at 35°C for 48 h (Candida spp. and Aspergillus spp.) and 72 h for Cryptococcus neoformans and 30°C for 7 days (dermatophytes). In addition, two reference antifungal compounds, amphotericin B (Sigma Aldrich) and fluconazole (Pfizer), were used to control the sensitivity of the tested microorganisms. All tests were performed in Roswell Park Memorial Institute medium (RPMI 1640 medium). For each strain tested, both growth conditions and sterility of the medium were checked in two control tubes. The innocuity of DMSO was also checked at the highest tested concentration. All experiments were performed in triplicate and repeated if the results differed.The serial doubling dilution of the essential oil was prepared in dimethyl sulfoxide (DMSO), with concentrations ranging from 0.32 to 20 μg/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2.Raw 264.7 (ATCC number: TIB-71), a mouse macrophage cell line kindly supplied by Dr Otília Vieira , was cultured in Iscove's Modified Dulbecco's Eagle Medium supplemented with 10% noninactivated fetal bovine serum, 100 U/mL penicillin, and 100 The anti-inflammatory activity of the oils from samples S1 and S2 was evaluated in the mouse macrophage cell line Raw 264.7.6 cells/well in 48-well culture plates, allowed to stabilize for 12 h, and then incubated with culture medium (control), or stimulated with 1 μg/mL LPS, or with 1 μg/mL LPS in the presence of different concentrations of the essential oil during 24 h. Briefly, 170 μL of culture supernatants was collected and diluted with equal volume of the Griess reagent (0.1% (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride and 1% (w/v) sulphanilamide containing 5% (w/v) H3PO4) during 30 min, in the dark. The absorbance at 550 nm was measured using an automatic plate reader . Nitrite concentration was determined using a sodium nitrite standard curve.The production of NO was measured by the accumulation of nitrite in the culture supernatants, using a colorimetric reaction with the Griess reagent . The celμg/mL streptomycin, at 37°C in a humidified atmosphere of 95% air and 5% CO2. In the experiments, cells were monitored by microscope observation in order to detect any morphological change.The human keratinocyte cell line HaCaT, obtained from DKFZ (Heidelberg), was kindly supplied by Dr Eugénia Carvalho . Keratinocytes were cultured in Dulbecco's Modified Eagle Medium (high glucose) supplemented with 4 mM glutamine, 10% heat inactivated fetal bovine serum, 100 U/mL penicillin, and 100 Cell viability was assessed for the oils from samples S1 and S2 using the mouse macrophage cell line Raw 264.7 and the human keratinocyte cell line HaCaT.6 cells/well, cultured in 48-well microplates) were incubated in a final volume of 600 μL, allowed to stabilize for 12 h, and then incubated for 24 h with different concentrations of the essential oil. After adding 60 μL of MTT solution (5 mg/mL in PBS) to each well, the cells were further incubated at 37°C for 15 min, in a humidified atmosphere of 95% air/5% CO2. Supernatants were then discarded and 300 μL of acidified isopropanol (0.04 N HCl in isopropanol) was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using an ELISA automatic microplate reader  at 570 nm, with a reference wavelength of 620 nm.Assessment of cell viability was made through a colorimetric assay, using MTT . The HaCμL of culture supernatants for NO measurement, 43 μL of MTT solution (5 mg/mL in PBS) was added and cells were incubated at 37°C for 15 min, in a humidified atmosphere of 95% air and 5% CO2. Supernatants were then discarded, and 300 μL of acidified isopropanol (0.04 N HCl in isopropanol) was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed as described above.In the case of the cell line Raw 264.7, after collection of 170 P < 0.01. The statistical tests were applied using GraphPad Prism, version 5.02 .All the experiments were performed in duplicate, being the results expressed as mean ± SEM of three independent experiments. The means were statistically compared using one-way ANOVA, with a Dunnett's multiple comparison test. The differences between the means were considered significant for values of The essential oil of the six samples was obtained in yields of 1.2–2.8% (w/w), based on the dry weight of the plant.The qualitative and quantitative composition of the oils is presented in α-thujone 1.2–3.3% and β-thujone 0.9–9.9%).In total, 25 compounds were identified accounting for 92.7–99.0%. The oils did not show a significant difference in composition between them with the oxygen containing monoterpenes representing the major percentage (72.1–82.1%), with slight variations in the amounts of the main compounds: 1,8-cineole (39.5–50.3%) and camphor (8.8–25.0%). In these samples, thujones, characteristic compounds of sage oils, are present in very low amounts , 69.60% ± 7.16 (0.64 μL/mL), 84.60% ± 0.34 (0.32 μL/mL), and 88.58% ± 1.11 (0.16 μL/mL) for S1 (μL/mL), 58.53% ± 2.72 (0.64 μL/mL), 78.68% ± 1.19 (0.32 μL/mL), and 86.46% ± 1.52 (0.16 μL/mL) for S2  for S1  and to 2) for S2 .μL/mL of S1 decreased MTT reduction to 10.69% ± 1.44, while for concentrations of 0.64, 0.32, and 0.16 μL/mL, the oil did not show significant cytotoxicity in macrophages, with values of 96.97% ± 3.88, 103.70% ± 0.96 and 104.9% ± 0.92, respectively. For S2, in the concentration of 1.25 μL/mL, the MTT reduction decreased to 46.50% ± 2.12, while for concentrations of 0.64, 0.32, and 0.16 μL/mL, the oil did not show significant cytotoxicity in macrophages, with values of 99.85% ± 0.44, 102.40% ± 0.36 and 102.0% ± 0.29, respectively, , 47.25% ± 0.30, and 51.66% ± 1.66 (1.25 μL/mL), respectively. However, incubation of HaCaT with 0.64, 0.32 and 0.16 μL/mL of the oil, during 24 h, showed no cytotoxic effect, with values of 91.01% ± 1.62 and 96.55% ± 0.54 (0.64 μL/mL), 101.10% ± 0.79, and 100.50% ± 1.10 (0.32 μL/mL), 106.20% ± 3.58, and 103.80% ± 1.70 (0.32 μL/mL).As shown in Figures A cell-free control was performed in order to exclude nonspecific effects of the oils on MTT (data not shown).S. officinalis is highly influenced by genetic and environmental factors, organ age, climate conditions, and seasonality [α-thujone (18–43%), β-thujone (3–8.5%), camphor (4.5–24.5%), 1,8-cineole (5.5–13%), humulene (0–12%), α-pinene (1–6.5%), camphene (1.5–7%), limonene (0.5–3%), linalool (free and esterified (1% maximum)), and bornyl acetate (2.5% maximum). The essential oil composition of sonality . Due to sonality  describeα-thujone is much lower (1.2–3.7%) and for 1,8-cineole (39.5–50.3%) is much higher.The essential oil of the Jordan samples (S1–S6) does not show a significant difference in composition between them and match these intervals for the majority of the compounds; however, the relative amount for α- and β-thujones increase gradually during the vegetative period. Our samples were collected in April, and they possess very low amount of thujones and a quite high amount of 1,8-cineole.These results are in agreement with the revision of Grausgruber-Gröger et al. 2012 , where tα-thujone > 1,8-cineole > β-thujone; (ii) camphor > α-thujone > β-thujone > 1,8-cineole; (iii) β-thujone > camphor > 1,8-cineole > α-thujone; (iv) 1,8-cineole > camphor > α-thujone > β-thujone; and (v) α-thujone > camphor > β-thujone > 1,8-cineole. These samples from Jordan belong to the fourth group, with 1,8-cineole (39.5–50.3%) > camphor (8.8–25.0%) > α-thujone (1.2–3.7%) > β-thujone . This chemotype is not very usual.In 1990, Tucker et al.  categoriα-thujone higher toxicity than β-thujone [μL/mL. The absence of keratinocyte toxicity is crucial to make an essential oil suitable for inclusion in topical formulations. Indeed, there is a lack of knowledge addressing keratinocytes cytotoxicity induced by essential oils and a current screening in our lab points that keratinocytes are very sensitive to several essential oils, which highlights the use of safe bioactive concentrations of S. officinalis essential oil for topical applications.Thujones have been reported to be toxic to both brain and liver cells, presenting -thujone , 39. Thein vitro anti-inflammatory activity of S. officinalis essential oil. The oils significantly inhibited NO production elicited by LPS in macrophages which demonstrates its strong anti-inflammatory potential. However, sample 2 (S2) has a slightly higher activity than sample 1 (S1), what can be related to the chemical composition. Although the samples possess similar chemical composition, they are distinguished by the content of camphor. Moreover, the higher amount of camphor in S2 did not affect the cell viability.Macrophages produced a variety of proinflammatory mediators, such as nitric oxide, upon activation with the pro-inflammatory stimulus LPS. Since NO is a well-established inflammatory marker, the inhibition of its production might be a useful strategy to treat inflammatoryrelated diseases. Therefore, we also evaluated, for the first time, the Candida and Aspergillus strains, particularly for Trichophyton rubrum and Epidermophyton floccosum with MIC of 0.64 μL/mL. Among the tested yeasts, Cryptococcus neoformans was the strain that showed more susceptibility with MIC of 0.64 μL/mL.Concerning the antifungal properties of the essential oil, the results presented here demonstrate that the dermatophyte strains showed more susceptibility to these oils when compared with Although the MIC and MLC results varied among tested organisms, in various cases the MIC was equivalent to the MLC, indicating fungicidal activity of the oils . S. officinalis oils do not affect mammalian macrophages and keratinocytes viability making them suitable to be incorporated in skin care formulations for cosmetic and pharmaceutical purposes.Taking together, our results demonstrated that bioactive concentrations of


Some compounds showed relatively high cytotoxicity. Especially compounds 5b exhibited the strongest cytotoxicity against HepG-2 and Spca-2 cells (IC50 was 8.82 and 9.01 μM), respectively. All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. Further experiments proved that 5b could disturb the cell cycle in HepG-2 cells and induce apoptosis. In addition, the binding properties of a model conjugate 5b to DNA were investigated by methods . Results indicated that 5b showed moderate ability to interact ct-DNA.Several rhein-phosphonate derivatives ( A nuetc. -α-(substituted phenyl)methyl}phosphonates 2, obtained by reacting of substituted aromatic ketones 1 with ammonium acetate and O,O′-dialkyl phosphite, were converted easily to O,O′-dialkyl α-amino-α-(substituted phenyl)methyl phosphonate 4 via hydrolysis /[GelRed] ratio of 8:1.In competitive binding experiments, GelRed and ct-DNA solutions were pre-incubated for 30 min to ensure sufficient interactions. The concentration ratio of GelRed to DNA was set at [GelRed]/[DNA] = 1:10 to ensure sufficient binding sites of DNA for GelRed. The emission spectra of the GelRed–ctDNA system in the absence and presence of 5b to GelRed fluorescence can be quantitively estimated by their respective quenching constant, Kq, which were derived from the Stern-Volmer quenching equation. The Kq value for 5b was 3.2 × 103, exhibiting similar intensive intercalation to DNA as rhein (3.94 × 103) (5a (2.02 × 103) and 5c (0.753 × 103) (5b to DNA was similar to GelRed /[DNA] ratio of 1:10, the CD absorption of ct-DNA showed an obvious decrease in the intensities of both the negative and positive absorption bands  with cytotoxic effects were investigated for the effects on the apoptosis of HepG-2 cells. To clarify the mechanism of rhein derivative-induced cell death, we determined both early and late apoptosis using annexin V-FITC and PI (Propidium iodide) labeling of live cells. Annexin V binds to phosphatidylserine, which is exposed on the cell membrane and is one of the earliest indicators of cellular apoptosis. PI is used as a DNA stain for both flow cytometry to evaluate cell viability or DNA content in cell cycle analysis and microscopy to visualize the nucleus and other DNA containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells. In the present study, the apoptotic cell rates were determined for the HepG-2 cells after stimulation for 24 h by the compounds at different concentrations. The graphical values of the results were given in 5a and 5b induced apoptosis in 84.4% and 70.4% of the HepG-2 cells. Compounds 5a and 5b are of special interest as these compounds seem to drive HepG-2 cells directly into early apoptosis. Meanwhile, we also investigated for the effects on the apoptosis of HUVEC cells. The result showed that the compounds we have synthesized had no significant apoptotic effects on HUVEC cells.Many anthraquinones such as rhein and emodim induce apoptosis ,33. Flow5a or 5b for 48 h at different concentrations. G2-phase populations of 7.23%, 11.08%, 18.05% for 5a at 20 μM, 30 μM, 40 μM were observed compared with the G2-phase population of 4.27% for untreated cells. For compound 5b, G2-phase populations of 8.52%, 14.56%, 18.23% for 5b at 10 μM, 20 μM, 30 μM were observed compared with the G2-phase population of 4.27% for control (−) cells.The cell cycle is the series of events that take place in a cell leading to its division and duplication (replication). The cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis). The G1 stage is the stage when preparation of energy and material for DNA replication occurs. The S stage is the stage when DNA replication occurs. The G2 stage is the stage when preparation for the M stage occurs. The M stage is “mitosis,” and is when nuclear and cytoplasmic division occurs. Flow-cytometric analysis further confirmed tumor cell apoptosis as shown in All chemicals (reagent grade) used were commercially available. NMR spectra were measured on a BRUKER AVANCE AV500 spectrometer using TMS as the internal standard. The mass spectra were obtained on a BRUKER ESQUIRE HCT spectrometer. Melting points were determined using an X-4 apparatus and were uncorrected. Elemental analyses were performed on a Vario Micro Cube Elemental instrument and were within 0.4% of the theoretical values.5General Procedure for the Preparation of Rhein α-Aminophosphonic Acids Conjugates 2, to which the diethyl phosphite (5 mmol) was added and the resulting solution was stirred for 6 h at 80 °C. After that, hydrochloric acid (4 mmol) in 30 mL ether was added to the reaction mixture, which was stirred for 2 h at 0 °C to give the precipitate 3. The precipitate was filtered