molecules

Article
Phytochemical Investigations, Antioxidant and Insecticidal
Properties of Essential Oil and Extracts from the Aerial Parts of
Pelargonium graveolens from Morocco
Zakya M’hamdi 1 , Federica Davì 2,3 , Mohammed Elhourri 1 , Ali Amechrouq 1 , Fabio Mondello 2 ,
Francesco Cacciola 4 , Roberto Laganà Vinci 5 , Luigi Mondello 5,6 , Natalizia Miceli 2, *
and Maria Fernanda Taviano 2

1 Laboratory of Molecular Chemistry and Natural Substances, Faculty of Science, Moulay Ismail University,
B.P. 11201, Zitoune, Meknes 50050, Morocco; zakyamhamdi1997@gmail.com (Z.M.);
med.elhourri@gmail.com (M.E.); a.amechrouq@umi.ac.ma (A.A.)
2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
98166 Messina, Italy; federica.davi@studenti.unime.it (F.D.); fabio.mondello@unime.it (F.M.);
mtaviano@unime.it (M.F.T.)
3 Foundation “Prof. Antonio Imbesi”, University of Messina, 98122 Messina, Italy
4 Department of Biomedical, Dental, Morphological and Functional Imaging Sciences, University of Messina,
98125 Messina, Italy; francesco.cacciola@unime.it
5 C/o Messina Institute of Technology (MeIT), Department of Chemical, Biological, Pharmaceutical and
Environmental Sciences, Former Veterinary School, University of Messina, 98168 Messina, Italy;
lmondello@unime.it (L.M.)
6 Chromaleont s.r.l., C/o Messina Institute of Technology (MeIT), Department of Chemical, Biological,
Pharmaceutical and Environmental Sciences, Former Veterinary School, University of Messina,
98168 Messina, Italy
* Correspondence: nmiceli@unime.it; Tel.: +39-090-676-6530

Abstract: The essential oil and the aqueous and ethanolic extracts obtained from the aerial parts of
Citation: M’hamdi, Z.; Davì, F.; Pelargonium graveolens cultivated in Morocco were studied for their antioxidant and insecticidal activ-
Elhourri, M.; Amechrouq, A.; ity against rice weevils (Sitophylus oryzae). The total phenolic content of the extracts was determined
Mondello, F.; Cacciola, F.; Laganà by a spectrophotometric method and the phenolic compounds were extensively characterized by
Vinci, R.; Mondello, L.; Miceli, N.;
HPLC-PDA/ESI-MS. To evaluate antioxidant potential, three in vitro assays were used. In the DPPH
Taviano, M.F. Phytochemical
test, the ethanolic extract was the most active, followed by the aqueous extract and the essential oil.
Investigations, Antioxidant and
In the reducing power assay, excellent activity was highlighted for both extracts, while in the Fe2+
Insecticidal Properties of Essential Oil
chelating activity assay, weak activity was observed for both the essential oil and the ethanolic extract
and Extracts from the Aerial Parts of
Pelargonium graveolens from Morocco. and no activity for the aqueous extract. Concerning insecticide activity, the toxicity of the essential
Molecules 2024, 29, 4036. https:// oil and the extracts was tested against rice weevils; the lethal concentrations LC50 and LC99 were
doi.org/10.3390/molecules29174036 determined, as well as the lethal time required for the death of 50% (LT50 ) and 99% (LT99 ) of the
weevils. The essential oil had the highest activity; 100% mortality of S. oryzae was observed around 5,
Academic Editor: Artur M. S. Silva
9, and 8 days for the essential oil and the aqueous and ethanolic extracts, respectively.
Received: 8 July 2024
Revised: 10 August 2024 Keywords: Pelargonium graveolens L’Hér.; HPLC-PDA/ESI-MS; antioxidant activity; botanical
Accepted: 17 August 2024 insecticide; Sitophilus oryzae L.; phenolic compounds
Published: 26 August 2024

1. Introduction
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland. The main problems affecting food during production, storage, and distribution are
This article is an open access article deterioration due to oxidation and attacks by pests. To protect foods from these effects,
distributed under the terms and many synthetic chemicals are widely used, causing injury to non-target organisms as well
conditions of the Creative Commons as human and environmental health problems [1].
Attribution (CC BY) license (https:// The use of plant-derived compounds instead of synthetic additives may be desirable,
creativecommons.org/licenses/by/ and there has been considerable interest in the isolation and development of new natural
4.0/). bioactive compounds. Phytochemicals are considered attractive due to their low cost,

Molecules 2024, 29, 4036. https://doi.org/10.3390/molecules29174036 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 4036 2 of 17

availability in large quantities from raw materials, biodegradability, and safety to human
health and the environment [2]. In this direction, plant extracts and essential oils (EOs)
stand out for their effectiveness throughout the world, while exploring the bioactivity
of phytochemical compounds has proved to be an effective and more feasible means
of controlling zoonotic diseases and reducing the microbial resistance index [3,4]. In
recent years, EOs have effectively controlled stored product pests [5], as they contain
monoterpenoid compounds that are toxic to insects by damaging their nervous systems [6].
The rice weevil (Sitophilus oryzae L.) is one of the most destructive pests of stored
cereals and processed cereal products worldwide [7]. Indeed, several research studies have
focused on the insecticidal and repellent activities of essential oils (EOs) and extracts from
many plant species against rice weevils [8,9].
Pelargonium graveolens L’Hér. or “Geranium pink”, belonging to the Geraniaceae fam-
ily, is a perennial aromatic shrub native to South Africa, Zimbabwe, and Mozambique, and
widely cultivated in Russia, Egypt, Algeria, Morocco, Congo, Japan, Central America, and
southern Europe (Spain, Italy, and France) [10]. This species is also used as a decoration
and as a remedy in African, European, Chinese, Iranian, Indian, and Arabic traditional
medicine [11,12]. It is well-known for its fragrance, and its EO, rich in geranial, (Z)-rose
oxide, isomenthone, and linalool, is widely used as a pharmaceutical, cosmetic, and flavor-
ing agent, as well as in folkloric foods and aromatherapy industries [13]. Geranium EO
has historically been used to treat dysentery, hemorrhoids, inflammation, heavy menstrual
flows, and even cancer. In French folk medicine, it is employed against diabetes, diar-
rhea, gallbladder problems, gastric ulcers, jaundice, liver problems, sterility, and urinary
stones [14]. The pounded leaves are used to treat skin diseases (wounds and sores); the leaf
decoction or infusion is employed against gastrointestinal disorders (constipation, intestinal
cramps, and dysentery), hyperglycemia, and to relieve inflammatory and pain-associated
ailments (i.e., headache and neuralgia), as well as those of the respiratory system (cold and
cough). The decoction of the root is utilized against fever and tuberculosis; whereas the
root infusion works against diarrhea and backache [15]. Several studies have confirmed
that P. graveolens has a wide range of pharmacological effects, including anti-inflammatory
and anticancer [16], anti-parasitic [17], anti-tuberculosis [18], and analgesic [19] effects.
The plant has also been reported to have antimicrobial activity against many pathogenic
bacteria and fungi [20,21]. Many chemical constituents such as volatile compounds, ter-
penoids, flavonoids, coumarins, phenolic acids, and tannins have been isolated from this
species [12]. The research on P. graveolens is intensively focused on the chemical compo-
sition of the EO, mostly characterized by monoterpenes and sesquiterpenes (oxygenated
and non-oxygenated). Oxygenated monoterpenes exist in a higher concentration than non-
oxygenated monoterpenes, and the predominant ones are β-citronellol, geraniol, linalool,
and isomenthone. Oxygenated sesquiterpenes are less abundant than non-oxygenated
ones, including δ-selinene, β-caryophyllene, guaia-6,9-diene, and α-humulene [22–24].
Previously, some co-authors of this work characterized the chemical composition of the
essential oil obtained from the aerial parts of P. graveolens grown in Er-Rachidia, Morocco.
Using GC/MS analysis, epi-γ-eudesmol (16.67%), geraniol (12.54%), β-citronellol (12.34%),
citronellyl formate (7.70%), geranyl tiglate (5.21%), and linalool (4.06%) were found to
be the major compounds [25]. In continuation of the previous study, the present work
was undertaken to investigate the antioxidant and insecticidal properties of the essential
oil, as well as of the ethanolic and aqueous extracts from the aerial parts of this species.
The antioxidant properties were examined by means of different in vitro systems: DPPH
scavenging, reducing power, and ferrous ion (Fe2+ )-chelating activity, and the insecticidal
activity was evaluated against S. oryzae. In addition, the phenolic content of the ethanolic
and aqueous extracts was determined by a Folin–Ciocalteu assay and characterized by
HPLC-PDA/ESI-MS analysis.
Molecules 2024, 29, 4036 3 of 17

