Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols. TM, for clinical use in 1985 marked a new era in the therapeutic sector, opening the door for novel cancer, autoimmune, and viral disease treatments [The FDA approval of the first monoclonal antibody, Orthoclone OKT3eatments ,3. With eatments . Howevereatments ,7, monobeatments . Single-eatments ,12. Theseatments , are dereatments . Nanobodeatments . They exeatments ,17, thereatments , and foreatments . Recentleatments ,22. Moreeatments  or chromeatments .Conventionally, nanobodies are selected from immune phage libraries ,26,27. UHere, we present an alternative method for the generation of VHH that involves the grafting of the complementarity determining regions (CDRs) from already existing, non-camelid antibodies to VHH frameworks, followed by affinity maturation using synthetic phage libraries. To date, myriad antibodies and fragments thereof have been generated against manifold antigens. This existing assortment represents an ideal CDR donor repertoire for our approach. CDR grafting is a powerful technique for transferring binding specificities to other antibody frameworks with desired properties. This is typically done to stabilize or humanize antibodies intended for medical use ,42,43.In the context of nanobodies, universal scaffolds have been identified, enabling the generation of robust or humanized VHH variants ,45,46. SHere, we propose the grafting of CDRs from pre-existing antibodies of non-camelid origin to VHH frameworks as the basis for the generation of nanobodies . Since tIn this study, we evaluated a two-step design approach for the generation of VHH involving the grafting of CDR from non-camelid donors to VHH frameworks, followed by affinity maturation using a synthetic phage library. As model antigens, we chose the Botulinum neurotoxin A (BoNT/A) and the small molecule fluorescein. Antibodies and antibody fragments against both targets have been generated, and we used the VH domains of scFvs binding to the light chain of BoNT  or to fE. coli, purified via affinity chromatography (−1 for the BoNT/A-LC and 100 µg mL−1 for the fluorescein binder), indicating weak VHH–antigen interactions. Nevertheless, we hypothesized that, similar to immune libraries, sequence randomization of weak binders might enrich binders with higher affinity, mimicking an in vivo germinal center reaction. We tested this hypothesis by designing a synthetic VHH library for affinity maturation via phage display. To the best of our knowledge, synthetic libraries have been successfully used for the selection of protein binders [The grafted VHH were produced in tography , and thetography B. Notewo binders , but notWe designed our synthetic VHH library mainly focusing on CDR3, which is usually the most diverse and important antigen-binding loop in VHH. Additionally, we randomized residues in CDR2. We systematically compared the corresponding amino acids with sequence logos  generateResidue 58 (residue 69 according to the AHo numbering scheme) shows the highest diversity in CDR2, however, the sequence logo did not comprise a histidine at this position. Therefore, we used the NNC-codon at this position providing 16 amino acids . We used the same codon at position 100i (135 according to AHo). Furthermore, we avoided stop codons in our sequence. We applied a two-step panning approach for the selection of fluorescein-specific VHH. In the first round of panning, we enriched the library for binders using immobilized fluorescein-conjugated BSA and by elution with trypsin. In the second round, we first eluted competitively with 10 µM, then with 1 mM fluorescein and obtained a 100- and 75-fold enrichment of phages eluted from selection versus BSA-control wells, respectively. We screened for binders by ELISA and identified a hit in constructs eluted with 1 mM fluorescein  [Similar to naïve libraries, these libraries are not pre-enriched for antigen-binders and thus require large sizes (10 clones) ,39,65. F clones) . Thus, V clones) .In the case of scFv, it has been shown that different types and sizes of antigens  require different topographies of the paratope . PerssonThe isolated VHH-D4 clone bound specifically and with nanomolar affinity to fluorescein and thus supported the convenience of our two-step approach for generating functional VHH, even against more difficult targets such as small molecules.We believe that this approach complements immune, naïve, and synthetic libraries for the generation of protein as well as hapten binders. Conveniently, the number of existing antibodies provides a huge repository of CDRs for the design of focused synthetic VHH libraries. Such methods that facilitate the development of VHH with the desired specificities and frameworks represent valuable approaches to advance nanobody-based research and the medical potential of VHH-based therapeutics.E. coli codon usage for bacterial protein production. The VHH-Flu graft was ordered as gBlocks Gene Fragments . The VHH-BoNT/A graft was constructed by assembly PCR. Details on plasmid cloning are summarized in The designed VHH amino acid sequences were converted to nucleotide sequences with E. coli BL21(DE3) SHuffle T7 Express . Strep-tag-BoNT/A-LC was produced in E. coli BL21(DE3) pLysS . Cells were grown in LB medium, supplemented with 100 µg/mL ampicillin, and in the case of Strep-tag-BoNT/A-LC, additionally with 36 µg/mL chloramphenicol, in shake flasks at 37 °C and 150 rpm. At OD600 = 0.6–0.8, protein production was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for 4 h (VHH) or 25 °C overnight (Strep-tag-BoNT/A-LC). Bacteria were harvested at 6000× g for 10 min and resuspended in 35 mL Ni Lysis buffer . The cells were disrupted by sonication at 60% amplitude and 0.5 sec/1 sec pulse/pause intervals for 10 min on ice  and centrifuged at 30,000× g for 30 min at 4 °C. Soluble VHH in the supernatant was either purified via Ni-NTA  or protein A  affinity chromatography. The cleared lysates were applied to gravity-flow columns (2 mL bed volume), equilibrated with Ni Lysis buffer. The columns were washed twice with Ni wash buffer  or once with 15 mL PBS (protein A). Proteins on the Ni-NTA material were eluted with 8 mL Ni elution buffer . Protein A-bound VHH was eluted with 0.1 M glycine, pH 3.0. The pH was neutralized with 1 M Tris-HCl, pH 8.0. Strep-tag-BoNT/A-LC was purified via Strep-Tactin®XT  using gravity flow columns (1 mL bed volume) and equilibrated with Strep-buffer . After washing with 5 mL Strep-buffer, the protein was eluted with 3 mL Strep-buffer supplemented with 50 mM biotin.VHH were produced in 10 mg/mL bovine serum albumin  was incubated with a 20-fold molar excess of fluorescein isothiocyanate  at RT for 2 h. Uncoupled FITC was removed by gel filtration using a dextran desalting column , followed by dialysis in a SnakeSkin dialysis tubing  against PBS.w/v) SDS-gels, followed by Coomassie staining.Protein concentrations were determined by Bradford assay  using a dilution series of BSA as a protein standard. For evaluating protein production and purification, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted using 12 or 15% (v/v) Tween-20 (PBST) and blocked with 300 µL PBST supplemented with 1% (w/v) BSA (blocking buffer) for 1 h at RT. After three washing steps with PBST, a 100 µL sample was added and incubated for 1 h at RT. The wells were washed three times and probed with 100 µL primary anti-His antibody  for 1 h at RT. After washing three times, 100 µL secondary anti-mouse IgG-HRP  was added. After 1 h incubation at RT, the wells were washed three times and bound VHH was detected by addition of 100 µL 0.5 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)  in 50 mM citric acid pH 4.0 supplemented with 0.05% (v/v) H2O2. The increase in absorbance at 405 nm was measured in a microplate spectrophotometer .Antigen-binding was analyzed by ELISA. ELISA plates  were coated with 1 µg Flu-BSA, 5 µg BoNT/A-LC, or 1 µg BSA (negative control) per well at RT overnight. The wells were washed three times with 300 µL PBS supplemented with 0.05% . The assay temperature was set to 30 °C. Aminopropylsilane (APS) biosensors were equilibrated with PBS and loaded with Flu-BSA or BSA (10 µg/mL in PBS) for 300 s. After blocking with assay buffer (PBS supplemented with 1% (w/v) BSA) , association of the VHH (1:1 dilution series 0–600 nM in assay buffer) was performed for 300 s, followed by dissociation in assay buffer for 300 s. Control experiments with BSA-coated biosensors showed no association with the highest applied VHH concentration. Assays conducted in the absence of VHH on Flu-BSA-coated biosensors served as reference and were subtracted from the data. Association and dissociation curves were fitted globally according to a 1:1 bimolecular interaction model (On- and off-rates of the fluorescein-specific VHH were determined by bio-layer interferometry using the Octeton model B. Based NcoI/NotI), purified via the High Pure PCR Cleanup Micro kit , followed by transformation of E. coli XL-1 blue  using an electroporator  with Gene Pulser® electroporation cuvettes  at 2.5 kV for 5 ms. Transformed bacteria were grown on LB agar supplemented with 1% (w/v) glucose and 100 µg/mL ampicillin. A library size of 1.05 × 107 clones was estimated by plating dilutions of each transformation. Phages were produced by inoculating 50 mL 2xYT medium supplemented with 2% (w/v) glucose and 100 µg/mL ampicillin to an OD600 = 0.1. Bacteria were grown at 37 °C and 150 rpm. At OD600 = 0.5, the cells were incubated with 2.5 × 1011 KM13 helper phages for 30 min at 37 °C without shaking. The medium was exchanged by centrifugation at 3000× g for 30 min and resuspending the cell pellet in 500 mL 2xYT supplemented with 100 µg/mL ampicillin and 50 µg/mL kanamycin. Phages were produced at 30 °C and 150 rpm overnight. The phage-displayed VHH library was prepared by pelleting the bacteria at 10,000× g and 4 °C for 30 min, mixing 400 mL supernatant with 100 mL phage preparation solution (20% (w/v) PEG-8000, 2.5 M NaCl) and incubation on ice for 1 h. Phage particles were spun down at 10,000× g and 4 °C for 30 min and resuspended in 8 mL PBS. After addition of 2 mL phage preparation solution, the phage suspension was incubated for 30 min on ice and spun down at 10,000× g and 4 °C for 30 min. The pellet was resuspended in 4 mL PBS, supplemented with 15% (v/v) glycerol. Remaining bacterial debris was removed by centrifugation at 10,000× g and 4 °C for 10 min. The obtained phage titer was 9 × 109 cfu/µL.The synthetic Flu-VHH library was designed by randomizing CDR2 and CDR3 as described in the main text see . The lib12/mL phages were incubated with 0.2% (w/v) BSA for 30 min at RT with rotation. Nunc Maxisorp 96-well plates  were coated with 1 µg/well Flu-BSA or BSA (negative control), blocked with blocking buffer  Tween-20 and 1% (w/v) BSA), washed with TBST  Tween-20) and incubated with 100 µL phage suspension for 2 h at RT with agitation. Wells were washed 15 times with TBST. In the first round of panning, phages were eluted by incubation with 100 µL trypsin (0.25 mg/mL) for 30 min. Trypsin was then inactivated with 0.1 mg/mL 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride . In the second round of panning, phages were competitively eluted, first with 10 µM fluorescein for 30 min, then with 1 mM fluorescein for 1 h. E. coli TG1 cells were infected with eluted phages and grown in LB, supplemented with 100 µg/mL ampicillin and 2% (w/v) glucose at 37 °C and 150 rpm overnight. To estimate the enrichment of phages eluted from the selection wells versus control wells (BSA coating), the phages were titrated. For this, E. coli TG1 cells  were infected with dilutions of phages and the number of colonies grown on LB agar supplemented with 100 µg/mL ampicillin and 1 % (w/v) glucose were determined. The enrichment factor was calculated by dividing the phage titer obtained from the selection wells by the titer obtained from control wells.For phage panning, 10w/v) glucose, and 10% (v/v) glycerol at 37 °C overnight. 