الفهرس 
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Abstract
Summary
One of the most destructive insect to many crops in Africa and Asia is the desert locust Schistocerca gregaria (Orthoptera: Acrididae) this due to its gluttonous feeding and invasions in swarms. The desert locust is not inhabited in Egypt but migrated from Sudan, Yemen, or Saudi Arabia. In November 2004, both Upper and Lower Egypt were destructively attacked by huge plagues of desert locusts causing great agricultural damage and 40% of agricultural crops were lost.
Detection and prevention is the main control method used against the Desert locust. Organophosphoruses are the current used pesticides that control locus due to the banning of organochlorines. The widespread uses of such synthetic pesticides has many disadvantages, such as the increased costs, handling hazards, concerns about insecticide residues, development of insect resistance to insecticides and great threats to both human and environmental health. The botanical control agents are biodegradable, pest-specific, relatively harmless to non-target organisms and consequently harmless to the environment. Botanical insecticides, (secondary metabolites) are easy to use, have broad-spectrum activity and relative specificity in their mode of action. They are also safe to animals and environment.
Insect glutathione S-transferases (GST) have been shown to be active toward numerous electrophilic xenobiotics and resistance of insects to in the resistance of organophosphate, pyrethroid and chlorine is attributed to GST. Plant extracts that are high in polyphenols are known to have important inhibitory effect on GST.
This study is the first concerned with the examination of the level of the antioxidant enzymes, total glutathione (GSH) concentration and its related enzymes in different developed instars and different organs of S. gregaria. It was also concerned with the examination of the effect of some plant extracts (having different bioactive compounds) on the activity of GST extracted and purified from S. gregaria fat body and ovary with a particular focus on the development of botanical inhibitors for this enzyme system.
Results could be summarized in the following:
1- Glutathione concentration and the specific activities of GST, glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) were determined in the different developed instars (1st-5th) crude homogenates. The concentration of GSH increased more than 10 folds with developing of the instars to reach the highest level in the fifth instar, while the specific activities of GST and CAT decreased from the 1st instar to the 5th instar. The specific activities of GPx and GR increased from the 1st to the 2nd and then decreased from the 2nd to the 5th instar.
2- The catalytic activity of GST, GR, GPx, CAT and GSH level were determined in the S. gregaria fore gut, med gut, hind gut, ovary and fat body crude homogenates. GST exhibited the highest significant value in the fat body homogenate. The highest GSH concentration was detected in ovary and the lowest value detected in the fat body. The hind gut homogenate showed the significantly highest GPx activity while the lowest GPx activity was detected in the fat body. The highest GR activity was detected in the hind gut, while the ovary exhibited the lowest values of GR activity. The significantly highest catalase activity was detected in fat body compared to the other tested organs homogenates. GST of the examined S. gregaria organ homogenates exhibited different affinities towards some GST substrates 1,2-epoxy-3-(ρ-nitro phenoxy) propane (EPNP), cumene hydroperoxide (CuOOH), p-nitro phenyl bromide (NPB) and styrene oxide (SO).
3- Phenols are very important plant constituent due to their hydroxyl groups which have radical scavenging ability. Total phenolic (TPC) and flavoniod contents (TFC) of twelve plants belong to different families namely; Anethum graveolens, Petroselinum crispum, Coriandrum sativum, Foeniculum vulgare, Pimpinella anisum, Cuminum cyminum, Daucus carota, Ammi majus, Apium graveolens seeds, (Family Umbelliferae). Tamarindus indica seed and Glycyrrhiza glabra roots, (Family Leguminosae) and Hibiscus sabdariffa flower, (Family Malvacceae) were determined. Eight plants were selected according to their high TPC and TFC for examination of their effects on the GST activity of crude homogenates of S. gregaria organs at three different concentrations of their aqueous and ethanolic extracts. The results showed that: The extracts of G. glabra and H. sabdariffa were the most effective inhibitors for the ovary and the fat body GST activity. T. indica and G. glabra aqueous extracts were the most effective plants on GST of S. gregaria med gut homogenate. Among all the studied plants, H. sabdariffa aqueous and ethanolic extracts were the most plant having inhibition effect on hind gut GST activity.
Among the eight studied plants, four plants (G. glabra, H. sabdariffa, C. cyminum and T. Indica) were chosen as the most effective plants on GST activity (low IC50 values) of S. gregaria organs.
4- Determination of polyphenol, flavonoid, anthocyanin contents, total hydrolysable tannins, condensed tannins and antioxidant capacities in aqueous and ethanolic extracts of the four plants were carried out. T. indica extracts exhibited the highest phenolic, total flavonoid, hydrolysable tannins and condensed tannins contents, while, lowest contents values were observed in C. cyminum. H. sabdariffa extracts showed the highest percentage of anthocyanin content relative to TPC. The antioxidant capacity examined values using 1, 1 Diphenyl-2-picryl-hydrazyl scavenging activity and 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging method showed that the ethanolic and aqueous extracts of T. indica seed have a powerful antioxidant capacity (IC50=0.034 and 0.096 mg dry plant/ml, respectively) compared to the other plant extracts.