2. Results and Discussion

2.1. Phytochemical Investigations
2.1.1. Determination of Total Phenolic Content
Polyphenols are strong antioxidants widely distributed in nature in the form of sec-
ondary plant metabolites. They are classified into different subclasses based on the arrange-
ment and the number of phenolic rings present, as well as the functional groups associated
with these phenolic rings. Their antioxidant property is due to their ability to scavenge free
radicals, donate hydrogen atoms or electrons, or chelate metal cations [26–28].
In the present work, the total phenolic content of the aqueous and ethanolic extracts
of P. graveolens was estimated spectrophotometrically by the Folin–Ciocâlteu method,
extensively used to quantify polyphenols in plant-derived extracts, as well as foods and
drinks [29,30].
The results, reported in Table 1, show that the total phenolic content was found to be
higher in the ethanolic extract, resulting in more than double that of the aqueous extract.

Table 1. Quantitative determination of total phenolic content (TPC), free radical scavenging activity
(DPPH assay), reducing power, and ferrous ion-chelating activity of essential oil and ethanolic and
aqueous extracts obtained from the aerial parts of Pelargonium graveolens.

Pelargonium TPC DPPH Reducing Power Chelating Activity Fe2+

graveolens (mg GAE/g Extract) IC50 (mg/mL) ASE/mL IC50 (mg/mL)
EO ND >2 a 21.77 ± 2.17 a >2 a
Aqueous extract 156.42 ± 0.73 a 0.13 ± 0.01 b 3.01 ± 0.03 b NA
Ethanolic extract 385.09 ± 2.09 b 0.05 ± 0.01 c 1.92 ± 0.04 b >2 a
BHT BHT EDTA
Standard -
0.07 ± 0.01 d 1.44 ± 0.02 b 0.007 ± 0.001 b
Values are expressed as the mean ± SD (n = 3). ND: Not determined. NA: Not active. a–d Different letters within
the same column indicate significant differences between mean values (p < 0.0001).

The total phenolic content of the extracts turned out to be higher than that previously
reported for various extracts obtained from P. graveolens. Ćavar and Maksimović [23] found a
much lower phenolic content in the aqueous extracts (hydrosols) obtained from leaves and stems
of this species cultivated in Bosnia (34.88 ± 2.00 and 102.44 ± 1.63 mg GAE/g, respectively). A
comparative study undertaken by Pradeepa et al. [31] on P. graveolens leaves collected in
India showed that ethanolic extract, obtained by Soxhlet, had the highest total phenolic
content (123.75 ± 8.25 mg GAE/g), followed by acetone (107.25 ± 4.25 mg GAE/g) and
then methanolic (100.65 ± 4.90 mg GAE/g) and aqueous (24.75 ± 5.62 mg GAE/g) extracts.
A similar work was conducted on extracts of leaves and flowers of P. graveolens from
Tunisia using different solvents; the most abundant content was found in leaf and flower
80% methanol extracts (142.71 ± 3.83 mg GAE/g and 129.2 ± 2.60 mg GAE/g, respectively),
followed by 80% ethanol extracts (136.54 ± 1.2 mg GAE/g and 118.05 ± 2.1 mg GAE/g,
respectively) and water extracts (92.77 ± 2.50 and 55.44 ± 1.30 mgGAE7g, respectively) [32].
In another work conducted on aerial parts, aqueous extracts were obtained by the infusion
and decoction of this species from Tunisia, and the phenolic content was found to be
27.05 ± 0.53 and 31.20 ± 0.58 mg GAE/g, respectively [33].
The extracts investigated in this study were obtained by using the Soxhlet extraction
technique, which is known to offer numerous advantages such as high yields with a much
lower volume of solvent. From comparisons with previous studies, it is evident that this
technique (using ethanol as a solvent) represents an efficient system to recover a high
content of phenolic compounds; notably, the extracts obtained from P. graveolens from
Morocco are a richer source of phenolic compounds than those from the same species
grown in other geographical areas.
Molecules 2024, 29, x FOR PEER REVIEW 4 of 17
Molecules 2024, 29, 4036 4 of 17

2.1.2.
2.1.2. Identification
Identification ofof Phenolic
Phenolic Compounds
Compounds by by HPLC-PDA/ESI-MS
HPLC-PDA/ESI-MS
Analysis
Analysis of the phenolic profile of the aqueous and
of the phenolic profile of the aqueous and ethanolic
ethanolic extracts
extracts obtained
obtained from
from
aerial
aerial parts of P.
parts of P. graveolens
graveolenswas
wascarried
carriedout
outby
byusing
usinghigh-performance
high-performanceliquid
liquidchromatogra-
chromatog-
raphy coupled
phy coupled to to a photodiode
a photodiode array
array andand electrospray
electrospray ionization
ionization massmass spectrometry.
spectrometry. A
A total
total of thirty-three phenolic compounds were detected (Figure 1A,B
of thirty-three phenolic compounds were detected (Figure 1A,B and Table 2). and Table 2).

Figure
Figure 1.
1. HPLC-PDA
HPLC-PDA chromatograms
chromatograms of of the phenolic compounds,
the phenolic compounds, extracted
extracted at
at 330
330 nm.
nm. Aqueous
Aqueous
extract (A) and ethanolic extract (B) of Pelargonium graveolens. For peak identification, see Table 2.
extract (A) and ethanolic extract (B) of Pelargonium graveolens. For peak identification, see Table 2.

