Chemical and Biological Evaluation of Amazonian Medicinal Plant Vouacapoua americana Aubl.

Vouacapoua americana (Fabaceae) is an economically important tree in the Amazon region and used for its highly resistant heartwood as well as for medicinal purposes. Despite its frequent use, phytochemical investigations have been limited and rather focused on ecological properties than on its pharmacological potential. In this study, we investigated the phytochemistry and bioactivity of V. americana stem bark extract and its constituents to identify eventual lead structures for further drug development. Applying hydrodistillation and subsequent GC-MS analysis, we investigated the composition of the essential oil and identified the 15 most abundant components. Moreover, the diterpenoids deacetylchagresnone (1), cassa-13(14),15-dien-oic acid (2), isoneocaesalpin H (3), (+)-vouacapenic acid (4), and (+)-methyl vouacapenate (5) were isolated from the stem bark, with compounds 2 and 4 showing pronounced effects on Methicillin-resistant Staphylococcus aureus and Enterococcus faecium, respectively. During the structure elucidation of deacetylchagresnone (1), which was isolated from a natural source for the first time, we detected inconsistencies regarding the configuration of the cyclopropane ring. Thus, the structure was revised for both deacetylchagresnone (1) and the previously isolated chagresnone. Following our works on Copaifera reticulata and Vatairea guianensis, the results of this study further contribute to the knowledge of Amazonian medicinal plants.

Antimicrobial and cytotoxic effects of the Copaifera reticulata oleoresin and its main diterpene acids.

ETHNOPHARMACOLOGICAL RELEVANCE: The oleoresin of Brazilian Copaifera reticulata is a traditional remedy used for the treatment of skin and urinary tract infections, respiratory diseases, rheumatism, ulcer and tumours; thus, playing an important role in the primary health care of the indigenous population. AIM: As most previous pharmacological tests used the crude oleoresin and only a few studies so far dealt with enriched fractions or pure chemically defined compounds, the aim of this study was to systematically evaluate the antimicrobial and cytotoxic properties of the Copaifera reticulata oleoresin and to assign traditional uses to specific secondary metabolites. MATERIALS AND METHODS: The oleoresin, as well as its neutral and acidic fractions were tested for their activity against six cancer cell lines, two clinically relevant bacterial strains, and two dermatophytes. Both fractions were analysed by GC-MS and UHPLC-ELSD, respectively. The antibacterial acidic phase was further fractionated by preparative chromatography to purify and characterize the compounds responsible for the observed pharmacological effect. RESULTS: Whereas no cytotoxic activity was detected, the crude oleoresin and its acidic fraction showed antibacterial activity against gram-positive bacteria Enterococcus faecium (IC50 values 4.2 and 4.8 µg/mL, respectively) and methicillin-resistant Staphylococcus aureus (MRSA, IC50 values 5.3 and 7.2 µg/mL, respectively). Purification of the acidic fraction of the C. reticulata oleoresin yielded two dicarboxylic diterpene acids and the four main diterpene acids, comprising three different diterpene scaffolds. Interestingly, the activity was not restricted to a particular diterpene-type but rather depended on the compounds' lipophilicity, with the most active constituent showing IC50 values of 1.6 (E. faecium) and 2.5 µg/mL (MRSA), respectively. Furthermore, ent-polyalthic acid, the major diterpenoid, was significantly active against dermatophytes with IC50 values of 6.8 µg/mL (Trichophyton rubrum) and 4.3 µg/mL against (T. mentagrophytes). CONCLUSION: The present study proved the antimicrobial effects of the C. reticulata oleoresin and its diterpenoid constituents, confirming its wide use in folk medicine for the treatment of skin and urinary tract infections. The inhibitory activity of copaiba diterpenoids against dermatophytic fungi as well as the gram-positive bacteria E. faecium and MRSA is being reported for the first time, providing potential lead structures for the treatment of these clinically relevant bacterial strains.

Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR.

In this study, we present the quantitation of eight diterpene acids in the oleoresin of Copaifera reticulata Ducke by UHPLC-ELSD and quantitative HSQC (heteronuclear single quantum correlation spectroscopy). UHPLC was performed using reversed phase material and external calibration and showed RSD values of ≤ 3% (repeatability) and ≤ 4% (precision), and mean recovery rates of 91.2-104.8%. LOQs were determined with 10 and 20 µg/mL, and LODs with 4 and 8 µg/mL, respectively. For the qHSQC method, calibration curves of eight different NMR cross-peaks (furylic, endo- and exocyclic methine signals, exocyclic methylene and methyl signals) were established and normalized with dimethyl terephthalate, which served as internal standard. This approach allowed the direct quantification of four major and one minor diterpene, whereas simple calculation procedures led to the contents of the remaining minor compounds. Comparison with the results of the UHPLC assay showed good agreement for seven of the eight diterpene acids. In terms of precision, the qHSQC method was advantageous for the quantification of the three main compounds, whereas UHPLC-ELSD was superior in the determination of the minor components. In contrast to previous reports, kolavenic acid was identified as a major diterpene acid in the oleoresin of Copaifera reticulata, with amounts of 4.0 ± 0.3%.