Leishmaniasis is a disease that affects millions of people and it is an important public health problem. The drugs currently used for the treatment of leishmaniasis present undesirable side effects and low efficacy. In this study, we evaluated the in vitro activity of Melampodium divaricatum (MD-EO) and Casearia sylvestris (CS-EO) essential oils (EO) against promastigote and amastigote forms of Leishmania amazonensis . Sesquiterpenes E -caryophyllene (56.0%), germacrene D (12.7%) and bicyclogermacrene (9.2%) were identified as the main components of MD-EO , whereas E -caryophyllene (22.2%), germacrene D (19.6%) and bicyclogermacrene (12.2%) were the main constituents of CS-EO. CS-EO and E -caryophyllene were active against promastigote forms of L. amazonensis (IC 50 24.2, 29.8 and 49.9 µg/mL, respectively). However, MD-EO, CS-EO and E -caryophyllene were more active against amastigote forms, with IC 50 values of 10.7, 14.0, and 10.7 µg/mL, respectively. E -caryophyllene presented lower cytotoxicity against macrophages J774-A1 (CC 50 of 62.1 µg/mL) than the EO. The EOs and E -caryophyllene should be further studied for the development of new antileishmanial drugs.

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Rationale Clerodane‐type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti‐inflamatory. There are several mass spectrometry (MS)‐based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin‐like compounds. Methods Casearin‐like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time‐of‐flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. Results Casearin‐like compounds presented a predominance of sodium‐ and potassium‐cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3, both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. Conclusions Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2, R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin‐like compounds present in complex extracts of Casearia species.

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Casearia sylvestris is an endemic tree of the Latin America that the essential oil (EO) has anti‐inflammatory and gastroprotective actions. This study evaluates the chemical composition of the EO from the volatile fractions of in natura, fresh, and dried C. sylvestris var. sylvestris and var. lingua leaves. For both varieties, the dried leaves presented higher EO yield as compared to fresh leaves. The major EO chemical components were (E)‐caryophyllene, α‐humulene, germacrene D, bicyclogermacrene, spathulenol, caryophyllene oxide, and humulene epoxide II. In both varieties, the content of sesquiterpene hydrocarbons decreased and oxygenated sesquiterpenes increased on going from in natura to fresh and dried leaves, which indicated that leaf drying and hydrodistillation modified the volatile composition. The results also suggested that bicyclogermacrene and (E)‐caryophyllene were oxidized during processing, to generate spathulenol and caryophyllene oxide, respectively. C. sylvestris varieties and in natura, fresh, and dried leaves differed in terms of the chemical composition of volatiles, which could affect the EO biological activities.

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory and antiulcerogenic activities. The clerodane diterpenes casearin B (cas B) and caseargrewiin F (casg F) are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of cas B and casg F were not previously investigated. We aimed to assess the stability of cas B and casg F in physiological conditions and their metabolism in human liver microsomes (HLM). The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of cas B and casg F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism is not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed log P in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with KM values of 61.4 μM and 66.4 μM and Vmax values of 327 and 648 nmol/min/mg of protein for cas B and casg F, respectively. Metabolism parameters in HLM were extrapolated to predict human hepatic clearance and suggest that casg F and cas B have a high hepatic extraction ratio. In conclusion, our data suggest that casg F and cas B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.

The Casearia sylvestris Sw. dried leaf essential oil (EO) contains sesquiterpenes as the main components. However, the volatile components in the in natura leaves remain unknown. This study compares the volatile chemicals in the in natura leaves and dried leaf EO of two C. sylvestris populations from Atlantic Forest and Cerrado. The volatile compounds were directly analysed by thermal desorption (TD) coupled to gas chromatography mass spectrometry (GC-MS); the dried leaf EO composition was determined by GC-MS. All the identified compounds were sesquiterpenes, and the major components were (E)-caryophyllene, bicyclogermacrene, ?-elemene, spathulenol, and caryophyllene oxide. In both populations, the sesquiterpene hydrocarbon content and the oxygenated sesquiterpene content respectively decreased and increased on going from the in natura leaves to the dried leaf essential oil, indicating that drying and/or hydrodistillation modified the volatile chemical composition by generating oxidation artifacts. Results suggested that (E)-caryophyllene and bicyclogermacrene may be oxidized during the process to yield caryophyllene oxide and spathulenol, respectively. The two C. sylvestris populations also differed in terms of volatile chemical composition.