In particular, most of them belonged to the flavonoid class, while only eight were
phenolic acids. Of the
the flavonoids,
flavonoids, eight
eight were
were kaempferol
kaempferol derivatives, seven werewere quercetin
quercetin
myricetin derivatives. The
derivatives, and four were myricetin The eight
eight phenolic
phenolic acids
acids were
were gallic
gallic acid,
acid,
caffeoylglucaric acid,
caffeoylglucaric acid, caftaric
caftaric acid,
acid, feruloylglucaric
feruloylglucaric acid,
acid, caffeoylquinic
caffeoylquinic acid,
acid, caffeic
caffeic acid,
acid,
caffeoylhydroxycitric acid, and rosmarinic acid.
caffeoylhydroxycitric
The results
resultsofofthe
theHPLC
HPLC analysis
analysis of P. P. graveolens
of graveolens extracts
extracts havehave shown
shown qualitative
qualitative and
and quantitative
quantitative differences
differences in the
in the phenolic
phenolic content.
content. Analysis
Analysis of ofthe
theethanolic
ethanolicextract
extract dis-
dis-
played 1717detected
detectedcompounds.
compounds.The main
The compounds were quercetin
main compounds were hexosyl-rhamnoside
quercetin hexosyl-
Molecules 2024, 29, 4036 5 of 17

(9.09 ± 0.049 mg/g; peak 19), quercetin hexosyl-rhamnoside (8.63 ± 0.083 mg/g; peak 20),
quercetin (5.45 ± 0.002 mg/g; peak 32), quercetin hexosyl-pentoside (4.41 ± 0.056 mg/g;
peak 17), and quercetin 3-O-pentoside (3.09 ± 0.034 mg/g; peak 25). The remaining de-
tected compounds were less than 2 mg/g, and two compounds were detected but not
quantified. On the other hand, analysis of the aqueous extract of P. graveolens revealed
28 compounds, of which the major compounds were rosmarinic acid (8.59 ± 0.017 mg/g;
peak 31), quercetin hexosyl-rhamnoside (4.44 ± 0.004 mg/g; peak 19), quercetin hexosyl-
pentoside (4.36 ± 0.006 mg/g; peak 17), caffeoylglucaric acid (3.39 ± 0.011 mg/g; peak
2), kaempferol hexuronide and kaempferol hexosyl-pentoside (2.84 ± 0.010 mg/g; peak
22 and 23, respectively), quercetin hexoside (2.63 ± 0.034 mg/g; peak 21), and caffeoyl
glucuronide (2.09 ± 0.044 mg/g; peak 4), while the other compounds were less than
2 mg/g.
Very few studies have investigated the phenolic composition of P. graveolens [34–36];
our results agree with those reported by Androutsopoulou [35] and Al-Sayed [36], who
found quercetin and kaempferol derivatives to be the main phenolics detected in leaf
extracts of P. graveolens from Greece and Egypt, respectively. Notably, this is the first work
reporting an extensive characterization of the phenolic profile of aerial parts of this species
growing in Morocco.

Table 2. Semi-quantification of phenolic compounds in aqueous and ethanolic extracts of the aerial
parts of Pelargonium graveolens through LC-PDA/ESI-MS analysis. Quantification of phenolic com-
pounds was reported in mg/g of dried extract ± SD (n = 3).

Peak N. Compound tR (min) UV max (nm) [M − H]− Aqueous Extract Ethanolic Extract Ref.
1 Gallic acid 2.91 270 169 0.60 ± 0.000 - Std.
2 Caffeoylglucaric acid 5.74 326 371, 179 3.39 ± 0.011 - [37]
3 Unknown 6.32 279 395, 197 X - -
4 Caffeoyl glucuronide 7.35 288, 312 355 2.09 ± 0.044 - -
5 Caftaric acid 7.94 325 311, 179 1.13 ± 0.035 - [38]
6 Feruloylglucaric acid 9.06 325 385, 193 0.72 ± 0.011 - -
7 Sinapoylglucose 9.36 281, 322 385, 223 0.30 ± 0.010 - -
8 Caffeoylglucose 9.77 323 341, 179 0.30 ± 0.003 - -
9 Unknown 9.90 312 293 X X -
10 Caffeoylquinic acid 10.80 324 353, 191, 179 1.11 ± 0.002 0.23 ± 0.016 Std.
11 Caffeic acid 10.96 322 179 0.92 ± 0.012 - Std.
12 Unknown 11.01 282 325 - X -
13 Caffeoylhydroxycitric acid 11.14 312 369 0.48 ± 0.003 - -
14 Myricetin hexoside 22.40 260 sh, 354 479, 317 - 1.16 ± 0.000 [37]
15 Myricetin rhamnosyl-hexoside 23.42 262 sh, 353 625, 479, 317 1.06 ± 0.004 1.90 ± 0.022 [37]
16 Quercetin hexuronide 24.38 276, 343 477, 301 0.37 ± 0.011 - [37]
17 Quercetin hexosyl-Pentoside 25.26 255, 353 595, 463, 301 4.36 ± 0.006 4.41 ± 0.056 [37]
18 Myricetin 3-O-rhamnoside 27.24 263, 348 463, 317 0.96 ± 0.006 1.49 ± 0.003 [35]
19 Quercetin hexosyl-rhamnoside 28.23 254, 353 609, 463, 301 4.44 ± 0.004 9.09 ± 0.049 [37]
20 Quercetin hexosyl-rhamnoside 29.53 256, 352 609, 463, 301 1.34 ± 0.041 8.63 ± 0.083 [37]
21 Quercetin hexoside 29.68 254, 352 463, 301 2.63 ± 0.034 - [37]
22 Kaempferol hexuronide 30.34 261, 347 461, 285 - -
2.84 ± 0.010
23 Kaempferol hexosyl-pentoside 30.79 265, 345 579, 447, 285 0.65 ± 0.008 [39]
24 Kaempferol hexosyl-rhamnoside 30.82 266, 347 593, 447, 285 0.81 ± 0.000 - [37]
25 Quercetin 3-O-pentoside 31.86 255, 353 433, 301 1.71 ± 0.008 3.09 ± 0.034 [35]
26 Kaempferol 3-O-glucoside 32.36 264, 344 447, 285 0.70 ± 0.001 1.71 ± 0.016 Std.
7 Kaempferol hexosyl-rhamnoside 34.93 265, 343 593, 447, 285 0.37 ± 0.017 - [37]
28 Kaempferol galactoside 35.16 264, 344 447, 285 0.92 ± 0.015 3.29 ± 0.033 [37]
29 Myricetin 35.99 252 sh, 370 317 - 1.38 ± 0.017 Std.
Molecules2024,
Molecules 29,x4036
2024,29, FOR PEER REVIEW 6 6ofof17
17

