Larvicidal and repellent potential of Ageratum houstonianum against Culex pipiens

Abstract

Mosquitoes are unquestionably the most medic arthropod vectors of disease. Culex pipiens, usually defined as a common house mosquito, is a well-known carrier of several virus diseases. Crude ethanol extracts of different organs of Agratum houstonianum are tested with Culex pipiens Linnaeus (Diptera: Culicidae) to determine their larvicidal, antifeedant, and repellency effects. Alongside biochemical analysis, the activity of the AChE, ATPase, CarE, and CYP-450 is detected in the total hemolymph of the C. pipiens larvae to examine the enzymatic action on the way to explain their neurotoxic effect and mode of action. Through HPLC and GC-MS analysis of the phytochemical profile of A. houstonianum aerial parts is identified. The larvicidal activity of aerial parts; flower (AF), leaf (AL), and stem (AS) of A. houstonianum extracts are evaluated against the 3rd instar larvae of C. pipiens at 24-, 48-and 72-post-treatment. A. houstonianium AF, AL, and AS extracts influenced the mortality of larvae with LC50 values 259.79, 266.85, and 306.86 ppm, respectively after 24 h of application. The potency of AF and AL extracts was 1.69-and 1.25-folds than that of AS extract, respectively. A high repellency percentage was obtained by AF extract 89.10% at a dose of 3.60 mg/cm 2. A. houstonianium AF prevailed inhibition on acetylcholinesterase and decrease in carboxylesterase activity. Moreover, a significant increase in the ATPase levels and a decrease in cytochrome P-450 monooxegenase activity (− 36.60%) are detected. HPLC analysis prevailed chlorogenic and rosmarinic acid as the major phenolic acids in AL and AF, respectively. GC-MS analysis of A. houstonianum results in the identification of phytol as the major makeup. Precocene I and II were detected in AF. Linoleic, linolenic, and oleic acid were detected in comparable amounts in the studied organs. Overall, results suggest that the A. houstonianum flower extract (AF) exhibits significant repellent, antifeedant, and larvicidal activities.

Full text

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Larvicidal and repellent potential
of Ageratum houstonianum
against Culex pipiens
Doaa El Hadidy
1, Abeer M. El Sayed
2*, Mona El Tantawy
1, Taha El Alfy
2,
Shaimaa M. Farag
3 & Doaa R. Abdel Haleem
3
Mosquitoes are unquestionably the most medic arthropod vectors of disease. Culex pipiens, usually
dened as a common house mosquito, is a well-known carrier of several virus diseases. Crude ethanol
extracts of dierent organs of Agratum houstonianum are tested with Culex pipiens Linnaeus (Diptera:
Culicidae) to determine their larvicidal, antifeedant, and repellency eects. Alongside biochemical
analysis, the activity of the AChE, ATPase, CarE, and CYP-450 is detected in the total hemolymph
of the C. pipiens larvae to examine the enzymatic action on the way to explain their neurotoxic
eect and mode of action. Through HPLC and GC–MS analysis of the phytochemical prole of A.
houstonianum aerial parts is identied. The larvicidal activity of aerial parts; ower (AF), leaf (AL), and
stem (AS) of A. houstonianum extracts are evaluated against the 3rd instar larvae of C. pipiens at 24-,
48- and 72-post-treatment. A. houstonianium AF, AL, and AS extracts inuenced the mortality of
larvae with LC50 values 259.79, 266.85, and 306.86 ppm, respectively after 24 h of application.
The potency of AF and AL extracts was 1.69- and 1.25-folds than that of AS extract, respectively.
A high repellency percentage was obtained by AF extract 89.10% at a dose of 3.60 mg/cm2. A.
houstonianium AF prevailed inhibition on acetylcholinesterase and decrease in carboxylesterase
activity. Moreover, a signicant increase in the ATPase levels and a decrease in cytochrome P-450
monooxegenase activity (− 36.60%) are detected. HPLC analysis prevailed chlorogenic and rosmarinic
acid as the major phenolic acids in AL and AF, respectively. GC–MS analysis of A. houstonianum results
in the identication of phytol as the major makeup. Precocene I and II were detected in AF. Linoleic,
linolenic, and oleic acid were detected in comparable amounts in the studied organs. Overall, results
suggest that the A. houstonianum ower extract (AF) exhibits signicant repellent, antifeedant, and
larvicidal activities.
Mosquitoes considered vectors to a wide variety of serious human diseases. e Culex pipiens is widely distributed
in Egypt causing nuisance to humans and transmits several viral diseases1. It is the vector of West Nile virus2,
Ri Valley fever virus3, Wuchereria bancroi4, yellow fever5, lariasis6 and other major public health problems
worldwide which cause a signicant human and animal mortality and morbidity in addition to sever economic
losses. e mosquito control mainly based on the application of synthetic insecticides as larvicides or as adult
repellents7. e chemical insecticides have adverse impacts on the health and environment beside to the devel-
opment of resistance8. ere is global interest in developing natural products as alternatives to conventional
insecticides for mosquito control9. Many plant species have been screened for their repellent and insecticidal
property10. Family Asteraceae contained many plant species which have been described for their medicinal and
insecticidal purposes11. Ageratum houstonianum Mill. belonging to this family is a medicinal plant and possesses
antimicrobial activity10. ere are some previous reports on the insecticidal activities12 of the dierent extracts of
leaves of A. houstonianum as well as repellency against mosquitoes13. Furthermore, A. houstonianum has found
to be a potent source of natural antioxidants14. Several classes of compounds were reported from A. houstoni‑
anum15–19. However, a literature survey has shown that there is no report on the phytochemicals of ethanolic
extracts of dierent aerial parts (leaves, stems and owers) of the Egyptian A. houstonianum which prompted
authors to investigate the secondary metabolite proles of the dierent organs under study. is study was
planned to evaluate the larvicidal activity, repellant and antifeedant eciency of ethanolic extracts of dierent
OPEN
1Department of Medicinal Plants and Natural Products, National Organization for Drug Control and Research
(NODCAR), 51-Wezaret El-Zeraa St, Giza 12611, Egypt. 2Department of Pharmacognosy, Faculty of Pharmacy,
Cairo University, Kasr El Aini 11562, Egypt. 3Department of Entomology, Faculty of Science, Ain Shams University,
Cairo 11566, Egypt. *email: abeer.ali@pharma.cu.edu.eg
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aerial parts of A. houstonianum against C. pipiens larvae and adult. As well as study their enzymatic action to
explain their neurotoxic eect and mode of action. Alongside investigation of the lipoidal and polyphenolic
phytochemical prole through GC–MS and HPLC analysis were carried out respectively, to shed light on the
bioactive components of dierent organs of A. houstonianum to which the biological activities may be attributed.