and washed with ether (3 × 15 mL), then it was added to 10 mL sodium hydroxide solution (10%) and stirred for 30 min at room temperature. Extraction with dichloromethane (3 × 25 mL) and evaporation of the solvent gave the oils of 4, which was purified by column chromatography on silica gel with ethyl acetate. To a mixture of rhein and HOBt in DMSO (15 mL), the appropriate EDAC was stirred for 15 min at 0 °C. Then 4 was added dropwise with constant stirring at 0 °C, the mixture was stirred at ambient temperature for another 10 h, then washed with brine, dried with anhydrous sodium sulfate and evaporated. The residue was purified by column chromatography on silica gel with petroleum ether/ethyl acetate  to give the pure title compounds 5 as yellowish solids.A suspension of aromatic ketones (10 mmol) and ammonium acetate (11 mmol) was stirred for 6 h at reflux. The reaction mixture was filtered to give the white precipitate O,O′-Diethyl{[2-acetamido] (4-tolyl) ethyl} phosphonate (5a). Yield 78%. m.p. 132~134 °C. 1H-NMR (500 MHz CDCl3) δ (ppm):0.88 , 1.31 , 2.20  = 16.0 Hz, 3H, CH3), 2.33 , 3.72–4.05 , 7.16–8.16 , 11.99 , 12.06 . 31P{H} NMR δ (ppm): 24.18. ESI-MS m/z: 560.2 (M+Na)+. Anal. Calc. (for C28H28NO8P+H2O): C, 62.57; H, 5.25; N, 2.61; Found: C, 60.57; H, 5.44; N, 2.52.O,O′-Diethyl{[2-acetamido] (Phenylethyl) ethyl} phosphonate (5b). Yield 71%. m.p. 119~121 °C. 1H-NMR (500 MHz CDCl3) δ (ppm): 0.87 , 1.39 , 1.83  = 16.0 Hz, 3H, CH3), 2.32–2.81 , 4.24 , 6.52 , 7.13–8.04 , 11.95 , 12.01 . 31P{H} NMR  δ (ppm): 26.67. ESI-MS m/z: 574.2(M+Na)+. Anal. Calc. (for C29H30NO8P+H2O): C, 63.15; H, 5.48; N, 2.54; Found: C, 61.16; H, 5.66; N, 2.46.O,O′-Diethyl {[2- acetamido] (phenyl) methyl} phosphonate (5c). Yield 78%. m.p. 141~143 °C. 1H-NMR (500 MHz CDCl3) δ (ppm): 1.23 , 1.30 , 2.23  = 15.9 Hz, 3H, CH3), 3.71–4.06 , 7.26–8.16 , 11.98 ,12.06 . 31P{H} NMR  δ (ppm): 24.07. ESI-MS m/z: 546.2 (M+Na)+. Anal. Calc. (for C27H26NO8P+H2O): C, 61.95; H, 5.01; N, 2.68; Found: C, 59.89; H, 5.21; N, 2.59.in vitro human cancer cell lines were used: HepG 2 (human epidermoid larynx carcinoma), Hela (Henrietta Lacks strain of cancer cells), Hct-116 , CNE , Spca-2 (human lung adenocarcinoma cell line), HUVEC . The cell lines  were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, The Academy of Sciences of China.The following l-Glutamine and 4,500 mg/L Glucose, supplemented with 10% (v/v) fetal bovine serum . Spca-2 cells were cultured in RPMI-1640 medium, containing 2.05 mM l-Glutamine without Calcium nitrate, supplemented with 10% (v/v) fetal bovine serum . The cell culture media was supplemented with penicillin/streptomycin at 100 Units/mL as adherent monolayers. Cell cultures were kept in a humidified incubator with 5% CO2 at 37 °C. Stock solutions were prepared in dimethyl sulfoxide (DMSO) and further dilutions were made with fresh culture medium. The concentration of DMSO in the final culture medium was 1%, which had no effect on the cell viability.HepG-2, Hela, Hct-116, CNE, HUVEC cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (Thermo), containing 4.0 mM i.e., a reduced form of MTT) generated from viable cells were dissolved by adding 100 μL DMSO in each well. The plates were swirled gently for 10 min to dissolve the precipitate, and quantified by measuring the optical density (OD) of the plates at a wavelength of 490 nm on plate reader (TECAN infinite M1000). Each concentration was repeated in three wells and the same experimental conditions were provided for all compounds and MTT analysis was repeated three times for each cell line.Chemosensitivity was assessed using 3--2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, exponentially growing HepG-2 , Hela , Hct-116 , CNE , HUVEC  and Spca-2  were seeded into 96-well plates and treated with indicated concentrations of samples for 48h, and then 10 mL of MTT (10 mg/mL) was added. After incubation for 4 h at 37 °C, the purple formazan crystals  and then resuspended in 1× Binding Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1 × 106 cells/mL. The transfer of 100 μL of the solution (1 × 105 cells) to a 5 mL culture tube was made, and 5 μL of FITC annexin V  and 5 μL propidium iodide (PI) were added to each tube. The cells were gently vortexed and incubated for 30 min at RT (25 °C) in the dark. Two hundred μL PBS were added to each tube. Analysis was performed with the system software . Lower left quadrant, viable cells (annexin V−/PI−); lower right quadrant, early apoptotic cells (annexin V+/PI−); upper right quadrant, late apoptotic cells (annexin V+/PI+); upper left quadrant, necrotic cells (annexin V−/PI+). The percentage of cells positive for PI and/or annexin V-FITC was reported inside the quadrants.Apoptosis was discriminated with the annexin V-FITC/propidium iodide test. Cells were seeded at 2 × 10The cell lines were treated with indicated concentrations of compounds. After being incubated for 48 h, cells were washed twice with ice-cold PBS, fixed and permeabilized with ice-cold 70% ethanol at −20 °C overnight. The cells were treated with 100 μg/mL RNase A at 37 °C for 30 min after washed with ice-cold PBS, and finally stained with 1 mg/mL propidium iodide (PI) in the dark at 4 °C for 30 min. Analysis was performed with the system software .−3 M ct-DNA stock solution was stored at 4 °C for no more than 5 days before use. The synthesized conjugate (5b) was prepared as 2 × 10−3 M DMSO stock solutions for DNA binding studies. The final working solutions of the complexes for DNA binding studies were diluted by TBS and the DMSO content was less than 10%. For UV-vis absorption experiments, the working solutions of the complexes were kept constant at 25 μM. The ct-DNA stock solution was increasingly added until a saturation state was achieved. After each addition, the solution was allowed to incubate for 5 min before the absorption spectra were recorded. Kb as the equilibrium DNA binding constant and s as the binding site size were determined by non-linear fitting according to The 2 × 10ɛa is the molar extinction coefficient of the compound bound with DNA, ɛf is the extinction coefficient of the free compound, and ɛb is the extinction coefficient of the compound fully bound to DNA. ɛa, ɛf and ɛb are all calculated from the Lambert-Beer’s law (ɛ = A/[compound]). To determine ɛb, it is assumed that the compounds are fully bound with DNA at the point where no further hypochromicity is observed under the addition of ct-DNA, and the apparent absorbance Ab is regarded as the absorbance of only the bound compounds. Ct is the constant concentration of the compounds, and s is the number of binding sites in base pairs. A solution containing 2 × 10−4 M DNA and 2 × 10−5 M GelRed ([DNA]/[GelRed] = 10:1) was prepared for GelRed-DNA competitive binding studies. Fluorescence emission spectra were recorded under slit width as 10 nm/10 nm for Ex/Em, respectively. The quenching constant for comparing the efficiency of fluorescence quenching, i.e., Kq, of each compound was obtained by the linear fit of plotting I0/I versus [Q], according to the classic Stern-Volmer equation: I0/I = 1 + Kq × [Q]  is the concentration of quencher. In the fluorescence polarization experiment, each sample was pre-incubated for 