Table 2. Cont.
29 Myricetin 35.99 252 sh, 370 317 - 1.38 ± 0.017 Std.
30 N.
Peak Kaempferol
Compound3-O-pentoside tR (min)36.53 265,(nm)
UV max 345 [M417,
− H]285
− 0.34 ±Extract
Aqueous 0.003 0.70 ± 0.009
Ethanolic Extract [35]
Ref.
30
31 Kaempferol 3-O-pentoside
Rosmarinic acid 36.53 40.13 265, 328
345 417,
359,285161 0.34 ±±0.003
8.59 0.017 0.70 -± 0.009 [35]
[40]
31 40.13 328 359, 161 8.59 ± 0.017
32 Rosmarinic acid
Quercetin 51.69 254, 369 301 - 5.45 ± -0.002 [40]
Std.
32 Quercetin 51.69 254, 369 301 - 5.45 ± 0.002 Std.
33 Kaempferol 65.07 265, 366 285 - 1.48 ± 0.007 Std.
33 Kaempferol 65.07 265, 366 285 - 1.48 ± 0.007 Std.
X: detected but not quantified; sh: wavelength shoulder.
X: detected but not quantified; sh: wavelength shoulder.

2.2. Antioxidant Activity

2.2. Antioxidant Activity
The antioxidant properties of the aqueous and ethanolic extracts and EO of P. grave-
The antioxidant properties of the aqueous and ethanolic extracts and EO of P. graveolens
olens were established using three in vitro tests to evaluate the different mechanisms
were established using three in vitro tests to evaluate the different mechanisms through
through
which the which
diversetheantioxidant
diverse antioxidant
compounds compounds
contained contained in the phytocomplexes
in the phytocomplexes could exert
could
their effect. The primary antioxidant properties were evaluated by aevaluated
exert their effect. The primary antioxidant properties were DPPH assay, by abased
DPPH on
assay, based on hydrogen atom transfer (HAT) and single-electron
hydrogen atom transfer (HAT) and single-electron transfer (SET) mechanisms and reducing transfer (SET) mecha-
nisms
power,andand reducing power,
a SET-based and the
assay; a SET-based
ferrous ion assay;
(Fe2+the ferrous ion
)-chelating (Fe2+)-chelating
activity assay was activity
utilized
assay was utilized to determine the secondary
to determine the secondary antioxidant properties. antioxidant properties.
The
Theresults
resultsof ofthe
theDPPH
DPPHtest,test,utilized
utilizedtotodetermine
determine the
thescavenging
scavengingpropertiespropertiesof offree
free
radicals, are shown in Figure 2A. Both aqueous and ethanolic extracts
radicals, are shown in Figure 2A. Both aqueous and ethanolic extracts exhibited excellent radical exhibited excellent
radical scavenging
scavenging activity; activity; the ethanolic
the ethanolic extract at the extract
lowestat concentrations
the lowest concentrations
(0.0625 to 0.250 (0.0625
mg/mL) to
0.250
showedmg/mL)
a highershowed a higher
effect than effect than
the reference the reference
standard standard
BHT, reaching its BHT,
maximum reaching its maxi-
activity, above
mum activity,
90%, at above 90%,ofat0.250
the concentration the concentration
mg/mL. On theofother 0.250hand,
mg/mL. Onshowed
the EO the other hand,
very lowthe EO
activity.
showed very low activity. This is also confirmed by the calculated IC
This is also confirmed by the calculated IC50 values equal to 0.05 ± 0.011 mg/mL for ethanolic50 values equal to 0.05
±extract,
0.011 mg/mL
which isfor ethanolic
better than BHT extract,
(IC50 =which
0.07 ±is0.01
better than BHT
mg/mL), (IC50 by
followed = 0.07the ±aqueous
0.01 mg/mL),
extract
followed by the aqueous extract (IC = 0.13 ± 0.01 mg/mL)
(IC50 = 0.13 ± 0.01 mg/mL) and EO (IC50 > 2 mg/mL) (Table 1). Figure 2B shows
50 and EO (IC 50 > 2 mg/mL) (Tablethe
1). Figure
results of 2B
theshows
reducingthe power
results assay.
of the Excellent
reducing reducing
power assay. Excellent
capabilities were reducing capabili-
highlighted for
ties
the were highlighted
ethanolic and aqueousfor theextracts
ethanolic and aqueous
compared to theextracts
reference compared
standardtoBHT. the reference
Ethanolic
standard
extract fromBHT.the Ethanolic
1 mg/mL extract from the 1was
concentration mg/mL
moreconcentration
active than the was more active
standard. than
However,
the
no standard.
statistically However,
significant nodifference
statistically significant
between differencevalues
the ASE/mL between the ASE/mL
of aqueous values
and ethano-
of
licaqueous
extractsand (3.01ethanolic
± 0.03 and extracts
1.92(3.01
± 0.04 ± 0.03 and 1.92respectively)
ASE/mL, ± 0.04 ASE/mL, respectively)
compared to thecom- BHT
(1.44 ±
pared to 0.02
the BHT
ASE/mL)(1.44 ±was0.02found,
ASE/mL) was found,
as shown as shown
in Table in Table
1. Instead, the 1.EO Instead,
showed theweak
EO
showed
reducing weak
power reducing ± 2.17(21.77
(21.77power ASE/mL). In the Fe2+
± 2.17 ASE/mL). In chelating
the Fe2+ chelating
activity activity
assay, the assay,
EO
andEO
the theand
ethanolic extractextract
the ethanolic showed low activity
showed compared
low activity to the to
compared reference
the referencestandard EDTA
standard
(Figure
EDTA 2C), also
(Figure 2C),demonstrated
also demonstrated by ICby values
50 IC > 2>mg/mL
50 values 2 mg/mLfor forboth
both(Table(Table1). On the
1). On the
contrary, the
contrary, the aqueous
aqueous extract
extract showed
showed no no activity.
activity.

Figure 2. Cont.
Molecules 2024,
Molecules 2024, 29,
29, 4036
x FOR PEER REVIEW 7 of 17
7 of 17

Figure Freeradical
Figure 2. Free radicalscavenging
scavengingactivity
activity (DPPH
(DPPH assay)
assay) (A),(A), reducing
reducing power
power (B), (B),
and and ferrous
ferrous ion-
chelating activity
ion-chelating (C) of
activity (C)EOof and
EO ethanolic and aqueous
and ethanolic extracts
and aqueous obtained
extracts from from
obtained aerialaerial
parts parts
of Pelar-
of
gonium graveolens.
Pelargonium Values
graveolens. are expressed
Values as the
are expressed asmean ± SD±(nSD
the mean = 3).
(n = 3).