4 µL of the overnight cultures were transferred to fresh 2xYT medium, supplemented with 100 µg/mL ampicillin, 0.1% (w/v) glucose, and incubated at 37 °C with and 200 rpm. After 2 h, 1 mM IPTG was added and the cultures were incubated for additional 5 h at 37 °C and 200 rpm. The cells were pelleted at 3000× g for 10 min and the supernatants were removed. Pellets were frozen at −20 °C overnight, thawed and resuspended in PBS. The suspension was centrifuged at 3000× g for 10 min and 1:2 dilutions of the supernatants were tested by ELISA.ELISA was conducted to screen for fluorescein-binding VHH. For this, single colonies of phage-infected cells were picked and grown in a 96-well plate with 2xYT, supplemented with 100 µg/mL ampicillin, 2% (


The identity of ZnFLC were confirmed by elemental analysis, IR spectrum, and single-crystal X-ray diffraction. The antifungal activities of ZnFLC was screened in 19 Candida species strains using the microdilution checkerboard technique. The minimum inhibitory concentration (MIC80) value of ZnFLC is 4 μg/mL on the azole-resistant clinical isolates of C. albicans HL973, which is lower than the positive control, FLC. The mechanism of ZnFLC against C. albicans HL973 showed that ZnFLC damaged the fungal cell membrane and reduced the ergosterol content. The expression of ERG1, ERG7, ERG11 ERG27, and ERG28, which have effects on the synthesis of ergosterol, were all significantly upregulated by ZnFLC.The rise in the number of fungal infections is requiring the rapid development of novel antifungal agents. A new polyoxovanadate functionalized by Zn-fluconazole coordination complexes, Zn Candida species including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei are posing serious nosocomial threats to patient populations 6− anions, [Zn3(FLC)6]6+ and the isolated H2O molecules  1.597(5)–1.702(4) Å, V-O12  2.086(4)–2.325(4) Å, and V-Ob (bridge) 1.687(4)–2.029(4) Å. The bond valence sum calculations give the values of all vanadium sites are in the 5+ oxidation state. As shown in 2O3]. The Zn(2) cation is six-coordinated by six nitrogen atoms from different FLC ligand with Zn(2) − N(10) = 2.129(6) Å, Zn(2) − N(10)#2 = 2.129(6) Å, Zn(2) − N(7)#2 = 2.220(6) Å, Zn(2) − N(7) = 2.220(6) Å, Zn(2) − N(6) = 2.277(6) Å, and Zn(2) − N(6)#2 = 2.277(6) Å, respectively  and ν(V-Obridge) of polyoxoanion, shown in −1 are assigned to the water molecules.The IR spectrum of ZnFLC has the characteristic asymmetric stretching vibration peaks at 956, 918, 815, 743, 670, 651, 631, 597, 515, and 456 cmCandida spp. As shown in 50 and MIC80 values of 0.5–64 μg/mL and 1–128 μg/mL were slightly higher than those of FLC. Because the FCZ-resistant clinic isolate HL973 was sensitive to ZnFLC, further studies were concentrated on the antifungal activities and the mechanism of ZnFLC against C. albicans HL973.The MICs of ZnFLC and FLC were evaluated for standard and clinical strains of Candida species HL973, HL27, HL981, HL946, HL963, HL981, HL996, ATCC 750, ATCC 22019, and ATCC 90028 strains were treated with equivalent doses of Zn(oac)2, NaVO3 and FLC in ZnFLC (wt/wt %) at the MIC80 (4 μg/mL). As shown in 2 and NaVO3 had similar growth rates, indicating that Zn(oac)2 and NaVO3 had minimal effects. The FLC had inhibitory efficacy but lower than that of ZnFLC. The result indicated that the antifungal activities of ZnFLC are not from the simple mixture of metal cations and the FLC.C. albicans HL973 cells in the presence of various concentrations of ZnFLC and FLC was evidenced once more by MTS method. As shown in C. albicans HL973 cells of ZnFLC have a significant reduction than that in the negative control group (p < 0.05). The inhibition ratio of ZnFLC-treated C. albicans HL973 cells at 48 h with the concentrations of 4, 8, 16, 32, 64, and 128 μg/mL of ZnFLC reached the peak value 77.44, 89.43, 95.71, 96.10, 96.57, 97.64, and 97.89%, respectively. The antifungal activities of ZnFLC are dose-dependent. The result is similar to that from the broth microdilution method.The inhibitory of the C. albicans HL973 at different times are given in p < 0.05).The growth inhibition effect in the presence of various concentrations of ZnFLC, negative control, and FLC groups on the C. albicans HL973 were plated on 6-well plates at a density of 1.0 × 106 cells/mL. After a 24 h or 48 h in period of incubation, the fluorescence microphotographs of HL973 cells treated with (0.5%) DMSO, ZnFLC, and FLC for 24 h and stained with AO/EB were shown in Candida cells in applied concentrations (up to 0.5%) did not cause any adverse effects. In the presence of ZnFLC or FLC, large EB-positive areas (red staining) were evident, whereas the yeast cells were AO positive (green), indicating they were viable. ZnFLC was more effective at killing HL973 yeast cells than that of FLC.The C. albicans HL973, the ergosterol contents, one kind of critical components in C. albicans, were determined by HPLC. The results showed that the retention time of ergosterol was about 12.90 min (p < 0.05) (R2 = 0.995). The treatment of C. albicans HL973 with ZnFLC and FLC resulted in a reduction of 92.02% and 42.53% of ergosterol content (p < 0.05), shown in C. albicans HL973 had the similar was through inhibition of ergosterol biosynthesis.In order to explore the reason of ZnFLC inhibiting the 2.90 min . After 2 < 0.05) . The staC. albicans HL973 cells were exposed to ZnFLC at 8 μg/mL values for 24 h, their total RNA was extracted, and cDNA was synthesized by reverse transcription. This cDNA was then used as a template for a series of real-time PCRs. The results showed that the expression of ERG1, ERG7, ERG11, ERG27, and ERG28 were significantly upregulated with the fold change relative to control of 18.11 ± 0.96, 11.19 ± 0.47, 14.39 ± 3.06, 8.07 ± 1.19, and 9.19 ± 0.28, respectively, as shown in C. albicans treated with azole  assay was performed according to manufacturer’s instructions . The cells were incubated in the dark for another 25 min at 37 °C. Then, using a multichannel pipette remove 80 μL of the resulting colored supernatant from each well and transfer into the corresponding wells of a new microtiter plate. Measured microplate absorbance at a wavelength of 490 nm on a microplate reader .The antifungal activity of ZnFLC on the terature . BrieflyC. albicans HL973 was prepared in YPD (yeast peptone dextrose) liquid medium overnight at 35 °C. C. albicans HL973 was diluted at the starting inoculum of 1 × 106 cells/mL in glass tubes. Different concentrations of the compound  and FLC  were added into tubes. At predetermined time points  after incubation in an orbital shaker (about 180 rpm) at 35 °C, a 100 μL aliquot was removed from every solution and was at 600 nm with microtiter plate reader (Thermo LabSystems Multiskan MK3), and background optical densities were subtracted from that of each well.Rejuvenation of C. albicans HL973 cells with acridine orange (AO) and ethidium bromide (EB) was done as described previously with some modifications. Fungal strains were seeded at a density of 1.0 × 106 cells/mL in 6-well plates. Then cells were treated with 64 μg/mL of ZnFLC and equivalent dose of FLC (58 wt/wt %) in ZnFLC for 24 h and 48 h at 37 °C. The cells were rinsed with PBS and stained with AO (100 μg/mL) and EB (10 μg/mL) for 1 min at room temperature in the dark. The cells was observed using the fluorescence microscopy .living/dead staining of C.albicans HL973 were treated with 16 μg/mL of ZnFLC and equivalent dose of FLC in ZnFLC (58 wt %) at 35 °C for 24 h. The cells were centrifuged and washed with PBS. A 0.5 g wet weight of cell mixed with PBS and fresh saponifier was saponified at 80 °C for 1 h and extracted by petroleum ether. Then the extract was volatilized to dryness at 60 °C. The dry residues were dissolved by 0.5 mL methanol and filtered through 0.45 μm micro membrane. Quantification of ergosterol in samples with or without the drugs was determined by comparing peak areas of samples to a standard curve generated from HPLC-grade ergosterol. A standard curve of HPLC-grade ergosterol consisted of 0.001, 0.004, 0.015, 0.0625, and 0.25 mg/mL. Ergosterol contents were analyzed using LC-20AB prominence Liquid Chromatograph  including Shimadzu C18 column . Eluent was methanol/water . Flow rate was 1 mL/min. Temperature was 35 °C. A SPD-20AV prominence UV–vis detector (Shimadzu) was used to detect UV at 282 nm [t 282 nm .C. albicans HL973 treated with ZnFLC. Total RNA was extracted from C. albicans HL973 incubated with or without 8 μg/mL of ZnFLC and equivalent dose of FLC (58 wt/wt %) in ZnFLC for 24 h using the hot phenol method. Reverse transcription was conducted in a total volume of 20 μL with Prime script RT reagent kit. Real-time PCR reactions were performed with SYBR Green I, using qTOWER 2.0 PCR system . The primer sequences used in real-time PCR were listed in −△△Ct method.Real-Time PCR was used to measure the transcriptional expressions of the genes involved in ergosterol biosynthesis of 3(FLC)6V10O28·10H2O exhibited potent anti-candidal effect via inducing membrane disruption. Therefore, we propose that Zn3(FLC)6V10O28·10H2O is a potential candidate in the development of a novel antimicrobial agent. Future research may focus on attempting to explore the novel antimicrobial POM nano-delivery system or synthesize new POM-based compounds.In summary, a new compounds Zn


