الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 143 |  |  |
| | | from | 143 | | |
Abstract
The present study was done to evaluate the effects of C. Procera leaves solvent extracts (ethanol, acetone and hexane) against 2nd and 4th instar larvae of S. Littoralis and determine LC50 of each extract to study the biochemical effects on total protein and some enzyme activity (ALT, AST, GST and AchE) of larvae homogenate and make Gas chromatography- mass spectrum (GC-MS) analysis for the most effective solvent extract.
Toxicity tests
Toxicity tests of solvent extracts of Calotropis procera against S. littoralis larvae were carried out using five concentrations (20, 10, 5, 2.5 and 1.25 %) were carried out from each crude extract (ethanol, acetone and hexane) of C. procera. Newly active 4th instar and 2nd instar larvae were selected and starved for 4 hours, then fed on treated leaves by the dipping technique, the results Showed that the LC50 values of the C. procera leaves acetone extract for the 2nd instar larvae of S. littoralis were 8.571, 5.733 and 4.366%, and for ethanol extract were 10.48, 7.96 and 5.36 %, where for hexane extract were 16.04, 11.185 and 9.96% after 24, 48 and 72h. after treatments, respectively. The LC50 values of the C. procera acetone extract for the 4th instar larvae of S. littoralis were 12.33, 7.5 and 5.36% and for ethanol extract were 15.3, 8.44 and 6.32% where for hexane extract were 17.426%, 11.827% and 11.2% after 24, 48 and 72h. after treatments, respectively. The all solvent extracts of C. procera leaves showed a noticeable toxicity on S. littoralis larvae, the acetone extract was more toxic than ethanol and hexane extracts in both 2nd and 4th instar larvae. In addition, results showed that the toxicity of C. Procera leaves solvent extracts to 2nd instar larvae was more effective than the 4th instar larvae.
Biochemical changes in S. littoralis induced by different solvent extracts of Calotropis procera
The effect of acetone, ethanol and hexane extracts of Calotropis procera based on LC50 values on (total protein, AST, ALT, GST and AchE) for the 2nd and 4th instar larvae of S. littoralis after 48h. The total protein were determined by the method of (Bradford, 1976), the results showed that, there was significant decreased (P<0.05) in all treatments acetone, ethanol and hexane extracts of C. procera leaves (1.93, 2.26 and 2.27 mg/g. body wt., respectively) compared with control (3.18 mg/gm. body wt.) for 2nd instar larvae. Total protein was significantly decreased (P<0.05) in all treatments acetone, ethanol and hexane extracts of C. procera (2.23, 2.54 and 2.37 mg/gm. body wt., respectively) compared with control (4.22 mg/g. body wt.) for 4th instar larvae.
Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT) activities were assayed according to the method of Reitman and Frankel (1957). The results revealed that there was a significant decrease in AST in acetone, ethanol and hexane extracts (18.68, 22.30 and 27.713 U/L, respectively) comparing with the control (31.1633 U/L), while ALT activities, there was a significant decrease for acetone, ethanol and hexane extracts (10.7600, 14.2767 and 17.976 U/L, respectively) compared to control 25.41 U/L for 2nd instar larvae.
The results showed decreasing activities of AST of 4th instar larvae of S. littoralis (21.076, 27.68 and 31.91 U/L, respectively) compared with control 35.893 U/L, while for ALT activities, there was a significant decrease in acetone, ethanol and hexane extracts (12.1, 21.08 and 25.94 U/L, respectively) compared with control 29.88 U/L.
Glutathione S-transferase (GST) catalyzes the conjugation of reduced glutathione (GSH) with 1-chloro 2, 4-dinitrobenzene (CDNB) via the -SH group of glutathione. The conjugate, S-(2, 4-dinitro-phenyl)-L-glutathione could be detected as described by the method of Habig et al.(1974).