The results
results of
of the
the antioxidant
antioxidant teststests indicate
indicate that
that the
the aqueous
aqueous andand ethanolic
ethanolic extracts
extracts
showed
showed excellent
excellent primary
primary antioxidant
antioxidant properties;
properties; on on the contrary,
contrary, the
the EOEO has
has shown
shown weak
antioxidant properties, both primary and secondary. secondary. The The primary
primary antioxidant
antioxidant properties
properties
could be mainly attributed to the phenolic compounds detected in the extracts by HPLC-
PDA/ESI-MS analysis. Flavonoids
Flavonoids and andphenolic
phenolicacids,
acids,the
thelargest
largestclasses
classes ofofplant phenolics,
plant phenol-
are
ics, effective antioxidants;
are effective antioxidants;the the
antioxidant
antioxidant activity of these
activity compounds
of these compounds is mainly
is mainlyduedueto
their redox
to their redoxproperties
properties andandchemical
chemical structure,
structure,which
whichcontribute
contributetototheir
theirability
abilitytoto inhibit
inhibit
lipoxygenase
lipoxygenase and and scavenging
scavengingfree freeradicals
radicals[41–43].
[41–43].TheThebest
bestradical
radical scavenging
scavenging activity
activity of
of the ethanolic extract could be related to the presence of the flavonols
the ethanolic extract could be related to the presence of the flavonols quercetin and myri- quercetin and
myricetin and their
cetin and their derivatives,
derivatives, whose whose antioxidant
antioxidant properties
properties havewidely
have been been widely demon-
demonstrated
strated [44–46]. These compounds were found in larger quantities
[44–46]. These compounds were found in larger quantities in the ethanolic extract thanin the ethanolic extract
the
than the aqueous
aqueous one. one.
Several
Several previous
previous works indicated P.
works indicated P. graveolens
graveolens asas aa potential
potential source
source of of antioxidant
antioxidant
compounds.
compounds. Referring to the literature, studies on the antioxidant activity of
Referring to the literature, studies on the antioxidant activity of this
this species
species
were conducted mainly on the essential oil, showing a strong antioxidant
were conducted mainly on the essential oil, showing a strong antioxidant effect, effect, whichwhich
does
not agree with our results [10,32,47–49]. On the contrary, our findings
does not agree with our results [10,32,47–49]. On the contrary, our findings are similar to are similar to those
reported by Ćavar
those reported et al. [23],
by Ćavar et al.showing very weak
[23], showing veryreactivity in the scavenging
weak reactivity of DPPH
in the scavenging of
radicals in the essential oils from the air-dried leaves and stems of P.
DPPH radicals in the essential oils from the air-dried leaves and stems of P. graveolens.graveolens.
Furthermore, Dimitrova et al. [50] and Ennaifer et al. [33,51] reported the remarkable
Furthermore, Dimitrova et al. [50] and Ennaifer et al. [33,51] reported the remarkable
antioxidant capacity of aqueous extracts of this species. El Aanachi et al. [13] showed
antioxidant capacity of aqueous extracts of this species. El Aanachi et al. [13] showed the
the activities of extracts from aerial parts (n-hexane, dichloromethane, and methanol) of
activities of extracts from aerial parts (n-hexane, dichloromethane, and methanol) of P.
P. graveolens by various antioxidant assays, including DPPH scavenging, reducing power,
graveolens by various antioxidant assays, including DPPH scavenging, reducing power,
and iron chelation. Strong antioxidant activity was demonstrated by the extracts, particu-
and iron chelation. Strong antioxidant activity was demonstrated by the extracts, particu-
larly the methanol extract, which was the most powerful.
larly the methanol extract, which was the most powerful.
Molecules 2024, 29, x FOR PEER REVIEW 8 of 17

Molecules 2024, 29, 4036 8 of 17

2.3. Insecticidal Activity on Adult Sitophilus oryzae

2.3.The
Insecticidal
EO of P. Activity
graveolenson at
Adult Sitophilus
different oryzae
concentrations (4, 8, 12, and 16 µL/L of air) signif-
icantly The
affected
EO of P. graveolens at different concentrations (4,batches,
the survival of S. oryzae adults. In the treated this16
8, 12, and survival
µL/L ofranged
air) sig-
between
nificantly affected the survival of S. oryzae adults. In the treated batches, this batch,
1 and 10 days for the concentration of 16 µL/L of air, whereas in the control survival
this parameter
ranged varied
between between
1 and 3 and
10 days for12thedays. The toxicityofof16EO
concentration depends
µL/L of air,onwhereas
the concen-
in the
tration
controland duration
batch, of exposure
this parameter (Figure
varied 3). The
between survival
3 and 12 days.times
Theoftoxicity
50% ofofthe EOadults
dependsex-on
posed to different concentrations
the concentration and durationofofEO varied from
exposure one3).
(Figure dayTheto around
survivalfive days,
times of whereas
50% of the
in adults
the control
exposedbatch, the adults
to different lived for an of
concentrations average of 12from
EO varied days. The
one dayTLto and TL99
50 around were
five days,
negatively
whereas correlated
in the controlwith the concentrations
batch, the adults livedofforEOantested (Table
average 3).days.
of 12 The toxicological
The TL50 andpa- TL99
rameters of the EOcorrelated
were negatively tested are with
shown theinconcentrations
Table 4. After of
three
EOdays
tested of(Table
treatment,
3). Thethetoxicological
LC50 and
LCparameters of thevalues
99 concentration EO tested
wereare shown
19.22 µL/LinandTable 4. After
76.42 µL/L,three days of treatment, the LC50
respectively.
and LC99 concentration values were 19.22 µL/L and 76.42 µL/L, respectively.

Witness(a) 4µl/L(a) 8µl/L(b) 12µl/L(b) 16µl/L(b)

Survival probability (%)

1
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Days after treatment
Figure
Figure 3. Survival
3. Survival of adult
of adult Sitophilus
Sitophilus oryzae
oryzae treated
treated with
with thethe
EOEO of Pelargonium
of Pelargonium graveolens.
graveolens. Survivors
Survivors
with the same lower-case letter did not differ statistically from one another (Scheffé test, p ≤p 0.05),
with the same lower-case letter did not differ statistically from one another (Scheffé test, ≤ 0.05),
while thethe
while others were
others different.
were different.

Table 3. TL
Table 50 and
3. TL TL99TLof99Sitophilus
50 and oryzae
of Sitophilus adults
oryzae exposed
adults to Pelargonium
exposed graveolens
to Pelargonium essential
graveolens oil. oil.
essential

Concentrations
Concentrations(µL/L)
(µL/L) TLTL
50
50
r > rr >(0.05; 2) 2)
r (0.05; TL99TL99 r > rr (0.05; 2) 2)
> r (0.05;
0 0 6.89
6.89 13.65
13.65
4 4 5.58
5.58 −0.89
−0.89 11.05
11.05 −0.89
−0.89
8 8 3.18
3.18 6.306.30
1212 2.71
2.71 5.365.36

Table
Table 4. Toxicity
4. Toxicity parameters
parameters of of essential
essential oiloil
of of Pelargonium
Pelargonium graveolens
graveolens onon Sitophilus
Sitophilus oryzae.
oryzae.