Papaver nudicaule (Papaveraceae). These glucosidic compounds belong to the small group of indole/flavonoid hybrid alkaloids. Here we describe in vivo and in vitro experiments which substantiate the strongly pH-dependent conversion of pelargonidin glucosides to nudicaulins as the final biosynthetic step of these alkaloids. Furthermore, we report the first synthesis of nudicaulin aglycon derivatives, starting with quercetin and ending up at the biomimetic fusion of a permethylated anthocyanidin with indole. A small library of nudicaulin derivatives with differently substituted indole units was prepared, and the antimicrobial, antiproliferative and cell toxicity data of the new compounds were determined. The synthetic procedure is considered suitable for preparing nudicaulin derivatives which are structurally modified in the indole and/or the polyphenolic part of the molecule and may have optimized pharmacological activities.Nudicaulins are yellow flower pigments accounting for the color of the petals of  Papaver nudicaule L. (“Iceland poppy”) petals was first reported in 1939 and named nudicaulin [The yellow pigment of dicaulin , and theP. nudicaule and that their fusion is possible without enzymatic catalysis.Biosynthetic studies ,5 revealNudicaulins can be considered flavonoidal indole alkaloids. Compared to the large class of monoterpenoidal indole alkaloids, the flavonoidal indole alkaloids are a small group with only a few known compounds, such as lotthanongine  and yureP. nudicaule cultivar was carried out to prove that the free indole is incorporated into the nudicaulin molecule. In orange petals, a mixture of red pelargonidin glucosides and yellow nudicaulins is responsible for the color. Under natural conditions, the low level of indole [An in vivo experiment using a petal of the orange f indole  probablyP. nudicaule petals at pH values 4 and 3 differed completely. At a pH value higher than or equal to 4, spectra show a strong maximum at 348 nm and a weak maximum at 510 nm. If the pH value is lower than or equal to 3, the maxima are around 450 and 337 nm. In comparison, the absorption spectrum of a petal measured under in vivo conditions has a broad maximum around 422 nm and two shoulders at 331 and 360 nm , the flavonol analogue of cyanidin, instead of the more expensive kaempferol. Cyanidin possesses an additional hydroxyl group in ring B compared to pelargonidin, the actual precursor of the nudicaulins in P. nudicaule  was permethylated with Me2SO4 under alkaline conditions in acetone/water [O-methylquercetin (4) with LiAlH4 in THF afforded 3,5,7,3′4′-penta-O-methylcyanidin (5) [5 reacted with indole and yielded a racemic mixture of permethylated nudicaulin diastereomers 6. The CD spectrum  . In the spectrum  confirmef indole .O-methylcyanidin (5) as an educt for the polyphenolic part of the nudicaulin molecule and a series of substituted indoles  as educts for the nitrogen-containing part, compounds 7 to 11 were obtained. According to the 3′,4′-dihydroxyphenyl ring of starting compound 3, the skeleton of all synthetic compounds differs from that of the native nudicaulins by an additional hydroxyl group (3′-OH). The structures were confirmed by the characteristic absorption between 460 and 480 nm, mass spectrometry, and 1D and 2D NMR spectroscopic data.Using 3,5,7,3′4′-penta-6, 10 and 11 showed the highest antiproliferative effects against HUVEC (50) values for compound 6 were 1.3 μmol·L−1 against HUVEC and 1.1 μmol·L−1 against K-562 cells, the values for compound 10 were 2.2 μmol·L−1 and 1.0 μmol·L−1 and the values for compound 11 were 2.0 μmol·L−1 and 1.0 μmol·L−1, respectively. Cytotoxicity varied between 3.4 μmol·L−1 (compound 6) and 5.7 μmol·L−1 (compound 10). Compounds 7, 8 and 9 exhibited lower antiproliferative activity against HUVEC and K-562 cells, and lower cytotoxicity against HeLa cells. The antiproliferative and cytotoxicity data from all tested compounds are shown in Compounds st HUVEC  and K-56st HUVEC  cells anst HUVEC  cells. T6, 7 and 9–11 despite the application of high concentrations of 1 mg·mL−1. Only compound 8 exhibited marginal activity against Bacillus subtilis, Staphylococcus aureus (MRSA and non-MRSA variant) as well as Mycobacterium vaccae. Detailed results are shown in No antimicrobial activity against the different bacterial and fungal strains was found for compounds P. nudicaule and Meconopsis cambrica [P. nudicaule petal cell sap revealed that the structure of the nudicaulins might also depend on pH as the spectra varied substantially as pH changed. A spectrum comparable to the one of the living flower was seen only around pH 2. This finding suggests that the vacuoles of P. nudicaule petals exhibit a strongly acidic pH value. Even though a spontaneous last step of the nudicaulin biosynthesis seems to be possible under these conditions, subcellular compartmentation studies need to be carried out, because both precursors are present simultaneously during petal development without any nudicaulins being produced [Nudicaulins are indole/flavonoid hybrid alkaloids that until now have only been known as natural products from the petals of some Papaveraceous plants such as cambrica ,17. Theicambrica ,5, but tcambrica , have toproduced ,20.P. nudicaule cultivars. Since the glucose moieties of native nudicaulins were considered nonessential for potential bioactivity, we aimed to synthesize aglycon derivatives instead of glucosides. Furthermore, to avoid the instability of the molecules caused by the hemiacetal structure, the hydroxyl group in position 11 had to be protected by methylation, as were the other hydroxyl groups of the cyanidin part of the molecule. Thus, we were successful in the first synthesis of non-glycosylated nudicaulins.In view of the activity of other indole alkaloids, the synthesis of nudicaulins appeared a promising way to obtain new compounds with interesting properties, e.g., potential antiproliferative activity. To synthesize nudicaulin derivatives, we chose a biomimetic approach, taking advantage of the spontaneous fusion of anthocyanins with the indole moiety that occurs under strongly acidic conditions. The synthetic route mimics the final step of nudicaulin biosynthesis as it occurs in the petals of the yellow and orange Optimizing the reaction conditions  is assumed to result in improved yield for the final step of the reaction. In general, our work provides easy synthetic access to flavonoidal indole alkaloids.6–11 showed high antiproliferative activity against HUVEC and K-562 cells and cytotoxic activity against HeLa cells. Compounds 6–11 possessed an activity against K-562 cells similar to doxorubicin, an established chemotherapeutic agent. The antiproliferative activity against HUVEC cells and cytotoxicity against HeLa cells was slightly lower than against K-562 cells  and visualized by UV light (254 nm).Solvents and reagents were purchased from Sigma-Aldrich, Deisenhofen, Germany and used without further purification. All reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm Merck silica gel sheets 60-Fv/v)); solvent B, methanol (MeOH); and solvent C, acidified MeOH (0.1% TFA (v/v)). The column temperature was 25 °C, the flow rate was 1 mL·min−1, and the detection wavelengths were 211, 254, 281, 351 and 460 nm. Method 1 included isocratic elution with 30% B/70% A for 20 min followed by a 35-min gradient from 30% to 65% of B with a subsequent washing step (65% to 100% solvent B in 10 min/100% solvent B for 10 min) and equilibration to starting conditions (100% to 30% solvent B in 5 min/30% solvent B for 10 min). Method 2 consisted of a linear binary gradient from 0 to 76% of solvent B in solvent A in 38 min with a subsequent washing step (76% to 100% solvent B in 2 min/ 100% solvent B for 5 min) and equilibration to starting conditions (100% to 0% solvent B in 5 min/0% solvent B for 10 min). Method 3 included a linear binary gradient from 50 to 100% of solvent C in solvent A in 35 min with a subsequent washing step (100% solvent C for 5 min) and equilibration to starting conditions (100% to 50% solvent B in 5 min/50% solvent B for 10 min).HPLC 1: The analytical Agilent 1100 HPLC system consisted of a degasser G1322A, binary pump G1312A, autosampler G1313A, and photodiode array detector (PDA) G1315B  equipped with an EC250/4 Nucleodur C18 HTec column . Solvent A was acidified water (0.1% trifluoroacetic acid (TFA) (−1. The binary solvent system consisted of acidified water (0.1% formic acid (FA) (v/v), solvent A) and acidified MeOH (0.1% FA (v/v), solvent B). Method 4 included a linear binary gradient from 70–100% B in 15 min with a subsequent washing step (100% solvent B) and fraction collection using intensity and time.HPLC 2: Preparative HPLC separations were carried out using a Shimadzu Prominence HPLC system , controlled by the Shimadzu LCSolution ver. 1.21, and consisting of a DGU-20A5 degasser, LC-20AT gradient pump, SIL-10AP autosampler, CTO-20A column oven, SPD-20A UV detector, FRC-10A fraction collector and CBM-20A system controller. A Nucleodur VP250/10 Nucleodur C18 HTec column  was used at 25 °C with a flow rate of 3.5 mL·minv/v); solvent A) and acidified methanol (0.5% TFA (v/v); solvent B). The flow rate was 30 mL·min−1. Method 5 consisted of a linear binary gradient from 0–10% B in 34 min, followed by a linear binary gradient from 10–100% in 2 min with a subsequent washing step (100% solvent B) and fraction collection using intensity.LC 3: Flash chromatography was carried out by a Biotage Isolera Spectra One  equipped with Biotage Snap-Ultra cartridges  and a PDA detector in λ-al detection mode. The binary solvent system consisted of acidified dichloromethane (0.5% TFA (1H and 100.61 MHz for 13C) equipped with 5 mm BBFO probe  or a Bruker Avance III HD 500 MHz spectrometer (operating at 500.13 MHz for 1H and 125.75 MHz for 13C) equipped with a 5 mm TCI cryoprobe. Structures were assigned with the aid of 1H NMR, 13C NMR, APT, 1H,1H-COSY, HSQC, 1H,13C-HMBC, and 1H,15N-HMBC spectra. CDCl3 (for compounds 4 and 5) or MeOH-d4/1% TFA (for nudicaulin derivatives 6–11) were used as solvents. Chemical shifts were referenced to the residual solvent peaks at δH 7.26 and δC 77.0 for CDCl3 and δH 3.31 and δC 49.15 for MeOH-d4. Data acquisition and processing were accomplished using TopSpin 3.2 . Standard Bruker pulse sequences as implemented in Bruker TopSpin were used for data acquisition.NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer , controlled by the Bruker Compass control suite  together with Bruker OTOFControl version 4.0 . The samples were analyzed in the positive ionization mode in the mass range UV/Vis absorption spectra were recorded with a Jasco V-550 spectrophotometer  between 200 and 700 nm. Blank sample measurements were used for baseline correction.CD spectra were recorded on a Jasco J-810 spectropolarimeter . All compounds were measured in MeOH using a quartz cuvette of 1 mm width. Spectra were recorded between 200 and 600 nm and were accumulated 