RESUMO
Ginkgo biloba seeds are widely used as a food and traditional medicine in China. In the present study, a novel antioxidant protein named GBSP was purified from Ginkgo biloba seeds. The protein (GBSP) was purified by homogenization of Ginkgo biloba seed powder in saline solution, 70% ammonium sulphate precipitation, filtration on a DEAE-Cellulose52 anion exchange column, gel filtration on a Sephadex G-50 column, and preparative chromatography on a C(18) column using RP-HPLC. GBSP showed an apparent molecular weight of 18 kDa by SDS-PAGE and MALDI-TOF/MS analyses. The amino acid sequence obtained by MALDI-TOF/TOF MS analysis showed GBSP was a novel protein, as no matching protein in was found the database. The protein exhibited significant antioxidant activities against free radicals such as DPPH, ABTS and superoxide anion and showed higher activity than α-tocopherol in a linoleic acid emulsion assay system. Furthermore, GBSP exhibited notable reducing power and a strong chelating effect on Cu(2+) and Fe(2+). Therefore, the present study demonstrates, for the first time, that this novel protein from Ginkgo biloba seeds is an excellent antioxidant.
Assuntos
Antioxidantes/isolamento & purificação , Ginkgo biloba/química , Proteínas de Plantas/isolamento & purificação , Sementes/química , Antioxidantes/química , Antioxidantes/farmacologia , Benzotiazóis/farmacologia , Compostos de Bifenilo/farmacologia , China , Eletroforese em Gel de Poliacrilamida , Emulsões/química , Ácido Linoleico/antagonistas & inibidores , Ácido Linoleico/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Picratos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ácidos Sulfônicos/farmacologia , Superóxidos/farmacologia , alfa-Tocoferol/antagonistas & inibidores , alfa-Tocoferol/metabolismoRESUMO
Japanese horse chestnut seed extract (HCSE) dose-dependently inhibited the autooxidation of linoleic acid (IC(50): 0.2 mg/ml), and the inhibition was almost complete at a concentration of 1 mg/ml. The HCSE scavenged DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals and superoxide anions with EC(50)s of 0.65 and 0.21 mg/ml, respectively. However, it had no effect on hydrogen peroxide. The HCSE inhibited the genotoxicities of furylfuramide, N-methyl-N-nitrosourea, methyl methanesulfonate, mitomycin C, 2-aminoanthracene and aflatoxin B1 at a concentration of 1 mg/ml or more. Total polyphenol content of the HCSE was 21 mg/g (13 mg/g-seeds). These results indicate that the Japanese horse chestnut seed is an antioxidative and antimutagenic botanical resource.
Assuntos
Aesculus/química , Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Sementes/química , Aflatoxina B1/metabolismo , Antracenos/metabolismo , Relação Dose-Resposta a Droga , Furilfuramida/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácido Linoleico/antagonistas & inibidores , Metanossulfonato de Metila/metabolismo , Metilnitrosoureia/metabolismo , Mitomicina/metabolismo , Fenóis/metabolismo , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. OBJECTIVE: We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. DESIGN: Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. RESULTS: Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. CONCLUSIONS: Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.