RESUMO
Tyrosinase is known for an enzyme that plays a key role in producing the initial precursor of melanin biosynthesis. Inhibition of the catalytic reaction of this enzyme led to some advantage such as skin-whitening and anti-insect agents. To find a natural compound with inhibitory activity towards tyrosinase, the five flavonoids of kushenol A (1), 8-prenylkaempferol (2), kushenol C (3), formononetin (4) and 8-prenylnaringenin (5) were isolated by column chromatography from a 95% methanol extract of Sophora flavescens. The ability of these flavonoids to block the conversion of L-tyrosine to L-DOPA by tyrosinase was tested in vitro. Compounds 1 and 2 exhibited potent inhibitory activity, with IC50 values less than 10 µM. Furthermore, enzyme kinetics and molecular docking analysis revealed the formation of a binary encounter complex between compounds 1-4 and the enzyme. Also, all of the isolated compounds (1-5) were confirmed to possess antioxidant activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Quempferóis/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Sophora/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Flavonoides/química , Flavonoides/isolamento & purificação , Células Hep G2 , Humanos , Quempferóis/química , Quempferóis/isolamento & purificação , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Three previously undescribed dihydronaphthalenones, 7-hydroxycatalponol, (4S)-3,4-dihydro-4-hydroxy-2-[(2R)-2,3-dihydroxy-3-methylbutylidene]naphthalen-1(2H)-one, and (6S)-5,6-dihydro-6-hydroxy-2,2-dimethyl-2H-benzo[h]chromen-4(3H)-one and one phthalide, (±)-3-(5-hydroxy-5-methyl-2-oxohex-3-en-1-yl)isobenzofuran-1(3H)-one, were isolated from the wood of Catalpa ovata G. Don (Bignoniaceae), together with six known compounds. The structures of the previously undescribed compounds were elucidated by interpretation of 1D and 2D NMR data. The absolute configurations of the dihydronaphthalenones were deduced by analysis of the ECD data and application of Mosher ester methodology. All isolates were investigated for their cytoprotective effects against hydrogen peroxide (H2O2)-induced oxidative damage in HepG2 cells. Moreover, the mRNA expression levels of antioxidant enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinine oxidoreductase 1 (NQO1) in HepG2 cells were examined by RT-PCR analysis. As a result, catalponol and epi-catalponol showed antioxidant activities via directly scavenging of intracellular ROS and inducing the antioxidant enzymes in vitro.
Assuntos
Antioxidantes/farmacologia , Bignoniaceae/química , Naftalenos/farmacologia , Compostos Fitoquímicos/farmacologia , Madeira/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Estrutura Molecular , Naftalenos/química , Naftalenos/isolamento & purificação , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Isoegomaketone (IK) was isolated from Perilla frutescens, which has been widely used as a food in Asian cuisine, and evaluated for its biological activity. We have already confirmed that IK induced the HO-1 expression via Nrf2 activation in RAW264.7 cells. In this study, we investigated the effect of IK on the mechanism of HO-1 expression. IK upregulated HO-1 mRNA and protein expression in a dose dependent manner. The level of HO-1 mRNA peaked at 4 h after 15 µM IK treatment. To investigate the mechanisms of HO-1 expression modulation by IK, we used pharmacological inhibitors for the protein kinase C (PKC) family, PI3K, and p38 MAPK. IK-induced HO-1 mRNA expression was only suppressed by SB203580, a specific inhibitor of p38 MAPK. ROS scavengers (N-acetyl-L-cysteine, NAC, and glutathione, GSH) also blocked the IK-induced ROS production and HO-1 expression. Furthermore, both NAC and SB203580 suppressed the IK-induced Nrf2 activation. In addition, ROS scavengers suppressed other oxidative enzymes such as catalase (CAT), glutathione S-transferase (GST), and NADH quinone oxidoreductase (NQO-1) in IK-treated RAW264.7 cells. Taken together, it can be concluded that IK induced the HO-1 expression through the ROS/p38 MAPK/ Nrf2 pathway in RAW264.7 cells.
RESUMO
Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Expressão Gênica , Planticorpos/genética , Planticorpos/metabolismo , Animais , Glicosilação , Espaço Intracelular , Camundongos , Células Vegetais/metabolismo , Planticorpos/química , Planticorpos/imunologia , Planticorpos/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico , Nicotiana/genética , Nicotiana/metabolismoRESUMO
The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 µL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 µL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.