5 times. Baseline correction was performed on the basis of a MeOH blank sample.Papaver nudicaule L. plants were raised from seeds of ‘Summer Breeze Yellow’ and ‘Summer Breeze Orange’  in soil  in the glasshouse facilities of the Max Planck Institute for Chemical Ecology, Jena, Germany. The glasshouse chamber was equipped with Phillips Sun-T Agro 400 Na lights to support an illumination period of 14 h. Temperatures ranged from 19–21 °C in the night and from 21–23 °C during the day with a relative air humidity of 50% to 60%. Plants were irrigated for 6 min every day.The P. nudicaule cultivar was placed in an aqueous indole solution  for 20 min until its color changed to yellow. Afterwards, the petal and an untreated control petal were extracted with methanol–water mixture 1:1  and centrifuged  for 20 min, after which the supernatant was used for HPLC analysis .In vivo: One petal of the orange −1 tissue) by Bertin Minilys Homogenizer  beads, 5000 rpm). After 10 min of ultrasound and 10 min of centrifugation , 100 µL of the supernatant was mixed with 10 µL of an aq. indole solution (1 mg·mL−1), vortexed and measured by HPLC 1 after 10 min and 70 min (method 2). Additionally, 100 µL of the extract was mixed with 1 µL of hydrochloric acid (37%), intensifying the color. The pH value decreased from 5.8 to 1.2 . Afterwards, 10 µL of aq. indole solution (1 mg·mL−1) was added, and after vortexing and 10 or 70 min of reaction time, the samples were measured by HPLC 1 (method 2).In vitro: Petals from the orange cultivar were homogenized in deionized water (10 µL mgP. nudicaule cultivar and diluted with deionized water (1:2). UV/Vis absorption spectra were recorded in a quartz cell with a thickness of 10 mm. The pH of the sample as well as the blank were adjusted with hydrochloric acid and sodium hydroxide solution (1 mol·L−1) using the pH-electrode HI1093B . To obtain a spectrum of a petal, the tissue was cut in pieces and its transparency was increased by vacuum infiltration in deionized water [pH dependence of nudicaulin UV/Vis absorption: The cell sap was pressed from the petals of the yellow ed water . AfterwaO-methylquercetin (4) was obtained from quercetin (3) by methylation with Me2SO4 in acetone/water 2:1 according to a reported procedure [4 was purified by recrystallization from CH2Cl2/EtOAc (1:6). Reduction of compound 4, using LiALH4 in dry THF under argon atmosphere as described by Kimura et al. [O-methylcyanidin (5). Analytical data of compounds 4 and 5 matched those reported in the literature [3,5,7,3′4′-Penta-rocedure . Compouna et al. , affordeterature ,22.O-methylcyanidin (1 eq.) was dissolved in a 1:1 solution of MeOH/water at room temperature. The pH of the resulting solution was set to 2.5 by adding 0.1 M HCl aq., followed by a methanolic indole solution (3.0–5.5 eq.). The reaction mixture was stirred for 2.5 h and was stopped by the evaporation of MeOH. The aq. residue was spiked with FA (4 mL) and extracted with DCM (4 × 25 mL). The combined extracts were dried over MgSO4, and the solvent was evaporated in vacuo until dryness. Purification was done by LC 3, method 5. Final purification was done by preparative HPLC 2, method 4. Evaporation of the solvent by a N2 stream and drying in vacuo yielded the desired nudicaulin. Purity was verified by HPLC 1, method 3.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and indole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 472.1755 m/z, (M + H)exp: 472.1772 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 210 nm, 472 nm.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and 5-methylindole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 486.1911 m/z, (M + H)exp: 486.1913 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 209 nm, 469 nm.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and 6-methylindole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 486.1911 m/z, (M + H)exp: 486.1929 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 206 nm, 473 nm.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and 7-methylindole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 486.1911 m/z, (M + H)exp: 486.1925 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 211 nm, 478 nm.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and 6-fluoroindole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 490.1660 m/z, (M + H)exp: 490.1668 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 212 nm, 395 nm, 478 nm.3,5,7,3′,4′-Penta-O-methylcyanidin  dissolved in MeOH/water mixture  and 5-hydroxyindole  reacted according to the general procedure. The title compound was obtained as a yellow solid . MS: (M + H)calc: 488.1704 m/z, (M + H)exp: 488.1708 m/z, UV/Vis (0.1% TFA-MeOH) λmax: 203 nm, 465 nm.3,5,7,3′,4′-Penta-1H-NMR data of compounds 6 to 11, see 13C-NMR data, see For 6–11 were used in a dimethylsulfoxide solution (conc. 10 mg·mL−1). Five replicates were investigated for each assay. Cell toxicity assays were conducted using a HeLa cell line, and antiproliferative activity was assayed using HUVEC and K-562 cell lines. All cell lines are from the Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Adolf-Reichwein-Straße 23, 07745 Jena, Germany. The test substances were dissolved in dimethylsulfoxide before being diluted in the respective medium of the cells to concentrations between 0.1 and 100 μmol·L−1. The adherent cells were harvested at the logarithmic growth phase after soft trypsinization using 0.25% trypsin in phosphate-buffered saline (PBS) containing 0.02% ethylenediaminetetraacetic acid (EDTA). For each experiment, approximately 10,000 cells were seeded with 0.1 mL culture medium per well of the 96-well microplates. HeLa cells were pre-incubated for 48 h prior to the addition of the test compounds, which were carefully diluted on the subconfluent monolayers. Incubation was then conducted in a humidified atmosphere at 37 °C and 5% CO2. In the case of K-562 cells, the number of viable cells in every well was determined using the CellTiter-Blue1 assay [To determine cell toxicity and antiproliferative activity, nudicaulin derivatives 6–11 were used in a methanolic solution (concentration 1 mg·mL−1). Antibacterial bioassays were performed in accordance with Krieg et al., 2017 [Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Mycobacterium vaccae. The bacteria were cultivated on standard I nutrient agar (NA I) in Petri dishes at 37 °C. Antifungal bioassays were conducted at 30 °C using strains of Sporobolomyces salmonicolor and Penicillium notatum, which were cultivated on malt agar (MA), and Candida albicans, which was cultivated on yeast morphology agar (YMA). After inoculation, a disc (9 mm in diameter) was removed from the center of the Petri dish and 50 μL of the test solution was added to the cavity. After 18 h of incubation at the respective temperatures, the inhibition areolas were measured. Ciprofloxacin (5 µg·mL−1 in deionized water) and amphotericin B (10 µg·mL−1 in DMSO/MeOH 1:1) were used as reference compounds against bacterial and fungal strains, respectively.To determine their antimicrobial activity, nudicaulin derivatives l., 2017  using st


Justicia, Phyllanthus, Haplophyllum and Linum species. This cyclolignan exhibits a wide array of biological properties ranges from piscicidal to antifungal, antiviral and antibacterial activities. Activity against Trypanosoma brucei makes justicidin B a potential antiprotozoal agent for the treatment of neglected tropical diseases. Pharmacological properties like antiplatelet, anti-inflammatory and bone resorption inhibition have been also attributed to justicidin B. This compound is a potent cytotoxic substance on several cell lines, especially chronic myeloid and chronic lymphoid leukemia. Pharmacological values, natural variation, as well as biotechnological production of justicidin B by plant cell, tissue and organ culture are also described in this review. Chemical characteristics and chromatographic methods to identify justicidin B and its biosynthetic pathway have been discussed. Different approaches to the total synthesis of justicidin B are compared. This review would shed light on the role of justicidin B as an intriguing natural compound and provides a chance to optimize conditions for industrial applications.Adverse effects and drug resistance to the current onchopharmacologicals have increased the demand for alternative novel therapeutics. We herein introduce justicidin B, an arylnaphthalen lignan isolated from different plant origins, especially  These inter-un1) , classifIn this review, the distribution, biosynthesis, biotechnological production, chemical, biological and pharmacological properties of justicidin B are described. Analytical methods for the isolation and quantification of the molecule are discussed and various strategies for the chemical synthesis of justicidin B are compared. Peer reviewed articles on justicidin B were retrieved from Google Scholar, Pubmed, Scopus and ISI Web of Knowledge until from 1960 to April 2016.21H16O6: colorless solid; mp 234–237 °C + (100), 335 (4), 319 (6), 305 (11), 291 (8), 277 (19), 167 (18), 163 (17); IR (KBr) 1760, 1620, 931, 873, 811 cm−1 . He. He8) (410) (11) followed by esterification with compound 12 to give compound 13 set the stage for the key cyclization step, a microwave-assisted intramolecular dehydro Diels-Alder cyclization, to directly afford justicidin B and isojusticidin B (14) in high yield and 2.3:1 ratio of isomers favoring justicidin B.In 2014, Kocsis et al. reported an efficient four-step route to justicidin B starting from commercially available dimethoxycinnamic acid (10)  58]. Re. Re10) (H. patavinum grown in hormone free Murashige and Skoog (MS) medium (12 h photoperiod), as well as callus and suspension cultures .,77.L. pe enzymes ,79. Then alcohol 5 derived enzymes . The ste enzymes . While (-re face . This reectively .16) to secoisolariciresinol (18) via lariciresinol (17). It has been shown in several plants that there are at least two copies of the plr gene in the genome of the corresponding species [8S,8′S-(−)-pinoresinol via 8S,8′S-(−)-lariciresinol to 8S,8′S -(+)-secoisolariciresinol the other gene is responsible for encoding an enzyme which converts 8R,8′R-(+)-pinoresinol via 8R,8′R-(+)-lariciresinol to 8R,8′R-(−)-secoisolariciresinol. In vitro assays of PLR-Lp1, which involves in the biosynthetic pathway of justicidin B  showed that both enantiomers of pinoresinol were converted to (+)- and (−)-lariciresinol. Similar behavior was observed in the formation of (+)- and (−)-secoisolariciresinol. However, PLR-Lp1 displayed preference for the (+)- enantiomer (RR configuration) in the first reaction step and (–)- enantiomer (SS configuration) in the second reaction step. Thus, PLR-Lp1 was introduced as the first PLR with opposite