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Acaricidal and Antioxidant Activities of Anise Oil (Pimpinella anisum) and the
Oil’s Effect on Protease and Acetylcholinesterase in the Two-Spotted Spider
Mite (Tetranychus urticae...

Article in Agriculture · February 2022

DOI: 10.3390/agriculture12020224

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 agriculture

Article
Acaricidal and Antioxidant Activities of Anise Oil (Pimpinella
anisum) and the Oil’s Effect on Protease and Acetylcholinesterase
in the Two-Spotted Spider Mite (Tetranychus urticae Koch)
Salwa M. El-Sayed 1, *, Nevin Ahmed 2 , Samy Selim 3, * , Areej A. Al-Khalaf 4 , Nihal El Nahhas 5 ,
Shams H. Abdel-Hafez 6 , Samy Sayed 7 , Heba M. Emam 8 and Mervat A. R. Ibrahim 1

1 Department of Biochemistry, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, P.O. Box 68,
Hadayek Shoubra, Cairo 11241, Egypt; [email protected]
2 Plant Protection Department, Faculty of Agriculture, Benha University, Benha 13736, Egypt;
[email protected]
3 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University,
Sakaka 72341, Saudi Arabia
4 Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428,
Riyadh 11671, Saudi Arabia; [email protected]
5 Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria 21526, Egypt;
[email protected]
6 Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
[email protected]
7 Department of Science and Technology, University College-Ranyah, Taif University, P.O. Box 11099, Taif 21944,





 Saudi Arabia; [email protected]
8 Department of Plant Protection, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima,
Citation: El-Sayed, S.M.; Ahmed, N.;
Cairo 11241, Egypt; [email protected]
Selim, S.; Al-Khalaf, A.A.; El Nahhas,
* Correspondence: [email protected] (S.M.E.-S.); [email protected] (S.S.)
N.; Abdel-Hafez, S.H.; Sayed, S.;
Emam, H.M.; Ibrahim, M.A.R.
Abstract: The two-spotted spider mite, Tetranychus urticae, also known as the red spider, is one of the
Acaricidal and Antioxidant Activities
most harmful pests in agriculture and causes large losses of many crops. These mites have rapidly
of Anise Oil (Pimpinella anisum) and
developed a resistance to many chemical pesticides in recent years. In this study, the essential oil
the Oil’s Effect on Protease and
Acetylcholinesterase in the
of seeds of the anise plant (Pimpinella anisum) was extracted by hydrodistillation, and the chemical
Two-Spotted Spider Mite (Tetranychus composition of the oil was analyzed. The antioxidant activity of the volatile oil was determined
urticae Koch). Agriculture 2022, 12, by the DPPH radical scavenging assay. The acaricidal activity of the anise oil, a natural acaricide,
224. https://doi.org/10.3390/ was evaluated for its ability to protect green bean plants from mite injury. The two-spotted spiders
agriculture12020224 were spread on green bean seedlings for 1 week; then, different plants were sprayed with different
concentrations of anise oil (10, 20, 30, or 40 µL/L). Our results revealed that anethole was the major
Academic Editor: Roy Kennedy
component of anise oil, at 53.23%. The acaricidal effect of the various concentrations on T. urticae was
Received: 13 January 2022 recorded after 24, 48, and 72 h of treatment. Our findings suggest that anise oil showed significant
Accepted: 28 January 2022 acaricidal activity against T. urticae in a dose- and time-dependent manner. Anise oil at a concentration
Published: 3 February 2022
of 40 µL/L killed 96.0% of the red spiders after 72 h. Also, all concentrations of anise oil inhibited
Publisher’s Note: MDPI stays neutral acetylcholinesterase, and the spiders’ protease activity declined when the plants were treated with 30
with regard to jurisdictional claims in or 40 µL/L of anise oil. The concentrations of 10 and 20 µL/L did not significantly affect the protease
published maps and institutional affil- activity of T. urticae mites. We can conclude that anise oil exhibited acaricidal activity against T. urticae
iations. and that this was highly correlated with the inhibition of acetylcholinesterase and protease activities
in the mites.

Keywords: Tetranychus urticae; acaricidal activity; Pimpinella anisum; anise oil; AChE; protease
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
1. Introduction
Attribution (CC BY) license (https:// The two-spotted spider mite, Tetranychus urticae Koch (Acari family Tetranychidae),
creativecommons.org/licenses/by/ is a harmful pest that damages many crops, including vegetables, fruits, and ornamental
4.0/). plants [1]. It is also the most common plant-eater of the family Tetranychidae [2], attacking

Agriculture 2022, 12, 224. https://doi.org/10.3390/agriculture12020224 https://www.mdpi.com/journal/agriculture

