A New Lignan from Annona squamosa L. (Annonaceae) Demonstrates Vasorelaxant Effects In Vitro.

Esquamosan, a new furofuran lignan, has been isolated by bio-guided assays from the methanolic extract of the leaves of Annona squamosa L., and its structure was elucidated by spectroscopic methods. Esquamosan inhibited the rat aortic ring contraction evoked by phenylephrine in a concentration-dependent manner and showed an inhibitory effect on vasocontraction of the depolarized aorta with high-concentration potassium. The vasorelaxant effect by esquamosan could be attributed mainly to the inhibition of calcium influx from extracellular space through voltage-dependent calcium channels or receptor-operated Ca2+ channels and also partly mediated through the increased release of NO from endothelial cells. The ability of esquamosan to modify the vascular reactivity of rat aortic rings incubated with high glucose (D-glucose 55 mM) was then evaluated, and this furofuran lignan reverted the endothelium-dependent impairment effect of high glucose in rat aortic rings. The antioxidant capacity of esquamosan was assessed using DPPH and FRAP assays. Esquamosan exhibited a similar antioxidant capacity compared to ascorbic acid, which was used as a positive control. In conclusion, this lignan showed a vasorelaxant effect, free radical scavenging capacity, and potential reductive power, suggesting its potential beneficial use to treat complex cardiometabolic diseases due to free radical-mediated diseases and its calcium antagonist effect.

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain.

L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

Acetylcholinesterase inhibitory activity, anti-inflammatory, and neuroprotective potential of Hippeastrum psittacinum (Ker Gawl.) herb (Amaryllidaceae).

Hippeastrum psittacinum, Amaryllidaceae, is used in traditional medicine as a purgative, aphrodisiac, and anticough remedy. The ethanol extract (EE) and alkaloid-rich fractions (ARF) from H. psittacinum bulbs were evaluated for their acetylcholinesterase (AChE) inhibition. The EE cytotoxic and anti-inflammatory effects in RAW 264.7 cells, and the neuroprotective and genotoxic activities in SH-SY5Y cells, were also estimated. Fifteen alkaloids were identified in the EE by gas chromatography-mass spectrometry. ARFs were less active for AChE inhibition than EE. The viability of both cell lines was higher than 70% with EE concentrations below 25 µg/mL. The EE decreased nitrite release in RAW cells stimulated with lipopolysaccharide, showing values of 83, 67, and 53% at 6.25, 12.5, and 25 µg/mL, respectively. Furthermore, the EE partially protected SH-SY5Y cells from hydrogen peroxide-mediated deleterious effects by approximately 50% at the same concentrations. The micronucleus assays showed that the extract caused chromosomal missegregation at concentrations above 12.5 µg/mL. The in silico analyses showed that some alkaloids presented properties of permeation of the blood-brain barrier and the intestine. Our findings present new evidence of the potential of H. psittacinum potential as an AChE inhibitor, as well as an anti-inflammatory and neuroprotective agent.

Assessment of anti-cholinesterase activity and cytotoxicity of cagaita (Eugenia dysenterica) leaves.

Eugenia dysenterica ex DC Mart. (Myrtaceae) is a Brazilian tree with pharmacological and biological properties. The aqueous leaf extract, rich in polyphenols, was tested in the human neuroblastoma cell line SH-SY5Y to evaluate its effect on cell viability. The extract and two isolated compounds were also assessed for the potential inhibitory activity on acetylcholinesterase, an enzyme related to Alzheimer's disease. A simple chromatographic method using Sephadex LH-20 was developed to separate catechin and quercetin from the aqueous leaf extract of E. dysenterica. Identification was carried out by spectroscopic techniques IR, UV, and 1H and 13C NMR. The IC50 values were obtained by constructing dose-response curves on a graph with percentage inhibition versus log of inhibitor concentration and compared with physostigmine, a well-known AChE inhibitor. The extract was toxic for SH-SY5Y cells at concentrations higher than 7.8 µg/ml given for 24 h. The decline in SH-SY5Y cell viability appears to be related to its antiproliferative activity. The extract also showed relatively moderate acetylcholinesterase inhibitory activity of 66.33% ± 0.52% at 1.0 mg/ml with an IC50 value of 155.20 ± 2.09 µg/ml. Physostigmine, quercetin, and catechin showed IC50 values of 18.69 ± 0.07, 46.59 ± 0.49, and 42.39 ± 0.67 µg/ml, respectively.

Cytotoxicity of universal, self-etching and etch-and-rinse adhesive systems according to the polymerization time.