enantiospecificity toward pinoresinol and lariciresinol. Although, the involvement of PLR-Lp1 in the biosynthesis of justicidin B has been demonstrated by RNAi approach; presence of at least a second more enantiospecefic PLR encoding gene, in the genome of L. perenne for the biosynthesis of justicidin B has been postulated [An NADPH-dependent bifunctional enzyme named pinoresinol lariciresinol reductase (PLR-Lp1) converts pinoresinol  by the enzyme secoisolariciresinol dehydrogenase (SDH) [O-methylations have been less investigated. Formation of justicidin B has been proposed from 7(8)- 7′(8′)-tetrahydrojusticidin B (20) and 7(8)-dihydrojusticidin B (collinusin) (7) [1) to diphyllin (22) [Km value about 3.9 ± 1.3 µM. Diphyllin is then glycosylated to become more water soluble for storage in the vacuole.Secoisolariciresinol is converted to matairesinol (se (SDH) . Later ssin) (7) . A cytoclin (22) . The enz+ = 365 [High Performance Liquid Chromatography (HPLC) is a robust technique to identify and isolate natural products . Using t+ = 365 . Other fJ. procumbens [n-hexan:ethylacetate:methanol:water 1.3:1:1.3:1.0, v/v as solvent system A and 2.5:1.0:2.5:1.0, v/v as solvent system B. Column temperature was 25 °C, flow rate adjusted at 3 mL/min and rotation speed was 1000 rpm. Justicidin B was eluted after 68 min running of solvent A.Conventional methods for the isolation and purification of secondary metabolites are time consuming and generally results in sample loss. Preparative HSCCC has been reported by Zhou et al. to separate justicidin B with higher recovery from other lignans in ocumbens . Stepwisf value for justicidin B was about 0.6 when silica gel TLC plates 60 F254 (20 × 10 cm) were scanned by fluorescence at 366 nm after developing in chloroform: methanol 99:1 as the mobile phase.HPTLC analysis has been established as a sensitive and accurate method for the isolation and quantification of justicidin B . Rf valuAspergillus fumigatus, Candida albicans and Aspergillus flavus  with minimum inhibitory concentration (MIC) ≥1, 4 and 12 µg/mL, respectively [A. fumigatus is half of the potency of miconazole or amphotericin B, one should consider the necessity of alternative chemotherapeutics against pathogenic fungi which are resistant to the current medications. Cryptococcus neoformans and Blastoschizomyces capitatus were both resistant to justicidin B [Rhodotorula acuta (MIC = 3 µg/mL), Pichia angusta (MIC = 0.8 µg/mL), C. neoformans (MIC = 0.5 µg/mL), Aspergillus niger (MIC = 0.2 µg/mL) and Botrytis fabae (MIC = 0.4 µg/mL) [Fungal infections have frightening influence on human health. One of the greatest challenges in fungal therapy is the numerous side effects of antifungal drugs . Justiciectively . Althougicidin B . Accordi4 µg/mL) . HoweverC. albicans shows that justicidin B has the strongest anti-Candida activity in comparison to (+)-pinoresinol (furofuran-type lignan) [A. niger and A. flavus. After phyllamyricin C (6-methoxyjusticidin B) (MIC = 4 µg/mL), justicidin B is the second strong antifungal agent against Fusarium oxysporum (MIC = 8 µg/mL) [Comparing the antifungal activity of justicidin B with its precursors against  lignan) , laricir lignan)  and seco lignan) . In Comp lignan)  justicid8 µg/mL) . There mMechanism of antiviral activity of lignans is diverse and ranges from tubulin binding to reverse transcriptase inhibition . JusticiBacillus cereus, Staphylococcus aureus, Pseudomonas aeroginosa and Escherichia coli [E. coli, P. aeroginosa, Bacillus subtilis, B. cereus, S. aureus, Micrococcus luteus, Mycobacterium smegmatis and Corynebacterium xerosis was 0.5, 0.2, 2.0, 2.5, 1.0, 0.5, 5.5 and 7 µg/mL, respectively [Contradictory reports exist on the antibacterial activity of justicidin B. Gertsch et al. did not detect any antibacterial effect from justicidin B against hia coli . In a seectively . No antiTrypanosoma brucei transmitted by tsetse fly is the cause of trypanosomiasis (sleeping sickness) in African populations [T. brucei rhodesiense was observed for justicidin B with IC50 = 0.2 µg/mL using melarsoprol (IC50 = 0.003 µg/mL) as the positive control [Trypanosoma cruzi [T. cruzi with IC50 = 2.6 µg/mL in comparison to benznidazol (IC50 = 0.27 µg/mL) as the positive control [T. cruzi [T. brucei rhodensis [50 > 5 µg/mL in comparison to chloroquine (IC50 = 0.12 µg/mL) as the positive control [ulations . Melarsoulations . This trulations . Therefo control . Chagas ma cruzi . Justici control . AlthougT. cruzi , justicihodensis . Gertsch control .Oryzias latipes fish was comparable with rotenone where no fish survived with a concentration as low as 0.032 ppm after 24 h [Musca domestica, Periplanata americana, Prodenia litura and Panonychus citri has been observed [100 was measured around 1.5 µg/mL after 25–50 min when Gertsch et al. treated adult zebra fish (Brachydanio rerio) with justicidin B [Piscicides are poisonous chemicals to fish. The piscicidal activity of justicidin B against ter 24 h . The medter 24 h . Toxicitobserved . Ichtyot50 = 8.0 ± 1 µM) is significantly higher than aspirin (IC50 = 20.3 ± 2.1 µM) and less than indomethacin (IC50 = 0.21 ± 0.04 µM) [50 = 104.8 ± 25.3 µM [Inhibitors of platelet aggregation play a significant role in the prevention of atherosclerosis and thrombosis development . Several0.04 µM) . Althoug 25.3 µM . Strong  25.3 µM .50 = 12.5 µM [Inflammation is involved in various disease states, such as arthritis, atherosclerosis, sepsis, multiple sclerosis and cancer. Several inflammatory mediators including reactive nitrogen oxide (NO) or cytokines like TNF-α and IL-12 are synthesized by macrophages. Justicidin B was able to inhibit LPS-INFγ induced NO and cytokine production from peritoneal murine macrophage with IC 12.5 µM . Justici 12.5 µM ,116. The 12.5 µM .50 = 3.3 µg/mL) [50 in the range of 1.2 × 10−2–7.3 × 10−2 µg/mL has been reported for justicidin B towards 9 KB (a subline of HeLa) cell line. LC50 of justicidin B on Artemia salina (brine shrimp) has been reported to be as much as 1.1 µg/mL [Adverse effects and drug resistance to the current onchopharmacologicals have increased the demands for alternative novel therapeutics. In 1984, Pettit et al. showed that justicidin B inhibits the growth of NCI murine P388 lymphocytic leukemia (ED3 µg/mL) . An ED50.1 µg/mL . Later, .1 µg/mL . Justici.1 µg/mL .Gertsch et al. determined the cytotoxicity of justicidin B in neoplastic cells (HeLa and Jurkat T cells) and primary cell cultures (L-6 and PBMCs) . Justici50 of justicidin B after 48 h was 45.4 µM on K562 cells, which is at least seven times higher than Vasilev et al’s. report. Effect of justicidin B on L1210 and P388D1 cell lines after 48 h has shown that justicidin B has higher activity than etoposide towards L1210 cells  cells have shown less sensitivity to etoposide . Justici10 cells .2−) radical to protect cells from peroxidation injury. Rao et al. could not detect any significant scavenging activity of justicidin B against superoxide anion with concentrations up to 100 µM [Superoxide dismutase (SOD) partitionates the superoxide  . DiphyllThe transcription factor NF-κB involves in the regulation of many genes that code for mediators of apoptosis and tumorigenesis. NF-κB is composed of homo- and heterodimeric complexes of the Rel family containing p50, p65 (RelA), c-Rel, p52 and RelB. The best-characterized form of NF-κB is the p65/p50 heterodimer . In chroNo in vivo toxicity has yet been reported for justicidin B. Justicidin B was injected intraperitoneally (i.p.) to H-albino mice by Ilieva et al. . A maximBone resorption inhibitors are therapeutic agents for patients with excess calcium in the blood or bone cancer. In 1996, justicidin B was introduced as a bone resorption inhibitor . Twenty The effect of justicidin B on the detoxification of pollutants has been also tested . Incompl50 = 65.0 ± 1.8 µM [Free radical reactions cause damage to macromolecules and results in various disease states. Antioxidant properties of justicidin B has been tested against DPPH radical. Justicidin B displayed moderate scavenging activity with IC± 1.8 µM .C. albicans, A. niger, A. flavus and F. oxysporum are distinguished properties of justicidin B. Antiprotozal effects of justicididin B against trypomastigote forms of T. brucei rhodensis is one of the most prominent activities of justicidin B over other lignans. In comparison to several other derivatives, justicidin B showed a dominant anti-inflammatory activity. Stronger anti-platelet effects of justicidin B in comparison to aspirin might be beneficial for the patients to whom aspirin is either contraindicated or does not result in therapeutic effects.A wide array of biological activities has been ascribed to justicidin B. The cytotoxic, antifungal and antiprotozoal effects demonstrate that justicidin B plays a major role as the chemical defense machinery of the corresponding species. Pharmaceutically valuable characteristics of justicidin B  resulted in considerable interest toward its production. Multiple myeloma as a debilitating malignancy is usually refractory and multidrug resistance. The annual treatment of leukemia costs more than $2 billion only in USA still more than half of the patients expire. Cytotoxic effect of justicidin B on 23 different cell lines has been compared. In comparison to the aryltetralin lignans, which are currently used for the treatment of malignancies, the strongest activity of justicidin B was against L1210 the lymphocytic leukemia cells and RPMI8226 the multiple myeloma cell line. This provides insights for introduction of a new compound into the clinical trial studies as a replacement or additive therapy. In addition to the specific cytotoxicity of justicidin B for L1210 and RPMI8226 cells, antifungal activity against In this review, we focused on the in vivo and in vitro distribution of justicidin B in plant families of Acanthaceae, Euphorbiaceae, Linaceae and Rutaceae. Reliable, robust and selective analysis of justicidin B by chromatographic methods was described. Reports which have been published featuring short and efficient total synthesis of this interesting natural product focusing on the construction of naphthalene ring as the key step in the synthetic strategy were compared. Cationic cyclization and intramolecular Diels-Alder reaction were the most affordable synthetic strategies. Difficulties in the total synthesis of justicidin B reveal how crucial is to establish and screen in vitro cell, tissue and organ cultures to optimize justicidin B production. Elucidation of the unknown enzymes and encoding genes in the biosynthetic pathway of justicidin B provides the opportunity to genetically manipulate and improve plants, producing high yields of the corresponding metabolite, which is economically feasible for scale up purposes.