Agriculture 2022, 12, 224 2 of 13

3877 host crops of both greenhouse and field crops [1]. The two-spotted spider mite
is polyphagous; it can feed on hundreds of plants, including many that are important
for the economic well-being of countries. In controlling this pest, the main option is
the use of synthetic acaricides, but they are not always efficient, because this species
has a high ability to develop resistant populations [3], and because these acaricides are
not selective for predatory mites [4]. The improper use of these agents can result in
environmental and food contamination, especially of fruits and vegetables meant to be
consumed when freshly harvested [5]. Chemical acaricides such as organophosphorus
compounds, synthetic pyrethroids, and amitraz are used to control mites. Recently, many
acaricides have been replaced with newer, safer agents owing to the toxicity resistance
of and environmental damage caused by earlier agents [6]. Several essential oils were
evaluated for their acaricidal activities. Essential oils consisting of volatile secondary
metabolites, mostly terpenes [7], act as botanical insecticides [8,9], some of which are
marketed as commercial ingredients for pesticides [10]. Many essential oils are used
as insecticides due to their direct effects, biodegradability, and low level of toxicity to
mammals [10–12]. Also, these natural compounds have very low toxicity in humans,
making them good alternatives to synthetic acaricides. These essential oils, including anise
oil, contain many natural compounds that have acaricidal activity [8,9]. The insecticidal
and acaricidal activities of anise oil could be attributed to their terpenoidal content [7].
As previous studies have shown, the toxicity of anise oil against insect pests is due to its
active ingredients, such as (e)-isoeugenol, limonene, linalool, and α-pinene [13,14]. Amini
et al. [14] identified the components of essential oils and showed that Pimpinella anisum L.
had the most fumigant toxicity against the storage pests. Anise insecticidal activity was
also studied by Tunç and Sahinkaya [15], who verified some anise acaricide activity against
Tetranychus cinnabarinus Boisd. On fumigation tests, Lucca et al. [16] reported that an
important potential insecticidal characteristic of anise was that it repelled moths. Anethole
phenylpropanoid, the most important component of anise, was very efficient in controlling
Aedes aegypti and Culex pipiens L. mosquitoes [17]. The volatile essences (monoterpenes)
attract insects, mainly sucking insects, due to their nutrimental attributes [18]. Koul
et al. [19] reported that the efficiency of essential oils such as anise is variable and depends
on the presentation, application, and concentration of the metabolite used. In the attempt
to identify alternatives with less environmental impact for the control of the two-spotted
spider mite, this study aimed at evaluating the acaricidal effect of anise oil on females of
this species, including the maximal lethal concentrations of the oil. It also investigated the
relationship between the acaricidal activity of anise oil and its inhibitory effect on spider
protease and acetylcholinesterase (AChE) activities.

2. Materials and Methods

2.1. Materials
The seeds of the anise plant (P. anisum L.) were purchased from the local market in Cairo,
Egypt. The substances 1,1-diphenyl-2-picrylhydrazyl (DPPH); butylated hydroxyanisole (BHA);
5,5-dithiobis (2-nitrobenzoic acid) (DTNB); acetylthiocholine iodide (ATCh); and bovine serum
albumin (BSA) were purchased from the Sigma–Aldrich Chemical Co (Taufkirchen, Germany).
All reagents used throughout this study were of analytical grade.

2.2. Extraction of Essential Oil of Anise

The essential oils of dried anise seeds (200 g) were isolated by steam distillation for
3 h using a glass Clevenger-type apparatus. The extracted yellow-colored volatile oils were
dried over anhydrous Na2 SO4 and were kept at 4 ◦ C in dark glass vials for further analysis.

2.3. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

After evaporation, the extracted oil residue was dissolved with 3 mL ethyl acetate and
1 mL transferred to GC vial for GC/MS analysis. Gas chromatography-mass spectrometry
was used for the analysis of various components of anise volatile oil that were present in
Agriculture 2022, 12, 224 3 of 13

modest quantities, in addition to the analysis of the main components of anise essential
oil. The identification of components was based on a comparison of their mass spectra and
retention time with those of the authentic compounds and by computer matching with the
NIST and WILEY libraries as well as by comparison of the fragmentation pattern of the
mass spectral data with those reported in the literature. The analysis was carried out using
a GC (Agilent Technologies 7890A, Poway, CA) interfaced with a mass-selective detector
(MSD, Agilent 7000, Poway, CA) equipped with a polar Agilent HP-5 ms (5%-phenyl
methyl poly siloxane) capillary column (30 m × 0.25 mm i.d. (In diameter) and 0.25 µm
film thickness). The carrier gas was helium with a linear velocity of 1 mL/min. The injector
and detector temperatures were 200 ◦ C and 250 ◦ C, respectively. Volume injected was 1 µL
of the sample. The MS operating parameters were as follows: ionization potential 70 eV,
interface temperature 250 ◦ C, and acquisition mass range 50–800 [20].