Results
Determination of the total phenolic contents. Quantitative determination of phenolic contents
of A. houstonianum AL, AS, and AF extracts were determined. It was observed that the ethanolic extract of
the AF have the highest total phenolic content, followed by the AL then the AS with values of 5.65, 4.82 and
3.39µg GAE/mg respectively. Flower was the richest extract in the avonoid contents with value 5.07µg QE/mg.
Whereas the leaves have half the avonoid content of the ower.
HPLC analysis and identication of phenolic compounds. HPLC analysis of 70% alcoholic extract of
AL, AS, and AF were expressed as (mg/g) extract and complied in Table1 and the chromatograms are presented
in Fig.S1. Allowed identication and quantication of several phenolic acid and avonoids. It was observed
that the total identied phenolic acids in extract of AL, AS, and AF were 8.35, 2.64 and 12.89mg/g extract,
respectively. Chlorogenic acid is the major one among the total phenolic acids by7.12, 5.19, 1.99mg/g in AL, AF
and AS, respectively. Rosmarinic acid was also detected at high concentration in the AF 7.303mg/g while it was
detected in small amount in the AL and AS 0.77 and 0.49mg/g, respectively. On the other hand, 14 avonoids
were identied 1.32, 0.48 and 6.43mg/g extract for the AL, AS and AF, respectively. Rutin was detected at high
concentration as 0.92, 0.52, and 0.33mg/g in AL, AF and AS respectively. Also, apigenin was found in the owers
extract at a concentration of 1.79mg/g.
GC/MS analysis of the lipoidal contents. It was concluded that the yield of lipoidal matter of leaves,
stems, and owers were (3.3%, 1.2% and 4.7%), respectively. e percentage of the unsaponiable matter (USM)
were (58.80%, 55.20% and 58.10%) and FAME were (38.20%, 33.70% and 40.40%) in the extracts of leaves,
stems, and owers, respectively. GC/MS analysis leads to identication of 30, 26 and 31components represent-
ing (99.27%, 99.33% and 97.50%) of the n-hexane extract yield of leaves, stems and owers respectively (Table2,
Fig.S2). It was observed that: unsaponiable matter was composed of hydrocarbons, alcohols, ketones, alde-
hydes, esters, acids, phenols, sterols, chromenes, quinones, lactones and epoxides. e hydrocarbons repre-
sented (19.06%, 10.77 and 15.24%) of the USM of leaves, stems, and owers respectively. e main of which was
5-Octadecene (3.07%) in leaves, 3-Eicosene (2.78%) in stem and in owers was Tridecane, 5-methyl (6.92%).
Table 1. HPLC analysis of the alcoholic extracts of leaves, stems, and owers of A. houstonianum. L leaves, S
stems, F owers, – not identied.
Peak no. Retention time (min) Identied compound
Conc. (mg/g extract)
L S F
1 3.9 Gallic 0.08 0.01 0.01
2 7.7 Protocatechuic 0.09 0.09 0.03
3 12.1 p-hydroxybenzoic 0.08 0.00 0.08
4 12 Gentisic – – –
5 15.2 Catechin –––
6 16.5 Chlorogenic 7.12 1.99 5.20
7 17.2 Caeic 0.07 0.02 0.13
8 19.3 Syringic 0.01 – 0.04
9 21.2 Vanillic 0.01 – 0.04
10 28.9 Ferulic 0.01 – 0.00
11 30.7 Sinapic 0.02 – 0.02
12 35 p-coumaric 0.08 0.01 0.05
13 34.5 Rutin 0.93 0.34 0.53
14 38.2 Apigenin-7-glucoside 0.13 0.08 3.85
15 39.2 Rosmarinic 0.78 0.49 7.30
16 46.9 Cinnamic 0.01 0.02 0.01
17 49.5 Qurecetin 0.02 – 0.03
18 55.2 Apigenin 0.17 0.06 1.80
19 55.9 Kaempferol 0.02 0.02 0.15
20 59 Chrysin 0.04 0.00 0.08
Total identied phenolic acids 8.35 2.64 12.89
Total identied avonoids 1.32 0.49 6.43
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Peak no. Rt (min) Identied compound Molecular formula Base peak Molecular ion peak (M+)
Yield (%)
L S F
1 13.91 3-Butylcyclohexanone C10H18O 55 154 0.79 – –
2 19.20 5-Tetradecene C14H28 55 196 – 0.66 –
3 19.21 3-Tridecene C13H26 41 182 1.54 – –
4 21.16 2,6-Dibutyl-2,5-cyclohexadiene-1,4-dione C14H20O241 220 – – 0.40
5 21.37 2,6-Di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one C15H24O257 236 – – 0.65
6 22.42 2-Allyl-5-t-butylhydroquinone C13H18O2191 206 – 1.49 –
7 22.43 Phenol,2,4bis (1,1dimethyl ethyl) C14H22O 191 206 1.94 – –
8 22.46 7-Methoxy-2,2,8-trimethyl chromene C13H16O2189 204 – – 0.65
9 23.96 3-Hexadecene C16H32 55 224 – 1.73 –
10 23.97 7-Hexadecene C16H32 55 224 2.98 – –
11 25.23 Caryophyllene oxide C15H24O 41 220 – – 3.21
12 25.80 2H-1-Benzopyran,6,7-dimethoxy-2,2-dimethyl (precocene II) C13H16O3205 220 22.08 13.26 19.20
13 26.20 7-t-Butyl-3,3-dimethyl-1-indanone C15H20O 201 216 – – 6.05
14 26.29 α-Bisabolol C15H26O 43 222 – – 0.54
15 26.32 Heptadecane C17H36 57 240 1.90 1.24 –
16 26.38 Ledene oxide C15H24O 43 220 – – 0.52
17 27.12 Methyl 1,5-di-tert-butylbenzene-3-carboxylate C16H24O2233 248 – – 1.28
18 28.10 Loliolide C11H16O343 196 – 1.12 0.49
19 28.28 5-Octadecene C18H36 55 252 3.07 1.91 –
20 28.41 Octadecane C18H38 43 254 0.61 – –
21 28.84 Erythro-(cis)(1,4),(cis)(1′,4′)-4,4′-Dihydroxybicyclooctyl C16H30O267 254 – 0.67 –
22 28.85 Cyclooctenone, dimer C16H24O255 248 0.72 – –
23 28.90 α-Bisabolene epoxide C15H24O 43 220 – – 0.44
24 28.97 11,13-Dihydro-11áH-arbusculin B C15H22O2219 234 1.09 1.43 –
25 29.23 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 81 296 2.21 1.41 4.93
26 29.38 2-Pentadecanone,6,10,14-trimethyl C18H36O 43 268 2.05 4.69 2.13
27 30.15 2-Methoxymethyl-4,4-dimethyl-5-phenyldihydropyran C15H20O243 232 – – 0.83
28 30.73 2-Methylhexadecanal C17H34O 58 254 – 0.82 –
29 31.02 Hexadecanoic acid, methyl ester C17H34O274 270 – – 0.38
30 32.18 5-Eicosene C20H40 55 280 2.51 2.78 0.48
31 32.27 Eicosane C20H42 57 282 – 0.82 –
32 32.28 Pentadecanoic acid, 2,6,10,14-tetramethyl, methyl ester C20H40O243 312 0.95 – –
33 32.99 Nerolidol C15H26O 41 222 0.99 – –
34 33.02 Geranyl linalool C20H34O 69 290 – – 1.35
35 33.22 Cembrene C20H32 68 272 – 0.77
36 33.83 Acetic acid, 3,7,11,15-tetramethyl-hexadecyl ester C22H44O257 340 0.82 – –
