RESUMO
Ruscogenin (RUS) is a steroidal sapogenin found in Ruscus aculeatus and Ophiopogon japonicus with several pharmacological activities. In the work reported herein, a novel method termed competitive fluorescence-linked immunosorbent assay (cFLISA) based on monoclonal antibodies (mAbs) coupled with quantum dots (QDs) was developed for the quick and sensitive determination of RUS in biological samples. The mAbs against RUS were conjugated with CdSe/ZnS QDs by the crossing-linking reagents and an indirect cFLISA method was developed. There was a good linear relationship between inhibition efficiency and logarithm concentration of RUS which was varied from 0.1 to 1000 ng/mL. The IC50 and limit of detection (LOD) were 9.59 ng/mL and 0.016 ng/mL respectively, which much lower than the enzyme-linked immunosorbent assay (ELISA) method. The recoveries in plasma and tissues were ranged from 82.3% to 107.0% and the intra- and inter-day precision values were below 15%. The developed cFLISA has been successfully applied to the measurement of the concentrations of RUS in biological samples of rats, and showed great potential for the tissue distribution study of RUS. The cFLISA method may provide a valuable tool for the analysis of small molecules in biological samples and such an approach could be applied to other natural products.
Assuntos
Anticorpos Monoclonais/química , Compostos de Cádmio/química , Corantes Fluorescentes/química , Fluorimunoensaio/métodos , Pontos Quânticos/química , Compostos de Selênio/química , Espirostanos/análise , Compostos de Zinco/química , Animais , Anticorpos Monoclonais/imunologia , Reagentes de Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática/métodos , Fluorescência , Humanos , Limite de Detecção , Tamanho da Partícula , Ratos Sprague-Dawley , Espirostanos/imunologia , Propriedades de SuperfícieRESUMO
A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known compounds, its structure was established as (3ß,5α,6ß,25R)-3-{(O-ß-D-glucopyranosyl-(1â3)-ß-D-glucopyranosyl-(1â2)-O-[O-ß-D-glucopyranosyl-(1â3)]-O-ß-D-glucopyranosyl-(1â4)-ß-D-galactopyranosyl)oxy}-6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune response against ovalbumin antigen.
Assuntos
Adjuvantes Imunológicos , Eritrócitos/efeitos dos fármacos , Cebolas/química , Saponinas/química , Saponinas/farmacologia , Espirostanos/química , Espirostanos/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Hemólise , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Conformação Molecular , Ovalbumina/imunologia , Saponinas/imunologia , Espirostanos/imunologia , Esteroides/químicaRESUMO
Ruscogenin is a major bioactive steroidal aglycone found in the Chinese medicine, Ophiopogon japonicus. We have developed a quantitative determination of ruscogenin with an indirect enzyme-linked immunosorbent (ELISA) assay using polyclonal antibodies against ruscogenin conjugated with bovine serum albumin. This assay was highly sensitive, and it had considerably less cross-reactivity to diosgenin and sarsasapogenin, but high cross-reactivity to ruscogenin glycosides. The assay was successfully used for the measurement of ruscogenin concentrations in crude Chinese medicines and in biological samples from a pharmacokinetics study of ruscogenin.
