Cultura de tecidos vegetais in vitro de Vismia japurensis Reichardt (Hypericaceae)

Abstract

Vismia japurensis is known for its ecological importance, as well as for the production of substances with biological activities. Thus, the present work aimed to establish in vitro V. japurensis and the chemical and biological evaluation of seedling extracts. Disinfestation tests were carried out with fungicide solution, 70% ethanol and sodium hypochlorite; for germination, Murashige and Skoog (MS) and Wood Plant Medium (WPM) media were evaluated with different concentrations of gibberellic acid (GA3). After establishment, the seedlings were multiplied using MS, MS with 50% N, and WPM, with different concentrations of 6-benzylaminopurine (BAP) and kinetin (KIN). Callus formation was induced using different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (ANA), BAP and KIN in MS, MS with nitrate reduction and WPM media. Microextraction of seedlings was performed with methanol, and the chemical content of seedlings grown in MS and WPM were compared by comparative thin layer chromatography (CCDC) and hydrogen nuclear magnetic resonance (1H NMR). A new cultivation in MS medium was carried out to obtain a greater mass, after 60 days in medium without addition of hormones, the seedlings were removed, washed and lyophilized, being subsequently extracted with hexane, methanol and water. The extracts were analyzed by CCDC and 1H NMR. The hexane extract was subjected to chromatographic fractionation in an open column, and the fractions of interest were purified by high performance liquid chromatography (HPLC). The structural determination of the isolated substance was performed using spectroscopic methods (1H NMR, including two-dimensional techniques: heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC) and correlation spectroscopy (COZY); and by mass spectrometry. The hexane extract was evaluated for allelopathic, antimicrobial and angiogenic activity, and the dichloromethane (DCM) and ethyl acetate (AcOEt) phases were evaluated for antimicrobial activity. As a result, in the disinfestation and germination tests, the seeds showed low contamination, and a higher percentage of germination in WPM medium supplemented with GA3. The multiplication had a larger number of shoots when supplemented with BAP for all evaluated culture media. As for the analysis of the chemical profile by CCDC and 1H NMR it was possible to verify the presence of anthraquinones, triterpenes and flavonoids in the hexanic extract, in the AcOEt phase evidence of flavonoids, and in the DCM phase, anthraquinones. From the phytochemical study of the hexane extract of the seedlings of V. japurensis, 1,8,10-trihydroxy-3-methyl-9(10H)-anthracenone (IUPAC/UIQPA) was isolated, which, according to the bibliographic survey is unprecedented. As for biological activities, the hexane extract in the MIC of 1000 μg/mL presented inhibition in the growth of Staphylococcus aureus, the DCM phase was active against: Aeromonas hydrophila, Citrobacter freundii, Escherichia coli and Serratia marcescens. As for angiogenic activity, hexane extract reduced the percentage of vessels in chicken eggs. For allelopathic activity, the hexane extract of the seedlings in vitro inhibited the growth of Lactuca sativa, a more pronounced activity than that of leaves and branches. This is the first study regarding the establishment and chemical and biological evaluation carried out with this species in vitro, and it is also the first isolation report of this anthrone.