Effect of polyphenols extracted from Punica granatum and Acacia saligna plants on glutathione S-transferase of the cattle tick Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae).

Ticks are hematophageal ectoparasites that transport major pathogens around the world. Glutathione S-transferases (GST) are involved in resistance to acaricide and redox balancing during the life cycle of the tick. The inhibition of tick GST enzymes by certain phenolic compounds, such as phenolic acids and tannins, can be a promising approach to tick control. The objective of this study was to evaluate the effects of Punica granatum red peel and Acacia saligna leaf extracts on Rhipicephalus (Boophilus) annulatus GST activity in order to reduce the resistance of cattle to acaricide. The results showed that P. granatum ethanol extract (70%) contained the highest total phenol content (350 ± 1.2 µM GAE g-1), the highest condensed tannin content (270 ± 1.3 µM CE g-1) and the highest hydrolysable tannin content (70 ± 5.0 µM TAE g-1). Adult immersion test with a dosage of 100 mg ml-1 of A. saligna ethanol extracts had a significant mortality of 50% and 75% after 24 h and 96 h, respectively (p < 0.01). A simple and reproducible procedure was established to purify the whole R. annulatus GST (wRaGST) while a full-length cDNA of GST was cloned from a cDNA library of the local Egyptian cattle tick R. (B.) annulatus (rRaGST). Aqueous extracts of P. granatum inhibited both wRaGST and rRaGST with values of IC50 = 0.114 and 0.07 µg ml-1, respectively, compared to A. saligna extracts (IC50 values = 2.08 and 1.35 µg ml, respectively). These inhibitory effects were attributed to the presence of a high tannin concentration (≥ 80%). HPLC analysis indicated the presence of gallic acid and catechin in both extracts, in addition to the rutin, which was only observed in A. saligna extracts. The addition of a tannin inhibitor, polyethylene glycol, suggested the existence of other phenolic compounds in combination with catechins responsible for inhibiting the activity of these extracts. Non-competitive behaviour of catechins may be helpful in preventing, or at least delaying, the development of chemical acaricide resistance in R. annulatus.

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.

Molecular cloning of Ra-sHSPI, a novel member of the HSP20 family from Rhipicephalus annulatus salivary glands.

Infestation of cattle by ticks of Rhipicephalus spp. results in severe veterinary and economical losses. Identification of novel proteins from tick salivary glands will enhance our understanding of several aspects of tick physiology and will aid in the development of anti-tick vaccines. Small heat shock proteins (HSPs) have important roles in infection and immunity, especially between invertebrate vectors and mammalian hosts while initially performing their molecular chaperone activity. Here, we report the identification of a small HSP gene from the salivary glands of Rhipicephalus annulatus ticks through immunoscreening of the corresponding cDNA expression library. The identified cDNA contained a 742bp sequence with 543bp open reading frame. It was subsequently cloned, expressed and successfully purified under both native and denaturing conditions. Sequence analysis and functional investigations showed that the protein belongs to the HSP20 family, hence the annotated name Ra-sHSPI. Indeed, recombinant Ra-sHSPI showed two typical in vitro activities of holdase chaperones, including thermal protection of bacterial cellular extracts and the recombinant HindIII at elevated temperatures. Moreover, the recombinant Ra-sHSPI showed strong immunogenic effect in animal model. These results pave the way toward further investigation of Ra-sHSPI role in ticks feeding and its potential use as protective antigen.

Purification and biochemical characterization of glutathione S-transferase from Down syndrome and normal children erythrocytes: a comparative study.

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was determined in ten DS and ten healthy children matched for age (3-10 years). DS group exhibited significantly lower GST value (2.7 units/gHb) as compared to controls (6.6 units/gHb) (40.9%). GST activity was significantly decreased to 40.9% in the DS group as compared to controls. Also GSH concentration was significantly decreased to 60.6% in the DS group compared to the controls. Glutathione transferase was purified from erythrocytes of normal and DS pooled blood samples by affinity chromatography with specific activity of 23.7% and 7.9%, respectively. The effect of freezing and thawing, storage time of freezing and GSH concentration on the stability of the enzyme were examined. Normal GST exhibited a pH optimum at pH 7 followed by sharp decrease, however DS GST exhibited pH optimum between pH 7.5 and 8. The Km values for 1-chloro-2,4-dinitrobenzene (CDNB) and GSH were 0.205 mM and 0.786 mM, respectively, for normal GST, and 0.318 mM and 1.307 mM, respectively for DS GST. The activation energy (Ea) was calculated to be 2.25 and 4.25 cal/mol for normal GST and 3.8 cal/mol for DS GST. Normal and DS GST were inhibited by the same inhibitors (hematin, bromosulfophthalein and cibacron blue), but with different degree. On kinetic basis, the individuals with lower overall GST activity and slight differences in some kinetic characters are at greater risk from xenobiotic contamination as compared to those with higher overall GST activity observed in normal individuals.