40 min before the fluorescence polarization was recorded under the condition of 595 nm emitting wavelength with 350 nm exciting wavelength, and slit width was set as 5 nm/5 nm for Ex/Em, respectively. CD absorption spectra of DNA were measured in TBS at a 100 nm/min scan rate in the wavelength range from 200 to 400 nm, with 1 × 10−4 M DNA in the absence and presence of each compound of 2 × 10−5 M, respectively. The CD signal of TBS was taken as the background and subtracted from the spectra. All the spectroscopic experiments were performed at 25 °C.where [DNA] is the DNA concentration in nucleotides, Kq × [Q] , where It-test with the significance level p ≤ 0.05 using SPSS.The data were processed by the Student’s 1H and 31P-NMR spectroscopy. It was detected that the compounds 5a and 5b had selective and significant cytotoxic effects on HepG-2 cells, respectively. Especially compounds 5b exhibited the strongest cytotoxicity against HepG-2 and Spca-2 cells with IC50 8.82 and 9.01 μM, respectively. In addition, it was found that 5a and 5b disturbed the cell cycle and induced apoptosis in HepG-2 cells and the toxic of all conjugates were lower than rhein. The binding properties of 5b to DNA examined by various methods indicated that 5b interacted with DNA. Therefore, these compounds may be considered as the agents with high potential anti-cancer activity and appear to be good candidates for more advanced screening.Cancer is a leading cause of death worldwide and hence studies to find anti-cancer agents for its treatment continue to grow in importance. In the present study, as potential anti-cancer agents, rhein-phosphorus derivatives were synthesized and characterized by mass spectrometry,


To investigate the antibacterial effect and the effect on the material properties of a novel delivery system with Irgasan as active agent and methacrylated polymerizable Irgasan when added to experimental dental resin composites.a, polymerization shrinkage, contact angle Θ, total surface free energy γS and its apolar γSLW, polar γSAB, Lewis acid γS+and base γS- term as well as bacterial viability were determined. Significance was p < 0.05.A delivery system based on novel polymeric hollow beads, loaded with Irgasan and methacrylated polymerizable Irgasan as active agents were used to manufacture three commonly formulated experimental resin composites. The non-modified resin was used as standard (ST). Material A contained the delivery system providing 4 % (m/m) Irgasan, material B contained 4 % (m/m) methacrylated Irgasan and material C 8 % (m/m) methacrylated Irgasan. Flexural strength (FS), flexural modulus (FM), water sorption (WS), solubility (SL), surface roughness Ra. Only A had lower FM than ST. Θ of A and C was higher and γSAB of A and B was lower than of ST. Materials A to C had higher γS+ than ST. The antibacterial effect of materials A to C was significantly increased when compared with ST meaning that significantly less vital cells were found.The materials A to C were not unacceptably influenced by the modifications and achieved the minimum values for FS, WS and SL as requested by EN ISO 4049 and did not differ from ST what was also found for RDental resin composites with small quantities of a novel antibacterially doped delivery system or with an antibacterial monomer provided acceptable physical properties and good antibacterial effectiveness. The sorption material being part of the delivery system can be used as a vehicle for any other active agent. Several attempts have been made to modify dental resin composites to avoid or at least to diminish pellicle and bacterial adherence which is considered to be important in early plaque formation -7. MainlMore recent literature presented an entirely new approach to obtain dental resin composites with low SFE, antimicrobial effect and acceptable physical properties ,5. NovelA. naeslundii, A. viscosus, S. oralis, S. mitis, S. sanguinis) after 8 or 24 hours, respectively, on these materials. One material contained the polymer hollow beads loaded with the antibacterially effective Irgasan phenol, The goal of the present investigation was, based on the new concepts of the aforesaid literature ,5, to exa were determined. Curing was done with a quartz-tungsten halogen device  performing an irradiance of 931 ± 90 mW cm-2 which was checked periodically with the bluephase meter . Four experimental resin-based restorative materials were prepared  and 2 usIrgasan  was diss1H-NMR (CDCl3): δ = 1.94  = 1.28 Hz, 3H, methyl -CH3); 5.69  = 1.47 Hz, 1H, acryl =CH2); 6.18  = 1.05 Hz, 1H, acryl =CH2); 6.86  = 8,73 Hz, 1H, aryl CH); 6.89  = 8,73 Hz, 1H, aryl); 7.14  = 8.73 Hz, 4J = 2.47 Hz, 1H, aryl); 7.18  = 8.73 Hz, 4J = 2.47 Hz, 1H, aryl); 7.25  = 2.33 Hz, 1H, aryl); 7.41  = 2.46 Hz, 1H, aryl) ppm.13C-NMR (CDCl3): δ = 18.21 (methyl -CH3); 119.91 (aryl); 120.66 (aryl); 124.59 (acryl =CH2); 125.69 (aryl C-Cl); 126.93 (aryl); 128.03 (aryl); 128.1 (aryl); 129.23 (aryl C-Cl); 129.46 (aryl C-Cl); 130.33 (aryl); 134.81 (acryl C=C); 142.06 (aryl -ether C-O); 146.5 (aryl -ester C-O); 151.05 (aryl -ether C-O); 164.61 (ester C=O) ppm.2), 2983 , 2875 , 1741 , 1636 , 1098  cm-1.FT-IR (diamond): ν = 3092 , 189 (10), 69 (100), 41 (14).GC/MS m/z (%): 361 (3), 360 (20), 358 (56), 357 (8), 356 (60) [M-1, Model 106.L, Test GmbH, Erkrath, Germany).Ten specimens (25 ± 2 x 2 ± 0.1 x 2 ± 0.1 mm) were made from each material according to ISO 4049  and cure2) and flexural modulus by E = (L3/ 4bh3) x (F/Y) both expressed in MPa with F = maximum strength, L = distance between the rests, b = width of the specimen, h = height of the specimen, and F/Y = slope of the linear part of the stress-strain curve.Flexural strength was calculated by σ = (3FL) /  / V and solubility by Wsl = (m1 - m3) / V both expressed in µg mm-3.Ten disks  were made from each material according to ISO 4049  and curea was determined . Each specimen was measured five times at different distances and in different directions  from its center. For each measurement, the stylus was automatically moved five times forward and backward along the same path. The data were filtered with a cut-off (Λc) of 0.8 mm (Gauss profile-filter) following DIN EN ISO 4288 [After contact angle measurement the specimens were polished again and their surface roughness RISO 4288 . 