2.4. DPPH Free Radical Scavenging Activity

The capacity of essential anise oil to scavenge the DPPH (1,1-diphenyl-2-picrylhydrazyl)
radical was determined according to the method described in [21]. Butylated hydrox-
yanisole (BHA) was used as a reference. In the DPPH method, 500 µL of freshly prepared of
DPPH solution (50 µM in absolute ethanol) was mixed with 300 µL of anise oil (10–50 µL/L)
and left in the dark for 30 min. Then, the absorbance of the mixture was recorded at 517 nm.
The capability to scavenge the DPPH radical (% inhibition) was calculated using the fol-
lowing equation: % inhibition = [(Ac − At )/Ac ] × 100. Where Ac is the absorbance of the
reaction without sample (control) and At is the absorbance of test samples.

2.5. Mite Rearing

One adult female mite was transferred by a fine camel hair brush to a sweet leaf
disc (1 mm, in diameter), preserved on a humid cotton wool pad in a Petri dish, and left
for 24–48 h to allow it to lay eggs. The deposited eggs were preserved under laboratory
conditions at 27 ± 2 ◦ C, 60 ± 5% R.H. (Realtive Humidity) and 16 L: 8 D photoperiod until
hatching. The newly hatched larvae were transferred singly to fresh sweet potato leaves to
follow their development. Distilled water containing 0.01% Tween 80 was used to prepare
the five dilutions of anise oil as an emulsifier and to make it slightly sticky. Distilled water
containing 0.01% Tween 80 was used as a control [22].

2.6. Acaricidal Activity of Anise Oil against Tetranychus Urticae under Laboratory Conditions
Four concentrations of the essential oil were used in the experiment (10, 20, 30, and
40 µL/L), and an untreated control group was prepared under laboratory conditions of
27 ± 2 ◦ C and 60 ± 5% relative humidity to evaluate the eggs and adult females of the
two-spotted spider mite. The concentrations of the essential oil were chosen based on
previous studies and the effectiveness of similar plants on other spiders. Thirty adult
females and 120 eggs of T. urticae were chosen and used per three replicates. Each group
was transferred to a leaf disc of 4 cm2 in area, which was placed upside down on moist
cotton wool in a Petri dish. Each leaf disc had 5 female mites and 20 mite eggs on it. Two
mL of each concentration of the anise oil was sprayed onto the surface of the leaf discs
using a hand-held atomizer at a distance of 25 to 30 cm. The replicates of the eggs and adult
females and the control were sprayed with distilled water containing 0.01% Tween 80. After
24, 48, and 72 h, the numbers of live and dead adult females were counted. The numbers of
hatching eggs also were counted over 6 days by a dissecting microscope. The percentage of
mites that had died was calculated and corrected according to Abbott’s formula [23].
The values of LC50 and LC90 were determined using the mortality regression lines
drawn according to the Finney, 1952 [24] method and the Sigmaplot program version 2.0
software [25].
Agriculture 2022, 12, 224 4 of 13

2.7. Determination of Enzyme Activities

Adult females of the two-spotted spider mite were homogenized at 4 ◦ C in a 0.066 M,
pH 7.5 sodium phosphate buffer with 0.2 percent (v/v) of Triton X-100. The mixture was
centrifuged at 10,000× g at 4 ◦ C for 15 min. The protease and acetylcholinesterase activity
were measured in supernatants.

2.8. Assay of Protease Activity

The activity of proteases in the whole mite was measured using the Ortego et al. [26]
method. A working solution of bovine serum albumin (BSA) was made by diluting the
standard BSA solution of 1 mg/mL to 0.1 mg/mL. In 5 test tubes 200 µL, 400 µL, 600 µL,
800 µL, and 1 mL of the BSA working solution was taken and named 1–5. By adding
distilled water, the volume was kept at 1 mL. Only distilled water was used to produce
a control. 500 µL of the crude extract was collected by Bradford method, and 500 µL of
sterile distilled water was added to obtain a final volume of 1 mL. The Bradford reagent
was added in 5 mL increments, and the absorbance was measured spectrophotometrically
at 595 nm after 5 min of incubation. To assess the proteolytic activity of the crude extract,
50 µL of BAS (1 mg/mL) standard solution and roughly 20 µL of crude extract were used.
The BSA and enzyme were mixed together, and the volume was kept at 200 µL by adding
phosphate buffer. After 30 min, 2.3 mL of Bradford reagent was added to the mixture. After
5 min, the absorbance was measured against a blank using a spectrophotometer at 595 nm,
with two controls: enzyme only and BSA only. The activity of the protease was determined
using the Bradford equation [27]. At pH 9.0 at 60 ◦ C, one unit of protease activity is defined
as the amount of enzyme that produces 1 µmol of tyrosine per minute.