37 33.91 9,15-Octadecadienoic acid methyl ester C18H32O241 294 – – 13.78
38 33.93 1-Hexadecanol C16H34O 55 242 – 1.58 –
39 34.31 2-Nonadecanone C19H38O 58 282 – – 2.34
40 34.53 Phytol C20H40O 71 296 38.28 52.10 19.39
41 34.96 Palmitaldehyde, diallyl acetal C22H42O284 338 1.92 1.83 –
42 35.43 1-Propene-1,2,3-tricarboxylic acid, tributyl ester C18H30O6112 342 2.12 – –
43 35.75 1-Hexacosanol C26H54O 43 382 – 2.93
44 35.76 10-Heneicosene C21H42 55 294 2.51 – –
45 35.85 Docosane C22H46 43 310 0.63 – 0.58
46 36.28 Phytol acetate C22H42O243 338 0.65 0.85 –
47 37.17 Tributyl acetylcitrate C20H34O8185 402 1.07 – –
48 37.51 Tricosane C23H48 57 324 1.33 – –
49 37.58 Tridecane,5-methyl C14H30 43 198 – – 6.92
50 38.51 4,8,12,16-Tetramethyl heptadecan-4-olide C21H40O299 324 – 1.11 0.5
51 39.03 1-Docosanol C22H46O 43 326 – 2.27
52 39.15 Hexatriacontane C36H74 57 506 – – 4.43
53 40.66 Pentacosane C25H52 57 352 0.81 1.22 –
54 41.59 1,2-Benzenedicarboxylic acid, dioctyl ester C24H38O4149 390 0.71 – –
55 42.41 Trans-Geranylgeraniol C20H34O 69 290 – – 0.39
Continued
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Alcohols were the major identied class of compounds of USM of the leaves, stems and owers representing
(41.48%, 60.96% and 26.60%, respectively).
GC/MS analysis of saponiable matter of A. houstonianum (Table3, Fig.S3) revealed the identication of 16,
21 and 22 components representing 96.51%, 97.14% and 98.42%, of the total FAME of leaves, stems and owers,
respectively. It was observed that: e unsaturated fatty acids constitute the major makeup in the stem, leaves,
and owers (63.27, 56.25, 54.65%), respectively. Omegas 6 and 3 were detected in comparable amounts as the
major makeup. On the other hand, palmitic acid was the major one (32.72%).
Larvicidal bioassay. e larvicidal activity of aerial parts of A. houstonianium extracts were evaluated
against the 3rd instar larvae of C. pipiens at 24-, 48- and 72-post-treatment and the data represented in Table4.
e mortality rate of larvae increased with increase time of exposure and concentrations for all extracts. e
results indicated that the extracts of A. houstonianium ower, leaf and stem inuenced the mortality of larvae
with LC50 values 259.79, 266.85 and 306.86ppm, respectively, aer 24h of application. e ower extract showed
high potency compared with leaf and stem extracts at the 1st, 2nd and 3rd day of exposure. e toxicity indexes
of leaf and stem extracts decrease gradually with time. At 72h post-treatment, the toxicity indexes of leaf and
stem extracts were 73.91 and 59.10, respectively. e potency of ower and leaf extracts were 1.69 and 1.25 folds
than stem extract, respectively. e slope values were low which indicate the homogeneity of the tested popula-
tion.
Repellency/antifeedant action of A. houstonianum Mill. ower, leaf and seed extracts against
the adult Culex pipiens. e overall, the repellency of the A. houstonianum ower, leaf and seed extracts
tested and DEET gave a variable degree of repellency (Table5). At a dose (1.8mg/cm2), potent repellency (100%)
was obtained by DEET through the 4h post treatment, the other 3 extracts exhibited < 89.1% repellency within
the 4h post-treatment; the relative repellency was increased as the dose increased, where highest repellency
% was obtained by ower extract (89.1%) at a dose 3.6mg/cm2 decreased to 73.3% at a dose 1.8mg/cm2 aer
4h from treatment, while the lowest repellency % was obtained by leaf extract (86.2%) at a dose 3.6mg/cm2
decreased to 49.6% at a dose 1.8mg/cm2 aer 4h post-treatment.
Biochemical activity. Activity of the enzymes, AChE, ATPase, CarE and CYP-450 were detected in the
total hemolymph of the C. pipiens larvae treated with LC50 of A. houstonianium ower, leaf and stem extracts
were shown in Table6. AChE activity was signicantly inhabited in C. pipiens larvae, the obtained inhibition
ratios of enzymatic activity ranging from − 57.86% (ower), − 40.979% (leaf) to − 15.95% (stem). It was noticed
that both ower and leaf extracts have high inhibition ecacy against acetylcholinesterase than stem extract.
All tested extracts led to decrease in the amount of CarE which more obvious with ower extract than other
extracts. It was 43.12, 47.30 and 53.05 (ug Meb/min/mg protein) for ower, leaf, and stem, respectively, as com-
pared with control 61.01 (ug Meb/min/mg protein).
Results given in Table6 indicated that the tested extracts increase the amount of ATPase which was clearly
detected in ower extract treatment compared with control. Amount of ATPase were 78.81, 69.16 and 63.93
(umoles Pi/min/mg protein) for extracts of ower, leaf, and stem, respectively, while it was 60.6 (umoles Pi/
min/mg protein) with control. A signicant reduction in CYP-450 activity was obtained by treatment with all
extracts whereas the ower extract showed the high reduction (− 36.606%) compared with leaf (− 22.14%) and
stem (− 20.87%) extracts.
Discussion
Chlorogenic acid is one of the most abundant benecial polyphenols in plants and is well known as nutritional
antioxidant in plant -based foods. Apart from its dietary antioxidant activity, it has been proven to be an ecient
defense molecule against a broad range of insect herbivores20. Increased eciency of bio-insecticides is achieved
by using chlorogenic acid as a synergistic bacterium. Chlorogenic acid has chemical defense against insects
ascribed to its prooxidant eect by binding of the highly reactive chlorogenoquinone with nucleophilic–NH2
and –SH groups in proteins and amino acids21. is reduces the bioavailability of amino acids consequently
decreases digestibility of dietary proteins so, it considered as eective deterrent or anti-feedant22.
