الفهرس 
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Abstract
Mosquitoes are considered the main vector of malaria, dengue fever, lymphatic flariasis, rift valley fever, Japanese encephalitis, yellow fever, dengue haemorrhagic fever and viral encephalitis, cause extensive human morbidity and mortality and are a major economic burden within disease-endemic countries.
Among the types of mosquitoes spread in Alexandria Governorate, Culex pipiens mosquitoes, which are referred to as a vector of filaria parasites and some other viruses, and one of the most important characteristics of their greed for food on humans, especially indoors, and one of the most important reasons for choosing this type is that it is the most prevalent in the area of study in Alexandria Governorate. The aim of this study was to prepare, characterize, and evaluate of nano-structural impregnated fabric and skin treatment formulation against vectors of diseases using synthetic and natural compounds.
5.1.1. Larvicidal activity
5.1.1.1 Larvicidal activity of pyrethroids and their emulsifiable concentrates (ECs) and nanoemulsions (NEs)
Deltamethrin, α- Cypermethrin, lambda-cyhalothrin, and permethrin NEs were prepared based on obtained optimal conditions as a follow: a.i (0.5 %), DMSO (44%), Tween 80 (15%), water (40.5%), sonication pulses (9 cycle/sec) at sonication power (15 kHz) for 15 min. The droplet size of α-cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin NEs were 90.26, 172.00, 168, and 72 nm, respectively, while the PDI values were 0.337, 0.827, 0.448, and 0.295, respectively. The viscosity values of α-cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin NEs were 60.15, 74.67, 53.76, and 50.68 mPa.s, respectively, whereas the pH measurements were 8.51, 7.84, 8.20, and 8.17, respectively. The prepared NEs revealed negative values of zeta potential, when the TEM micrographs of demonstrated the spherical shape, uniform shape, size, and nanometric droplet diameter. It can be noted that the technical form exhibited the lowest larvicidal activity. Where, the EC of all tested insecticides slightly improved the toxic action against the larvae. However, all insecticides’ NE showed significantly high toxicity (1.5-2-fold) compared to the technical and EC. This finding led to a significant decrease in the field application rate by half-value, resulting in low environmental pollution and hazards.
5.1.1.2. Larvicidal activity of essential oils (EOs) their ECs and NEs
The components of each EO were identified by GC/MS analysis. The major component of cinnamon EO detected in the oil, was (E)-cinnamaldehyde (80.09%). While the main identified components of cypress EO were α-pinene (29.63%). The main components of lavender EO was linalool (16.48 %). The analysis of lemon eucalyptus EO revealed that the β-citronellal (71.61%), was the major constituent. β-Cymene was the major constitute (28.47%) of tea tree oil.
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The oils of cinnamon, cypress, lavender, lemon eucalyptus, and tea tree were prepared in NEs form according to optimized method as follow: pure oil (10%), Tween 80 (10%), and water (80%) at sonication pulses 9 pulses/sec and 15 kHz for 5 min.
The droplet sizes of cinnamon, cypress, lemon eucalyptus, lavender, and tea tree NEs were 95.67, 71.67, 104.55, 211.07, and 70.67 nm, respectively, while the PDI values were 0.328, 0.319, 0.433, 0.536, and 0.422, respectively. There are no significant differences between the viscosities of the prepared NEs, also, all had pH values around 6. The prepared NEs showed high negative values of zeta potential. Their microscopic images showed the spherical and uniform shape. The data showed that the tea tree oil has the most significant effect with LC50 of 60.02 and 57.10 mg/L after 24 and 48 h of exposure, respectively. While cypress oil proved the lowest toxicity with LC50 values 202.24 and 180.70 mg/L after 24 and 48 h, respectively. However, lavender oil does not show any effect against the larvae at the tested concentrations (LC50 > 2500 mg/L). Cinnamon NE showed larvicidal activity (LC50 = 134.20 mg/L) compared to their EC formulation (LC50 = 147.52 mg/L) and EO (LC50 = 154.08 mg/L) after 24 h of exposure. Cypress NE gave the LC50 value of 114.12 mg/L that more significant than 185.41 mg/L for EC form, and 202.24 mg/L for pure EO after 24 h of the experiment. NE of lemon eucalyptus exhibited larvicidal effect with LC50 values = 154.06 while its pure form gave 154.06 mg/L after 24 h of exposure. In tea tree oil, the LC50 values of NE and EO were 53.74 and 60.02 mg/L after 24 h of exposure, respectively.
