Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2.

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver-Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Phenological stage effect on phenolic composition and repellent potential of Mentha pulegium against Tribolium castaneum and Lasioderma serricorne

Objective: To elucidate for the first time the phenological stage effect on polyphenol, flavonoid and proanthocyanidin contents of pennyroyal (Mentha pulegium) as well as their antioxidant capacities and insecticidal potentials against Tribolium castaneum and Lasioderma serricorne. Methods: Crude methanol extracts from pennyroyal (Mentha pulegium) aerial parts were evaluated for their antiradical, reducing power activities and repellent potentials against selected coleopteran insects during different phenological stages. Results: Phenolic contents of pennyroyal aerial parts and their antioxidant effects were significantly dependent on maturity stage of the plant. The maximum of phenol production was reached during the full flowering stage. Apigenin was the major phenol in Mentha pulegium with an amount of 6.01 mg/g of dry vegetable matter during this period. Nevertheless, at fructification stage, the highest antioxidant activity was not related to high phenolic content. The repulsive effect of pennyroyal extract showed that Lasioderma serricorne was more susceptible to pennyroyal extract than Tribolium castaneum with corresponding median repellent dose values of 0.124 and 0.006 mg/mL respectively. Moreover, great differences in insect repulsion depending on extract concentrations, exposure time and developmental stage was observed. For the lowest concentration (0.125 mg/mL), the repellent effect against Tribolium reached its maximum (90%) during the fructification stage (after 24 h of exposure) while this repellent effect was 80% during the vegetative stage. Conclusions: Due to the strong insecticidal potential of pennyroyal extracts, this study highlights the therapeutic properties of this plant and encourages its use as a safer, environmental-friendly and efficient insecticide in food industry.

Antioxidant antileishmanial cytotoxic and antimicrobial activities of a local plant Myrtus nivellei from Algeria Sahara

Objective To study the phytochemical constituents and in vitro biological activities of hydromethanolic extract and fractions from Algerian Sahara Myrtus nivellei (M. nivellei) collected in Hoggar region and to identify the active fraction that can act as an alternative of commonly used antibiotics and as antileishmanial or antioxidant agents. Methods Phytochemical screening of M. nivellei aerial parts was realised according to the literature. Extract was firstly prepared by using aqueous methanol then fractionated with ethyl acetate and butanol solvents. Total phenolics, tannis and flavonoids, of the hydromethanolic extract and their fractions were determined by Folin–Ciocalteu method as gallic acid equivalents and by aluminium chloride as rutin equivalent respectively. Extract and fractions were tested for their antimicrobial and antiparasital activities against standard bacteria using agar diffusion method and two kinds of leishmania visceral and cutaneous. The antioxidant activities were realized using phosphomolybdenum, FRAP and DPPH tests. Results Preliminary phytochemical screening exhibited the presence of flavonoids, tannins, saponins, and alkaloids. The experimental results showed that plant extract and fractions were high in phenolic compounds and exhibited an important role as antioxidant, antimicrobial and had a moderate antileishmanial activity. Conclusions These observations lead us toward more studies in this field, so that we can get more benefits from our local Algerian medicinal plants.

Protective effect of grape seed and skin extract on cerebral ischemia in rat: implication of transition metals.

Ischemic stroke is a leading cause of long lasting disability in humans and oxidative stress an important underlying cause. The present study aims to determine the effect of short term (seven-days) administration of high dosage grape seed and skin extract (GSSE 2.5 g/kg) on ischemia/reperfusion (I/R) injury in a rat model of global ischemia. Ischemia was induced by occlusion of the common carotid arteries for 30 min followed by one-hour reperfusion on control or GSSE treated animals. I/R induced a drastic oxidative stress characterized by high lipid and protein oxidation, a drop in antioxidant enzyme defenses, disturbed transition metals as free iron overload and depletion of copper, zinc and manganese as well as of associated brain enzyme activities as glutamine synthetase and lactate dehydrogenase. I/R also induced NO and calcium disruption and an increase in calpain activity, a calcium-sensitive cysteine protease. Interestingly, almost all I/R-induced disturbances were prevented by GSSE pretreatment as oxidative stress, transition metals associated enzyme activities, brain damage size and histology. Owing to its antioxidant potential, high dosage GSSE protected efficiently the brain against ischemic stroke and should be translated to humans.

Acute effects of a partially purified fraction from garlic on plasma glucose and cholesterol levels in rats: putative involvement of nitric oxide.

Garlic has been extensively used as a medicinal plant. Most of its numerous beneficial effects such as antioxidant, antibacterial, antitumoral involve sulfur-derived amino acids. In the present work, we reevaluated the acute effects of aqueous extract of garlic on plasma glucose and cholesterol levels in normal rats. Control (vehicle H2O) or garlic extract-treated group at 100-120 mg protein/kg body wt were intraperitoneally injected (IP) and glucose, cholesterol, insulin and nitric oxide metabolites levels were determined after a short-term duration of 6 h. We confirmed that garlic contained an active fraction, exerting both glucose and cholesterol-lowering activity. The glucose-lowering effect was triggered by an increase in insulinemia. Preliminary study indicated that the active agent was different from S-allyl-cysteine-sulfoxide, the active principle implicated in hypoglycaemic and hypolipidemic effects of garlic or arginine. The mechanism of action seemed to involve nitric oxide (NO), which increased time and dose-dependently. The garlic effects were abolished by diphenyleneiodonium chloride (DPI = 1 mg/kg body wt), a specific inhibitor of NO production, suggesting the involvement of constitutive nitric oxide synthase.