Table 2. GC/MS analysis of the unsaponiable matter (USM) of n-hexane extract of the leaves, stem and
owers of A. houstonianum. L leaves, S stem, F owers.
Peak no. Rt (min) Identied compound Molecular formula Base peak Molecular ion peak (M+)
Yield (%)
L S F
56 43.62 Heptacosane C27H56 57 380 – – 1.10
57 45.49 Squalene C30H50 69 410 1.17 – –
58 48.92 Octacosane C28H58 57 394 – – 0.5
59 51.10 Stigmasta-5,22-dien-3-ol (Stigmasterol) C29H48O 55 412 0.55 0.52 0.49
60 52.48 22,23-Dihydro stigmasterol (β-sitosterol) C29H50O 43 414 0.45 0.41 0.5
61 52.88 Lupeol C30H50O 43 426 0.82 0.13 1.82
Total identied compounds 99.27 99.33 97.50
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Table 3. GC/MS analysis of the fatty acid methyl ester matter (FAME) of n-hexane extract of the leaves, stems
and owers of A. houstonianum.
Peak no. Rt (min) Identied compound Molecular formula Base peak Molecular ion peak (M+)
Yield (%)
L S F
1 5.97 9-octadecenoic acid, methyl ester (Oleic acid, methyl ester) C19H36O255 296 – 13.66 –
2 7.97 Dodecanedioic acid, dimethyl ester C14H26O455 258 – 5.65 –
3 8.58 Tetradecanoic acid, methyl ester (methyl myrictate) C15H30O274 242 1.34 – 3.61
4 9.30 16-Octadecenoic acid, methyl ester C19H36O255 296 – – 0.14
5 10.25 Hexadecanoic acid, 15-methyl-, methyl ester C18H36O274 284 – 0.28 –
6 10.75 Hexadecanoic acid,2,3-dihydroxypropyl ester C19H38O455 330 0.14 – –
7 10.80 Pentadecanoic acid, methyl ester C16H32O274 256 – – 0.28
8 10.86 Tetradecanoic acid, 12-methyl, methyl ester C16H32O274 256 – – 0.61
9 12.54 9-Hexadecenoic acid, methyl ester (methyl palmitoleate) C17H32O255 268 0.49 – 0.47
10 12.65 13,16-Octadecadienoic acid, methyl ester C19H34O255 294 – – 0.95
11 13.10 Hexadecanoic acid, methyl ester (palmitic acid, methyl ester) C17H34O274 270 32.72 22.74 34.27
12 13.24 5-Hexenoic acid, methyl ester C7H12O274 128 – – 9.39
13 14.77 Hexadecadienoic acid, methyl ester C17H30O267 266 – – 0.44
14 15.23 Hexadecenoic acid, 15-methyl, methyl ester C18H36O274 284 0.38 – 0.83
15 15.69 Pentadecanoic acid, 14-methyl, methyl ester C17H34O274 270 0.16 – –
16 16.86 9,12-Octadecadienoic acid, methyl ester (linoleic acid methyl
ester) C19H34O267 294 29.13 24.40 15.29
17 16.93 9,12,15-Octadecatrienoic acid, methyl ester (linolenic acid,
methyl ester) C19H32O279 292 25.95 16.57 25.96
18 17.40 Octadecanoic acid, methyl ester (stearic acid, methyl ester) C19H38O274 298 4.16 0.25 –
19 17.42 Heptadecanoic acid,
9-methyl, methyl ester C19H38O274 298 0.82 –
20 18.26 9-Octadecenoic acid, ethyl ester C20H38O255 310 – 0.98 –
21 18.31 1-Propene-1,2,3-tricarboxylic acid, tributyl ester (tributyl
aconitate) C18H30O6112 342 0.49 – –
22 18.81 Octadecanoic acid, ethyl Ester (Stearic acid, ethyl ester) C20H40O288 312 – 2.46 –
23 18.95 4,7-Octadecadiynoic acid, methyl ester C19H30O2105 290 0.09 – –
24 19.54 8-Methyl-9-tetradecen-1-ol acetate C17H32O243 268 – – 0.16
25 19.76 Oxiraneundecanoic acid, 3-pentyl, methyl ester C19H36O355 312 – – 0.27
26 19.90 6,9,12-Octadecatrienoic acid, methyl ester C19H32O241 292 – – 0.15
27 20.23 1,1′Bicyclopropyl-2-octanoic acid, 2′-hexyl, methyl ester C19H36O273 322 – – 0.77
28 20.44 Tributyl acetylcitrate (citroex A) C20H34O8185 402 0.38 – –
29 20.87 9-Octadecenoic acid, methyl ester (oleic acid, methyl ester) C19H36O255 296 – – 1.27
30 21.54 Eicosanoic acid, methyl ester (arachidic acid, methyl ester) C21H42O274 326 0.57 – 2.05
31 21.56 Arachidonic acid, ethyl ester C22H36O279 332 – 0.37 –
32 21.98 Hexadecanedioic acid, 3-methyl, dimethyl ester C19H36O474 328 – – 0.34
33 22.49 9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester
(1-mono linolenin) C21H36O479 352 – – 0.09
34 22.75 10-Heptadecen-8-ynoic acid, methyl ester C18H30O279 278 – 5.17 –
35 25.42 Docosanoic acid, methyl ester C23H46O274 354 0.29 0.84
36 25.87 1,2-Benzenedicarboxylic acid, dioctyl ester (dioctyl phthalate) C24H38O4149 390 0.1 0.35 –
37 26.52 8,11-Octadecadienoic acid, methyl ester C19H34O267 294 – 1.05 –
38 29.23 17-Octadecynoic acid, methyl ester C19H34O274 294 – 0.35 –
39 31.35 Triacontanedioic acid, dimethyl ester C32H62O498 510 – 0.37 –
40 32.81 Docosanoic acid, methyl ester C23H46O274 354 – 0.28 –
41 32.99 Tricosanoic acid, methyl ester C24H48O274 368 – 0.34 –
42 33.31 15-Tetracosenoic acid, methyl ester C25H48O255 380 – 0.37 –
43 33.59 Heneicosanoic acid, methyl ester C22H44O274 340 – 0.34 –
Total identied compounds 96.51 97.14 98.42
Saturated fatty acids 40.26 33.87 43.77
Unsaturated fatty acids 56.25 63.27 54.65
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High performance liquid chromatography (HPLC) and quantitative determination of phenolic contents of
A. houstonianum showed that the ethanolic owers extract was the richest extract in the avonoid and total
polyphenolic contents followed by the leaves then the stems, which interpreted the high potency of owers
extract than leaves followed by stem. is high potency was due to the synergism of its bioactive compounds
which detected in high levels than in leaves and stems extracts. Where, the owers extract exhibited high activ-
ity against C. pipiens larvae with approximately 2-folds than leaves and stems. e same results were detected
for repellency and antifeedant eects against the C. pipiens adults. Where the repellency % obtained by ower
extract was (89.1%) at a dose 3.6mg/cm2 indicating a good repellent property. Also, antifeedant activity and the
maximum protection was obtained by ower extract with 90% of unfed females.