2) – (1 / D1)) x (1 / D1) x 100.Polymerization shrinkage was determined according to the Archimedes’ principle . After weighing the specimens in air and in water, their densities were calculated in g cm-3 by D = (A/(A-B)) x (D0-DL) + DL with D = density of sample, A = weight of sample in air, B = weight of sample in water, D0 = density of water, and DL = air density (0.0012 g cm-3). Firstly the density D1 of the uncured material  was determined. Next, disks  were prepared, polymerized for 40 s from each side and the density D2 was measured. The polymerization shrinkage in % was calculated by ΔV = . Ten pictures of each drop were photographed (magnification 25-fold) in one second  and analyzed .LLW, polar Lewis acid γL+ and Lewis base γL- terms of the test liquids were taken from the literature [S, their apolar term γSLW, polar term γSAB, acid term γS+ and base term γS- were calculated according to the equation of van Oss et al. [L = 2(√(γSLW γLLW) + √(γS- γL+) + √(γS+ γL-)) and with γSAB = 2√(γS+ γS-) . To determine contact angles and SFE, ten discs  of each material were prepared and polymerized for 40 s from each side. Their surfaces were polished with fine and superfine polishing-discs  for 1 minute each with a grinding pressure of 40 - 50 g and 10000 rpm . Contact angle Θ was measured one time on each specimen. Measurements with aqua dest. (laboratory product) were done before and after three weeks water storage at 37°C. Measurements with glycerol , ethylene glycol  and diiodomethane  were solely done after three weeks water storage at 37°C. SFE was calculated from the Θ measured after water storage. Apolar Lifshitz-van der Waals γs et al.  by (cosΘStreptococcus sanguinis (strain 20068), Streptococcus oralis , Actinomyces viscosus (strain 43329), and Actinomyces naeslundii  were used to determine cell viability. Bacteria were exposed on agarplates and incubated for 48 hours at 37 °C (actinomyces under anaerobe conditions). Overnight cultures of the streptococci were established in sterile trypticase soy broth  supplemented with yeast extract  at 37°C. Single colonies of the actinomyces were cultivated in sterile actinomyces broth  for 24 hours at 37 °C. The bacteria were harvested by centrifugation , washed twice with sterile 0.9 % NaCl solution and re-suspended again in sterile 0.9 % NaCl. Optical density of the suspensions was adjusted to 1iter at 600 nm , which corresponded to a microbial concentration of 5 x 108 cells ml-1.Unstimulated human saliva was collected from ten healthy non-smoking subjects . Approval from the ethics committee was obtained . The participants gave their consent verbally, as they all were part of the academic faculty staff of the department. Documentation of the oral consent was done by making a list of prospective saliva donors. All donors who subsequently gave their oral consent were checked-off on this list. The oral consent was explicitly judged as adequate, and the need for written informed consent from the participants was waived by the ethics committee. The saliva samples were all mixed with each other, so that they could not be identified anymore after sampling. The saliva was centrifuged  and the supernatant was sterile filtered  and subsequently heated to 56 °C for 30 minutes . Finally the samples were pooled and stored at -20 °C. Prior to incubation samples were defrosted and 1:1 diluted with PBS .2 were measured and no significant decrease of the output was observed. The cured specimens were stored for 14 days in water at 37°C, disinfected  and then one side was wet-polished (sterile water) with fine and superfine polishing-discs  for 1 min, with a grinding pressure of 40 - 50 g and 10000 rpm . Afterwards they were placed with the polished side up in a well , incubated with 250 µl of the diluted saliva for 2 hours at 37 °C and finally washed twice with a sterile 0.9 % NaCl solution.Thirty disks  of each material were made and cured 40 s each side . The output of the curing device was routinely checked . Irradiances of 931 ± 90 mW cm-2, actinomyces at anaerobic conditions), then washed with 0.9 % NaCl solution and air-dried. Vital and non-vital cells were determined  by measuring the fluorescence emission  on the blinded specimens. Fluorescent microscopic images of four randomly selected sites of each specimen (magnification 400 fold) were captured  with different fluorescence filter sets . Vital and non-vital cells were calculated by counting pixel per colour .Three specimens (polished sides) for each material / bacterium / measurement point were used and incubated with 350 µl bacterial suspension for 8 hours or 24 hours, respectively, at 37 °C . T-Test was used to identify differences of the contact angle between dry and wet storage and to find differences between the 8 hour and 24 hour bacteria counts. Multivariate ANOVA and post hoc Scheffé’s test were calculated to identify differences between the test materials’ SFEs . Statistical significance for all tests was considered as p < 0.05.S of material B was lower than of ST and of C  and the polar terms of the surface free energy γSAB of A and B were lower than of ST and of C . The acid terms of the surface free energy γS+ of materials A to C were higher than of ST  and γS+ of B was higher than of C (p = 0.026).A. naeslundii on material B (p = 0.015) and for A. viscosus and S. sanguinis on materials B and C than on ST (p < 0.0005 to p = 0.002). Higher total bacteria counts were observed for A. viscosus and S. mitis on material A . Less vital cells were found for A. viscosus, S. oralis and S. sanguinis on materials A to C (p < 0.0005 to p = 0.031) but for A. naeslundii only on A (p = 0.011) and B (p = 0.003) and for S. mitis only on B and C than on ST . More non-vital bacteria were counted for each individual species on material A . Materials A, B and C did not differ among each other in the vital cells of S. sanguinis after 8 hours. S. oralis had less vital cells (p = 0.002) on material A than on C.For the overall bacteria the total count of material A was higher but of B and C lower than of ST . Materials A to C had less vital cells and A had more non-vital cells than ST . Considering the individual species the total bacteria count was lower for actinomyces were found on materials B and C compared with ST (P = 0.006 to p = 0.014) This was also observed for S. mitis and S. sanguinis (p < 0.0005 to p = 0.039), but not for S. oralis. Less vital actinomyces also existed on A, B and C (p < 0.0005 to p = 0.003) but for S. mitis and S. sanguinis less vital cells existed only on B and C (p = 0.002 to p = 0.011) and for S. oralis only on A (p = 0.044). Less vital actinomyces lived on material C (p < 0.0005 to p = 0.002) but more total (p = 0.033) and more non-vital (p = 0.005) A. naeslundii were on material B. Total and non-vital S. mitis cells were only influenced by material A (p = 0.006 and p = 0.009) resulting in a decrease. The vital cells of S. oralis decreased on ST, B and C (p = 0.016 to p = 0.046) but of S. sanguinis only on ST and C (P = 0.024 and p = 0.002).For the overall bacteria consideration the total bacteria count decreased only on material C compared with 8 hours (p = 0.012). Materials B and C showed less vital (p < 0.0005 to p = 0.048), ST and B showed more non-vital (P = 0.001 and p = 0.017) but A showed less non-vital cells (P = 0.005). Considering the individual species less total Materials A, .B and C did not differ among each other in the vital cells of the overall bacteria, of the actinomyces species, of S. mitis and S. oralis S. sanguinis showed more vital cells (p = 0.009) on materials A and C. Two examples of the vital fluorescence pictures can be seen in Figures 3 and 4.a on bacterial adherence was discussed thoroughly in the literature [a ≤ 0.2 µm was judged to have negligible effect [a is assumed not to be a factor. To mimic the salivary pellicle [S. mutans or Lactobacilli, as these bacteria can also attach to the tooth/restoration surface directly. Based on the literature [2 were assumed to be appropriate for the present study. The preparation of ST and the experimental dental composites ,5, the dterature ,36,54-59e effect ,56-58, tpellicle  the