2.9. Assay of Acetylcholinesterase (AChE) Activity

Acetylcholinesterase was determined according to Wu and Miyata [28] with ATCh
iodide as a substrate in the presence of 5,5-dithiobis (2-nitrobenzoic acid) DTNB in a 0.066 M
phosphate buffer, pH 7.8, at 25 ◦ C. Absorption was measured at 412 nm. The reaction
mixture (2.0 mL) consisted of 0.6 mM ATCh, 0.4 mM DTNB and 0.05 mL aliquot of the
enzyme solution.

2.10. Molecular Docking of E-Anethole in AChE

The molecular docking was performed using AutoDock 4 [29]. The system was prepared
in PyMOL (Schrödinger), using the plug-in developed by Daniel Seeliger (https://github.com/
ADplugin/ADplugin, accessed on 15 August 2021). Acetylcholinesterase and Cathepsin L
were obtained from the structure of the AChE (6XYU) [30] and Cathepsin L (3F75) [31]
complexes. A 54 × 60 × 54 Å grid box with 29.488 × 71.531 × 12.27 grid point spacing
of 0.375◦ A in the case of Acetylcholine esterase and a 56 × 54 × 40 Å grid box with
24.97 × 18.609 × 35.567 grid point spacing of 0.375◦ A in the case of Cathepsin L were
employed. The ligands structure was drawn using the PubChem draw structure tool
(https://pubchem.ncbi.nlm.nih.gov/#draw=true, accessed on 15 August 2021). The default
parameter set of autodock4 was used to generate 10 docking poses. The pose with the best
energy score was selected as the most representative.

2.11. Statistical Analysis

The collected data were calculated as means and standard deviations from three
replicates and were analyzed using SPSS statistical software (IBM SPSS Statistics, version 23,
New York, NY, USA). The differences between treatments were compared using one-way
analysis of variance (ANOVA) according to method of Tamhane and Methods, 1977 [32],
post hoc LSD test was also performed at p ≤ 0.000.

3. Results
The results of the chemical composition study of anise seeds are shown in Table 1.
The components of anise oil were analyzed using gas chromatography–mass spectrometry
Agriculture 2022, 12, 224 5 of 13

(GC-MS) to determine the active constituents of anise seed oil. The results were as follows:
There are a total of 28 components found in anise oil (see Table 1). The major constituent of
anise oil is trans-anethole (53.23%), followed by estragole (13.52%) and longifolene (6.08%).

Table 1. Chemical composition (%) in essential oil of Pimpinella anisum analyzed by GC-MS.

No Peak RT 1 (Min) Component Names %2

1 5.215 α-Pinene 0.59
2 4.621 E-β-Ocimene 0.93
3 5.802 D-Limonene 1.00
4 6.171 γ-Terpinene 0.60
5 6.421 3-Carene 0.93
6 6.901 Linalool 0.77
7 6.958 α-Santalol 0.70
8 7.545 Estragole 13.52
9 8.147 Z-Anethole 1.17
10 8.734 E-anethole 53.23
11 9.099 α-Guaiene 0.88
12 9.238 2-Allyl-4-methylphenol 0.59
13 9.372 (−)-Aristolene 1.47
14 9.501 Caryophyllene 1.26
15 9.718 Aromandendrene 0.56
16 9.803 γ-Elemene 1.07
17 9.976 α-Himachalene 1.94
18 10.218 Longifolene 6.08
19 10.358 Thujopsene 1.26
20 10.489 Cedrene 0.80
21 11.031 Ledene 1.42
22 11.354 Isospathulenol 0.71
23 11.826 E-Sesquisabinene hydrate 0.66
24 12.683 E-Isoeugenol 4.81
25 13.048 Phenol, 2-methoxy-4-(1-propenyl)- 0.66
26 13.676 Acetophenone, 20 ,50 -dimethoxy- 0.71
27 17.561 Geranyl isovalerate 0.99
28 20.336 Heptacosane 0.72
1 Retention time; 2 Compound percentage.

The minor constituents in the essential oil of anise seed were cis anethole, D-limonene,
(−)-aristolene, caryophyllene, γ-elemene, α-himachalene, longifolene, thujopsene and ledene.
While seventeen components were present at less than 1%. These were: α-pinene (0.59%),
T-β-ocimene (0.93%), γ-terpinene (0.6%), 3-carene (0.93%), linalool (0.77%), α-santalol (0.7%),
α-guaiene (0.88%), 2-allyl-4-methylphenol (0.59%), aromandendrene (0.56%), cedrene (0.8 %),
isospathulenol (0.71%), trans-sesquisabinene hydrate (0.66%) trans-isoeugenol (4.81 %), phenol,
2-methoxy-4-(1-propenyl)- (0.66%), acetophenone, 20 ,50 -dimethoxy- (0.71%), geranyl isovaler-
ate (0.99%) and heptacosane (0.72%).
There are usually considerable variations in the major active compounds within this
species. The major component of anise oil seen in the current study was trans-anethole,
which was similar to major component of many regions of the world. In current research,
the main compound of anise oil is trans-anethole followed by estragol.
To evaluate the antioxidant activity of anise oil, the free radical scavenging activity
against DPPH was determined. The DPPH assay (Figure 1) showed that the antioxidant
activity of anise oil at 10 and 20 µL/mL was approximately 36.02 ± 2.033% and 43.62 ±
1.071%, respectively, compared with BHA used as a reference. The antiradical activity
percentages of anise oil at 30, 40, and 50 µL/mL were 58.12 ± 1.238%, 68.42 ± 2.007%, and
77.58 ± 1.044%, respectively. The highest percentage of radical scavenging activity (77.58%)
was recorded for P. anisum at a concentration of 50 µL/mL, followed by a concentration of
40 µL/mL (68.42%). It was noticed that as the concentration of any anise extract increased,
Agriculture 2022, 12, 224 6 of 13

