Anti-inflammatory and antioxidant activity of the hot water-soluble polysaccharides from Anacyclus pyrethrum (L.) Lag. roots.

ETHNOPHAMACOLOGICAL RELEVANCE: the roots of Anacyclus pyrethrum (L.) Lag. (Family: Asteraceae) are used in Algeria to treat respiratory infections, to cure chronic head and nostrils catarrh, and to clear the brain by stimulating the free flow of nasal mucous. They contain a high quantity of hot water-soluble polysaccharides. AIMS OF THE STUDY: The study aims to evaluate the antioxidant and anti-inflammatory activity of polysaccharides extracted from Anacyclus pyrethrum roots (APPS). MATERIALS AND METHODS: The APPS were extracted using boiling water, separated from proteins by the Sevag method then precipitated with 90% ethanol. The antioxidant effect of crude APPS was evaluated using FRAP assay. To investigate the anti-inflammatory potential, mice were treated with crude polysaccharides (25, 50, and 100 mg/kg, i.p.) for 3 days (14th, 15th, and 16th day of the experimentation). Respiratory inflammation was induced by HDM (House Dust Mite), mice were sensitized intranasally with 25 µg HDM suspended in 10 µl NaCl (5 µl/nostril) on days 0 and 7 then challenged with 5 µg HDM on days 14, 15, and 16. Mice were sacrificed 24 h after the last challenge. The number of immune cells in the blood in NL (Nasal Liquid) and in BAL (Broncho Alveolar Liquid) was enumerated, the spleen was removed to calculate the relative spleen weight and to count splénocytes, lungs histopathological examination was carried out to confirm the protective effect of APPS. Structural characterization of APPS was identified using FTIR (Fourier-Transform Infrared Spectroscopy) and SEM (Scanning Electron Microscopy). RESULTS: The crude APPS possessed reducing power. In vivo assay, treatment with APPS causes a decrease in the number of blood leucocytes at all doses on the one hand, and in the relative spleen weight and splénocytes number on the other hand except at the dose of 50 mg/kg in which an enhancement of the number of splénocytes and immune cells in NL and BAL was significant. The histopathological examination showed clear protection of lung tissue damaged by HDM, after treatment with APPS mainly, at the dose of 50 mg/kg. CONCLUSION: Our data clearly showed antioxidant and anti-inflammatory activity of APPS on HDM-challenged mice induced lungs inflammation by equilibrating the inflammatory reaction mostly, with an optimal dose of 50 mg/kg.

Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000µM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

Anvillea garcinii extract inhibits the oxidative burst of primary human neutrophils.

BACKGROUND: Anvillea garcinii Coss. & Durieu (Anv) plant is used as a traditional North African medicine against several diseases associated with inflammation. At inflammatory sites, reactive oxygen species (ROS) produced in excess by activated phagocyte NADPH oxidase (NOX2) can accentuate inflammatory responses. Thus, we investigated if Anv-water soluble polysaccharides could modulate primary human neutrophil oxidative burst in vitro. METHODS: Human neutrophils were isolated from fresh whole blood and O2.- generation was measured by cytochrome c reduction assays. Western blots were used to analyse the translocation of PKC, p47phox (a key component of NOX2 activity) to neutrophil plasma membrane. Also, myeloperoxidase (MPO) release in the extracellular medium was studied by western blots. Flow cytometric analysis was used to detect CD11b membrane expression. RESULTS: Water soluble polysaccharides from Anv dose-dependently inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and phorbol myristate acetate (PMA)-induced O2.- generation by human neutrophils. Moreover, Anv-polysaccharides strongly inhibited PMA-induced PKCß and p47phox translocation to membranes and p47phox phosphorylation on Ser328, a main PKC target. In contrast, polysaccharides extract from Zygophyllum gaetulum plant, which is also used as a traditional North African medicine against inflammatory diseases, was ineffective on this PKCß-p47phox pathway. Further, Anv inhibited important neutrophil degranulation markers corresponding to myeloperoxidase (MPO) release and CD11b membrane expression. CONCLUSION: The process of down-regulating NADPH oxidase by polysaccharides extracts from Anv provides new insights into the mechanism of Anv's anti-inflammatory actions.