specpellicle . As alrepellicle . As we eterature -64 and ts < 30 mJ m-2 so that they were not really hydrophobic according to Vogler’s interpretation [sAB of materials A and B were significantly lower than of ST which even suggests a higher hydrophilicity. According to retation . The polS. mitis for 8 and 24 hours and S. sanguinis for 24 hours). The results also show that amounts of 4 % (m/m) were adequate to reduce the number of vital cells significantly  because no increased cell death was observed with 8 % (m/m) active agent in material C. S. oralis was the most resistant bacterium which did not differ in the vital cells from ST after 24 hours for material B and C and only material A had less vital cells for this observation time. This is the only case where the delivery system  is more effective than Methacryl-Irga in 4 % (m/m) concentration . Since materials A and B did not differ in their surface properties  strongly indicated that an antibacterial effect did not necessarily diminish bacterial adhesive forces. As shown by the results for materials B and C the chemistry of the antibacterial ingredient needs also to be considered. Although nearly insoluble in water, the surface concentration of the free Irgasan provided by the delivery system might be reduced during the manipulation processes of the specimens. This could not be happen with the immobilized polymerizable Methacryl-Irga.Considering the total bacteria counts it is very striking that significantly less cells were found on materials B and C than on ST and mostly also on material A. Again sAB was reported to create strong bacterial adhesion [sAB might lessen bacterial adhesive forces. Considering the total bacteria counts which might be an indicator for bacterial adhesion no relation was detected between the number of total bacteria and γsAB. sAB but material B had significantly less total bacteria for all species with the exception of S. oralis and material C with the same γsAB than ST had less total cells than ST and did not differ from B with lower γsAB. The differences of materials B and C for γsAB were not surprising because Irgasan is a very hydrophobic substance and its concentration was twice as much in material C than in material B. It is hardly to explain why γsAB of material A and B was highly significantly lower than of ST. It might be possible that the polar groups of Irgasan (Cl- and O-atoms) turned away from the hydrophobic parts of the matrix and levelled to the hydrophilic environment. An influence of SFE on bacterial adherence was not clearly found in this study. As already discussed previously  a high padhesion ,66, whicsAB.The much higher Methacryl-Irga concentration of material C might have overcompensated this effect which explains the increased γThe present study is certainly limitated by the fact that the antibacterial effects needs not only to be investigated in-vitro but also in-vivo. Furthermore, other antibacterial substance or combinations thereof should be included in the delivery system. To test combinations of the present antibacterial monomers or delivery systems with the bacteria-repellent formulations of previous investigations ,5 would Experimental dental resin composites modified with small quantities of a novel antibacterially doped delivery system or with an antibacterial monomer were described that provided acceptable physical properties and good antibacterial effectiveness. The sorption material being part of the delivery system can be used as a vehicle for any other, perhaps even a more effective, active agent. Based on the results of the study the null hypothesis has to be rejected in total.


Porphyromonas gingivalis (P.g.) LPS. The expression of Toll-like receptor 2 (TLR2), TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to  Porphyromonas gingivalis (P.g.) is the major periodontopathic bacteria [P.g. LPS) is thought to induce periodontitis through Toll-like receptors, TLR2 or TLR4 [P.g. LPS-induced oral innate immune responses through TLRs during chronic periodontitis [α, IL-1β, and expressing inducible NO synthase (iNOS). In contrast, M2 macrophages promote anti-inflammation and wound healing by secreting anti-inflammatory mediators, including IL-10 and TGF-β1, and upregulating arginase 1 (Arg 1) [Periodontitis is the most common adult chronic inflammatory disorder, which results in a consequence of the persistent systemic inflammatory responses , 2. Porpbacteria , 4, and  or TLR4 . As the dontitis , 6. Macrdontitis . M1 macr (Arg 1) , 9. In a (Arg 1) , periodo (Arg 1) –15. HoweBesides the physical role as the cerebrospinal fluid-blood barrier , 17, thePropolis is a resinous substance produced by honeybees as a defense against intruders. It has relevant therapeutic properties that have been used since ancient times. The chemical composition of propolis depends on the local flora at the site of collection , 29. ConP.g. LPS stimulation. The mean amounts of TNF-α and IL-1β secreted by leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean amounts of TNF-α and IL-1β secreted by microglia after treatment with the conditioned medium from P.g. LPS-treated leptomeningeal cells were significantly higher than those after treatment with P.g. LPS alone. These observations suggest that leptomeninges transduce inflammatory signals from peripheral macrophages to brain-resident microglia by secreting inflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1β production by leptomeningeal cells through inhibiting the nuclear factor-κB (NF-κB) signaling pathway. Together with our recent findings of direct inhibitory effects on the microglial inflammatory responses, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.In the present study, we have attempted to examine possible roles of leptomeninges in transducing inflammatory signals from peripheral macrophages to brain-resident microglia in response to P.g. LPS were purchased from InvivoGen . Propolis was purchased from Yamada Bee Farm Corporation , function blocking antibodies to TLR2, TLR4 and isotype control antibodies were purchased from eBioscience . Bay 11-7082, a specific NF-κB inhibitor, was purchased from Sigma-Aldrich . Antibodies of mouse anti-TLR2 (T2.5), mouse anti-TLR4 (HTA-125) were purchased from eBioscience . Mouse anti-phospho-IκBα, rabbit anti-IκBα, goat anti-TNF-α, and rabbit anti-actin were purchased from Santa Cruz Biotechnology . Mouse anti-iNOS (4E5) was purchased from Abcam . Rabbit anti-ionized calcium binding adaptor molecule (Iba1) was purchased from Wako Chem Industries Ltd .The gingival samples were obtained from patients undergoing periodontal surgery or extraction. The periodontal diagnosis of subjects with chronic periodontitis was established based on clinical and radiographic criteria defined by the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions . The samμm) were subjected to the immunohistochemical analyses [Gingival samples were immersed in the periodate lysine paraformaldehyde (PLP) fixative consisting of 0.01 M sodium metaperiodate, 0.075 M L-lysine-HCl, 4% paraformaldehyde, and 0.03% phosphate buffer (pH 6.2) for 6 h at 4°C. The specimens were cryoprotected 2 days in 30% sucrose in phosphate-buffered saline and then were embedded in an optimal