Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus glutathione S-transferase.

Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57µM compared to delphinidine-HCl (IC50=14.9±3.1µM). Gossypol had a weak inhibitory effect (IC50=43.7µM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034µM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13µM and 7.5µM, respectively. The activity of rRaGST was highest for CDNB (30.2µmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5µmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and competitively with respect to CDNB. While red pomegranate extracts inhibited rRaGST activity competitively with respect to GSH, uncompetitive inhibition was observed with respect to CDNB.

Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway.

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS-PAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (Δψ(m)), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of Δψ(m) besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.

Phytotherapeutic effects of Echinacea purpurea in gamma-irradiated mice.

Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbeckia. In this paper, we report the curative efficacy of an Echinacea extract in gamma-irradiated mice. E. purpurea was given to male mice that were divided into five groups (control, treated, irradiated, treated before irradiation & treated after irradiation) at a dose of 30 mg/kg body weight for 2 weeks before and after irradiation with 3 Gy of gamma-rays. The results reflected the detrimental reduction effects of gamma-rays on peripheral blood hemoglobin and the levels of red blood cells, differential white blood cells, and bone marrow cells. The thiobarbituric acid-reactive substances (TBARs) level, Superoxide dismutase (SOD) and glutathione peroxidase (GSPx) activities and DNA fragmentation were also investigated. FT-Raman spectroscopy was used to explore the structural changes in liver tissues. Significant changes were observed in the microenvironment of the major constituents, including tyrosine and protein secondary structures. E. purpurea administration significantly ameliorated all estimated parameters. The radio-protection effectiveness was similar to the radio-recovery curativeness in comparison to the control group in most of the tested parameters. The radio-protection efficiency was greater than the radio-recovery in hemoglobin level during the first two weeks, in lymphoid cell count and TBARs level at the fourth week and in SOD activity during the first two weeks, as compared to the levels of these parameters in the control group.