Regnault-Roger etal.23, showed that all phenolic compounds had toxicity to beetles, which paralyzed or
dead at the bioassay test, by their cumulative toxic eect. Vanillin and caeic and ferulic acids had a knockdown
Table 4. Larvicidal activity of A. houstonianum ower, leaf, and stem ethanol extracts on 3rd larval instar
of C. pipiens 24, 48 and 72h post-treatment. *(F.l.) Fiducially Limits *(χ2) Chi square value. *Slope of the
concentration-inhibition regression line ± standard error.
Extract (ppm)
Flower Leaf Stem
24h 48h 72h 24h 48h 72h 24h 48h 72h
LC25 (*F.l. at
95%) 159.40 (136.90–
179.15) 117.34 (95.69–
136.41) 89.22 (66.99–
108.92) 133.72 (104.95–
158.28) 122.65 (94.62–
146.67) 113.01 (85.86–
136.40) 158.083
(128.53–183.32) 166.74 (139.42–
190.34) 144.24 (115.10–
169.09)
LC50 (*F.l. at
95%) 259.79 (236.87–
283.76) 203.07 (180.70–
224.72) 168.043
(143.70–190.31) 266.85 (236.05–
301.19) 246.33 (216.65–
277.79) 227.34 (198.58–
256.47) 306.86 (273.57–
347.47) 302.42 (272.40–
337.71) 284.31 (252.57–
321.16)
LC90 (*F.l. at
95%) 657.16 (563.88–
809.40) 575.69 (492.12–
713.01)
559.488
(469.006–
717.732)
991.71 (764.78–
1472.53) 926.62 (720.53–
1356.55) 857.91 (674.00–
1233.78)
1082.13
(829.234–
1622.945)
937.37 (748.39–
1304.52)
1032.19
(793.75–
1539.25)
Slope ± SE 3.17 ± 0.26 2.83 ± 0.26 2.45 ± 0.25 2.248 ± 0.255 2.22 ± 0.25 2.22 ± 0.25 2.34 ± 0.26 2.61 ± 0.27 2.28 ± 0.25
χ25.68 4.94 0.92 5.05 5.72 6.47 3.34 1.29 3.90
Probability (P) 0.13 0.18 0.82 0.17 0.13 0.09 0.34 0.73 0.27
Toxicity index 100 100 100 97.36 82.43 73.91 84.66 67.14 59.1
Relative potency 1.18 1.489 1.69 1.14 1.227 1.25 1 1 1
Table 5. Repellency/antifeedant eect of A. houstonianum Mill. (Asteraceae) ower, leaf and seed ethanol
extracts on females of C. pipiens.
Plant parts Dose (mg/cm) No. of tested females No. of fed % No. of unfed % Repellenc y %
Leaf 3.6 48 6 12.5 42 87.5 86.3
1.8 52 13 25.0 39 75.0 72.6
Flower 3.6 40 4 10.0 36 90.0 89.1
1.8 42 11 26.2 31 73.8 73.3
Stem 3.6 33 10 30.3 23 69.7 68.2
1.8 25 12 48.0 13 52.0 49.6
DEET 1.8 25 0.0 0.0 25 100.0 100.0
Control – 23 21 91.3 2 8.7 0.0
Table 6. Eect of A. houstonianium ower, leaf, and stem extracts on the activity of acetylcholinesterase,
carboxylesterase, ATPase, and cytochrome P-450 monooxegenase in 3rd larval instar of C. pipiens. According
to Duncan’s multiple range test (P ≥ 0.05), Means with the same letters are not signicantly dierent. Each value
represents the mean of three replicates ± SD, SD Standard deviation.
Enzyme
Activity mean ± SE
ControlFlower Leaf Stem
Acetylcholinesterase (ug AchBr/min/mg
protein) 7.66 ± 2.7a (− 57.86%) 10.73 ± 1.3b (− 40.97%) 15.28 ± 1.05c (− 15.95%) 18.18 ± 4.2d
Carboxylesterase (ug Meb/min/mg
protein) 43.12 ± 10a (− 29.32%) 47.303 ± 12.5a (− 22.47%) 53.05 ± 5.5b (− 13.04%) 61.01 ± 10.8c
ATPase (umoles Pi/min/mg protein) 78.81 ± 0.11c (30.05%) 69.16 ± 0.10b (14.13%) 63.93 ± 0.06a (5.49%) 60.6 ± 0.12a
Cytochrome P-450 Monooxegenase (m
mol sub. oxidized/min/mg protein) 37.16 ± 0.85a (− 36.60%) 45.44 ± 2.05b(− 22.14%) 46.18 ± 0.98b(− 20.87%) 58.36 ± 2c
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eect, while rosmarinic acid, gallic acid, naringin and luteolin-7-glucoside had signicant toxic and attractive
eects. Rosmarinic acid was also detected at high concentration in the owers. Rosmarinic acid is an insecti-
cidal agent with high insecticidal activity at very low concentrations in 24h against aphids. Also, it is known to
reduce genotoxic eects induced by harmful chemicals so, it considered very safe to consumers24. e avonoid
rutin negatively aected the behavior, biology, and physiology of Spodoptera frugiperda and Helicoverpa zea by
prolonging the larval development time, reducing the larval and pupal weight, and decreasing the pupal viability.
e addition of dierent concentrations of rutin prolonged the life cycle of S. frugiperda; therefore, the use of
rutin is indicated in future studies evaluating the control of S. frugiperda25. e ower extract showed higher
total identied avonoids than leaves and stems. Flavonoids and iso-avonoids adversely aect insect growth,
development, and behavior by inuencing the steroid hormone systems. Some avonoids are highly toxic to
insect, while other act as feeding deterrents and repellency property25. e coumarin exhibited acute toxicity and
deteriorated the growth of red palm weevil larvae26 and showed antifeedant eects against Rhyzopertha dominica
F. and Oryzaephilus surinamensis L. and demonstrated that the insect used the energy generated from ingested
food to perform its physiological activities to ght the toxin (coumarin), therefore, aect the insect growth and
development27. So, the polyphenols act in dierent ways and at dierent rate. Some components acted progres-
sive toxicity while others had knockdown, repellent or anti-feedent eects.