Context:Pimenta dioica (L.) Merr. (Myrtaceae) is used in Costa Rican traditional medicine for women’s health. Our previous work showed that P. dioica extracts were oestrogenic.Objectives: This work identifies phytochemicals from P. dioica that are responsible for the plant’s oestrogen-like activities.Materials and methods:P. dioica leaves were collected in Costa Rica in 2005. Fractions resulting from chromatographic separation of a methanol extract were tested at 50 μg/mL in a competitive oestrogen receptor-binding assay. Active compounds were isolated by HPLC and identified by NMR and MS. Pure compounds were tested at 1 μM in the oestrogen-responsive SEAP reporter gene assay. The effects on cell viability, cytotoxicity and apoptosis were investigated in breast cancer (MCF-7 and SK-BR3) and gastric cancer (AGS and NCI-N87) cell lines using the ApoTox-Glo and Caspase-Glo assays and qPCR.Results: Quercitrin and three new chromones, including a 2-phenoxychromone, 6,8-di-C-methylcapillarisin (1) were isolated and identified. Compound 1 caused a 6.2-fold increase in SEAP expression at 1 μM (p < 0.05). This activity was blocked by the ER antagonist ICI 182,780. Compound 2 caused a 6.0-fold increase in SEAP, inhibited the growth of MCF-7, AGS and NCI-N87 cells , and induced apoptosis via caspase 8 and increased the Bax/Bcl-2 mRNA ratio in MCF-7 cells. Compound 3 was anti-oestrogenic in MCF-7 cells.Discussion and conclusions: Compounds from P. dioica have oestrogenic, anti-oestrogenic and cytotoxic effects that may explain the ethnomedical use of this plant. Menopause is defined as the termination of menstruation due to a reduction in the number and function of ovarian follicles, resulting in a decline in estradiol and progesterone production Gold . The menIn Costa Rica, women enter menopause around the same age as their American counterparts (∼50 years), and suffer from similar menopausal symptoms  Merr. (Myrtaceae),  was selected for further investigation based on its activity in preliminary screens for oestrogen-like effects , and two new glycosylated methyl chromones (2 and 3).In our previous work, we found that extracts of the leaves of this plant bound to both the ERα and ERβ isoforms of the oestrogen receptor with preference for ERβ, as well as induced transcription of an oestrogen responsive luciferase gene in stably transfected MCF-7 cells  and the University of Costa Rica (UCR) based on a Memorandum of Agreement signed by authorities from UIC and UCR.P. dioica were collected in 2005 at Finca La Isla in Playa Negra, Limon Province, Costa Rica, and extracts were prepared at the Center for Natural Products Research (CIPRONA) at the UCR. Leaves were dried in an oven at 37 °C and ground in a hammer-mill to a course particle size. The plant material (1 kg dry weight) was then extracted by maceration in 5 L methanol twice overnight. The extract was filtered and partially dried in vacuo followed by lyophilization. Herbarium specimens were identified by Jorge Laurito-Gomez at the UCR and deposited in the herbarium at UCR (voucher #BD101).The leaves of l-glutamine . AGS (CRL-1739) was obtained from ATCC, grown and maintained in Kaighn’s modification of Ham’s F-12 with l-glutamine (ATCC). NCI-N87 obtained from ATCC was grown and maintained in RPMI 1640 medium . All growth media were supplemented with 10% FBS  and 1% penicillin/streptomycin . The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. At 80% confluency, the cells were harvested by adding 0.25% trypsin/EDTA and counted by means of trypan blue and haemocytometer. These cells were then re-suspended at appropriate concentration and plated for cellular assays.Human gastric cancer cells, AGS and NCI-N87 were purchased from ATCC . Human breast adenocarcinoma cells, MCF-7 were a kind gift from Dr. Hyun-Young Jeong of the Department of Pharmacy Practice, UIC. It was grown and maintained in minimum essential medium Eagle with Earle’s salt and 4 cells in 100 µL/well while NCI-N87 was seeded at 5.0 × 104 cells in 100 µL/well in opaque-walled 96-well plate. Control wells containing medium (supplemented with 10% FBS and 1% penicillin/streptomycin) without cells to determine background luminescence were also prepared. The cells were left to attach overnight in the plate. Culture medium was aspirated and fresh medium added to the wells before reconstituted extracts of P. dioica  and isolated compounds  at 100, 50, 20, 10 and 5 µg/mL were added to experimental wells. Drug controls (5-fluorouracil and doxorubicin) and vehicle control (0.02% DMSO) wells were also prepared simultaneously, and the plate was incubated in a humidified incubator at 37 °C in an atmosphere of 5% CO2 for 72 h. At the end of the incubation period, the plate and its contents were equilibrated to room temperature for approximately 30 min. A 100 µL volume of CellTiter-Glo 2.0 Reagent  was added to each well. The contents were mixed for 2 min on an orbital shaker to induce cell lysis and the plate was incubated at room temperature for 10 min to stabilize the luminescent signal. Luminescence signal was read using the Synergy HT Plate reader  and Gen5 1.11 software. The IC50 defined as the concentration that causes 50% inhibition of cell growth after exposure to extract for 72 h was calculated using log (inhibitor) vs. normalized response analysis with GraphPad Prism 7 .MCF-7 and AGS cells were seeded at 2.5 × 104 cells in 100 µL/well in triplicate were set up. After overnight incubation to allow attachment of cells, 2 at its IC50 concentration of 55 µg/mL and vehicle controls were added to the appropriate wells at a final volume of 100 μL per well. Plates were incubated appropriately before addition of 20 μL of viability/cytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110 substrate to all wells and briefly mixed by orbital shaking (300–500 rpm for ∼30 s). Plates were incubated for 30 min at 37 °C and fluorescence was measured at 400ex/505em (viability) and 485ex/520em (cytotoxicity). A 100 μL volume of Caspase-Glo® 3/7 reagent was added to all wells, briefly mixed by orbital shaking (300–500 rpm for ∼30 s), and incubated for 30 min at room temperature. Luminescence was measured using the Synergy HT Plate reader  and Gen 5 1.11 software to detect caspase activation.Ninety-six (96) well assay plates containing MCF-7 cells in medium at a density of 1.0 × 104 cells in 100 µL/well in opaque-walled 96-well plates. The cells were left to attach overnight in the plate. Compound 2 at 55 µg/mL was added to the test wells. Caspase-Glo® 8 Reagents  were prepared according to manufacturer’s instruction and allowed to equilibrate to room temperature. The test plates containing treated cells as well as controls were removed from the incubator after 2 h of incubation and allowed to equilibrate to room temperature. A 100 μL volume of Caspase-Glo® 8 Reagent was added to each well of a 96-well plate containing 100 μL of cell culture medium (blank), non-treated cells (negative control) in culture medium, and treated cells in culture medium. The contents of wells were gently mixed using a plate shaker at 300–500 rpm for 30 s and incubated at room temperature for 1 h. The luminescence of each sample was read using the Synergy HT Plate reader  and Gen5 1.11 software.MCF-7 cells were seeded in triplicate at a density of 3.0 × 106 cells per well in a six-well plate and incubated overnight for cell attachment. Total RNA was extracted from cells that had been treated with 2 at 55 µg/mL after 2 h incubation using Trizol  according to the manufacturer’s instructions. Vehicle-treated control cells served as the negative control. The concentration and quality of RNA were determined by measuring absorbance at 260 nm and 280 nm on SynergyHT Take 3 session  and Gen5 1.11 software.MCF-7 cells were seeded at a density of 1 × 10T 1-step kit  as described by manufacturer using a Step-One Plus Real Time PCR System . Primer sequences were selected from previously published papers and shown in Total RNA was reverse transcribed and amplified using Power SYBR Green RNA-to-C1 was determined by testing at concentrations of 20–100 μg/mL. All tests were performed in triplicate, and the results were determined from three independent experiments.A competitive radioligand-binding assay using full-length ERβ was used in the bioassay-guided fractionation of the crude methanol extract. The assay was performed as previously described  and mass spectroscopy (MS). NMR analysis was performed on a Bruker DRX-400 400 MHz spectrometer  using DMSO-d6 as the solvent. Spectra were processed and analysed using Xwin NMR software. Both 1H and 13C 1D experiments were performed, and 2D experiments, such as 1H–1H COSY, HSQC and HMBC, were performed as necessary. The molecular weights were obtained by MS using a Shimadzu LCMSITTOF spectrometer  with MeOH as the solvent. Samples were analysed in both positive and negative polarity modes using the ESI method of ionization.The structures of 2 flasks. Forty-eight hours prior to transfection, the cells were maintained in phenol red free DMEM medium supplemented with 0.5% FCS, in a humidified atmosphere with 5% CO2 at 37 °C. For transfection, 0.5 × 105 cells were seeded per well of a 96-well plate in OPTI-MEM supplemented with dextran-charcoal stripped FBS. After 4 h, serum concentration was reduced to 1%. Prior to transfection, cells were washed with DMEM and incubated in phenol red-free OPTI-MEM without FBS as described by Nozawa et al.  gene that is induced through an oestrogen response element (ERE). MCF-7 (HTB-22) human breast cancer cells  were maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin in 75 cma et al. .1–3 with and without 10 nM E2 or 1 μM ICI 182,780. Estradiol was used as the positive control and DMSO as the negative control. Cells were treated for 12 h, then the medium was harvested, centrifuged and 25 μL from each well was transferred to a new 96 well plate. The SEAP activity was measured using the Great EscAPe SEAP Chemiluminescence High-Throughput Detection Kit  using a 96-well luminometer according to the manufacturer’s instructions. To assess novel mechanisms of action other than ER, we tested the extract and fractions in a HER2/neu tyrosine kinase assays  . Statistical significance was ascribed to the data when p < 0.05.Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test as the The compounds were isolated using liquid–liquid partition chromatography, column chromatography and semi-preparative HPLC .1H and 13C NMR and high resolution MS data with previously published data for this compound  was isolated as a yellow, amorphous powder and was identified by comparison of 1 (10.1 mg) was isolated as a white, amorphous powder. The molecular formula C17H14O6 was determined by high resolution MS ([M + H]+=315.0858 m/z), and the structure was determined to be 2-(p-hydroxyphenoxy)-6,8-dimethyl-5,7-dihydroxychromone (δ1H (ppm): 13.0 , 9.77 , 9.72 , 7.20 , 6.86 , 5.09 , 2.08 , 2.02 . δ13C (ppm): 183.6 (C-4), 167.6 (C-2), 159.4 (C-7), 156.2 (C-4′), 150.1 (C-9), 143.1 (C-1′), 122.0 (C-2′/6′), 116.5 (C-3′/5′), 107.5 (C-6), 101.9 (C-8/10), 86.7 (C-3), 8.0 (6-Me/8-Me).Compound chromone  based onδ C 159.4 ppm), suggesting that a hydroxy-substituted carbon, C-7, was positioned between the two methylated carbons, C-6 and C-8. The methyl group assigned to C-6 also correlated with C-5, which was assigned based on HMBC correlation with the hydroxyl proton at δ 13.0 ppm. Furthermore, NMR data for this compound are consistent with previously published NMR data for the 6,8-dimethyl-5,7-dihydroxyflavone syzalterin  from fraction 15, sub-fraction 5 (15.5). Based on the data from 1D and 2D NMR, MS analysis, comparison with data from 1, and study of the literature, 2 appears to be a new O-glycoside of syzalterin  (Compound flavone) . The aglflavone) . It is e+ at 461.1 m/z which is consistent with the molecular formula for syzalterin-4′-O-glucopyranose C23H24O10. δ1H  13.07 , 8.04 , 7.21 , 6.89 , 5.61 , 5.39 , 5.12 , 5.06 , 5.02 , 4.6 , 4.36 , 3.70 (m), 3.5–3.1 (m), 2.30 , 2.05 . δ13C  182.2 (C-4), 162.7 (C-2), 160.3 (C-7/4′), 156.1 (C-5), 152.6 (C-9), 128.1 (C-2′/6′), 124.4 (C-1′), 116.7 (C-3′/5′), 107.2 (C-6), 103.6 (C-3/10), 102.0 (C-8), 100.0 (C-1″), 77.2, 76.6, 73.2, 69.7, 60.7, 8.4 (8-CH3), 8.1 (6-CH3).MS analysis detected [M + H]3 was isolated as a white powder (3.9 mg) from fraction 15, sub-fraction 6 (15.6). Based on comparison of 1D and 2D NMR and MS data with that of 2, 3 is the 5-O-glycoside of 7-methoxy-6-methyl-3,5,4′-trihydroxyflavone , C-7 (160.6) and C-9 (156.2). A methoxyl group was assigned to C-7 based on HMBC correlations between a peak at 3.91 ppm in the 1H NMR spectrum and the 13C peak at 160.6. The protons at CH3-6 and H-8 also correlate with this peak at 160.6 supporting its assignment to position 7. As with 2, peak characteristics of glucose were clearly observed in the carbon spectrum (61.1–99.3 ppm), indicating a glycoside. The glycoside is linked through the oxygen atom at position 5 due to the fact that the characteristic resonance of the 5-OH (∼13 ppm as observed in 1 and 2) is not observed in the 1H NMR spectrum of 3.Compound yflavone . The molδ 1H  10.08 , 9.06 , 8.07 , 7.11 , 6.94 , 5.66 , 5.43 (s), 5.15 (s), 5.10 (s), 4.82 , 4.76 (s), 3.91 , 3.78 (m), 3.48 (m), 3.46–3.20 obscured by H2O, 3.16 (m), 2.30 (s). δ 13C 176 (C-4), 160.6 (C-7), 159.0 (C-4′), 156.2 (C-9), 153.9 (C-5), 144 (C-2), 129.2 (C-2′/6′), 122.1 (C-1′), 115.6 (C-3′/5′), 107.5 (C-6), 104.2 (C-10), 99.6 (C-8), 99.3 (C-1″), 77.8, 76.1, 73.8, 70.3, 61.1, 56.2 (MeO-7) and 8.0 (Me-6).1 was determined in vitro by competitive oestrogen receptor binding assay and functionalized cell-based, oestrogen-responsive reporter gene assay. Compound 1 demonstrated binding to ERβ with an IC50 of 0.39 mM. Treatment of transiently transfected MCF-7 cells with 1 at 1 μM significantly enhanced the expression of the oestrogen responsive SEAP reporter gene compared to the control . Treatment of MCF-7 cells with activity .2 at the IC50 for 24 h decreased the expression of Bcl-2 mRNA, but had little effects on Bax mRNA expression. The alteration in Bcl-2 expression significantly increased the Bax/Bcl-2 ratio favouring apoptosis  cell proliferation with an ICe kinase . Since Pe kinase . These dP. dioica not only bound to the oestrogen receptors, but also induced the transcription of oestrogen responsive genes in MCF-7 cells . For 1, these effects were reduced by the addition of the ER antagonist ICI 182,780, suggesting that despite relatively weak binding to ERβ, 1 is acting through the ER, and thus is a partial ERβ agonist. Interestingly, other researchers investigated the biological activity of the essential oil of allspice, and found that one of its chemical constituents, eugenol, bound to oestrogen receptors, but did not induce expression of oestrogen-responsive genes in Ishikawa cells  K.Heyne (Fabaceae) that bound both ER subtypes, but it did not induce transcription of oestrogen-responsive reporter genes through either receptor subtype. Researchers assessed the ER-binding and antiproliferative effects of several synthetic flavonoids containing an oxygen atom at carbon 2, which served as a ‘hinge’ to direct an ethoxy amine side chain to the ER binding site (Kim et al. In addition to up-regulating receptor expression, the ERα has a larger ligand-binding site than ERβ, and as a result some ligands that bind both receptor subtypes may activate ERα, but not ERβ (Pike et al. ge at C2 . Howeverge at C2  isolatedP. dioica extract, fractions and compounds in two breast cancer cell lines MCF-7 and SK-BR3. The extract and a 50% MeOH fraction inhibited the growth of both MCF-7 and SK-BR-3 breast cancer cells, but was less effective in MCF-7 cells that express only basal levels of HER2. However, since 2 induced apoptosis in MCF-7 cells, we also investigated some of the well known signalling factors involved in apoptosis, including the Bcl-2 and caspase family of proteins (Kugu et al. 2 enhanced both caspase 8 activity and decreased the expression of Bcl-2 mRNA, but had little effects on Bax mRNA expression. The reduction in Bcl-2 mRNA expression significantly increased the Bax/Bcl-2 ratio to be more favourable towards apoptosis in MCF-7 cells.One of the serious issues associated with the ingestion of oestrogens and phytoestrogens is the possibility of enhancing the oestrogen-responsive cancer risk in women with familial history of the disease or with active breast or endometrial cancers. It is well known that oestrogens regulate the proliferation of ERα positive breast cancer cells through the regulation of many gene networks and signalling pathways (Saxena and Dwivedi P. dioica acts as a HER2 inhibitor, but further research is needed to identify the active constituents.In addition to MCF-7 cells, the extract and 50% aqueous methanol fraction inhibited the growth of ER negative SK-BR3 cells. ERα negative breast cancer cells proliferate independently of oestrogen signalling through other signalling pathways such as the EGFR, and downstream PI3K/Akt/mTOR, NF-κB signalling pathways (Saxena and Dwivedi 2 inhibited AGS and NCI-N87 cell proliferation, suggesting potential effects against gastric cancer. Other researchers reported that polyphenols isolated from a P. dioica leaf methanol extract inhibited the proliferation of Hep-G2 liver cancer and HCT-116 colon cells, but had a lesser effect on MCF-7 cells (Zhang and Lockeschwar P. dioica is used in Costa Rica and other countries to manage gastritis, dyspepsia and other gastrointestinal disorders (Zhang and Lockeschwar P. dioica extracts and 2 inhibit the growth of H. pylori, it is also possible that allspice/pure compounds may be developed for gastric cancers.Finally, in gastric cancer cells, the extract, the 50% MeOH fraction and P. dioica with oestrogenic effects corroborates the Costa Rican traditional use in the treatment of menopausal symptoms. The inhibitory impact of the extract, fractions and pure compounds on breast and gastric cancer cells suggests that this plant has potential to be developed for the prevention or treatment of these cancers, and further suggests that this plant may not increase the risk of breast cancer in women using it to treat menopause.Allspice is used in Costa Rican ethnomedicine for the management of women’s reproductive health issues, menopause, gastritis and a number of other conditions. Literature on the safety and efficacy of this plant is limited. Isolation and identification of new compounds from the leaves of


The crystalline, microstructure, and piezoelectric properties of (1−x)PMN-xPT ceramics depending on the sintering temperature were intensively investigated and discussed. By optimizing the sintering temperature and compositions from the PMN-PT ceramics, the maximum value of the piezoelectric charge coefficient (33d) of 665pC/N, planar electromechanical coupling factor (pk) of 77.8%, dielectric constant (rε) of 3230, and remanent polarization (rP) of 31.67 μC/cm2 were obtained.The pyrochlore phase in ferroelectric and piezoelectric materials is the main obstacle device application due to its poor electrical properties. Especially, the pyrochlore phase is frequently observed in the perovskite-based metal-oxide materials including piezoelectric and ferroelectric ceramics, which are based on solid-state reaction methods for fabrication. To overcome these problems, advanced innovative methods such as partial oxalate process will be investigated. In this method, crystalized magnesium niobite (MN) and lead titanate (PT) powders will be coated with a certain amount of lead oxalate and, then, the calcination process can be carried out to form the PMN-PT without pyrochlore phase. In this study, (1− Among the several processing methods used to fabricate PMN-PT ceramics to form pure pervoskite phase, the Columbite precursor method has been widely used due to higher yield of perovskite phase content [3, prior to the reaction with lead. However, this Columbite method has demerits in the formation of pyrochlore phase during the sintering process. Also, several other fabrication methods such as sol-gel method, KCl molten salt method, and hot pressing have been performed and analyzed to improve piezoelectric properties by eliminating the pyrochlore phase. However, most of them leave a residual pyrochlore phase in the sintered ceramics [PbMg/3Nb2/3O3f 490 °C ,6. Especl phases ,8,9; it  around − °C with l phases ,18,19,20 content . The Colceramics ,23.The partial oxalate method is an innovative technique to remove the pyrochlore phase of lead-based ceramics. This method has been successfully investigated for the synthesis of PZT, PLZT and PMN ,25.3 structure using columbite precursor method, MN and PT powders were coated with the needed amount of lead oxalate followed by calcination to form the PMN-PT. (1−x)PMN-xPT ceramics were prepared using a partial oxalate method to remove the pyrochlore phase at various sintering temperatures. The influence of synthesis method on the dielectric, piezoelectric, and ferroelectric properties of PMN-PT ceramics was investigated. Also, we have investigated (1−x)PMN-xPT ceramics, focusing on the effects of MPB region according with PT content with respect to using applications. In this study, after making B-site compound of ABOx)PMN-xPT  ceramics were prepared using the partial oxalate method [2O5 , TiO2 , Pb(NO3)2 , and oxalic acid  powders were used as starting materials. The weighed powders  according to the stoichiometry of composition were mixed in high-purity ethanol  for 6 h by planetary ball milling with a zirconia ball, then calcined at 1100 °C for 4 h. The stoichiometric MN and TiO2 powders were mixed in high-purity ethanol for 6 h by planetary ball milling with zirconia balls, then calcined at 1100 °C for 4 h. The weight ratio between the balls and powders were 10:1. Until now, very rare papers were reported for the partial oxalate processing techniques for the PMN ceramics case. Also, there is no papers for the PMN-PT piezoelectric ceramics with improved piezoelectric properties by comparing existence of pyrochlore phase. The partial oxalate process was carried out at 90 °C because this temperature enhances the reaction with oxalic acid to form soluble Pb(MNT)OC2O4·H2O species. The processing steps of the oxalic process may be described by the following equations:OC2O4·H2O