DaysDaysafter
after χ2 Calculated
χ2 Calculated LC50LC(µL/L) (2) (2)
50 (µL/L) LCLC
99 (µL/L)
99 (µL/L)
(2) (2)
Slope±±
Treatment Slope SESE(1)(1) 2 (0.05; 2) = 5.991 [Confidence Interval] [Confidence Interval]
Treatment 2 <χ
<χ (0.05; 2) = 5.991 [Confidence Interval] [Confidence Interval]
1 3.03 ± 0.71 4.36 36.7836.78 215.88
215.88
1 3.03 ± 0.71 4.36 [30.03; 53.74] [109.33; 1312.13]
[30.03; 53.74] [109.33; 1312.13]
3 3.88 ± 0.81 3.70 19.22 76.42
19.22
[14.23; 23.25] 76.42171.002]
[53.353;
3 3.88 ± 0.81 3.70
[14.23; 23.25]
15.35 [53.353; 171.002]
56.40
4 4.11 ± 0.82 1.96 [10.96; 18.82] [41.22;
15.35 56.40108.74]
4 4.11 ± 0.82 1.96 13.79 35.55
5 5.66 ± 1.22 0.30 [10.96; 18.82] [41.22; 108.74]
[9.82; 16.78] [27.78; 59.75]
13.79 35.55
5 6 5.66
8.02± ±1.22
2.14 0.300.02 12.30 23.99
[9.82; 16.78]
[8.73; 15.07] [27.78; 59.75]
[18.89; 43.78]
12.30
(1) SE: Standard Error; (2) LC and LC : Lethal concentrations, respectively, for 50% and 23.99
99% of the individu-
6 8.02 ± 2.1450 99
0.02
als used. [8.73; 15.07] [18.89; 43.78]
(1) SE: Standard Error; (2) LC50 and LC99: Lethal concentrations, respectively, for 50% and 99% of the
Abd El-Salam [52] found that the EOs of Cymbopogon flexuosus and Melaleuca alternifolia
individuals used.
had potent toxicity against S. oryzae. The LC50 of these essential oils were, respectively, 31.0,
36.0, and 69.6 µL/L after three days of treatment, while the LC50 of the P. graveolens EO
studied was 19.22 µL/L, showing that S. oryzae was more sensitive to this oil. In addition,
 Abd El-Salam [52] found that the EOs of Cymbopogon flexuosus and Melaleuca alterni-
folia had potent toxicity against S. oryzae. The LC50 of these essential oils were, respectively,
Molecules 2024, 29, 4036 9 of 17
31.0, 36.0, and 69.6 µL/L after three days of treatment, while the LC50 of the P. graveolens
EO studied was 19.22 µL/L, showing that S. oryzae was more sensitive to this oil. In addi-
tion, Mesbah et al. [53] evaluated the S. oryzae contact toxicity of the EO from P. graveolens
andMesbah
preparedet al. [53] evaluatedThe
nanoemulsions. S. oryzae
the results contactthat
showed toxicity of the EO from
the nanoemulsion hadP. graveolens
the best ac-and
prepared nanoemulsions. The results showed that the nanoemulsion
tivity (LC50 = 2.29 ppm/cm ) against adult S. oryzae after 72 h, whereas the EO was
2 had the bestfound
activity
(LC = 2.29 ppm/cm 2 ) against adult S. oryzae after 72 h, whereas the EO was found to be
to be less toxic, (LC50 = 67.662 ppm/cm ). A study carried out by Jayakumar et al. [54] as-
50 2
lessthe
toxic, (LC50 =and67.662 2 A study carried out by Jayakumar et al. [54] assayed
sayed fumigant theppm/cm
repellent ).effect of geranium EO on S. oryzae and found a fu-
the fumigant
migant and weevils.
effect on rice the repellent
Seadaeffect
et al. of geranium
[55] evaluated EOthe S. oryzae
oncontact and found
toxicity a fumigant
of P. graveolens
effect on rice weevils. Seada et al. [55] evaluated the contact toxicity
and found that geranium oil had the highest repellent activity against S. oryzae, followed of P. graveolens and
by found
fennel that
and geranium
basil oils. oil
Thehad the highest
results repellent
of the study activity
carried out byagainst
Arab etS. al.
oryzae,
[56] followed
indicated by
fennel and basil oils. The results of the study carried out by Arab et al.
that geranium stripping oil was highly toxic against adult S. oryzae. after 24 h of exposure [56] indicated that
geranium stripping oil was highly toxic
(LC50 =1310.4 mg/L), in agreement with our findings. against adult S. oryzae. after 24 h of exposure
(LC50 = 1310.4 mg/L), in agreement with our findings.
The ethanolic extract of P. graveolens significantly affected the survival of adult S. ory-
The ethanolic extract of P. graveolens significantly affected the survival of adult S. oryzae.
zae. In the treated batches, weevil survival ranged from one to eleven days, whereas in the
In the treated batches, weevil survival ranged from one to eleven days, whereas in the
control batch, this parameter fluctuated between two and fifteen days. The toxicity of the
control batch, this parameter fluctuated between two and fifteen days. The toxicity of the
ethanolic extract depended on the concentration and duration of exposure (Figure 4). The
ethanolic extract depended on the concentration and duration of exposure (Figure 4). The
TL50 and TL99 were negatively correlated with the concentrations tested (Table 5).
TL50 and TL99 were negatively correlated with the concentrations tested (Table 5).

Witness(a) Dn/2(b) Dn(b) 2Dn(b) 4Dn(b)

1
Survival probability (%)

0.8
0.6
0.4
0.2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Days after treatment
Figure 4. Survival of adult Sitophilus oryzae treated with the ethanolic extract of Pelargonium graveolens.
Figure 4. Survival
Survivors with of
theadult
sameSitophilus oryzae
lower-case treated
letter with
did not the statistically
differ ethanolic extract of Pelargonium
from one graveo-test
another (Scheffé
lens. Survivors with the same lower-case letter
p ≤ 0.05), while the others were different. did not differ statistically from one another (Scheffé
test p ≤ 0.05), while the others were different.
Table 5. TL50 and TL99 of Sitophilus oryzae adults exposed to Pelargonium graveolens ethanolic extract.
Table 5. TL50 and TL99 of Sitophilus oryzae adults exposed to Pelargonium graveolens ethanolic extract.
Concentrations
Concentrations TL r > r (0.05; 2) TL r > r (0.05; 2)
(g/50 Seeds) TL50 50 r > r (0.05; 2) TL99 99 r > r (0.05; 2)
(g/50 Seeds)
0 7.71 13.65
0 7.71 13.65
Dn/2 5.53 −0.99 11.05 −0.99
Dn/2Dn 5.535.05 −0.99 11.056.30 −0.99
Dn2 Dn 5.054.62 6.305.36
2 Dn4 Dn 4.624.15 5.368.21
4 Dn 4.15 8.21
The toxicity parameters of the ethanolic extract of P. graveolens are summarized in
The6.toxicity
Table parameters
The calculated of concentrations
lethal the ethanolic extract
LC50 andof P.LCgraveolens are summarized
99 reveal that in
adults of S. oryzae
Table
are 6. Thesensitive
very calculated lethal
to this concentrations
extract. The extremeLC50values
and LC reveal
of99LC that
50 and LCadults
99 varyofaccording
S. oryzae to
arethe
very sensitive
duration to this extract.
of exposure (TableThe
6). extreme values of LC50 and LC99 vary according to
the duration of exposure (Table 6).
Molecules 2024, 29, x FOR PEER REVIEW 10 of 17

Molecules 2024, 29, 4036 10 of 17

Table 6. Toxicity parameters of ethanolic extract of Pelargonium graveolens on Sitophilus oryzae.