5.1.1.3. Larvicidal activity of monoterpenes their ECs and NEs
Cinnamaldehyde, citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate were prepared in NE forms according to the chosen technique as a follow: a.i (2.5%), DMSO (5%), Tween 80 (2.5%), and water (90%) at 9 pulses/sec for sonication and power 5 kHz for 5 min.
There is a significant difference between the droplet size values. The PDI of prepared NEs were 0.58, 0.41, 0.44, 0.49, and 0.34 for cinnamaldehyde, citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate, respectively. The viscosity values of the NEs were 6.93, 6.53, 6.41, 6.62, and 6.46 for cinnamaldehyde, citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate, respectively, while the pH values were 4.08, 5.50, 5.61, 6.58, respectively. Negative zeta potential of cinnamaldehyde, citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate NEs were -21.20, -15.78, -24.60, -25.80, and -18.10, respectively. TEM micrographs of NEs showed their spherical form, typical appearance, and nanometer size.
As a general, the resulted data showed that the T forms of α-terpinyl acetate and β-cymene were the most active compounds against C. pipiens larvae with LC50 values of 20.52 and 21.70 mg/L, respectively after 24 h of exposure. In contrast, (R)-linalool showed the least activity against larvae understudy with LC50 values 426.86 mg/L and 410.84 mg/L after 24 and 48 h of exposure, respectively. While cinnamaldehyde and citronellal gave LC50 values 94.46 and 174.23 mg/L, respectively after 24 h of exposure. Cinnamaldehyde NE gave the LC50 value of 78.93 mg/L that more than the EC formulation (85.38 mg/L) and the T form (94.46 mg/L) after 24 h of the experiment. It can be observed that the larvicidal behavior of the NE was the best. However, the cinnamaldehyde EC slightly enhanced the toxic action against the larvae. The findings show that changing of citronellal, β-cymene, (R)-linalool, and α-terpinyl acetate from T to EC or NE forms causes their efficacy to decrease and disappear.
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5.1.2. Adulticidal activity
5.1.2.1. Knockdown activity of EOs and monoterpenes against C. pipiens adults by fumigation technique
Results of EOs showed that lemon eucalyptus oil has the highest activity against C. pipiens adults with KT50 = 40.29 sec at 10 mg/L. At the same concentration, cypress oil caused KT50 = 53.03 sec followed by lavender oil, cinnamon oil, and tea tree oil with KT50 values = 84.72, 152.03, and 266.13, respectively. Also, at the same concentration (10 mg/L) all tested oils caused 100% mortality after 24 h of exposure except lavender oil causes 70% adult mortality. In the case of monoterpenes, linalool showed the highest effect with the lowest KT50 value = 12.73 sec. The effect of citronellal was a higher than α-terpinyl acetate. At the same concentration (10 mg/L) all tested compounds cause 100% mortality after 24 h of exposure.
5.1.2.2. Repellent activity of lemon eucalyptus oil and linalool nanocreams against C. pipiens adult by arm in cage technique
The data showed that lemon eucalyptus oil was stronger than linalool with RC50 values = 100.82 and 998.84 mg/L, for pure compounds while 10.03 and 68.11 mg/L, for NEs formulations, respectively. It’s obvious, the effect of converting pure compounds to NEs, which increases the activity of compounds by 10 times in EOs and 70 times in linalool. The concentrations caused 100% repellency for 6 h were 300 mg/L and 5000 mg/L for lemon eucalyptus oil and linalool, respectively. While NEs formulations caused 100% repellency at 30 and 200 mg/L for lemon eucalyptus oil and linalool, respectively.