the antioxidant activity also increased, possibly due to the increase in the concentration of
the active substances in the oil.

Figure 1. Free radical scavenging activity of essential anise oil (expressed as % inhibition) compared
to Butylated hydroxyanisole (BHA).

Table 2 shows the acaricidal activity of various concentrations of anise oil. The obtained
data indicated that five concentrations of anise oil showed remarkable acaricidal effects
after 24, 48, and 72 h of treatment. Anise oil treatments lead to 91.0 and 96.0% of adult
females of T. urticae killed after 48 and 72 h of treatment with concentrations of 40 µL/L.
The lowest percentages recorded were 27.30%, 28.10%, and 33.00%, respectively, at 10 µL/L
after 24, 48, and 72 h. Also, the data clearly indicated that the acaricidal activity of anise oil
was increased by increasing both the concentration of the oil and the length of time of the
treatment; there were significant differences in the mean values of the tested concentrations
on mortality percentages.

Table 2. The mortality percentage of T. urticae as affected by different concentrations of anise oil.

Anise Oil Concentration Mortality% of T. urticae

(µL/L) 24 h 48 h 72 h
10 27.3 ± 2.7 d 28.1 ± 1.0 d 33.0 ± 1.4 d
20 42.2 ± 3.9 c 43.9 ± 1.3 c 45.3 ± 1.8 c
30 70.1 ± 1.8 b 73.9 ± 4.1 b 87.0 ± 1.5 b
40 89.3 ± 6.6 a 91.0 ± 1.4 a 96.0 ± 2.2 a
Control 0.000 0.000 0.000
L.S.D 0.46 0.98 0.36
Each value is average of three replicated samples ± SD. Different letters refer to significant differences at p ≤ 0.000.

Results (Table 3) revealed that the mean numbers of eggs hatched was not recorded
on the first and second days after treatment for all concentrations of oil. Highly significant
differences were recorded on the fourth, fifth, and sixth days. Egg hatchability was observed
on the third, fourth, fifth, and sixth days for all concentrations. The lowest mean numbers
of eggs hatched were recorded at high concentrations of the oil; the findings for 40 µL/L
on the fifth and sixth days were 4.75 ± 0.28 and 1.25 ± 0.28 eggs. The highest values were
observed on the third and fourth days at a concentration of 10 µL/L and were 19.75 ± 0.40
and 19.5 ± 0.48 eggs, respectively.
Figure 2 shows the toxicity of the anise oil at different times against adult females of
T. urticae. The recorded LC50 and LC90 values of the anise oil after 72 h were 20.94 and
35.80 µL/L, respectively; the values after 48 h were 21.73 and 39.99 µL/L, respectively;
and the values after 24 h were 22.32 and 43.98 µL/L, respectively. These data revealed that
the adult females proved to be more susceptible to the toxic action of anise oil after 72 h,
followed by 48 h and 24 h. There was an inverse relationship between the LC50 and LC90
values and the toxicity of the anise oil.
Agriculture 2022, 12, 224 7 of 13

Table 3. Mean numbers of eggs hatched into larvae of T. urticae as affected by different concentrations
of anise oil.

Mean Number of Egg Hatchability ± SE after Detected Days

Conc. (µL/L)
1st 2nd 3rd 4th 5th 6th
10 0.00 0.00 19.75 ± 0.40 a 19.52 ± 0.48 a 17.25 ± 0.37 a,b 11.25 ± 0.31 b
20 0.00 0.00 19.25 ± 0.28 a 16.75 ± 0.38 b 15.25 ± 0.40 b 9.50 ± 0.29 b
30 0.00 0.00 16.25 ± 0.39 b 10.75 ± 0.35 c 9.50 ± 0.29 c 6.25 ± 0.20 c
c d d
40 0.00 0.00 10.75 ± 0.21 7.75 ± 0.28 4.75 ± 0.28 1.25 ± 0.28 d
Cont. 0.00 0.00 19.12 ± 1.00 a
L.S.D 1.17 1.52 3.33 1.79
Each value is average of three replicated samples ± SD. Different letters refer to significant differences at p ≤ 0.000.