LC50 (g/50 Seeds) (2) LC99 (g/50 Seeds) (2)

Days after χ2 Calculated
Slopeof±SE (1) [Confidence [Confidence
6. Toxicity parameters
TableTreatment ethanolic<χextract 2)Pelargonium
2 (0.05;of = 5.991 graveolens on Sitophilus oryzae.
Interval] Interval]
Days after (1) χ2 Calculated LC50 (g/50 Seeds) (2)
10.63 Seeds) (2)
LC99 (g/50329.52
Treatment1 Slope ±SE 1.56 ±<χ
0.71
2 (0.05; 2) = 5.9910.33 [Confidence Interval] [Confidence Interval]
[5.09; 922656.81] [109.33;1312.13]
1 1.56 ± 0.71 0.33 10.63 1.93 329.52
62.13
6 1.54 ± 0.77 0.23 [5.09; 922656.81] [109.33; 1312.13]
[0.00; 4.12] [53.35; 171.002]
6 1.54 ± 0.77 0.23 1.93 62.13
[0.00; 4.12] 1.38 7.507
[53.35; 171.002]
8 3.17 ± 1.20 2.01
1.38 [0.20; 2.13] [41.22;
7.507 108.74]
8 3.17 ± 1.20 2.01 [0.20; 2.13] 0.82 [41.22; 108.74]
3.86
9 3.47 ± 1.44 1.31 0.82 [0.03 ; 1.33] 3.86 59.75]
9 3.47 ± 1.44 1.31 [27.78;
[0.03; 1.33] [27.78; 59.75]
(1) SE: Standard Error; (2) LC50 and LC99: Lethal concentrations, respectively, for 50% and 99% of the
(1) SE: Standard Error; (2) LC and LC : Lethal concentrations, respectively, for 50% and 99% of the individu-
50 99
individuals
als used. used.

The
Theaqueous extractofofP.P.graveolens
aqueousextract significantly
graveolens affected
significantly affectedthethe
survival of adult
survival S. oryzae.
of adult S. ory-
In
zae. In the treated batches, weevil survival ranged from one to eleven days, whereasininthe
the treated batches, weevil survival ranged from one to eleven days, whereas the
control
controlbatch,
batch,this
thisparameter
parameterfluctuated
fluctuatedbetween
betweentwotwoand
andfifteen
fifteendays.
days.The
Thetoxicity
toxicityof
ofthe
the
aqueous
aqueousextract ofP.P.graveolens
extractof graveolensdepended
dependedon onthe
theconcentration
concentrationandandduration
durationofofexposure
exposure
(Figure
(Figure5).5).The
TheTL
TL5050 and
and TL
TL99 were negatively correlated with the concentrations tested
99 were negatively correlated with the concentrations tested
(Table 7).
(Table 7).

Witness (a) Dn/2 (b) Dn (b) 2Dn (b) 4Dn (b)

1
Survival probability (%)

0.8

0.6

0.4

0.2

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Days after treatment
Figure5.5.Survival
Figure Survivalofofadult
adultSitophilus
Sitophilus oryzae
oryzae treated
treated with
with thethe aqueous
aqueous extract
extract of Pelargonium
of Pelargonium graveo-
graveolens.
lens. Survivors with the same lower-case letter did not differ statistically from one another (Scheffé
Survivors with the same lower-case letter did not differ statistically from one another (Scheffé test
test p ≤ 0.05), while the others were different.
p ≤ 0.05), while the others were different.
Table 7. TL50 and TL99 of Sitophilus oryzae adults exposed to Pelargonium graveolens aqueous extract.
Table 7. TL50 and TL99 of Sitophilus oryzae adults exposed to Pelargonium graveolens aqueous extract.
Concentrations
Concentrations TL50 r > r (0.05; 2) TL99 r > r (0.05; 2)
(g/50 Seeds) TL50 r > r (0.05; 2) TL99 r > r (0.05; 2)
(g/50 Seeds)
0 7.53 14.90
0 7.53 14.90
Dn/2 5.24 −0.99 10.38 −0.99
Dn/2 5.24 −0.99 10.38 −0.99
Dn
Dn 4.90
4.90 9.96
9.96
2
2 DnDn 4.47
4.47 8.85
8.85
44DnDn 4.24
4.24 8.39
8.39

The toxicity
The toxicity parameters
parameters of of the
the aqueous
aqueous extract
extract of
of P.P.graveolens
graveolensare
aresummarized
summarized in in
Table 8. The calculated lethal concentrations LC 50 and LC 99 reveal that adults
Table 8. The calculated lethal concentrations LC50 and LC99 reveal that adults of S. oryzaeof S. oryzae
are more
are more sensitive
sensitive to
to this
this aqueous
aqueous extract.
extract. The
Theextreme
extremevalues
valuesofofLCLC50 and LC99 vary ac-
50 and LC99 vary
cording totothe
according theduration
durationofofexposure.
exposure.
Molecules 2024, 29, 4036 11 of 17

Table 8. Toxicity parameters of the aqueous extract of Pelargonium graveolens on Sitophilus oryzae.

Days after χ2 Calculated LC50 (g/50 Seeds) (2) LC99 (g/50 Seeds) (2)
Slope ±SE (1) [Confidence [Confidence
Treatment <χ2 (0.05; 2) = 5.991 Interval] Interval]

1 1.89 ± 0.86 0.80 11.34 192.63

[5.09; 922,656.81] [109.33; 1312.13]

2 1.83 ± 0.83 0.68 6.96 129.83

[0.00; 4.12] [53.35; 171.002]

3 1.50 ± 0.76 0.18 6.30 221.01

[0.20; 2.13] [41.22; 108.74]

8 2.20 ± 1.01 0.64 1.16 13.21

[0.03; 1.33] [27.78; 59.75]
(1)SE: Standard Error; (2) LC50 and LC99 : Lethal concentrations, respectively, for 50% and 99% of the individu-
als used.

Overall, the obtained results highlighted the strongest toxicity against S. oryzae for P.
graveolens EO. The strong insecticidal action of EO could depend on the presence of some
components contained in high amounts such as monoterpenoids [25]. These compounds
are severely poisonous to insects and have repellent and antifeedant qualities; for this
reason, they have been explored as possible pest control agents [57]. In particular, this effect
could depend mainly on geraniol, citronellol, and linalool detected in great concentrations
in the EO and whose toxicity against rice weevils has been demonstrated [56]. The findings
of the present study indicate that this EO can provide an alternative source of insect control
agents because it contains a range of bioactive chemicals, most of which are selective and
have little or no harmful effect on the environment and non-target organisms including
humans. EO-based formulations can be used as alternative tools in stored grain insect
management [58].
Interestingly, even the ethanolic and the aqueous extracts, rich in phenolics, exhibited
toxicity against rice weevils, with the former being more active than the latter. The effects
of plant extracts and their active constituents, including flavonoids and phenolic acids,
against stored product insect pests have been previously reported; indeed, several phenolic
compounds were found to possess insecticidal activity against S. oryzae [59,60]. As far
as we know, there are no data in the previous literature on the insecticidal activity of P.
graveolens extracts against S. oryzae.

