Differentiation of black and white pitch (Burseraceae) oleoresins: A mass spectrometry-based chemoethnotaxonomic study.

ETHNOPHARMACOLOGICAL RELEVANCE: In the quilombola communities of the municipality of Oriximiná (Pará State, Brazil), Protium spp. (Burseraceae) oleoresins are distinguished in black and white pitch. White pitch oleoresins may be superior to black pitch in terms of quality, but the criteria used for their differentiation are fairly subjective. AIM OF THE STUDY: This study was designed to provide a scientific rationale for the traditional differentiation of black and white pitch oleoresins based on their non-volatile fraction. MATERIALS AND METHODS: Black and white pitch oleoresin samples collected in quilombola territories in Oriximiná were analysed by GC-EI-MS and UPLC-APCI-MS. The feasibility of EI and APCI mass spectrometry-based pattern recognition methods PLS-DA and Random Forest Analysis (RFA) for black and white pitch oleoresins differentiation was demonstrated. RESULTS: The UPLC-APCI-MS method allowed the separation of 43 triterpenoids. Assessment of the triterpenoid fingerprints by GC-EI-MS led to the tentative identification of ursa-9(11),12-dien-3-ol as a potential marker for black pitch oleoresins. PLS-DA and RFA applied to the APCI-MS and EI-MS data gave good models for black and white pitch oleoresins classification. The most important ions for the classifications of black pitch oleoresins by APCI-MS/PLS-DA and APCI-MS/RFA likely represented triterpenoid acids. CONCLUSIONS: The triterpenoid pattern differs between black and white pitch oleoresins. The characteristic presence of ursa-9(11),12-dien-3-ol and triterpenoids acids in black pitch oleoresins, along with other field observations, suggest that black pitch oleoresins are actually aged white pitch oleoresins.

In vitro leishmanicidal activity of monoterpenes present in two species of Protium (Burseraceae) on Leishmania amazonensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis is a neglected disease that affects millions of people around the world. Parasite resistance and the toxicity to the current treatments lead to the search for new effective molecules. Plants are widely used in traditional and indigenous medicine to treat different diseases. The oleoresin of the genus Protium, which is rich in volatile compounds active against different microorganisms, is among these plants. AIM: The aim of this study was to evaluate the leishmanicidal potential of Protium altsonii (PaEO) and P. hebetatum (PhEO) (Burseraceae) oleoresins, as well as of three representative monoterpenes in their constitution: α-pinene, p-cymene and 1,8-cineole. MATERIALS AND METHODS: Protium altsonii (PaEO) and P. hebetatum (PhEO) oleoresins and three of their constituents were tested in vitro on promastigotes and amastigotes-infected macrophages in different concentrations. Their toxicity for macrophages was analyzed by XTT assay and phagocytic ability. It was evaluated the ability of the compounds to induce NO production on treated-macrophages using Griess reaction and the effect of them in lipid profile on treated-parasite through Thin Layer Chromatography. RESULTS: Our data showed that both essential oils have toxic effect on promastigotes and amastigotes of L. amazonensis in vitro in a dose-dependent manner. PaEO IC50 were 14.8 µg/mL and 7.8 µg/mL and PhEO IC50s were 0.46 µg/mL and 30.5 µg/m for promastigotes and amastigotes, respectively. Toxicity to macrophages was not observed at 50 µg/mL with both EOs. The compounds 1,8- cineole, α-pinene, and p-cymene inhibited amastigotes survival in a dose-dependent manner with IC50s of 48.4 µg/mL, 37 µg/mL, 46 µg/mL, respectively. Macrophage viability was around 90% even at 200 µg/mL and the phagocytic capacity was not altered in the treated-macrophages to up 50 µg/mL. The compounds were not able to modulate the nitric oxide production either at rest or LPS-activated macrophages. In addition, treated promastigote revealed an important change in their lipid profile after 48 h at 50 µg/mL in the presence of the compounds. CONCLUSIONS: The results indicate that oleoresins of Protium genus are potent against Leishmania and α-pinene, p-cymene and 1,8-cineole have anti-Leishmania properties that could be explored in synergistic assays in order to develop new drug candidates.