Phytol was the major makeup in stem (52.10%), leaves (38.28%), and owers 19.39%. Where, ketones rep-
resented by (4.65%, 4.69% and 5.12%) in the leaves, stems and owers USM, respectively, the main of which
was 2-pentadecanone, 6, 10, 14-trimethyl showing a yield of (2.05%) in the leaves and it is the only ketones
present in the stems, while the main of which in owers was 2-nonadecanone representing (2.34%). As well as
aldehyde presented as (1.92% and 2.65%) in the USM of leaves and stems respectively, the main of which was
palmitaldehyde diallyl acetal whose percentage was (1.83%) in stems and it is the only aldehyde detect in leaves.
Furthermore, esters represented as (6.32%, 0.85% and 1.66%) in the leaves, stems and owers USM, respectively.
Acid and sterols were detected in comparable percent in the dierent organs under investigation. GC–MS analysis
of the chloroform extract of Ageratum conyzoides whole plant prevailed 9,12-Octadecadienoic acid (12.48%), as
major identied compound which comparable to our nding28.
Chromone presented by precocene II which was detected in leaves and stem as 22.08% and 13.26% respec-
tively. While, in ower chromene I and II were detected. Chromone1 and 2 derivatives, detected in ower extract,
are a well-known allelochemical and showed good insecticidal potency against M. separata29. Moreover, they
have signicant larvicidal activity against C. pipiens30. Also, these derivatives have antioxidant activity and MAOs
inhibition activities31. ese results agree with the present results, the ower extract exhibited higher insecticidal
activity than stem and leaves against C. pipiens larvae.
Insecticidal eect of precocene II on the human body louse, Pediculus humanus was reported32. Essential
oil of A. houstonianum Mill. aerial parts and its constituent compounds (precocene I and II) have potential for
development into natural insecticides or repellents for control of insects in stored grains33. Precocene II inhibits
juvenile hormone biosynthesis by cockroach corpora allata invitro34. e precocenes (I and II), isolated from A.
houstonianum, showed anti-juvenile hormonal eects on metamorphosis, ovarian development, and embryonic
development also, exhibited larval mortality, the oviposition inhibition of ticks, Rhipicephalus microplus35. Fahmi
etal.36, were investigate the inuence of precocene II on the toxicological and biochemical parameters on the 4th
instar larvae of S. littoralis. Overall, phytol can be considered further for developing eective and eco-friendly
green insecticides against aphids37.
Whereas the ovicidal activity of A. houstonianum leaf extracts against the eggs of vector mosquitoes and to
develop additional tools for the control of mosquito-borne diseases previously reported by Tennyson etal.38. e
potential oviposition deterrent property of A. houstonianum crude leaf extracts detected in both laboratory and
eld studies designates the presence of phytocompounds that act as eective contact restraint39.
e insects have detoxication system to degrade toxic substances for the insect survival40. Metabolism of
toxic substances involves two phases. e rst phase is the cleavage of the substrate or addition of a polar group,
while the second phase is the addition of sulfate, phosphate groups, sugar, or amino acid to the resulted products
of 1st phase to increase hydrophilicity, consequently, facilitate excretion by the insect41. e most important
enzymes responsible for the detoxication of toxins are CYP-450 for oxidative degradation and CarE for hydro-
lytic degradation that involved in 1st phase42. e detoxication capabilities of enzymes could be modied due to
variations in gene expression43, consequently, variation of insect response to toxins44. e treatment of C. pipiens
larvae with owers, leaves and stem extracts inhibit the activity of CYP-450 and CarE activity with dierent
levels due to variations in their constituents. e coumarin targets CYP-450 genes causing masking/silencing its
expression that leads to high toxicity with low LD50 values against red palm weevil26. ese results agree with45
who reported that the Piper betle extract reduced the level of CYP-450 in W strain of Ae. aegypti. Also, the sub-
lethal dosage of A. conyzoides blocked the activity of CarE activity46. As well, the Sophora alopecuroides alkaloids
are involved in the inhibition of CarE activity in Aedes albopictus47. In general, the esterases activities of the H.
armigera larvae were signicantly inhabited by avonoid-treated diets25.
AChE has essential role as neurotransmitter in cholinergic synapses for insects48. Many insecticides inhibit of
AChE action that causes accumulation of acetylcholine (ACh) at the synaptic cle resulting in permanent neuro
excitation/stimulation, paralysis, ataxia, and eventual death49. e obtained results showed that the ower and
leaf extracts exhibited high inhibition eects against AChE than stem extract, that explained by Hussein etal.30,
who proved that chromone 1 and 2 signicantly inhibit the AChE activity in treated larvae of C. pipiens using
molecular docking simulation. Many plant secondary metabolites decrease the levels of CarE and AChE activity
of a wide range of insects50. e exposure of the A. aegypti larvae to the Sapindus emarginatus extract showed
signicant inhibition in the activities of AChE and CarE51, Similar reduction in AChE levels was observed by
azadirachtin application against Nilaparvata lugens52.
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ATPase plays a main role in intracellular functions and is a sensitive indicator of toxicity. It hydrolyzes
adenosine triphosphate (ATP) to release the energy substantial for the active transport of Na+ and K+ across
the cell membrane53. e metabolic detoxication mechanisms to toxins in insects consume high energy54. e
elevated activity of the ATPase is a responsive action to the activation of detoxication mechanisms as a defense
mechanism therefore, high energy demands55. Toxicity of botanical toxins to insects has been associated with
the overexpression of genes involved with ATPase synthesis and energy demand56, this concept interpreted the
enhancement of ATPase activity to reduce the damage caused by ower and leaves extracts, respectively, while
the stem extract did not greatly stimulate ATPase with low expression.
Plant extracts have been studied extensively for their insecticidal eect57. Phytochemicals such as phenolic
acid, avonoids, chromene, phytol and monoterpenes are known for their mosquito repellent and insecticidal
properties57. Ageratum houstonianum essential oil and extracts have been stated to have bioactive molecules58
with repellency and adulticidal action against the adult mosquitoes59. ere are various degrees of activity of
Ageratum sp. extracts against insects due to variation of active ingredients with a wide variety of insecticidal
properties60 which agree with the results obtained in our investigation.
Many publications on the phytochemistry of Ageratum sp. from many disparate countries have been dealt
with the various extracts with diversity in major and minor active constituents61. Petroleum ether extract of
A. conyzoides showed signicant larvicidal activity against the 4th larval instars, adult mortality and aected
percentages of oviposition deterrence index of females of three mosquito vectors. Beside to, these extracts harm-
less to aquatic mosquito predator Toxorhynchites splendens even at the prominent dosage (1000ppm)46. e A.
conyzoides ethanolic extract has acaricidal potency against acaricides- susceptible and resistant ticks infesting
bualoes and cattle, moreover, adversely aected egg laying capacity35.