Figure 2. Toxicity lines of anise oil on adult females of T. urticae after 24, 48, and 72 h under laboratory
conditions. (a) Toxicity lines after 24 h; (b) toxicity lines after 48 h; (c) toxicity lines after 72 h.

The data in Figure 3 show the effect of the anise oil on the protease and AChE activities
of the two-spotted spider mites. The results showed that treatment with a low concentration
of anise oil (10 µL/L) led to the activation of protease activity by about 2.26 µg/h/mg
protein, and that treatment with 20 µL/L of anise oil did not cause a significant change
in protease activity compared with the untreated control. On the other hand, treatment
with higher dose of the anise oil, that is, 40 µL/L, led to a significant reduction in protease
activity by 0.49 µg/h/mg protein. An experimental result (see Figure 3) indicated the
AChE activity of T. urticae after 48 h of exposure to various concentrations of anise oil. The
results clearly indicated that anise oil significantly inhibited AChE in a dose-dependent
manner. The inhibitory effect of a low dosage of anise oil (10 µL/L) on T. urticae AChE
was only about 7.3%. The highest reduction of AChE activity was recorded after 48 h of
treatment with 40 µL/L of anise oil; the AChE activity decreased by 34% compared with
the untreated control.

Figure 3. Enzyme activities of T. urticae subjected to different doses of anise oil. (a) Protease activity;
(b) acetylcholinesterase (AChE) activity and for each parameter, the mean values ± SD followed by a
different letter are significantly (p ≤ 0.05) different according to LSD.

To study the effect of anise oil on AChE and protease, the main compound in anise oil
was selected. Docking was performed with Autodock 4 [29]. Docking of anethole into the
active site of AChE (6XYU) [22] showed the presence of hydrophobic interactions between
Agriculture 2022, 12, 224 8 of 13

anethole; two catalytic residues, His480 and Ser238; three residues of the oxyanion hole,
Gly150, Gly151, and Ala239; and the choline-binding pocket Trp83 in the active site of
the enzyme. The structures of complexes of AChE with an anethole inhibitor show the
importance of these aromatic residues in ligand binding (Figures 4 and 5). The docking
of anethole into the active site of cathepsin L (3F75), as one of the protease enzymes in
T. urticae, showed the formation of a hydrogen bond between anethole and one residue,
Gly74, and hydrophobic interactions between anethole and two catalytic residues, Cys31
and His167, at the active site of the enzyme (see Figure 4).

Figure 4. Structures of the complex of AChE with substrate acetylecholine (a) and updated (b)
structures of the complex of AChE with E–anethole (major compound in anise oil). The docking
was performed with Autodock. The ligands are represented as sticks in green color; the residues
that interacted with the ligand hydrophobicly are represented as lines in pink color; residues that
interacted with the ligand with hydrogen bonds are represented as sticks in teal color; non-interactive
residues are represented as lines in grey and hydrogen bbonds are represented as yellow dashes.

Figure 5. Structures of E–anethole (a) and updated (b) structures of the complex of Cathepsin L with
E–anethole (major compound in anise oil). the ligands are represented as sticks in green color; the
residues that interacted with the ligand with hydrophobicly are represented as lines in pink color;
residues that interacted with the ligand with hydrogen bonds are represented as sticks in teal color;
non-interactive residues are represented as lines in grey and hydrogen bonds are represented as
yellow dashes.

4. Discussion
In recent years, natural pesticides have become important as alternatives to synthetic
pesticides due to the adverse effects of chemical control not only on environment wildlife
but also on human health [33]. A lot of studies have been done on the possibility of using
natural compounds in plants as an alternative to synthetic pesticides [34,35]. Studies
have focused on natural materials that do not add toxic substances to the environment,
decompose in a short period of time, and do not cause soil and water pollution [36]. There
have been many studies on the effects of extracts from plants obtained by different methods
on T. urticae [37]. Several essential oils and plant extracts have insecticidal properties
Agriculture 2022, 12, 224 9 of 13