5.1.2.3. Knockdown activity of permethrin and nano-permethrin impregnated fabrics against C. pipiens adults
Impregnated fabrics were treated with 10000 mg/m2 permethrin of each form. NE of permethrin showed 100% knockdown of C. pipiens adults using cone bioassay (after 10 min of exposure) and WHO tube (after 60 min of exposure) techniques. While impregnated fabrics with permethrin (EC) caused 60% and 95% knockdown against C. pipiens adults using WHO tube and cone bioassay techniques, respectively. Transforming the EC form of permethrin to a nano form led to increasing the adulticidal compound’s effect on C. pipiens adults. The cone bioassay test was more appropriate technique than WHO tube to determine the knockdown activity after 10 min of exposure.
5.1.2.4. Adulticidal activity of permethrin and nano-permethrin impregnated fabrics against C. pipiens adult
NE of permethrin showed 90 and 95% mortality of C. pipiens adults using WHO tube and cone bioassay techniques, respectively. While impregnated fabrics with permethrin (Pergon®, EC) caused 75% and 70% mortality against C. pipiens adults using WHO tube and cone bioassay techniques, respectively.
5.1.3. Biochemical studies of pyrethroids, EOs, and monoterpenes against larval enzymes
CaE and GST were activated while ATPase was inhibited as a result of exposed to different forms of pyrethroids. In case of, the exposure to EOs and monoterpenes, the AChE was inhibited, while GABA-T and ATPase were activated.
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5.1.4. Molecular docking of tested pyrethroids and monoterpenes against some larval enzymes
The pyrethroids showed binding affinity with CaE and GST more than ATPase. The docking scores with ATPase were ranged from -4.33 to -5.46 kcal/mol. While exhibited binding affinity ranged from -7.44 to -10.01 kcal/mol on the active sites of CaE.
The binding affinity of monoterpenes ranged from -3.74 to -4.52 kcal/mol on the active sites of AChE. While they exhibited low binding affinity towards the active sites of the ATPases. The monoterpens exhibited binding affinity with docking scores ranged from -3.33 to -5.43 kcal/mol on the active sites of GABA-T.
5.1.5. Biochemical and histological effects of dermal topical treatment of linalool, lemon eucalyptus and their NEs on albino rat
5.1.5.1. Effect of linalool, lemon eucalyptus and their NEs on the complete blood count (CBC) of treated albino rat
Albino rats were treated with 100 μL of linalool (2%) and lemon eucalyptus oil (0.3%) and their NEs for 5 days. Different treatments caused slightly increasing in WBCs compared with control (7.01 thousand). There is no obvious effect of tested treatments on RBCs and Hb. All treatments caused slightly non-significant decreasing in blood platelets count. There are no obvious effects of treatments on HCT and MCHC compared with control (43.87% and 27.63 g/dl, respectively). NEs of lemon eucalyptus oil and linalool did not show significant effects on MCH, while their pure compounds caused slightly decreasing compared with untreated sample. NEs of lemon eucalyptus oil and linalool did not cause clear effects on MCV, while their pure compounds caused slightly decreasing. The conversion of pure compounds to NEs decreasing their significant effects on theses blood parameters.
5.1.5.2. Effect of linalool, lemon eucalyptus and their NEs on kidney enzymes of treated albino rat
The results showed that different treatments significantly decrease the urea levels in blood serum. NE of linalool caused the greatest effect on urea level (30.40 mg/dl) compared with its level in control sample (38.00 mg/dl). While there are not any significant differences between other treatments. On the other hand, different treatments elevated creatinine levels in blood serum. The NE of lemon eucalyptus oil significantly increased creatinine level (0.48 mg/dl) in blood serum compared with its levels in control sample (0.35 mg/dl).