Materials and methods
Plant material. One kilogram of leaves, stems, and owers of A. houstonianum Mill., collected individually
during the owering season from April to September 2019 from herbs growing in El Orman botanical garden,
Giza square https:// goo. gl/ maps/ NnGub Z5FDn E8RJZ X8. e plant was authenticated by Dr. Reem Sameer, Pro-
fessor of Plant Taxonomy, Botany Department, Faculty of Science, Cairo University. A voucher specimen (No.
26. 3.2018) was kept at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Cairo University-
https:// goo. gl/ maps/ v6Psv Jp6KJ W52Pk H8. e use of plants in the present study complies with international,
national and/or institutional guidelines.
Preparation of plant extract. One hundred grams of the powdered leaves, stems, and owers of the plant
were separately extracted with about 1000ml of 70% ethanolic solution by using maceration till exhaustion then
ltered. e collected extract was completely dried under vacuum using rotatory evaporator at 40°C to yield a
residue of about 30g, 15g and 25g extracts for leaves, stems, and owers, respectively. e extract was kept in
tightly sealed containers to be used for the polyphenolic and biological study.
Preparation of the n-hexane extracts. e powdered dried leaves, stems, and owers (1000g, 165g
and 150g, respectively) of A. houstonianum were exhaustively extracted in a Soxhlet apparatus with n-hexane.
e extracts were evaporated under reduced pressure at 40°C to yield (35g, 2g and 7g) greasy, dark green
residue of leaves, stems, and owers, respectively. e residues were stored in a desiccator for lipoidal matter
investigation.
Preparation of the lipoidal matters. e lipoidal matters; unsaponiable matter (USM) and fatty acid
methyl esters (FAME) were prepared according to the method of Ichihara and Fukubayashi62, to identify the
lipoidal constituents and to determine their percentages in the n-hexane extracts of leaves, stems, and owers
of A. houstonianum.
Spectrophotometric determination of total phenolic contents. e polyphenol content was deter-
mined using the Folin-Ciocalteu reagent method according to Mruthunjaya and Hukkeri63, with some modi-
cations. e method involves the reduction of Folin Ciocalteau reagent (Sigma chemical, St.louis, Missouri,
USA) by phenolic compounds, with a concomitant formation of a blue complex, and the absorbance was read at
765nm using an UV–Vis spectrophotometer. e total polyphenolic content was expressed as gallic acid, using a
standard calibration curve. Each experiment was repeated in triplicate and the readings were mean values. Same
practice was repeated for the standard solution of gallic acid, and the calibration line was constructed. Based
on the absorbance, the concentration of phenolics was interpreted (mg/ml) from the calibration line; then the
contents of phenolics in extracts were articulated in the total phenolic contents as gallic acid correspondent (mg
of GAE/g of sample).
Spectrophotometric determination of total avonoid contents. Total avonoid content was
determined according to Atanassova etal.64, with some modications. e absorbances of the solutions were
measured at 510nm against blank using a spectrophotometer. Similar procedure was returned for the standard
solution of quercetin and the calibration graph was constructed. e content of avonoids in each sample was
articulate as quercetin, using a standard calibration curve as mg of QAE/g of sample).
HPLC analysis of the phenolic components. HPLC quantitative analysis of phenolic components was
performed according to method presented by Mizzi etal.65. Using an Agilent 1100 series LC System) equipped
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with a model G 1311 A quaternary solvent pump and degasser, a thermostatted column compartment (G1316A),
autosampler (G1329A) and a diode array detector—DAD (G1315B). e analytical column was Eclipse XDB-
C18 (150 × 4.6μm; 5μm) with a C18 guard column (Phenomenex, Torrance, C.A.). Mobile phase: e mobile
phase consisted of acetonitrile (solvent A) and 2% acetic acid in water (v/v) (solvent B). Gradient programmed
as follows: 100% B to 85:15 B: A, v/v in 30min. 85:15 B: A to 50:50 B: A in 20min, 50:50 B: A in 5min, 0:100 B:
A in 5min and 100% A to 100% B in 5min. Injection volume:50μl, Flow rate:0.8ml/min. Column temperature
30°C. Detector type DAD detector, wave length 280 and 330. For investigations of phenolic acids and avonoids,
National Research Center. Phenolic acid and standards from Sigma Co. were dissolved in the mobile phase and
injected into HPLC. Peaks were integrated both manually and using Agilent soware. Retention time and peak
area were used to calculate phenolic acids and avonoids concentrations by data analysis using Agilent soware
e data collect and analyses were carried out using the soware ChemStation Rev. A.10.02 Edition (copyright
Agilent Technologies, 1990–2003.
GC–MS analysis of lipid constituents. e prepared USM and FAME were analyzed by GC–MS. Using
ermo Scientic, trace GC Ultra/ISQ Single Quadrupole: MS, TG-5MS fused silica capillary column, coupled
to an electron ionization system, for analysis of lipoidal content, National Research Center. e GC/MS analysis
of the unsaponiable and saponiable fractions obtained from the powdered dried leaves, stems and owers
was carried out adopting the following conditions column typeTG-5MS fused silica capillary column. Column
internal diameter 30m, 0.251mm, 0.1mm lm thickness. Carrier gas is Helium. Flow rate 1ml/min. Sample
size 1 μl. Injection mode: split less. Temperature programming in USP 50°C (2min) then elevated to 150°C
at a rate of 7°C/min then to 270°C at a rate of 5°C/min (hold for 2min) then to 310°C at a rate of 3.5°C/min
and isothermally 10min. In FAME temperature programming is 50°C (4min) then elevated to 280°C at a rate
of 5°C/min and isothermally for 4min. Injector temperature 280°C. Ionization voltage70 eV. Scan mass range
50–500m/z. Identication of the components was achieved by library research database, Wiley mass spectral
database and by comparing their retention indices and mass fragmentation patterns with those of the available
references as well as, published data28.
Insect rearing. Maintenance of mosquito colony. e laboratory strain of C. pipiens was reared and main-
tained continuously for several generations in an insectary in Research and Training Center for Vectors of Dis-
eases (RTC), in Faculty of Science, Ain Shams University, using the standard procedures described by Kasap
and Demirhan66, under controlled conditions; 27 ± 2 °C and RH 75 ± 5%, and photoperiod 12:12 light: dark
hours7. e newly hatched larvae were fed on Tetramin. e pupae were collected and transferred to the rearing
screened wooden cages (25 × 25 × 25cm). Adults were provided daily with a 10% sucrose solution. e females
were allowed to feed a blood meal from a pigeon host.