including mites [38]. Promising results have been obtained in studies using plant extracts
and essential oils in the control of pest mites. Thus, for T. urticae, in trials with spraying on
females, Mentha spicata X suaveolens aqueous extract caused the death of 96% of females
after 120 h of application [39]. oEssential oil of Cymbopogon citratus Boisduval in non-contact
diffusion bioassay with oil application in filter paper in a closed container provided 100%
mortality at a dose of 19 × 10−3 µL/mL of air [40].
The variability in the volatile components of anise oil in our results appears to be largely
due to the stage of harvest and seasonal and environmental factors, as well as the method
of extraction. In anise oil, the main compound was E-anethole, followed by estragole.
Similarly, trans-anethole was previously reported as a major component of P. anisum [41,42].
These results are in agreement with previous investigations on anise essential oil [43,44].
Muthanna and Hiyam [45] identified trans-anethole as a major component of P. anisum
essential oil (26.97%), followed by estragole (20.50%). Haşimi et al. [46] determined that the
main components of anise essential oil were trans-anethole (52.94%), followed by isoanethole
(13.89%), caryophyllene oxide (8.55%), and caryophyllene (2.4%). Mohammed et al. [47]
reported that essential oil had a percentage of trans-anethole of 55.491%.
Anise oil showed the highest activity at a concentration of 50 µL/L. It was the least
effective radical scavenger at a concentration of 10 µL/L. This means that by increasing
the concentration of the essential oil, the antioxidant activity was increased. This may be
due to the increase in terpenes and active substances, which have a free radical scavenging
effect. The antioxidant activity of spices and herbs is attributed to the presence of volatile
oils and bioactive components [48–50].
According to [51,52], anise oil possesses antioxidant properties. This is due to the fact
that the seeds are rich in important minerals and compounds such as anethole, anisalde-
hyde, anise alcohol, acetophenone, pinene, limonene, and glycerol.
This work has shown that anise oil has acaricidal activity, possibly due to the active
substances present in the essential oil. The acaricidal activity assessment reported that
anise oil at 40 µL/L could fully control the two-spotted spider mites, with a mortality rate
after 72 h of 96.0%. The highest concentration of anise oil caused the highest mortality rate
for T. urticae after 24 h. The lowest mean number of eggs hatched at a high concentration
of 40 µL/L was recorded during the fifth and sixth days. This may be attributed to the
increase in the concentration of active substances, which also increased their effects on
egg hatching. The two-spotted spider mite can be controlled with essential oils which
possess odor-producing compounds including monoterpenes, sesquiterpenes, phenols,
oxides, esters, aldehydes, and ketones [53]. Plants use these active compounds to protect
against various arthropods such as insects and mites [54]. Vinicius et al. [5] reported
that the most promising extracts for T. urticae control were Origanum vulgare, Matricaria
chamomilla, and P. anisum; all produced a mortality rate above 75% in two replicates. The
volatiles emanating from the essential oil, especially carvacrol, borneol, cineol, terpineol,
and terpinene, have an acaricidal effect on T. urticae females, reaching a mortality rate of
100% as a function of concentration and exposure time [55]. In this study, a mortality rate of
96.0% was obtained when large amounts of essential oils were used. High mortality rates
have been reported before; for example, Tunc and Sahinkaya [56] reported a 100% mortality
rate of T. cinnabarinus and Aphis gossypii Glover when essential oils of Cuminum cyminum,
P. anisum, and Origanum syriacum were used in greenhouse conditions. In addition to
being toxic against the postembryonic stage of insects and mites, some essential oils have
oviposition-deterring activities; for example, three essential oils extracted from Laurus
nobilis, Myrtus communis, P. anisum, and Artemisia absinthum were toxic against the adults
and eggs of T. cinnabarinus under laboratory conditions [57].
The ways essential oils work vary with their neurotoxicity. This confirms why anise
oil inhibits the AChE growth in insects, degrading the waxy layers of insect cuticles,
which obstruct digestive enzymes such as protease and inhibit glutathione-S-transferase
(GST) [58]. Ahmed et al. [58] stated that the compounds (−)-terpinen-4-ol and γ-terpinene
were two main ingredients with insecticidal properties and affected insect enzymes such
Agriculture 2022, 12, 224 10 of 13