cutting temperature compound . Serial coronal frozen sections , and mouse monoclonal anti-iNOS  antibodies were mixed with rabbit polyclonal anti-Iba1 (1 : 500) antibody. After washing with PBS, the sections were incubated with a mixture of FITC-conjugated and rhodamine-conjugated secondary antibodies for 2 h at 24°C. After washing, the sections were mounted in the antifading medium Vectashield (Vector Laboratory) and examined by a confocal laser scanning microscope (CLSM) LSM510MET . CLSM images of individual sections were taken as a stack at 1 μm step size along z-direction with 20 × objectives , zoom factor 1.0. A rectangle (1024 × 1024 pixels) corresponding to the size of 450 × 450 μm was used as the counting frame. CLSM images were shown as the middle of the stacked images.The sections were hydrated and treated with 10% donkey serum for 1 h at 24°C and then were incubated with each primary antibody overnight at 4°C. The primary mouse monoclonal anti-TLR2 , mouse monoclonal anti-TLR4 , goat polyclonal anti-TNF-μg/mL). RAW264.7 cells which were purchased from the ATCC were cultured in Minimum Essential Medium Alpha  supplemented with 10% FBS, penicillin G (40 U/mL), and streptomycin (50 μg/mL); those cells were cultured at 37°C in a humidified atmosphere with 5% CO2.THP-1 cells which were purchased from the ATCC  were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum , 0.05 mM 2-mercaptoethanol, penicillin G (40 U/mL), and streptomycin (50 2) and incubated in Dulbecco's Modified Eagle's medium  containing 10% FBS, penicillin G (40 U/mL), and streptomycin (50 μg/mL) at 37°C in a humidified atmosphere with 5% CO2 for 7 days. At this time, any contaminated cells such as neuronal and glial cells were removed by shaking and were washed twice with Ca2+/Mg2+-free sterile isotonic buffer, pH 7.0, which consisted of 137 mM NaCl, 5 mM KCl, 0.7 mM KH2PO4, 25 mM glucose, 59 mM sucrose, 0.3% bovine serum albumin, penicillin (40 U/mL), and streptomycin (50 μg/mL). The purity of the leptomeningeal cells was more than 96% as determined by the immunostaining of fibronectin [Leptomeningeal cells were prepared from the brain of 3-day-old C57black/6N mouse. Dissected leptomeningeal tissues were plated on poly-D-lysine-coated culture dishes  [The c-myc-immortalized mouse microglial cell line, MG6 , was maintained in DMEM containing 10% FBS supplemented with 100 kes, NJ) , 37.P.g. LPS (1 μg/mL) for 24 h, leptomeningeal cells were incubated with the conditioned medium from P.g. LPS- or P.g. LPS-treated RAW264.7 cells (MCM) for 4 h, and MG6 were incubated with the conditioned medium from P.g. LPS- or P.g. LPS-treated leptomeningeal cells (LCM) for 24 h. The mRNA isolated from P.g. LPS-treated or nontreated cells were subjected to real-time quantitative RT-PCR. The total RNA was extracted with the Purelink RNA microkit  according to the manufacturer's instructions. A total of 800 ng of extracted RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA Master Mix . The thermal cycling was held at 50°C for 2 min, and then at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The cDNA was amplified in duplicate using TaqMan Universal PCR Master Mix  with an Applied Biosystems 7500/7500 Fast Real-Time PCR System. The data were evaluated using the 7500 software program . The primer sequences used were as follows: iNOS: 5′-GCC ACC AAC AAT GGC AAC A-3′ and 5′-CGT ACC GGA TGA GCT GTG AAT T-3′; Arginase-1: 5′-CGC CTT TCT CAA AAG GAC AG-3′ and 5′-CCA GCT CTT CAT TGG CTT TC-3′; TNF-α: 5′-ATG GCC TCC CTC TCA GTT C-3′and 5′-TTG GTG GTT TGC TAC GAC GTG-3′; IL-1β: 5′-CAA CCA ACA AGT GAT ATT CTC CAT G-3′ and 5′-GAT CCA CAC TCT CCA GCT GCA-3′; IL-10: 5′-ATG CTG CCT GCT CTT ACT GAC TG-3′ and 5′-CCC AAG TAA CCC TTA AAG TCC TGC-3′. For data normalization, an endogenous control (actin) was assessed to control for the cDNA input, and the relative units were calculated by a comparative Ct method. All real-time RT-PCR experiments were repeated three times, and the results are presented as the means of the ratios ± SEM.THP-1 and RAW264.7 cells were treated with P.g. LPS (1 μg/mL), leptomeningeal cells were treated with P.g. LPS (100 ng/mL), and the condition medium was collected at 6 h, 24 h, 48 h, and 72 h after P.g. LPS treatment. RAW264.7 were incubated with propolis (15 μg/mL) 1 h before P.g. LPS treatment, and the condition medium was collected at 48 h after treatment. The leptomeningeal cells were incubated with propolis (10 μg/mL) 1 h before P.g. LPS treatment, and the condition medium was collected at 6 h after treatment. In the separated experiments, RAW264.7, leptomeningeal cells, and MG6 were treated with TLR2 (10 μg/mL), TLR4 (10 μg/mL) antibodies or the control antibodies or Bay 11-7082 (20 μM) 1 h before P.g. LPS treatment. The condition medium was collected at the time points after the reagents treatment. TNF-α and IL-1β released from THP-1, RAW264.7, leptomeningeal cells, and MG6 were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) following the protocol provided by the manufacturer. The absorbency at 450 nm was measured using a microplate reader.THP-1 and RAW264.7 cells were treated with 3 cells/well) then incubated with various concentrations of propolis for 48 h. Cell viability was assessed using the Cell-Counting Kit-8 (CCK-8)  according to the manufacturer's instructions. Briefly, after propolis treatment, 10 μL CCK-8 was added to each well and incubated at 37°C for 2 h. The optical density was read at a wavelength of 450 nm with a microplate reader. Cell viability was calculated using the following formula: optical density of treated group/control group × 100%.RAW264.7 and leptomeningeal cells were seeded in 96-well plates for 24 h  treatment with or without propolis . The samples were electrophoresed in 12% SDS-polyacrylamide gels, and the proteins on SDS gels were transferred electrophoretically to nitrocellulose membranes. Following the blocking, the membranes were incubated at 4°C overnight under gentle agitation with each primary antibody: rabbit anti-IκBα (1 : 1000), mouse anti-pIκBα (1 : 1000) antibodies. After washing, the membranes were incubated with horseradish peroxidase (HRP-) labeled anti-rabbit  or anti-mouse  antibodies for 2 h at 24°C, then the protein bands were detected by an enhanced chemiluminescence detection system  using an image analyzer .RAW264.7 and leptomeningeal cells were cultured at a density of 5 × 10P < 0.05 was considered to indicate statistical significance .The data are represented as the means ± SEM. The statistical analyses were performed using a one-way or two-way analysis of variance (ANOVA) with a post hoc Tukey's test using the GraphPad Prism software package. A value of P.g. LPS through TLR2 and TLR4 [α, and iNOS corresponded well with those for Iba1 . We further determined the macrophage phenotypes after treatment with P.g. LPS using THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. In comparison to the nontreated cells, both the mean mRNA expression levels of TNF-α and iNOS were significantly increased in THP-1 cells after treatment with P.g. LPS (1 μg/mL). However, mean mRNA expression levels of IL-10 and Arg 1 were not significantly increased after treatment with P.g. LPS . The mean mRNA expression