This in vitro study evaluated in fibroblast cultures the direct cytotoxicity of universal, self-etching and etch-and-rinse adhesive systems according to the polymerization time. Paper discs were impregnated with adhesives and light-cured (10, 20 or 40 s). The discs were then immersed in culture medium to obtain the eluates for the experimental groups (A1-Single Bond 2; A2-Scotchbond Multi-purpose; A3-Clearfil SE Bond; A4 Scotchbond Universal). As a negative control, paper discs were immersed in culture medium only. After 24 h or 7 days, the eluate obtained was applied on fibroblast culture. Cell viability, cell morphology, membrane damage and the presence of residual monomers were evaluated by MTT assay, SEM, flow cytometry and high-performance liquid chromatography (HPLC), respectively. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (=0.05). All adhesive systems significantly reduced 33-51% cell metabolism when compared to the negative control, regardless of polymerization time, storage period and adhesive system. Moreover, the adhesives caused intense morphological alterations and cell membrane damage. Toxicity was directly related to the presence of residual monomers in the eluates. Residual monomers and additional components are capable of reducing mitochondrial activity, causing morphological alterations and disruption of the cell membrane in fibroblasts, regardless of the polymerization time. This study highlights that despite the more complex composition of the universal adhesive system, its biological response was not more toxic when compared with other systems, even when the shortest polymerization time was tested in cell culture.

Cytotoxicity of Universal, Self-Etching and Etch-and-Rinse Adhesive Systems According to the Polymerization Time

This in vitro study evaluated in fibroblast cultures the direct cytotoxicity of universal, self-etching and etch-and-rinse adhesive systems according to the polymerization time. Paper discs were impregnated with adhesives and light-cured (10, 20 or 40 s). The discs were then immersed in culture medium to obtain the eluates for the experimental groups (A1-Single Bond 2; A2-Scotchbond Multi-purpose; A3-Clearfil SE Bond; A4 Scotchbond Universal). As a negative control, paper discs were immersed in culture medium only. After 24 h or 7 days, the eluate obtained was applied on fibroblast culture. Cell viability, cell morphology, membrane damage and the presence of residual monomers were evaluated by MTT assay, SEM, flow cytometry and high-performance liquid chromatography (HPLC), respectively. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (=0.05). All adhesive systems significantly reduced 33-51% cell metabolism when compared to the negative control, regardless of polymerization time, storage period and adhesive system. Moreover, the adhesives caused intense morphological alterations and cell membrane damage. Toxicity was directly related to the presence of residual monomers in the eluates. Residual monomers and additional components are capable of reducing mitochondrial activity, causing morphological alterations and disruption of the cell membrane in fibroblasts, regardless of the polymerization time. This study highlights that despite the more complex composition of the universal adhesive system, its biological response was not more toxic when compared with other systems, even when the shortest polymerization time was tested in cell culture.

O presente estudo in vitro avaliou a citotoxicidade direta dos sistemas adesivos convencionais, autocondicionantes e universais de acordo com o tempo de polimerização em cultura de fibroblastos. Discos de papel foram impregnados com adesivos e fotoativados (10, 20 e 40 s). Os discos foram posteriormente imersos em meio de cultura para obtenção dos eluatos dos grupos experimentais (A1-Single Bond 2; A2-Scotchbond Multi-purpose; A3-Clearfil SE Bond; A4 Scotchbond Universal). Para o controle negativo, os discos de papel foram imersos somente em meio de cultura. Após 24 h ou 7 dias, o eluato obtido foi aplicado na cultura de fibroblastos. O metabolismo celular, morfologia, dano de membrana e presença de monômeros residuais foram avaliados por teste de MTT, MEV, citometria de fluxo e HPLC, respectivamente. Os dados foram analisados estatisticamente por Kruskal-Wallis e Mann-Whitney. Todos os sistemas adesivos reduziram significativamente o metabolismo celular em 33 a 51% quando comparados ao grupo controle, independente do tempo de polimerização, período de armazenamento e tipo de sistema adesivo. O eluato do adesivos causou ainda intensas alterações morfológicas e danos à membrana celular. A toxicidade foi diretamente relacionada à presença de monômeros residuais nos eluatos experimentais. Monômeros residuais e componentes adicionais dos sistemas adesivos foram capazes de reduzir a atividade mitocondrial, causar alterações morfológicas e danos à membrana citoplasmática de fibroblastos, independente do tempo de polimerização. Esse estudo evidencia que apesar de uma composição mais complexa do sistema adesivo universal, sua resposta biológica não apresentou maior toxicidade quando comparado aos demais sistemas, mesmo no menor tempo de polimerização quando testados em cultura celular.