Anti-inflammatory action of ethanolic extract and clerodane diterpenes from Casearia sylvestris

ABSTRACT The present study aimed to investigate the anti-inflammatory activity of ethanolic extract from Casearia sylvestris Sw., Salicaceae, leaves and to identify the compounds responsible for this activity. The ethanolic extract from C. sylvestris leaves was fractionated by solid phase extraction and the chemical composition of extract and fractions were assessed by chromatographic techniques. Casearin-like clerodane diterpenes were quantified in ethanolic extract (27.4%, w/w) and in fraction 2 of solid phase extraction (50.6%, w/w). Carrageenan-induced paw edema and carrageenan-induced pleurisy assays (rats) were used to evaluate anti-inflammatory activity of ethanolic extract, its fractions and clerodane diterpenes from C. sylvestris - caseargrewiin F and casearin B. The ethanolic extract was tested in the rat paw edema model and the doses tested (10 and 100 mg/kg) had no effect. In the pleurisy model, the extract doses of 300 and 500 mg/kg showed inhibitory effect. The fraction 2 of solid phase extraction (10 mg/kg), caseargrewiin F and casearin B (0.5 mg/kg) showed a significant reduction (p < 0.05) of the carrageenan-induced paw edema in rats compared to indomethacin. Gastric ulcers were not observed in animals treated with samples from C. sylvestris. In conclusion, the ethanolic extract from C. sylvestris, its enriched fraction of clerodane diterpenes, casearin B and caseargrewiin F exhibited anti-inflammatory activity on in vivo models in rats. Casearin-like clerodane diterpenes may be considered active chemical markers for C. sylvestris leaves. On the other hand, these diterpenes are promising compounds in the development of new drugs with anti-inflammatory action without gastric side effects.

Evaluation of morpho-anatomical and chemical differences between varieties of the medicinal plant Casearia sylvestris Swartz.

Casearia sylvestris Swartz (Salicaceae) has been used in traditional medicine and its leaf extracts have been exhibited important pharmacological activities. The species presents morphological, chemical and genetic variation. Two varieties are considered due external morphological differences: C. sylvestris var. sylvestris and var. lingua. There are difficulties in definition of these varieties. The objective of this work is to evaluate chemical and morpho-anatomical differences between C. sylvestris varieties that can be applied in their distinction for pharmaceutical or botanical purposes. Transverse and paradermic sections of leaves were prepared for morpho-anatomical, histochemical and quantitative microscopy (stomatal and palisade index) analyses. Diterpene profiles of the specimens were obtained by HPLC-DAD and TLC. Morpho-anatomical analyses demonstrated significant differences between the varieties only in paradermic sections: var. sylvestris--polygonal epidermic cell walls and hypostomatic; var. lingua--rounded epidermic cell walls and amphistomatic. No differences were observed for stomatal index; palisade index was found 2.8 for var. lingua and 3.9 for var. sylvestris. Chromatographic analyses confirmed previous results demonstrating that diterpene profile in varieties differs, with predominance of these metabolites in var. sylvestris. In conclusion, this work indicates that chromatographic analysis besides morpho-anatomical analysis can be applied in distinction of C. sylvestris varieties.

In vitro cytotoxic activity of Brazilian Middle West plant extracts

Cytotoxic activity of eight plant extracts, native from the Mid-West of Brazil comprising Cerrado, Pantanal and semideciduous forest, was evaluated for MDA-MB-435, SF-295, and HCT-8 cancer cell strains. A single 100 µg.mL-1 dose of each extract was employed with 72 h of incubation for all tests. Doxorubicin (1 µg.mL-1) was used as the positive control and the MTT method was used to detect the activity. Cytotoxicity of distinct polarities was observed in thirty extracts (46 percent), from different parts of the following species: Tabebuia heptaphylla (Vell.) Toledo, Bignoniaceae, Tapirira guianensis Aubl., Anacardiaceae, Myracrodruon urundeuva Allemão, Anacardiaceae, Schinus terebinthifolius Raddi, Anacardiaceae, Gomphrena elegans Mart., Amaranthaceae, Attalea phalerata Mart. ex Spreng., Arecaceae, Eugenia uniflora L., Myrtaceae, and Annona dioica A. St.-Hil., Annonaceae. Extracts of at least two tested cell strains were considered to be highly active since their inhibition rate was over 75 percent.

Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis.

Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (-)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0µM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC(50) of 0.4µM) and human peripheral blood mononuclear cells (PBMC, IC(50) of 1.2µM). After 24h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5µM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.

Classification of Aristolochia species based on GC-MS and chemometric analyses of essential oils.

Essential oils were obtained from roots of 10 Aristolochia species by hydrodistillation and analysed by GC-MS. A total of 75 compounds were identified in the analysed oils. Multivariate analyses of the chemical constituents of the roots enabled classification of the species into four morphological groups. These forms of analysis represent an aid in identification of further specimens belonging to these species.

Screening Brazilian C gasoline quality: application of the SIMCA chemometric method to gas chromatographic data.

A total of 2400 samples of commercial Brazilian C gasoline were collected over a 6-month period from different gas stations in the São Paulo state, Brazil, and analysed with respect to 12 physicochemical parameters according to regulation 309 of the Brazilian Government Petroleum, Natural Gas and Biofuels Agency (ANP). The percentages (v/v) of hydrocarbons (olefins, aromatics and saturated) were also determined. Hierarchical cluster analysis (HCA) was employed to select 150 representative samples that exhibited least similarity on the basis of their physicochemical parameters and hydrocarbon compositions. The chromatographic profiles of the selected samples were measured by gas chromatography with flame ionisation detection and analysed using soft independent modelling of class analogy (SIMCA) method in order to create a classification scheme to identify conform gasolines according to ANP 309 regulation. Following the optimisation of the SIMCA algorithm, it was possible to classify correctly 96% of the commercial gasoline samples present in the training set of 100. In order to check the quality of the model, an external group of 50 gasoline samples (the prediction set) were analysed and the developed SIMCA model classified 94% of these correctly. The developed chemometric method is recommended for screening commercial gasoline quality and detection of potential adulteration.

Optimisation of conditions for the extraction of casearins from Casearia sylvestris using response surface methodology.

Optimal conditions for the extraction of casearins from Casearia sylvestris were determined using response surface methodology. The maceration and sonication extraction techniques were performed using a 3 x 3 x 3 full factorial design including three acidity conditions, three solvents of different polarities and three extraction times. The yields and selectivities of the extraction of casearins were significantly influenced by acidity conditions. Taking into account all variables tested, the optimal conditions for maceration extraction were estimated to involve treatment with dichloromethane saturated with ammonium hydroxide for 26 h. Similar yields and selectivities for casearins were determined for sonication extraction using the same solvent but for the much shorter time of 1 h. The best results for stabilisation of the fresh plant material were obtained using leaves that had been oven dried at 40 degrees C for 48 h.