levels of TNF-α and IL-1β were significantly increased after 24 h treatment with LCM in comparison to those observed after treatment with P.g. LPS alone  in comparison to that by nontreated microglia. The amount of IL-1β secreted from microglia also reached peak at 48 h after treatment with P.g. LPS (data not shown). The P.g. LPS-induced secretion of TNF-α by microglia was significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody (P.g. LPS-induced leptomeningeal cells through TLR2.We have previously demonstrated that the leptomeninges are involved in the cytokine production by glial cells during chronic systemic inflammation –20. In oPS alone . We furtAD brain . The meaantibody . HoweverP.g. LPS-induced proinflammatory mediators by macrophages and leptomeningeal cells were examined, because propolis has antioxidative and anti-inflammatory effects [μg/mL on RAW264.7 macrophages (μg/mL on leptomeningeal cells after treatment with propolis (μg/mL on RAW264.7 and 10 μg/mL on leptomeningeal cells, respectively, for the following experiments. In comparison to the nontreated cells, pretreatment with propolis significantly inhibited TNF-α secretion by macrophages (P.g. LPS. The effects of propolis on the P.g. LPS-induced NF-κB activation were then examined, because NF-κB regulates the expression of proinflammatory mediators, including TNF-α and IL-1β. The expression of IκBα phosphorylation was significantly increased from 30 min in both RAW264.7 and leptomeningeal cells after treatment with P.g. LPS. Pre-treatment with propolis significantly inhibited the P.g. LPS-induced phosphorylation of IκBα in RAW264.7 cells (P.g. LPS-induced proinflammatory responses by inhibiting the NF-κB signaling pathway in both peripheral macrophages and leptomeningeal cells.Finally, the effects of propolis on the secretion of  effects , 32. Therophages  and the propolis . Thereforophages  and leptrophages  after tr.7 cells  and lept.7 cells . These oP.g. LPS-induced inflammatory signals from peripheral macrophages to brain-resident microglia, resulting in the induction of neuroinflammation. Furthermore, propolis was found to attenuate the secretion of P.g. LPS-induced proinflammatory mediators by leptomeningeal cells. To date, this is the first report to highlight that the leptomeninges serve as an important route for transducing peripheral inflammatory signals to the CNS during chronic periodontitis.The major findings of the present study are that leptomeninges transduce P.g. LPS-induced oral innate immune responses through TLRs during periodontitis [α, and iNOS in the gingival tissues of chronic periodontitis patients. However, proinflammatory M1 macrophages are not limited to the infected gingiva but also increased in the circulation during chronic periodontitis [P.g. LPS stimulation significantly increased the mean levels of mRNA expression of TNF-α and iNOS, but not those of IL-10 and Arg1, suggesting that macrophages are polarized to M1 phenotype in response to P.g. LPS during chronic periodontitis. Furthermore, the mean amounts of P.g. LPS-induced TNF-α and IL-1β secreted by macrophages were significantly suppressed by anti-TLR2 antibody, but not by anti-TLR4 antibody, further indicating that macrophages respond to P.g. LPS mainly through TLR2 [As the main population in inflammatory oral mucosa, macrophages phenotypes are known to determine dontitis . In the dontitis , 40. Theugh TLR2 , 41, 42,ugh TLR2 , 42.α and IL-1β in the circulation during periodontitis [α and IL1-β are associated not only with the cognitive decline but also with the progression of AD [P.g. LPS stimulates both THP-1 and RAW264.7 macrophages to secrete TNF-α and IL-1β. Furthermore, the mean mRNA expression levels of TNF-α and IL-1β in leptomenigeal cells were significantly increased as early as 4 h after treatment with MCM. These observations indicate that leptomenigeal cells could respond to proinflammatory mediators secreted from M1 macrophages during chronic periodontitis. Furthermore, P.g. LPS also stimulates leptomeningeal cells to produce TNF-α and IL-1β, which is consistent with previous studies using E coli LPS [α and IL-1β from 6 h after treatment with P.g. LPS. Considering the inhibitory effect of TLR2 antibody on P.g. LPS-induced production of proinflammatory mediators, leptomeningeal cells could respond to P.g. LPS more sensitively than peripheral macrophages through TLR2, but not through TLR4, even though TLR4 is also expressed in meningeal cells [Systemic inflammation and infections could worsen a number of CNS disorders , 43. Amodontitis , 40 alsodontitis –12. Theron of AD –47. Receon of AD –20. In tcoli LPS , 19 and coli LPS –50. Impoal cells , 23.α and IL-1β in an autocrine manner [α and IL-1β in microglia, suggesting that proinflammatory mediators secreted from P.g. LPS-treated leptomenigeal cells could subsequently activate microglia to generate neuroiflammation during chronic periodontitis. Furthermore, our present observations demonstrate that the mean levels of TNF-α and IL-1β secreted by microglia were significantly increased after treatment with P.g. LPS alone. Furthermore, P.g. LPS-induced secretion of proinflammatory mediators by microglia was significantly suppressed by anti-TLR2 antibody. These observations support the recent idea that P.g. LPS may be involved in the progression of AD [P.g. LPS is only one of the related factors of chronic periodontitis.Microglia are well-known key players of neuroinflammation , 27, whie manner , 52. Theon of AD . Furtheron of AD . Therefoon of AD , 55. Morα and IL-1β production by both RAW264.7 cells and leptomenigeal cells through inhibiting the NF-κB signaling pathway, because NF-κB is a critical transcription factor that encodes genes of proinflammatory mediators, including TNF-α and IL-1β [κB-dependent production of proinflammatory mediators by microglia. Recently, we have reported that the exaggerated neuroinflammatory responses evoked by microglia are responsible for an impairment of the hippocampal long-term potentiation in the middle-aged animals subjected to adjuvant arthritis [P.g. LPS-induced NF-κB-dependent proinflammatory pathway of leptomeningeal cells and microglia may prevent the age-dependent exaggerated neuroinflammatory responses in the CNS.Neuroprotective drug therapies have not yet translated well from the lab to the clinic because of an excessive focus of treatments on promoting the survival of neurons, with far less work on nonneuronal brain cells. Recently, leptomeninges have been focused on delivering compounds/genes to brain , 57. Thend IL-1β . These frthritis . TherefoP.g. LPS-induced inflammatory signals from peripheral macrophages, leptomeningeal cells and microglia during chronic periodontitis. Further investigations are necessary to clarify the involvement of other factors in neuroinflammation during chronic periodontitis.In conclusion, our present findings strongly suggest that the leptomeninges serve as an important route for transducing inflammatory signals from peripheral macrophages into brain-resident microglia by secreting proinflammatory mediators during chronic periodontitis. Propolis may benefit for preventing and reducing neuroinflammation in CNS disorders, including AD, by attenuating