Metabolism Characterization and Chemical and Plasma Stability of Casearin B and Caseargrewiin F.

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

Green Chromatographic Methods and in Vitro Pharmacological Analysis of Varronia curassavica Leaf Derivatives.

Varronia curassavica displays anti-inflammatory, antiulcerogenic, and antioxidant activities. Herein, we employed new UHPLC -UV green chromatographic methods for the analysis of in vitro antioxidant and anti-inflammatory activities of V. curassavica and its embryotoxicity in Zebrafish. Cordialin A, brickellin, and artemetin were purified from the ethanol (EtOH) extract of V. Curassavica leaves and identified using spectrometric techniques. In line with Green Analytical Chemistry principles, the proposed UHPLC methods involve the use of ethanol as organic modifier with low mobile phase consumption, and without sample pretreatment (OLE-UHPLC-UV). The application of the Agree and HPLC-EAT tools for greenness assessment yielded this pattern: HPLC-UV (reference)

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

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.

Aloe vera-loaded natural rubber latex dressing as a potential complementary treatment for psoriasis.

Psoriasis is a disease that causes keratinocytes to proliferate ten times faster than normal, resulting in chronic inflammation and immune cell infiltration in the skin. Aloe vera (A. vera) creams have been used topically for treating psoriasis because they contain several antioxidant species; however, they have several limitations. Natural rubber latex (NRL) has been used as occlusive dressings to promote wound healing by stimulating cell proliferation, neoangiogenesis, and extracellular matrix formation. In this work, we developed a new A. vera-releasing NRL dressing by a solvent casting method to load A. vera into NRL. FTIR and rheological analyzes revealed no covalent interactions between A. vera and NRL in the dressing. We observed that 58.8 % of the loaded A. vera, present on the surface and inside the dressing, was released after 4 days. Biocompatibility and hemocompatibility were validated in vitro using human dermal fibroblasts and sheep blood, respectively. We observed that ~70 % of the free antioxidant properties of A. vera were preserved, and the total phenolic content was 2.31-fold higher than NRL alone. In summary, we combined the antipsoriatic properties of A. vera with the healing activity of NRL to generate a novel occlusive dressing that may be indicated for the management and/or treatment of psoriasis symptoms simply and economically.

Baccharis trimera (Less.) DC leaf derivatives and eupatorin activities against Leishmania amazonensis.

Natural products have been largely explored as treatments for leishmaniasis, neglected diseases with few toxic therapeutic options, as scaffolds for the development of new drugs. Herein, derivatives from the aerial parts of Baccharis trimera (Less.) DC (extract and its fractions) were evaluated against Leishmania amazonensis and macrophage cells. The ethyl acetate extract was fractionated by solid-phase extraction, resulting in eight fractions (F1-F8). Fractions F3-4 were further separated into 149 subfractions; subfraction 148 (IC50-PRO = 1.56 ± 0.1 µg mL-1) was selected for purification and constituent(s) characterization by high-performance liquid chromatography, as well as 1H and 13C nuclear magnetic resonance spectroscopy. The flavonoid eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) was identified. This compound was 3.7 times more effective against intracellular amastigotes (IC50-AMA = 1.6 ± 0.1 µM) than amphotericin B and presented low cytotoxicity (CC50 > 100 µM), being almost 62 times more selective for the parasite, showing great potential in drug development for cutaneous leishmaniasis treatment.

Antileishmanial activity of Melampodium divaricatum and Casearia sylvestris essential oils on Leishmania amazonensis.

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 (IC50 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 IC50 values of 10.7, 14.0, and 10.7 µg/mL, respectively. E-caryophyllene presented lower cytotoxicity against macrophages J774-A1 (CC50 of 62.1 µg/mL) than the EO. The EOs and E-caryophyllene should be further studied for the development of new antileishmanial drugs.

In vitro and in vivo anti-Helicobacter pylori activity of Casearia sylvestris leaf derivatives.

ETHNOPHARMACOLOGICAL RELEVANCE: The number of bacterial strains that are resistant to multiple conventional antimicrobial agents is increasing. In this context, natural products have been widely used as a strategy to treat diseases caused by bacteria. Infections by Helicobacter pylori have attracted attention because they are directly related to severe gastric medical conditions. Casearia sylvestris Swartz, popularly known as guaçatonga, is largely employed to treat gastric disorders in Brazilian folk medicine. This plant species has aroused much interest mainly because it displays anti-inflammatory activity and can act as an antiulcer agent. AIM OF THE STUDY: To evaluate the in vitro and in vivo anti-H. pylori action of C. sylvestris leaf derivatives incorporated or not in a nanostructured drug delivery system. MATERIALS AND METHODS: The essential oil (obtained by hydrodistillation) and ethanolic extract (obtained by maceration) were obtained from C. sylvestris leaves. The ethanolic extract was submitted to fractionation through solid phase extraction and column chromatography, to yield the ethanolic fractions. Hydrolyzed casearin J was achieved by submitting isolated casearin J to acid hydrolysis. The derivatives were chemically characterized by nuclear magnetic resonance (NMR), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS) analyses. A nanostructured lipid system was used as drug delivery system. To assess the in vitro antibacterial activity of C. sylvestris leaf essential oil, ethanolic extract, and derivatives, microdilution, biofilm, and time-kill assays were performed against H. pylori ATCC 43504. Finally, the in vivo action was investigated by employing male Wistar rats experimentally infected with H. pylori. RESULTS: Many C. sylvestris leaf derivatives presented significant in vitro activity against H. pylori. Among the derivatives, fraction 2 (F2) was the most effective. In vivo tests showed that both the ethanolic extract and F2 decreased the ulcerative lesion size, but only the ethanolic extract eradicated H. pylori from the gastric lesions. Incorporation of plant derivatives in nanostructured lipid system blunted the in vitro action, as demonstrated by the microdilution assay. However, this incorporation improved the ethanolic extract activity against biofilms. CONCLUSION: C. sylvestris leaf derivatives are effective against H. pylori both in vitro and in vivo. According to phytochemical analyses, these derivatives are rich in terpenoids, which could be related to the anti-H. pylori action. Synergism could also underlie C. sylvestris efficacy judging from the fact that the sub-fractions and isolated compounds had lower activity than the extract. Incorporation in a nanostructured lipid system did not improve the activity of the compounds in our in vivo protocol.

Chemical variability between different organs of the medicinal plant Casearia sylvestris

The phytochemical profile of essential oils and extracts from Casearia sylvestris leaves, flowers and fruits have been investigated here. Leaf and flower extracts were prepared by sonication and analyzed by thin-layer chromatography and high-performance liquid chromatography. The phenolic content was determined by ultraviolet spectrophotometry. Leaves, flowers, and fruits essential oils were extracted by hydrodistillation. The highest extracts yields were 20.3 % (leaves) and 23.4 % (flowers) with ethanol 70 %. Essential oil extraction yields were 0.3 % (leaves) and 0.1 % (flowers and fruits). Bicyclogermacrene was the major component in all essential oil. Thin-layer chromatography suggests a chemical profile similar for leaves and flowers. The leaves and flowers phenolic content were similar (14.0 and 15.0 %, respectively). Chromatography analyses indicated the predominance of casearin clerodane diterpenes in leaves (λmax 232-235), whereas in flowers, diterpenes with a different standard diene in side-chain C13(16) and C14 (λmax 223-229). The different phytochemical profile of C. sylvestris flowers as compared to the leaves could be explored by the search for new bioactive components. This is the first report on the fruit and flower C. sylvestris essential oil composition. These data could be used as quality control of herbal medicine derived from C. sylvestris leaves.(AU)