Fat body represented the most important site for the detoxification and the major site of xenobiotics metabolism. It also actively participates in vitellogenesis (synthesis of vitellogenin, the precursor of the yolk protein). Female reproduction has been a major focus of insect’s research, as need to control of insect pest’s reproduction. The present study focused on fat body and ovary of S. gregaria organs. Our results showed that G. glabra and H. sabdariffa extracts were the most effective inhibitors for the ovary and the fat body GST activity. Therefore, these two effective plants were chosen for the following HPLC analysis.
5- HPLC analysis for H. sabdariffa showed the presence of gallic acid, cinnamic acid, chlorgenic acid, caffeic acid, rutin, delphinidine and cyanidin 3-O-glucoside in both ethanolic and aqueous extracts. Catachin was found only in aqueous extract while ferulic acid and qurecetin were found only in the ethanolic extract. Delphinidine and cyanidin 3-O-glucoside represented the major content of H. sabdariffa. HPLC analysis for G. glabra extracts showed that only rosmarinic acid, rutin, quercetin, kaempferol and 18 α-glycyrrhetinic were found in both ethanolic and aqueous extracts and the amount of 18 α-glycyrrhetinic acid was the highest one.
Main metabolic center of insects is the fat body and the most complex cellular functions occur in it. Also it is important site for the detoxification of xenobiotics as it contains very high levels of GST activity.
6- A simple method for the GST purification from S. gregaria fat body using affinity chromatography followed by ion exchange chromatography was carried out. The elution profile of S. gregaria fat body by ion exchange chromatography showed the presence of two GST activity peaks, designated GST1 and GST2 according to their elution. The enzyme activity of these peaks represented 6.8% and 0.56% of the total crude extract activity, respectively. Only GST1 proved to be homogenous as judged by native PAGE and SDS-PAGE. The purified fat body GST1 exhibited optimum activity at pH 8.0. The kinetic studies of fat body GST1 exhibit a distinct Michaelis-Menten constant with Km values equal 0.35 mM for GSH and 0.20 mM for CDNB.
7- Fat body GST acts readily on CDNB and also showed high selenium-independent glutathione peroxidase activity with CuOOH (represents 114% of its activity on CDNB). It has no detectable activity toward the substrates DCNB, ECA, BSP, EPNP, NPB and SO. Cibacron blue was the most potent inhibitor followed by BSP and ECA.
Both aqueous and ethanolic extracts of G. glabra have strong inhibitory effect on fat body GST1 activity with IC50= 0.0125 and 0.002 mg/g dry plant, respectively, while H. sabdariffa extracts inhibit GST1 activity with higher IC50 values (0.025mg and 0.0187 mg/g dry plant).
8-The major components in G. glabra were 18 α-glycyrrhetinic acid, quercetin and rutin and the major component in H. sabdariffa were cyanidin 3-O-glucoside chloride and delphinidine. Quercetin and delphinidine chloride exhibited strong inhibition with IC50 equal 2.23 and 3.88 µM, respectively. Rutin have less inhibitory effect while, 18 α-glycyrrhetinic and cyanidin 3-O-glucoside chloride acid had no effect. The inhibition type was found to be competitive for quercetin with CDNB and non competitive with GSH and it was non-competitive with both CDNB and GSH for delphinidine chloride.
9- Little knowledge is available about the role of GST in ovary of insect. The present study showed that, as the weight of insect increase the weight of ovary increase, also the gonad somatic index (GSI) increase. The number of eggs increases by increasing the GSI value. S. gregaria ovaries were divided into three groups according to their GSI. The results showed that the specific activities of GST, GR, GPx and CAT decreased gradually in ovary homogenates as GSI increased. GST and GR showed insignificant decrease in the second group and highly significant decrease in the third group.There was highly significant decrease in the specific activities of GPx and CAT in the second and third group. GSH concentration increased from the first group to the third group with increasing the GSI, this increase in GSH is highly significant.
10- The elution profile of S. gregaria ovary chromatography on GSH-Sepharose column showed the presence of only one peak for GST. The affinity purified GST proved to be homogenous as judged by native PAGE and SDS-PAGE. The affinity purified GST from S. gregaria ovary showed maximum activity at pH 8.0. It acts readily on CDNB and it exhibited peroxidatic activity on CuOOH (43% of that for CDNB). The specific activity with DCNB was 30% and it was 8.28% with ECA of that for CDNB. Cibacron blue was the most potent inhibitor followed by BSP and ECA, while the inhibition effect of hematin was the least.
Both ethanolic and aqueous extracts of G. glabra have strong inhibition on S. gregaria ovary GST, while ethanolic and aqueous extracts of H. sabdariffa inhibit GST activity with higher IC50 values. Quercetin and delphinidine chloride were exhibited strong inhibitions with IC50 equal 5 and 3.84 μM respectively. Rutin have less effect while, cyanidin 3-O-glucoside chloride and 18α-glycyrrhetinic acid have no inhibitory effect. Quercetin inhibit GST competitively with CDNB and non-competitively with GSH, while delphinidine chloride competitively inhibits GST with both CDNB and GSH