DEVELOPMENT AND VALIDATION OF AN HPLC-DAD ANALYTICAL METHOD TO QUANTIFY 5-METHOXYFLAVONES IN METHANOLIC EXTRACTS OF Vochysia divergens POHL CULTURED UNDER STRESS CONDITIONS
Vochysia divergens Pohl, known as "Cambara" in Brazil, is an invasive species that is expanding throughout Pantanal in Brazil, to form mono-dominant communities. This expansion is affecting the agricultural areas that support the typical seasonal flood and drought conditions of this biome. This article describes the development and validation of an HPLC-DAD analytical method to quantify 5-methoxyflavones in methanolic extracts of greenhouse-grown V. divergens associated with one of two endophytic fungal species Zopfiella tetraspora (Zt) or Melanconiella elegans (Me) and later subjected to water stress. The developed method gave good validation parameters and was successfully applied to quantify the flavones 3',5-dimethoxy luteolin-7-O-β-glucopyranoside (1), 5-methoxy luteolin (2), and 3',5-dimethoxy luteolin (3) in the target extracts. Inoculation of the plant with Zt decreased the concentration of flavone 1 in the extract by 2.69-fold as compared to the control. Inoculation of the plant with Zt or Me did not significantly alter the contents of flavones 2 and 3 in the extracts as compared to the control. Therefore, the aerial parts of germinated V. divergens plants inoculated with either Zt or Me responded differently in terms of the production of flavones. These results can cast light on the symbiosis between fungal microorganisms and V. divergens, which most likely influences the response of V. divergens to changes in the availability of water in Pantanal.