Avaliação química e biológica de fungos endofíticos isolados de Vismia japurensis (Hypericaceae)

Abstract

Endophytic fungi are known to inhabit the aerial and underground parts of plants without causing any apparent damage. These microorganisms have been the subject of studies and have shown promise in the production of bioactive substances, given that they can produce secondary metabolites with varied activities, just like their host plant. In this sense, the Hypericaceae family has species that produce biologically active substances, such as the species Vismia japurensis, which in chemical and biological studies demonstrated the ability to produce substances with antimicrobial, antioxidant, toxic, antimalarial activities, among others. This species, as well as other representatives of Hypericaceae, lack studies regarding their endophytic fungi. Therefore, this study aimed to evaluate the chemical and biological characteristics of the endophytic fungi of V. japurensis. Initially, healthy leaves of V. japurensis were collected at UFAM mini campus and, through disinfestation processes, 19 fungi of this species were isolated, 14 of which were cultivated in Sabouraud (SB) liquid medium and methanolic extracts of the mycelia were obtained. The extracts were analyzed by TLC, resulting in the inference of secondary metabolite classes. In the toxicity test against Artemia salina, extract VJ15 caused mortalities of 66% (1000 μg/mL) and 33% (500 μg/mL), being considered toxic and moderately toxic, respectively. In the antibacterial test, extract VJ15 showed good activity, inhibiting the Staphylococcus aureus strain by 69% (1000 μg/mL). Extracts VJ10 and VJ12 showed moderate activity, inhibiting the Morganella morganii strain by 51% and 58%, respectively. Through the results of the analyses by CCDC and biological tests, extract VJ15 was selected for species identification and cultivation on a larger scale (5L) under the same cultivation conditions as the smaller scale. The mycelium of VJ15 was subjected to identification by molecular tools and identified at the genus level as Diaporthe sp. The extract of VJ15 was partitioned to obtain two phases (ethyl acetate and hydromethanol). The acetate phase stood out in the analysis by CCDC, revealing all the used developers. In the toxicity test, neither phase of Diaporthe sp. presented toxic activity, however, they can be used in other bioassays. In the antibacterial activity, they also did not present inhibitory activity against S. aureus. These results suggest an antagonistic effect regarding the synergy of the secondary metabolites in the extracts when subjected to partition. The acetate phase was chosen for fractionation in a chromatographic column utilizing neutral alumina and silica gel 60 as the stationary phases. From the fractionation of the fraction F27-28 (F14-16) in silica gel 60, using the Nuclear Magnetic Resonance method, it was possible to isolate two alkaloids in a mixture: proline betaine (1) and glycine betaine (2). Therefore, this work demonstrates the importance of studying the endophytic fungi of V. japurensis, which may be producers of substances with skeletons different from the mother plant from which they were obtained.