The results showed a very significant decrease in GST activity (P<0.01) (107.13, 136.43 and 145.5167 U/g tissue, respectively) compared to the control 170.40 U/g tissue for 2nd instar larvae. Regarding to GST activity results, there was very significant decrease (p<0.01) for acetone, ethanol and hexane extract (145.5, 146.16 and 196.8 U/g tissue, respectively) compared with control 233.23 U/g tissue for 4th instar larvae.
acetyl cholinesterase (AchE) activity was measured according to the method described by (Simpson et al., 1964), using acetylcholinebromide ( AchBr) as substrate. The results showed that there was a very significant decrease ( p<0.001) in AchE activity for all extracts (acetone, ethanol and hexane), its values were 13.88, 87.66 and 103.77 µg Acetyl-choline bromide/ min/ml., respectively compared with control 188.05 µg Acetyl-choline bromide/ min/ml. for 2nd instar larvae. While for 4th instar larvae, there was very significant decreased for acetone, ethanol and hexane extract (110.28, 441.57 and 461.67 µg Acetyl-choline bromide/min/ml., respectively) compared with control 514.86 µg Acetyl-choline bromide/ min/ml.
The results of the present study showed that the solvent extracts of C. procera had a tangible effects on the enzyme activities of S. littoralis where there was a significant decrease in total protein that recorded in both 2nd and 4th instar larvae of S. littoralis, in addition to its effect on the decrease in the enzymes responsible for the transfer of the amine group in the insect (ALT, AST) and also the detoxification enzymes (GST, AchE), the acetone extract was the most effective.
Chemical constituents of Calotropis Procera acetonic extract.
where the acetone extract of C. procera showed more remarkable effect than ethanol and hexane against the 2nd and 4th instar larvae of S. littoralis it was demanded to identify the chemical constituents of that extract using GC-MS (Gas chromatography - Mass Spectrometry). The chromatogram revealed the presence of thirty components. The characterization and identification of compounds were confirmed by interpretation of their mass spectra fragmentations data and comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data software. GC-MS chromatogram of acetonic leaf extract showed 14 bioactive compounds were identified and appeared at retention time 5.58, 29.88, 30.09, 32.13, 33.88, 34.24, 35.00, 37.15, 46.96, 48.35, 50.12, 50.76, 54.04 and 54.25 min., respectively. Compound Docosane (C22H46) was the major bioactive compound present in acetone leaf extract of C. procera and was observed at retention time 32.13 min with peak area 27.70%, followed by Pregn-5-ene-3,11-dione 17,20:20,21-bis[methylenebis (oxy)]-cyclic 3-(1,2-ethanediyl acetal) (C25H34O7) and 5,7-Dimethoxy-4’-hydroxy flavanone (C17H16O5) with peak area (13.61%) and (6.08%), respectively. Additionally, the other compounds observed were Azadirachtin I (C32H42O12 ) (2.92%),Quercetin (C15H10O7) (1.86%), Corynan-17ol,18,19-didehydro-10,methoxy,acetate (ester)(C22H28N2O3) (1.60%), Acetylisocodeine (C20H25NO4) (1.50%), Docosanoic acid, methyl ester (C23H46O2) (1.49%), 2,7-Diphenyl-1,6-dioxopyridazino[4,5:2’,3’] pyrrolo [4’,5’d] pyridazine (C20H13N5O2) (1.45%), Heptadecane, 9-hexyl(C26H34O11) (1.40%), Oleanan-3-ol (C30H52O) (1.40%), Astaxanthin(C40H52O4) (1.33%), Lucenin2 (C27H30O16) (1.33%), Gibberellic Acid (C19H22O6) (1.31%) and 7,10,13-Hexadecatrienoic acid, methyl ester, (C17H28O2) (1.28%).
A popular concept assumes that plant secondary metabolites evolved in response to stress conditions or defense against herbivores, since they may serve as precursors of toxic substances.
Their insecticidal activity is probably due to their ability to complex with extracellular and soluble proteins and biomembranes. They also act as inhibitors for vital enzymatic pathways, in which the flavonoids specifically block steroid hydroxylase enzymes involved in the regulation of the molting process of insect pests which may lead to alteration of insect’s molting causing death.