Characterization of oxyphytosterols generated by ß-sitosterol ozonization.

ß-Sitosterol (ßSito) is the most abundant phytosterol found in elevated concentrations in vegetable oils, nuts, seeds, cereals, fruits, and in many phytosterol-enriched foods. Although the benefits, there is a concern in terms of food quality and health due to the increasing consumption of phytosterols and the possible adverse side effects of their oxidation products, oxyphytosterols. ßSito has a similar structure to cholesterol, with an unsaturated double bond at C5-C6, which is susceptible to oxidation by reactive oxygen species like ozone, generating oxyphytosterols. In this work we propose a mechanism of formation of three oxyphytosterols 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7- octahydroinden-4-yl] acetaldehyde (ßSec), (2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan- 2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4- yl] acetaldehyde (ßLac) and 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2- oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl) acetic acid (ßCOOH) generated by ozonization of ßSito, through their synthesis and molecular characterization. The cytotoxic effect of ßSito and its main oxyphytosterol ßSec was evaluated and both reduced the HepG2 cell viability.

Unilateral intranigral administration of ß-sitosterol ß-D-glucoside triggers pathological α-synuclein spreading and bilateral nigrostriatal dopaminergic neurodegeneration in the rat.

The spreading and accumulation of α-synuclein and dopaminergic neurodegeneration, two hallmarks of Parkinson's disease (PD), have been faithfully reproduced in rodent brains by chronic, oral administration of ß-sitosterol ß-D-glucoside (BSSG). We investigated whether a single injection of BSSG (6 µg BSSG/µL DMSO) in the left substantia nigra of Wistar rats causes the same effects. Mock DMSO injections and untreated rats formed control groups. We performed immunostainings against the pathological α-synuclein, the dopaminergic marker tyrosine hydroxylase (TH), the neuroskeleton marker ß-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using ß-galactosidase (ß-Gal) assay and active caspase-3 immunostaining, we assessed cell death mechanisms. Golgi-Cox staining was used to measure the density and types of dendritic spines of striatal medium spiny neurons. Motor and non-motor alterations were also evaluated. The study period comprised 15 to 120 days after the lesion. In the injured substantia nigra, BSSG caused a progressive α-synuclein aggregation and dopaminergic neurodegeneration caused by senescence and apoptosis. The α-synuclein immunoreactivity was also present within microglia cells. Decreased density of dopaminergic fibers and dendritic spines also occurred in the striatum. Remarkably, all the histopathological changes also appeared on the contralateral nigrostriatal system, and α-synuclein aggregates were present in other brain regions. Motor and non-motor behavioral alterations were progressive. Our data show that the stereotaxic BSSG administration reproduces PD α-synucleinopathy phenotype in the rat. This approach will aid in identifying the spread mechanism of α-synuclein pathology and validate anti-synucleinopathy therapies.

Anti-Trypanosoma cruzi Activity in vitro of Phases and Isolated Compounds from Excoecaria lucida Leaves.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. This illness is found mainly in 21 Latin American countries and an estimated 8 million people are infected worldwide. The unsatisfactory chemotherapy provokes severe toxicity and resistant strains. Medicinal plants constitute a promising source of new drugs and remedies against all kinds of disorders, mainly infectious diseases arousing interest worldwide. OBJECTIVE: The aim of this study is the isolation, structural identification and evaluation of the trypanocidal activity of samples present in the Excoecaria lucida Sw. leaves. METHODS: Total extract (TE) of E. lucida Sw. leaves was obtained by ethanol extract therefore fractionated sequentially with hexane, ethyl acetate and n-butanol, to obtain three phases: Hex, EA and But, respectively. Ellagic acid (EL1) was purified from both EA and But phases, while EL2; a 1:1 stigmasterol-3-O-ß-D-glucopyranoside plus sitosterol-3-O-ß-D-glucopyranoside mixture was obtained from the Hex phase. Activity assays were performed using bloodstream and intracellular forms of T. cruzi and cytotoxicity assays using L929 fibroblasts. RESULTS: The EL1 and EL2 samples were more active against bloodstream trypomastigote forms with EC50 of 53.0±3.6 and 58.2±29.0 µg/mL, respectively; at 100 µg/mL. These samples also showed 70% of inhibition of L929 cells infection. Toxicity assays demonstrated that after 96 h of treatment only the fractions Hex and EA presented detectable cytotoxicity. CONCLUSION: Ellagic acid, stigmasterol-3-O-ß-D-glucopyranoside and sitosterol-3-O-ß-Dglucopyranoside are reported for the first time in E. lucida Sw. leaves as well as their biological activity studies supporting further investigations for Chagas disease treatment.

Chronic effects of Pinus radiata and Eucalyptus globulus kraft mill effluents and phytosterols on Daphnia magna.

Two kraft pulp mill effluents were compared in terms of their chronic toxicity to Daphnia magna. One resulted from pulping Pinus radiata and the other came from a parallel processing of Pinus radiata and Eucalyptus globulus (mixed kraft pulp mill effluent). The concentration of phytosterols found in the mixed kraft pulp mill effluent was higher than in the effluent from Pinus radiata, with values of 0.1082 and 0.02 µg/L, respectively. The phytosterols per se are responsible for 12.9% and 8.1% of the deviation from the natural shape, while the kraft pulp mill effluents account for 25.6%-27.8% of shape deviation. The role of ß-sitosterol and stigmasterol is discussed in relation to endocrine disruption.

Antineoplastic activity of selected constituents of Duguetia glabriuscula.

The cytotoxic effects of seven constituents isolated from Duguetia glabriuscula were evaluated against Hep2 human larynx carcinoma cells. The cytotoxicity exhibited by beta-sitosterol was as strong as that of cis-platin. (+)-Alloaromadendran-10,14beta-diol caused inhibition of cellular growth with IC50 values lower than 25 microg/ml, a feature that was considered as revealing significant activity. Polycarpol showed borderline cytotoxicity, whereas the other compounds were inactive.