powders were dried at 120 °C, then calcined at 750 °C for 4 h. The (1−x)PMN-xPT powders were crushed and milled using high-purity ethanol as a ball milling for 12 h and calcined again at 750 °C for 4 h. The (1−x)PMN-xPT powders were pressed into a disc (12 mm diameter × 1.5 mm thickness) at a pressure of 294 MPa using polyvinyl alcohol (PVA) as a binder. After burning off the PVA  the pellets were sintered at 1200–1300 °C  for 4 h. The coated Pb(MNT)OCα radiation source. The microstructures of the ceramics were observed by field emission scanning electron microscopy (FE-SEM) . The frequency-dependent and temperature-dependent dielectric constant (rε) of these ceramics were analyzed. Polarization versus electric (P-E) hysteresis loops and strain versus electric (S-E) loops of the ceramic were then measured. The piezoelectric charge coefficient (33d) and planar electromechanical coupling factor (pk) were measured by the Berlin-court quasi-static meter  and impedance analyzer , respectively. The structures were examined by X-ray diffraction (XRD) analysis . The measurements were carried out from 20–70° by 0.02° differences. The wave-length of incident beam is around 1.54 Å from CuKx)PMN-xPT  ceramics sintered at 1275 °C. The XRD patterns were measured from 20 to 70° at 0.02° intervals for 0.5 h, respectively. Although the XRD figures were shown in the log scale in the Y -axis, any pyrochlore phase was not observed. As shown in the x)PMN-xPT from 44 to 46°. A PT composition of 0.250 mol shows the rhombohedral phase. Then, as the PT composition increased up to 0.400 mol, the (200) peak positions moved to the higher angle, and confirmed (002)/(200) peak splitting at PT composition of 0.400 mol. This indicated that mixed rhombohedral and tetragonal phases (MPB region) are present between the PT composition from 0.325 to 0.350 mol. Also, that composition could show the highest piezoelectric, dielectric, and ferroelectric properties compared with other specimens. x)PMN-xPT  ceramics sintered at 1275 °C and the 0.650PMN-0.350PT ceramics depending on a sintering temperature from 1200 to 1300 °C. After polishing cross-sections of all specimens, they were thermally etched for 10 min at a temperature of 100 °C lower than the sintering temperature.Platinum (Pt) coating using an ion-coater was carried out for 120 s, and all samples were measured under the same conditions at 7000 magnifications. 0.750PMN-0.250PT ceramics showed a relatively large grain size of approximately 6.75 μm compared with that of other composition ceramics. The increasing amount of PT substitution in the PMN composition can cause not only lattice distortion but also dwindle the grain size of these ceramics, as shown in the rε) and dielectric losses of the (1−x)PMN-xPT  ceramics sintered at 1275 °C and the 0.650PMN-0.350PT ceramics sintered from 1200 to 1300 °C depending on the frequency range. The dielectric properties were measured from 1 kHz to 1 MHz. The rε was calculated from the computed capacitance by an impedance analyzer through the following equation:C was the capacitance of the sample, d was the sample thickness, A was the area of the electrode, and ε0 was the dielectric constant of a vacuum (8.854 × 10−14 F/cm). Based on the equation, the rε of PT composition of 0.350 mol is higher than that of other compositions, with a maximum value of 3230 at 1 kHz. Then, it decreases when PT composition is above 0.350 mol. This tendency is attributed to the occurrence of phase transition that was previously verified by analysis of XRD pattern as shown in x)PMN-xPT  ceramics sintered at 1275 °C were decreased as increasing the PT composition, indicating that PT can usefully decrease dielectric loss. Additionally, we found that by increasing the sintering temperature up to 1300 °C, the rε value was increased by about 1.07 times at 1 kHz compared to that of 1200 °C as shown in the rε of 0.650PMN-0.350PT ceramic sintered at 1300 °C is around 3278 at 1 kHz. This result correlates to grain size and sintering temperature. As increasing the sintering temperature, grain size can cause not only a decrease of grain-boundary volume but also increase in the rε of 0.650PMN-0.350PT ceramic. The dielectric losses of the (1−33d) of the (1−x)PMN-xPT  ceramics sintered from 1200 to 1300 °C. The 33d increased with increasing PT composition up to 0.350 mol and sintering temperature up to 1275 °C, with a maximum 33d of 665 pC/N. pk) of the (1−x)PMN-xPT  ceramics sintered from 1200 to 1300 °C. These tendencies of pk were like those of 33d. The pk were calculated by using the following equation [af and rf are the anti-resonance and the resonance frequency, respectively. The maximum value of pk was 77.8% in 0.650PMN-0.350PT at 1275 °C. As a result, these increased 33d and pk were related with each composition and MPB region, as discussed in equation :(4) kp= rε) at 1 kHz for the (1−x)PMN-xPT  ceramics sintered at 1275 °C. The (1−x)PMN-xPT ceramic samples showed a temperature of maximum dielectric constant (mT) of 84.12, 102.33, 115.59, 129.84, and 150.07 °C for PT composition of 0.250, 0.300, 0.325, 0.350, 0.400 mol, respectively. As shown in mT increased with increasing contents of PT up to 0.400 mol, reaching a maximum value of 150.07 °C, because PT has a Curie temperature of 490 °C. Also, PMN-PT ceramics show ferroelectric properties at temperatures above mT unlike normal ferroelectric materials because of relaxor property. x)PMN-xPT  ceramics sintered at 1275 °C. The P-E hysteresis loops were measured by a Sawyer–Tower circuit under an applied electric field of 20 kV/cm at 0.1 Hz. With an increasing PT composition, the coercive electric field (cE) was decreased. Whereas saturation polarization (sP) and remnant polarization (rP) increased up to 39.42 μC/cm2 and 31.67 μC/cm2, respectively, and the P-E loop revealed the presence of a typical hysteresis loop of ferroelectric materials. Also, 0.600PMN-0.400PT ceramics showed large cE which is attributed to smaller grain size (ain size . It is wx)PMN-xPT  ceramics sintered at 1275 °C. The strain increased up to a PT composition of 0.350 mol and the maximum value of strain was 0.12%. However, upon further increasing the PT composition, the strain began to decrease. Negative strain behavior of all specimens was observed. This result agreed well with the previous data for rε, 33d, pk of (1−x)PMN-xPT ceramics sintered at 1275 °C.rP and cE decreased as the temperature increased and showed a ferroelectric property at temperatures above mT, as discussed in x)PMN-xPT ceramics with outstanding piezoelectric and ferroelectric properties have been investigated and analyzed based on a partial oxalate process. As mentioned in XRD patterns, all specimens showed pure pervoskite structure without the pyrochlore phase. At the 0.325 and 0.350 mol of PT near the MPB region between the rhombohedral-tetragonal phase, higher piezoelectric properties were observed. We observed the maximum value of the 33d of 665pC/N and pk of 77.8% from the 0.650PMN-0.350PT ceramic sintered at 1275 °C. The relaxor ferroelectric property of (1−x)PMN-xPT ceramics was confirmed through temperature-dependent rε and P-E loop. Therefore, these outstanding properties suggest that 0.650PMN-0.350PT ceramics sintered at 1275 °C could be suitable for various applications substitutes for PZT as the relaxor ferroelectric material.The (1−


A series of azo disperse dyes based on aminothienochromene were synthesized. The fastness properties of the dyed samples were measured. Most of the dyed fabrics tested displayed excellent washing and perspiration fastness and moderate light fastness. The chemistry of aminothiophenes has received much attention due to their facile availability through the versatile Gewald synthesis . The impAminothienochromene compounds are very useful as precursors for the synthesis of fused heterocyclic ring systems, which display an important range of biological and pharmacological activities ,12,13,14In view of these findings, and in continuation of our previous studies  on the so-hydroxyacetophenone with ethyl cyanoacetate and elemental sulfur in the presence of diethylamine as basic catalyst with a focused microwave heating oven as an energy source at 50 °C for 4 h, chromen-4-one (4). A mixture of o-hydroxyacetophenone , sulfur  and ethyl cyanoacetate , in methanol (2 mL) was irradiated by focused microwaves at 50 °C for 4 h in the presence of diethylamine . Completion of the reactions was monitored by TLC. The build-up of pressure in the closed reaction vessel was carefully monitored. After the irradiation, the reaction tube was cooled with high-pressure air through an inbuilt system in the instrument until the temperature had fallen below 50 °C. The mixtures were cooled and then poured into ice-water. The solids that formed were collected by using filtration and crystallized from benzene to give compound 4 as a yellow powder (48%), mp 199 °C (Lit. chromen-4-one (7a). Red crystals (79%), mp 239 °C (Lit. chromen-4-one (7b). Brown powder (75%), mp 293 °C (Lit. chromen-4-one (7c). Reddish brown crystals (68%), mp 281–283 °C (Lit. chromen-4-one (7d). Red crystals (77%), mp 304 °C (Lit. chromen-4-one (7e). Violet crystals (75%), mp 307 °C (Lit. [2), 1689 (CO) cm−1. 1H-NMR (DMSO-6d): δ = 7.42 , 7.46 , 7.65 . 7.86 , 7.95 , 8.32 , 8.86 . MS (EI) m/z= 366 , UV/Vis at maxλ(DMF) = 662 nm. Anal. Calcd for C17H10N4O4S: C, 55.73; H, 2.75; N, 15.29; S, 8.75. Found: 55.87; H, 2.91; N, 15.29; S, 8.41.°C  was obtained from El-Shourbagy Co., Cairo, Egypt. The fabric was treated before dyeing with a solution containing non-ionic detergent  and sodium carbonate (2 g/L) in a ratio of 50:1 at 60 °C for 30 min, then thoroughly washed with water and air dried at room temperature.Polyester 100%  is a sealed stainless steel dye pot of 300 mL capacity in the “Atlas-Germany” dyeing machine. An additional dispersing agent (Sodium lignin sulfonate) was added with a ratio to dye 1:1 and the pH of the bath was adjusted to 4.5–5 using glacial acetic acid. The dyeing was being carried out by raising the dye bath temperature from 20 °C to 130 °C at a rate of 7 °C/min and holding at this temperature for 60 min before rapidly cooled to 50 °C. The dyed fabrics was then rinsed with cold water, reduction-cleared using sodium hydroxide (2 g/L) and sodium hydrosulphite (2 g/L) at 80°C for 30 min and then the dyed samples thoroughly washed and air-dried.R = decimal fraction of the reflectance of the dyed fabric; oR = decimal fraction of the reflectance of the undyed fabric; K = absorption coefficient; S = scattering coefficient.The colorimetric parameters  of the dFastnesses to washing, perspiration, and light were tested according to reported methods .In summary, a series of monoazo disperse dyes were synthesized from 3-aminothienochromene. Synthesis of the aminothienochromene in a microwave oven afforded better yield and shorter reaction times. The dyes produced in this manner were then applied to polyester fabrics using the high temperature dyeing method at 130 °C. The dyed polyester fabrics, which displayed brown to violet hues, showed excellent washing and perspiration fastness and moderate light fastness.