as AChE and GST. This confirms the results of our study; anise seed oil affected various
biological parameters of T. urticae such as AChE and protease. Results showed that this
oil had a high degree of toxicity against the nervous system, as it inhibited AChE, and
the highest inhibition was at a concentration of 40 µL/L after 48 h. This is related to the
essential oil’s high concentration of E-anethole, which binds to the active site of AChE by
hydrophobic interactions (see Figure 3). These results are in agreement with the findings
of Ivanov et al. [59], who reported that anise hyssop essential oil showed an inhibitory
effect on AChE activity where the IC50 value equaled 19.25 mg/L. Anise oil concentrations
of 22.32, 21.73, and 20.94 ppm led to a mortality rate of 50% of T. urticae mites after 24,
48, and 72 h, respectively. The obtained LC50 and LC90 values of anise oil reflect high
acaricidal activity of anise oil against T. urticae. Rania [60] found insecticidal activity of
lupine extract, olive oil, marjoram oil, anise oil, and orange oil against two strains of A.
gossypii and Rhopalosiphum maidis (Fitch). Mead [61] stated that the toxic effect of C. citratus
oil against T. urticae was undoubtedly due to its component citral (which has two isomers,
geranial and neral). The AChE inhibitory activity is due to synergistic and antagonistic
interactions between the chemical constituents of anise oil and AChE. The major compound
in anise hyssop essential oil was estragol. This compound was reported to have a high
ability to inhibit AChE (IC50 0.337 µmol), followed by eugenol (IC50 40.32 µmol) [62]. The
results of our study indicated that anise oil affected the activity of protease; the exposure of
T. urticae adults to anise oil at concentration of 40 µL/L for 48 h had a significant effect on
the protease enzyme compared with the control. This may be attributed to the binding of
E-anethole to the active site of the enzyme by a hydrogen bond in the residue Gly74 and
the hydrophobic interactions between anethole and the catalytic residues Cys31 and His167
at the active site of the enzyme (Figure 4). On the other hand, the treatment of T. urticae
with a low concentration of anise oil (10 µL/L) did not cause inhibition of the protease
activity. Accordingly, it can be concluded that the increase in the anise oil concentration had
a paradoxical impact on the protease activity. In addition to the above findings, effective
compounds have been identified in the essential oils of thyme and anise and synergistic
activity has been shown [63]. Hummelbrunner and Isman [64] reported that monoterpenes
produce synergistic insecticidal effects. The Chaubey [65] study reported that α-pinene and
β-caryophyllene in a binary combination showed synergy, reduced the egg-laying capacity,
and prevented pupation and adult emergence in Tribolium castaneum. These results reported
earlier clearly support the results of the current study. In the current research, anise oil
(P. anisum) was more effective against T. urticae. The presence of bioactive components
in anise oil provides hope for the development of new natural insecticides that would be
economically and environmentally sound for the management of insect pests that affect
stored products. The present findings confirmed the findings of Athanase and Fedai [66]
and Ahmed et al. [58]. Gas chromatography analysis for volatile oils showed that bisabolol
oxide A (44.34%), carvone (70.29%), linalool (85.60%), and camphor (54.36%) were the main
components of chamomile, spearmint, coriander, and rosemary volatile oils, respectively,
and they may be responsible for controlling T. urticae. Anise oil contains most of these active
substances, and it had the same effect on T. urticae. In addition to the effect of the main
compound, E-anethole, on the inhibition of AChE and protease enzymes. The inhibition
was competitive for the active site.

5. Conclusions
In conclusion, anise oil caused high mortality rates in T. urticae mites at different
times with a concentration of 40 µL/L. The lowest mean number of eggs hatched was
recorded at the high concentration of 40 µL/L on the fifth and sixth days of the study. The
results showed that the activity of AChE was significantly inhibited at 40 µL/L, followed
by 30 µL/L. This is related to the essential oil’s high concentration of E-anethole, which
binds to the active site of the AChE enzyme by hydrophobic interactions. The activities
of protease decreased when mites were treated with 30 or 40 µL/L of anise oil. This is
related to the essential oil’s high concentration of active compounds such as E-anethole,
Agriculture 2022, 12, 224 11 of 13

–(e)-isoeugenol, limonene, linalool, and α-pinene. Therefore, we suggest that tested anise
oil can be used to control mites on green bean seedlings as an alternative to harmful
insecticides. Due to the devastating effects of synthetic insecticides on humans and the
environment, the use of anise oil as a natural pesticide is advised.

Author Contributions: Conceptualization S.M.E.-S., N.A., H.M.E., M.A.R.I.; methodology, S.M.E.-S.,

N.A., S.S. (Samy Selim), A.A.A.-K., N.E.N., H.M.E., M.A.R.I.; validation, S.M.E.-S., N.A., H.M.E.,
M.A.R.I.; formal analysis, S.M.E.-S., N.A., S.S. (Samy Sayed), H.M.E., M.A.R.I.; investigation, S.S.
(Samy Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed); resources, S.M.E.-S., N.A., S.S. (Samy
Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed), H.M.E., M.A.R.I.; data curation, S.M.E.-S.,
N.A., S.S. (Samy Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed), H.M.E., M.A.R.I.; writing—
original draft preparation, S.M.E.-S., N.A., H.M.E., M.A.R.I.; writing—review and editing, S.M.E.-S.,
N.A., S.S. (Samy Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed), H.M.E., M.A.R.I.; super-
vision, S.M.E.-S., N.A., S.S. (Samy Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed), H.M.E.,
M.A.R.I.; project administration, S.H.A.-H., S.S. (Samy Sayed), H.M.E., M.A.R.I.; funding acquisition,
S.M.E.-S., N.A., S.S. (Samy Selim), A.A.A.-K., N.E.N., S.H.A.-H., S.S. (Samy Sayed), H.M.E., M.A.R.I.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank Princess Nourah bint Abdulrahman University
Researchers supporting Project number (PNURSP2022R37), Princess Nourah bint Abdulrahman
University, Riyadh, Saudi Arabia. The authors would like to thank Taif University Researchers
Supporting Project number (TURSP-2020/92), Taif University, Taif, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.

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