Larvicidal bioassay. e 3rd arval instar of C. pipiens was treated with serial concentrations of A. houstonianum
ower, leaf and stem extracts according to the previous standard protocol67 with some modications. Five con-
centrations of A. houstonianum ower, leaf and stem extracts were prepared in ethanol for stock solution, while
serial concentrations (500, 400, 300, 200 and 100ppm) were diluted using distilled water to prepare 100ml of
each concentration. Distilled water only was used for control. Twenty larvae were transferred to each treatment
and control. Each treatment and control were replicated three times. Mortality was recorded aer 24-, 48- and
72-h post-treatment.
Repellency and antifeedant bioassay. e standard cages (20 × 20 × 20cm) were used to test the repellent activ-
ity of the extracts. Dierent amounts from each extract were dissolved in 2ml (distilled water with a drop of
Triton × 100) in 4 × 4cm cups to obtain the dierent concentrations. e concentration was directly applied
onto 5 × 6cm of the ventral surface of pigeon aer removing the abdomen’s feathers. Aer 10min of treatment,
pigeons were placed for 3h (from 6 to 9 PM) in cages containing the laboratory strain of starved C. pipiens
females. Control tests were carried out using water. Each test was repeated three times to get a mean value
of repellent activity68. Post treatment, the number of fed and unfed females was counted, and repellency was
recorded statistically by using Abbott formula69.
where A: the percentage of unfed females in treatment. B: the percentage of unfed females in control.
Biochemical analysis. Enzyme preparation. e whole 3rd instar larvae of C. pipiens treated with LC50
values were homogenized in distilled water (50mg/1ml). Homogenates were centrifuged at 8000 r.p.m. for
15min at 5°C in a refrigerated centrifuge. e deposits were discarded, and the supernatants were kept in a deep
freezer (2°C) till use as Amin70.
Acetylcholinesterase (AChE) activity assay. Acetylcholine bromide (AChBr) was used as substrate to detect the
AChE activity according to the method described by Simpson etal.71. 200µl enzyme solution were mixed with
0.5ml AChBr (3mm) and 0.5ml 0.067M phosphate buer (pH 7). e mixture tubes were incubated for 30min
at 37°C. en 1ml of alkaline hydroxylamine and 0.5ml of HCl were added. e mixture tubes were mixed well
and allowed to stand for 3min. 0.5ml of FeCl3 solution was added to the mixture tube and shaken vigorously.
e decrease in AChBr level resulted from the hydrolysis by AChE was read at 515nm.
The repellency %
=
(%A
−
%B
/
100
−
B%
)
×
100,
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ATPase activity assay. e total ATPase activity was estimated as described by Amaral etal.72. e main con-
cept of this method is estimation the amount inorganic phosphate (Pi) resulted from ATP hydrolysis by ATPase.
e enzyme was incubated at pH 7.5 and 37°C, in 0.5ml of a solution containing mixture of NaCl 150mM,
ATP.Na2-TRIS 5mM and KCl 15mM in histidine HCl-TRIS 30mM. ATP was added to start the reaction. e
mixture was incubated for 30min at 37°C, then 100 μl SDS (5%) was added to stop the reaction. e amount of
formed Pi was measured by phosphorus kit. ATPase activity was expressed in µmoles of Pi released per minute
per milligram protein.
Cytochrome P‑450 monooxegenase (CYP‑450) activity assay. P-nitroanisole O-demthylation was used to deter-
mine the CYP-450 activity according to Hansen and Hodgson73 method with some modications. e mixture
solution containing 1.5ml enzyme solution, 0.2ml NADPH, 1ml sodium phosphate buer (0.1M, pH 7.6),
50µg glucose-6-phosphate dehydrogenase and 0.2ml glucose-6-phosphate. p-nitroanisole in 10µl of acetone
was added to start the reaction and attain the nal concentration of 0.8mM. e nal mixture was incubated at
37°C for 30min then 1ml HCl (1N) was added to terminate the incubation period. p-nitrophenol was extracted
with 0.5N NaOH and CHCl3. e absorbance of NaOH solution was estimated at 405nm. An extinction coef-
cient of 14.28mM/cm was used to calculate 4-nitrophenol concentration.
Carboxylesterase (CarE) activity assay. Carboxylesterase activity was determined as described by method of
Simpson etal.71, and methyl n butyrate (MeB) used as substrate. e reaction solution containing 0.5ml MeB
(4mM), 200µl enzyme solution and 0.5ml 0.067M phosphate buer (pH 7). e mixture tubes were incubated
for 30min at 37°C. en, 1ml of alkaline hydroxylamine (equal volume of 3.5M NaOH and 2M hydroxylamine
chloride) was added to the mixture tubes followed by 0.5ml of HCl. e mixture tubes were mixed well and
allowed to stand for 3min. 0.5ml of FeCl3 solution was added to the mixture tube and shaken vigorously. e
decrease in MeBr level resulted from the hydrolysis by carboxylesterases was read at 515nm.
Statistical analysis. Lethal concentrations were determined at the 95% condence level were recorded in
probity regression line and LC50, and LC90, slope, standard error, and correlation coecient; and for the good-
ness of t (Chi square test) were calculated according to Finney74 and correction for control mortality was con-
ducted using Abbott’s formula according to Abbott69. e biochemical results were analyzed by one-way analysis
of variance (ANOVA) using CoStat system for Windows, Version 6.311 (CoHort soware, Berkeley, CA 94701)
https:// www. cohor tso ware. com/ costat. html. When the Anova statistics were signicant (P < 0.01), means were
compared by the Duncan’s multiple range test75.
Conclusion
Overall, results suggest that the ethanolic ratios of enzymatic activity ranging extracts of ower, leaves, and stem
of A. houstonianum exhibited a signicant repellent, antifeedant and larvicidal activities with dierent levels,
which may be attributed to chlorogenic, phytol, coumarin, rosmarinic acid, rutin, precocene I, and II compounds.
All these bioactive molecules act in dierent ways with various rates and synergist each other to exhibit the toxic-
ity action. Some components acted progressive toxicity while others had knockdown, repellent or anti-feedent
eects. e owers extract was rich with bioactive components which responsible for its high ecacy relative
to leaves and stem extracts. e tested extracts inhibited the activity of AChE, CYP-450 and CarE with various
levels, while the ATPase activity was enhanced. Dierent organs of A. houstonianum ethanol extracts could be
used as bio-agents for mosquito control.
Data availability
e datasets used and/or analysed during the current study available from the corresponding author on reason-
able request.
Received: 10 April 2022; Accepted: 7 December 2022
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Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis
were performed by D.E., A.M.E.S., D.A.H. and S.F. All authors read and approved the nal manuscript.
Funding
Open access funding provided by e Science, Technology & Innovation Funding Authority (STDF) in coopera-
tion with e Egyptian Knowledge Bank (EKB).
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 25939-z.
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