5.1.5.3. Effect of linalool, lemon eucalyptus and their NEs on liver enzymes of treated albino rat
All treatments caused significantly increasing in ALT concentrations. Technical linalool showed the highest activity caused increasing of ALT concentration from 44.67 U/L in control sample to 61.60 U/L in treated sample. While NE of linalool caused increasing of ALT concentration to 46.20 U/L. Lemon eucalyptus oil caused slightly increasing in ALT levels .All treatments elevated of AST concentrations in blood serum. In case of Alp assay, the results showed that, all treatments significantly decreased the activity of ALP enzyme except the NE of linalool which caused increasing of its enzyme activity.
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5.1.5.4. Effect of linalool, lemon eucalyptus and their NEs on AChE and ATPase of albino rat blood serum
All treatments including control samples increased AChE activity. Technical linalool was the most active compound which increased activity with 40.21%. The NE form of lemon eucalyptus oil increased its activity from 3.01% for pure oil to 27.86%. While NE form of linalool decreased its activation activity of AChE from 40.21% for technical form to 19.83%. The activation effect of linalool on ATPase was higher than lemon eucalyptus oil. Linalool caused enzyme activation with 30.44% for technical form and 26.67% for NE. While pure lemon eucalyptus oil had slightly activation effect (1.69%) against ATPase.
5.1.5.5. Histological effects of linalool, lemon eucalyptus and their NEs on treated skin
The observed effects of topical application of linalool, lemon eucalyptus oil, and their NEs on the skin of albino rat did not show any irritating or sensitizing effects.
5.2. Conclusion
from this study results it is concluded that:
1. The modification of traditional formulations of pyrethroids to NEs formulations increased their larvicidal properties.
2. The remarkable stable behavior of prepared pyrethroid NEs with adequate larvicidal activity at the lowest exposure concentration makes it a suitable and effective mosquito control agent.
3. Lambda-cyhalothrin showed the most larvicidal effect against C. pipiens.
4. All tested pyrethroid formulations caused a significant inhibition of ATPase enzyme and significant activation of GST activity.
5. All tested pyrethroid formulations caused slightly activation of CaE activity except their NEs which caused a significant activation.
6. All previous results and analysis of the docking results confirmed that lambda-cyhalothrin was the highest affinity binding on the teasted enzymes ATPase, CaE, and GST with the lowest energy docking scores.
7. Tea tree oil had the most significant effect while lavender oil does not show any effect against C. pipiens larvae at tested concentrations.
8. Cinnamon, cypress, lemon eucalyptus, and tea tree NEs could be considered as promising candidates for botanical larvicides against C. pipiens.
9. The NEs of EOs showed significantly and high toxicity compared to the pure oil and EC formulation against C. pipiens larvae.
10. Tea tree oil NE had the highest inhibition effect of AChE activity (59.62%) followed by the NEs of lemon eucalyptus oil (51.21% inhibition) and cinnamon oil (46.40% inhibition).
11. The effect of cypress oil on the AChE activity was the lowest.
12. NE formulations of EOs caused a high level of ATPase activation by 297.62, 224.68, 193.66, and 75.42% for cypress oil, tea tree oil, lemon eucalyptus oil, and cinnamon oil, respectively. 13-The technical form of α-terpinyl acetate and β-cymene were the most active monoterpenes against C. pipiens larvae.14. (R)-Linalool and citronellal showed the least effective against C. pipiens larvae.
15. The larvicidal activity of β-cymene, (R)-linalool, and α-terpinyl acetate was disappeared after their conversion to NEs form.
16. Cinnamaldehyde NE displayed a remarkable larvicidal effect.
17. The molecular docking study showed that the affinity binding of the monoterpenes on GABA-T was more than AChE and ATPase.
18. β-Cymene, α-terpinyl acetate, and cinnamaldehyde had a significant overall effect on C. pipiens larvae and could be used as promising natural mosquitocides.
19. β-Cymene showed the highest biniding affininty on the active sites ATPases and GABA-T, while α-terpinyl acetate showed the highest binding affinity on AChE enzyme.
20. The protein-ligand docking in the present study revealed that the tested compounds have an excellent theoretical binding affinity to the active sites of the target enzymes with low binding energy and high affinity for the functional pocket.
