Copaifera species (Fabaceae) comprises approximately 70 species of large trees, from which 16 can be found in Brazil. The oleoresins obtained from their trunk are widely used in Brazilian folk medicine, which display important antitumoral potential. Chemically, these oleoresins are mainly composed of a mixture of sesquiterpenes and diterpenes. In this paper we are describing the isolation and identification of 12 already known terpenes from oleoresins obtained from three different Copaifera species (C. multijuga, C. pubiflora and C. trapezifolia) and 2 novel diterpenes (ent-16-hidroxy-3,13 clerodadien-15,18-dioic acid and ent-labda-5,13-dien-15-oic acid) from C. trapezifolia. Both new compounds were identified by nuclear magnetic resonance (NMR) spectroscopic (1H and 13C NMR, correlation 1H-1H (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC)) and by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) analyses. The cytotoxic potential of these oleoresins, their main non-volatile compounds and their volatile compound fractions were evaluated against a panel of tumoral (MCF-7, ACP01, A549, HeLa) and normal cell lines (MCF-10A, GM07492-A) through XTT (tetrazolium salt) and SRB (sulforhodamine B) assays. The novel diterpene ent-labda-5,13-dien-15-oic acid displayed relevant cytotoxic effect against most of the cancer cell lines with mean inhibitory concentration (IC50) values ranging from 3.57 ± 1.12 to 22.56 ± 1.03 µg mL-1, and a high selectivity level in both assays.

In this work, a search for antimicrobial agents against multi-drug resistant (MDR) bacteria was undertaken. It involved two natural diterpenes of different basic skeletons, named ent-kaurenoic acid and ent-pimaradienoic acid, that were used as precursors to access 28 semi-synthetic derivatives that were also submitted to biological assays. All 30 substances were assayed against a set of seven clinically isolated MDR bacteria, including three Staphylococcus aureus strains. Results classified both natural compounds as promising bactericidal antimicrobial agents against these MDR bacteria, especially for their performance in time-kill curve assay, improving on the positive control vancomycin. Moreover, di-hydro ent-pimaradienoic acid, obtained as one of the derivatives, also displayed promising minimum inhibitory concentration (MIC) values against the microorganism set, proving optimum activity against S. aureus strains. Thereby, this work also yielded a promising semi-synthetic bacteriostatic antimicrobial agent against MDR S. aureus. Furthermore, the study was carried out with 30 structures in order to verify the veracity and scope of Urzúa’s hypothesis about the structure-activity relationship between diterpenes and antimicrobial action. Our results are 93% in agreement with this hypothesis.

Oleoresins from Copaifera species are extensively used in folk medicine in Brazil, which are employed mainly in the production of cosmetic formulations in Brazil, North America and Europe. Considering the lack of validated analytical methods for the analyses of diterpenes in Copaifera oleoresins, it was developed a validated and reliable reversed-phase high-performance liquid chromatography with photodiode array detection (RP-HPLC-PDA) method for the analysis of six diterpenes, including: (-)-polyalthic acid; (13E)-ent-labda-7,13-dien-15-oic acid; ent-8(17)-labden-15,18-dioic acid; (-)-copalic acid; (-)-3β-acetoxycopalic acid and (-)-3β-hydroxycopalic acid. These compounds were isolated from C. duckei, C. reticulata and C. multijuga oleoresins by chromatographic means. The analytical curves were linear with regression coefficients (r2) between 0.9903 and 0.9999. The limits of detection (LOD) and quantification (LOQ) values were 0.35 to 3.09 µg mL-1 and 1.05 to 9.36 µg mL-1, respectively. The method also displayed good precision and accuracy. The developed analytical method is reliable and a useful tool for the analysis of Copaifera oleoresin and its products.

The labdane diterpenes copalic acid, 3β-acetoxy-copalic acid, 3β-hydroxy-copalic acid and ent-agathic acid were isolated from Copaifera langsdorffii oleoresin. These four compounds were submitted to structural modifications by reduction with hydrogen/palladium, esterification with diazomethane, esterification with methanol/sulfuric acid and conversion into sodium salt, furnishing 15 compounds. All compounds were assayed in vitro against Mycobacterium tuberculosis (H37Rv, ATCC 27294). The four compounds displayed minimum inhibitory concentration (MIC) value of 125 µg mL-1, and were not considered active. A methylated derivative of compound 3β-hydroxy-copalic acid, and a sodium salt of copalic acid displayed MIC values of 25 µg mL-1 (71.7 µM) and 6.25 µg mL-1 (19.2 µM), respectively. The sodium salt of copalic acid stood out by displaying similar activity in comparison with streptomycin (MIC 6.25 µg mL-1) and a better activity compared to µM value of pyrazinamide (MIC 3.12 µg mL-1; 25.34 µM). Therefore, the methylated derivative of compound 3β-hydroxy-copalic acid and the sodium salt of copalic acid should be considered for further studies.

This work describes the phytochemical study of the extracts from aerial parts of Tibouchina candolleana as well as the evaluation of the antimicrobial activity of extracts, isolated compounds, and semi-synthetic derivatives of ursolic acid against endodontic bacteria. HRGC analysis of the n-hexane extract of T. candolleana allowed identification of b-amyrin, a-amyrin, and b-sitosterol as major constituents. The triterpenes ursolic acid and oleanolic acid were isolated from the methylene chloride extract and identified. In addition, the flavonoids luteolin and genistein were isolated from the ethanol extract and identified. The antimicrobial activity was investigated via determination of the minimum inhibitory concentration (MIC) using the broth microdilution method. Amongst the isolated compounds, ursolic acid was the most effective against the selected endodontic bacteria. As for the semi-synthetic ursolic acid derivatives, only the methyl ester derivative potentiated the activity against Bacteroides fragilis.