RESUMEN
Asian honeybee venom is widely used in traditional oriental medicine. Melittin is the main component of Asian honeybee venom. In the present study, an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) method was used for accurate qualitative and quantitative analyses of melittin in Asian honeybee venom. The results showed that the dynamic linear range of melittin was from 0.094 to 20 µg/mL, and the limit of quantification was 0.3125 µg/mL. The spiking recovery of melittin in honeybee venom ranged from 84.88% to 93.05%. Eighteen Asian honeybee venom samples in eighteen batches were collected from two different zones of China, and their melittin contents were measured. The contents of melittin in Asian honeybee venom samples was 33.9-46.23% of dry weight. This method proved a useful tool for the rapid evaluation of the authenticity and quality of Asian honeybee venom in terms of the melittin contents, and will contribute to a broader understanding of Asian honeybee venom.
Asunto(s)
Venenos de Abeja/química , Abejas , Cromatografía Liquida , Meliteno/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Animales , Límite de Detección , Reproducibilidad de los ResultadosRESUMEN
Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques.
Asunto(s)
Antivenenos/uso terapéutico , Venenos de Abeja/antagonistas & inhibidores , Diseño de Fármacos , Mordeduras y Picaduras de Insectos/tratamiento farmacológico , Proteínas de Insectos/antagonistas & inhibidores , Meliteno/antagonistas & inhibidores , Anticuerpos de Cadena Única/uso terapéutico , Animales , Antivenenos/genética , Antivenenos/metabolismo , Antivenenos/farmacología , Venenos de Abeja/química , Venenos de Abeja/enzimología , Venenos de Abeja/toxicidad , Técnicas de Visualización de Superficie Celular , Células Clonales , Quimioterapia Combinada , Edema/etiología , Edema/prevención & control , Hemólisis/efectos de los fármacos , Humanos , Mordeduras y Picaduras de Insectos/fisiopatología , Proteínas de Insectos/análisis , Proteínas de Insectos/toxicidad , Masculino , Meliteno/análisis , Meliteno/toxicidad , Ratones , Inhibidores de Fosfolipasa A2/farmacología , Inhibidores de Fosfolipasa A2/uso terapéutico , Fosfolipasas A2 Secretoras/antagonistas & inhibidores , Fosfolipasas A2 Secretoras/toxicidad , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Cadena Única/farmacología , Tejido Subcutáneo/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
A high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC-DAD-MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1-100 and 0.2-25 microg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 microg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.