21. Lemon eucalyptus oil has the highest activity against C. pipiens adults, followed by cypress oil, lavender oil, cinnamon oil, and tea tree oil by fumigation technique.
22. All tested oils caused 100% mortality after 24 h of exposure except lavender oil caused 70% mortality by fumigation technique.
23. Linalool showed the highest adulticidal effect while β-Cymene and cinnamaldehyde were significantly the lowest adulticidal effects against C. pipiens compared to other tested monoterpenes.
24. All tested monoterpenes caused 100% mortality after 24 h of exposure at the tested concentration against C. pipiens adults. 25. The repellent activity of lemon eucalyptus nano-cream was a higher than (R)-linalool against C. pipiens adults. 26. Converting lemon eucalyptus oil and (R)-linalool to NE forms increased their repellent effect, many times the original effect of these compounds. 27. The NEs formulations of tested oils and monoterpenes could be considered as promising candidates for botanical adulticides against C. pipiens. 28. As a repellent, nano-cream of lemon eucalyptus oil and (R)-linalool could be considered as promising ecofriendly alternatives against C. pipiens adults.
29. Linalool induced obvious mortality of mosquito’s adults but had lower larvicidal activity than the other compounds.
30. Nano permethrin impregnated fabrics showed knockdown effect was more than EC permethrin impregnated fabrics against C. pipiens adults.
31. The cone bioassay test was more appropriate technique than WHO tube to determine the knockdown activity after 10 min of exposure.
32. The WHO tube was more appropriate technique than cone bioassay test to determine the adulticidal effect after 60 min of exposure.33. Transforming the EC formulation of permethrin impregnated fabrics to NE form led to increase the adulticidal compound’s effect on C. pipiens adults.
34. There is no obvious effect of (R)-linalool, lemon eucalyptus oil, and their NEs treatments on RBCs, Hb, HCT, and MCHC.
35. The conversion of pure compounds to NEs decreasing their significant effects on RBCs count, HCT, MCV, MCH, and MCHC.
36. (R)-Linalool and lemon eucalyptus oil treatments significantly decrease the urea levels and elevated creatinine levels in blood serum, compared with its levels in untreated sample.
37. (R)-linalool and lemon eucalyptus oil treatments caused significantly increasing in ALT and AST concentrations in the blood serum.
38. All (R)-linalool and lemon eucalyptus oil treatments significantly decreased the activity of ALP enzyme except the NE of (R)-linalool which caused increasing of its enzyme activity.
39. (R)-linalool and lemon eucalyptus oil treatments increased AChE and ATPase activity in the blood serum.
40. The activation effect of (R)-linalool on ATPase was higher than lemon eucalyptus oil.
41. There is no obvious harmful side effects of the dermal topical treatments of (R)-linalool and lemon eucalyptus oil on some biochemical parameters of treated albino rat.
42. There is no obvious effects of the dermal topical treatments of (R)-linalool and lemon eucalyptus oil on the histological status of the treated skin of treated albino rat.
5.3. Recommendations
from the conclusion of the present study results, it is recommended that:
1. Because of the remarkable stable behavior of prepared pyrethroid NEs with adequate larvicidal activity at the lowest exposure concentration, these advantages make such new formulations are suitable and effective agents to mosquito control.
2. β-Cymene, α-terpinyl acetate, and cinnamaldehyde had a significant overall effect on C. pipiens larvae and could be used as promising natural larvicides.
3. The NEs of cinnamon, cypress, lemon eucalyptus, and tea tree NEs could be considered as promising candidates for botanical larvicides against C. pipiens. 4. The NEs of tested oils and monoterpenes could be considered as promising candidates for botanical adulticides against C. pipiens. 5. Nano-creams of lemon eucalyptus oil and (R)-linalool could be considered as promising ecofriendly repellents against C. pipiens adults.
6. NE of permethrin impregnated fabrics could be used as promising technique against C. pipiens adults.
7. There is a need to further studies to improve the repellency performance and durability of the treated fabrics for an extended time.
8. Insect repellent may be applied into the fiber or yarn during the spinning process of the fiber before the final textiles were produced.