High performance liquid chromatography determination of cucurbitacins in the roots of Wilbrandia ebracteata Cogn.

Abstract

RESUMO: "Determinação de cucurbitacinas em raízes de Wilbrandia ebracteata Cogn. por cromatografia líquida de alta eficiência". Raízes de Wilbrandia ebracteata Cogn. (Cucurbitaceae), tradicionalmente empregada no tratamento de doenças reumáticas, contém cucurbitacinas, sendo di-hidrocucurbitacina B a mais abundante, enquanto cucurbitacina B está presente em menor quantidade. Foi desenvolvido um método para determinação quantitativa destas cucurbitacinas. Os parâmetros selecionados foram: eluente isocrático acetonitrila/H 2 0 40:60, fluxo 1,2 mL/min. e detecção em 230 nm. Diversas formas de preparo da amostra foram testadas, sendo que extração com diclorometano sob refluxo forneceu o melhor resultado. O processo de validação incluiu: linearidade, exatidão, repetibilidade e precisão intermediária. A curva de calibração para a dihidrocucurbitacina B foi linear de 40.00 to 400 µg/mL, a recuperação foi 95,5±3.01%, a precisão intermediária, 1,64% e a repetibilidade variou entre 1,30 a 2,05%. A curva de calibração da cucurbitacina B foi linear de 4,00 to 240 µg/mL, a recuperação encontrada foi igual a 96,6±2.45%, a precisão intermediária, 2,29% e a repetibilidade variou entre 1,03 a 2,95%. Análise do mesmo espécime de W. ebracteata uma vez por ano de 2002 a 2005 revelou grande aumento no teor de cucurbitacina B após a raiz ter sido atacada por herbívoro. Unitermos:Wilbrandia ebracteata, Cucurbitaceae, cucurbitacina B, di-hidrocucurbitacina B, interação planta herbívoro, validação.ABSTRACT: Roots of Wilbrandia ebracteata Cogn., Cucurbitaceae, used in folk medicine for treatment of rheumatic disease, are rich in cucurbitacins. Dihydrocucurbitacin B is the most abundant cucurbitacin while cucurbitacin B is a minor component. A reverse-phase HPLC system was developed for simultaneous quantitative assay of these cucurbitacins in the roots. The optimised experimental conditions were acetonitrile/H 2 0 40:60, flow-rate 1.2 mL/min., detection at 230 nm and isocratic elution. A variety of sample preparation modes were tested and the extraction with dichloromethane under reflux gave better results. The validation process included linearity, accuracy, repeatability and intermediate precision. The calibration curve of dihydrocucurbitacin B was linear from 40.00 to 400 µg/mL, the recovery was 95.5±3.01%, the intermediate precision was found to be 1.64% and the repeatability varied between 1.30 to 2.05%. The calibration curve of cucurbitacin B was linear from 4.00 to 240 µg/mL, intermediate precision was found to be 2.29% and repeatability varied between 1.03 to 2.95%. Analysis of the same specimen of W. ebracteata every year from 2002 to 2005 revealed a great rise on the cucurbitacin B concentration after the root was attacked by an herbivore. Keywords:Wilbrandia ebracteata, Cucurbitaceae, cucurbitacin B, dihydrocucurbitacin B, plantherbivore interaction, accuracy.disease (Corrêa, 1984, Simões et al., 1986. The antiinflammatory activity of the dichloromethanic fraction has been demonstrated (Peters et al., 1997;Peters et al., 1999)...

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Production of Wilbrandia ebracteata extract standardized in flavonoids and dihydrocurcubitacin and assessment of its topical anti-inflammatory activity

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