Antibacterial substances from leaves of Protium spruceanum (Burseraceae): in vitro and in silico evaluation

Due to the increase of bacterial resistance, the search for new antibiotics is necessary and the medicinal plants represent its most important source. The aim of this study was to evaluate the antibacterial property of extract and fractions from Protium spruceanum leaves, against pathogenic bacteria. By means of diffusion and microdilution assays, the crude extract was active against the nine bacteria tested being the hydromethanolic fraction the most active. During phytochemical procedures, procyanidin (1) and catechin (2) were identified as the main antibacterial constituents of this fraction. In silico results obtained using PASSonline tool indicated 1 and 2 as having good potential to interact with different targets of currently used antibiotics. These results no indicated potential to none DNA effect and indicated the cell wall as mainly target. Electrophoresis result supported that had no DNA damage. Cell wall damage was confirmed by propidium iodide test that showed increased membrane permeability and by cell surface deformations observed in scanning electronic microscopy. The in vitro assays together with the in silico prediction results establish the potential of P. spruceanum as source of antibacterial compounds that acts on important bacterial targets. These results contribute to the development of natural substances against pathogenic bacteria and to discovery of new antibiotics.

Antibacterial activity and chemical compounds of leaves and branches of Protium hebetatum / Atividade anti bacteriana e compostos químicos das folhas e ramos de Protium hebetatum

ABSTRACT The extracts and fractions of leaves and branches of Protium hebetatum D. C. Daly (Burseraceae) were investigated for their antibacterial activity and chemical composition. The methanol extract of branches (EMG) was considered active against the Escherichia coli and the Proteus vulgaris, showing an inhibition zone of 13 mm, and was selected for bioassay-guided phytochemical fractionation. From the technique of broth microdilution, the extract was considered a moderate inhibitor against Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis, with a minimum inhibitory concentration (MIC) of 1 mg/mL. The dichloromethane fraction was considered a moderate inhibitor against S. aureus (MIC of 1 mg/mL) and a potent inhibitor against E. faecalis (MIC of 0.5 mg/mL). F1, F2, F5 and F6 from chromatographic column of dichloromethane fraction were considered moderate inhibitors against S. aureus (MIC of 1 mg/mL). Through analysis by a gas chromatography mass spectrometry, eighteen compounds were identified, from which thirteen (isoeugenol, p-vinylguaiacol, metoxyeugenol, coumarin, 5-hydroxy-scopoletin, 4,7-dihydroxy-6-metoxicromam-2-one, 4[(1E]-3-hydroxy-1-propenyl)-2-methoxyphenol, piperonal, scoparon, o-guaiacol, spathulenol, seringol and antiarol) are unprecedented in these species. We also identified the triterpenes α-amyrin and β-amyrin, the steroids stigmasterol and sitosterol and the coumarin scopoletin, which was closely linked to the antibacterial activity of the samples.

RESUMO Atividade antibacteriana e compostos químicos de folhas e galhos de Protium hebetatum. Extratos e frações de folhas e galhos de Protium hebetatum D. C. Daly (Burseraceae) foram investigados quanto sua atividade antibacteriana e composição química. O extrato metanólico dos galhos (EMG) foi considerado ativo contra Escherichia coli e Proteus vulgaris, apresentando um halo de inibição de 13 mm, sendo selecionado para um fracionamento fitoquímico biomonitorado. A partir da técnica de microdiluição em caldo o EMG foi considerado um inibidor moderado contra Staphylococcus aureus, Pseudomonas aeruginosa e Enterococcus faecalis, apresentando uma concentração inibitória mínima (CIM) de 1mg/mL. A fração diclorometânica foi considerada inibidora moderada contra S. aureus (CIM de 1 mg/mL) e inibidora potente contra E. faecalis (CIM de 0,5 mg/mL). F1, F2, F5 e F6 provenientes da fração diclorometânica foram consideradas inibidoras moderadas contra S. aureus (CIM de 1 mg/mL). Através da análise por cromatografia gasosa acoplada a espectrometria de massa, foram identificados dezoitos compostos, dos quais treze (isoeugenol, p-vinilguaiacol, metoxieugenol, cumarina, 5-hidroxi-escopoletina, 4,7-dihidroxi-6-metoxicromam-2-ona, 4[(1E]-3-hidroxi-1-propenil)-2-methoxifenol, piperonal, escoparona, o-guaiacol, espatulenol, seringol e antiarol) foram identificados pela primeira vez nesta espécie. Foram também identificados os triterpenos α-amirina e β-amirina, os esteroides estigmasterol e sitosterol e a cumarina escopoletina, que estão intimamente ligados à atividade antibacteriana da espécie.