3. Materials and Methods

3.1. Plant Material and Extraction Procedure
The aerial parts of P. graveolens were harvested in May 2020 in the ksar Tizgaghine,
20 km from Tinjdad, in the region of Er-Rachidia, Morocco (31◦ 55′ 55′′ N, 4◦ 25′ 28′′ W). The
plant was identified and confirmed by Professor Benkhnigue Ouafae at the Botanics and
Plant Ecology Department of the Scientific Institute of Rabat, Morocco. The plant was
deposited in the herbarium under the voucher number RAB 114766. The plant material
was dried in a dry ventilated place for one month, then ground with an electric mill and
kept in the shade in closed premises. A total of 30 g of powdered plant material was put
in a cartridge and extracted with 250 mL of extraction solvent (ethanol or water) using a
Soxhlet extractor for 6 h. Then, the solvent was evaporated using a rotary evaporator. The
extraction yield of ethanolic and aqueous extracts was 18.26 and 22.25%, respectively.
The essential oil was extracted by hydro-distillation; 100 g of dry plant material was
placed in 1.5 L of distilled water heated to 100 ◦ C in a Clevenger-type apparatus. Distillation
was performed for three hours after the first drop of distillate had been collected. The
essential oil was dried with anhydrous sodium sulfate and stored at +4 ◦ C in the dark. The
extraction yield of the essential oil was 0.21%.

3.2. Phytochemical Investigations

3.2.1. Determination of Total Phenolic Content
The total phenolic content of the aqueous and ethanolic extracts was determined by
the Folin–Ciocâlteu colorimetric method as previously reported [61]. The results were
Molecules 2024, 29, 4036 12 of 17

obtained from the average of three independent determinations and expressed as mg gallic
acid equivalent (GAE)/g extract (dw) ± standard deviation (SD).

3.2.2. Phenolic Compounds Analysis by HPLC-PDA/ESI-MS

Analysis of phenolic compounds of the aqueous and ethanolic extracts was performed
using high-performance liquid chromatography coupled with a photodiode array detector
and electrospray ionization mass spectrometry (HPLC-PDA/ESI-MS) (Shimadzu, Kyoto,
Japan). Chromatographic separation was carried out on an Ascentis Express C18 column
(150 × 2.1 mm, 2.7 µm; Merck Life Science, Merck KGaA, Darmstadt, Germany) using, as
the mobile phase, 0.1 % (v/v) acid formic in water (mobile phase A) and 0.1 % (v/v) acid
formic in acetonitrile (mobile phase B). The gradient elution applied was: 0 min (0 % B),
10 min (10 % B), 20 min (11 % B), 30 min (15 % B), 50 min (18 % B), 65 min (23 % B), 70 min
(100 % B), and 75 min (100 % B) at a flow rate of 0.5 mL/min. The column temperature
was 30 ◦ C and the injection volume was 2 µL. UV detection wavelengths were in the
range of λ =190–400 nm. Positive and negative ion mass spectra were set as follows: scan
range: m/z 100–800, nebulizing gas (N2 ) flow rate: 0.5 mL/min, drying gas (N2 ) flow
rate: 15 L/min, interface temperature: 350 ◦ C. LabSolutions software ver. 5.92 (Shimadzu,
Kyoto, Japan) was used to control the LC-PDA-ESI-MS system and for data processing.
The identification of phenolic compounds was made by comparison of retention times
and UV–visible and mass spectra, and with co-standard injection data and data from the
literature when available.

3.3. Antioxidant Activity

3.3.1. DPPH Test
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used to determine the free radical
scavenging activity of P. graveolens extracts and EO, according to the method of Ohnishi
et al. [62], using butylated hydroxytoluene (BHT) as the reference standard. The results
were obtained from the average of three independent experiments, and are reported
as mean radical scavenging activity (%) ± SD and mean 50% inhibitory concentration
(IC50 ) ± SD.

3.3.2. Reducing Power Assay

The reducing power of P. graveolens extracts and EO was determined using the Fe3+ -
Fe2+ transformation method, according to the protocol of Oyaizu [63], using Ascorbic acid
and BHT as reference standards. The results were obtained from the average of three
independent experiments, and are expressed as mean absorbance values ± SD and ascorbic
acid equivalent/mL (ASE/mL) ± SD.

3.3.3. Ferrous Ions (Fe2+ ) Chelating Activity Assay

The chelating activity of P. graveolens extracts and EO was measured by evaluating
their ability to inhibit the formation of the Fe2+ -ferrozine complex, according to the method
previously reported by Kumar et al. [64]. The results, obtained from the average of three
independent experiments, are reported as the mean inhibition of ferrozine–(Fe2+ ) complex
formation (%) ± SD and IC50 ± SD.

3.4. Insecticidal Activity

3.4.1. Sitophilus oryzae Strain
The insects were derived from a strain isolated from wheat grains infested with
S. oryzae. The grains were collected from a farmer in the Meknes region. The strain was
grown in the laboratory in a ventilated room at 25–28 ◦ C and 70% humidity. Mass rearing
was carried out in glass jars with mesh lids, filled with durum wheat grains, to which a
sufficient number of S. oryzae insects of undetermined sex were added. The pots were then
left at room temperature. After one or two weeks of infestation, the adults were removed
from the grains.
Molecules 2024, 29, 4036 13 of 17

3.4.2. Effect of the Essential Oil on Adult Sitophilus oryzae

Pelargonium graveolens EO oil fumigant was used in 2.5 L hermetically sealed transpar-
ent plastic boxes as an exposure chamber to test the essential oil’s toxicity against adult S.
oryzae, using a modified version of the techniques outlined by El Idrissi et al. (2014) [65].
Five Petri dishes are placed in each box (ensuring five repetitions). Each repetition consists
of ten S. oryzae adults. Five Petri dishes were placed, each replicate consisting of ten S.
oryzae adults. The tests were carried out under rearing conditions. The EO was spread on
Whatman-type filter paper, which was placed inside the exposure chamber. Four doses
were applied: 4 µL, 8 µL, 12 µL, and 16 µL, and an untreated batch was used as a con-
trol. Mortality was monitored by counting dead insects from the first day of treatment
until death.

3.4.3. Effect of Ethanolic and Aqueous Extracts on Adult Sitophilus oryzae

The method outlined by Riffi et al. (2019) [66] was used to assess the fumigant effect of
P. graveolens extracts against adult S. oryzae. Ten wheat burrows were introduced into Petri
dishes containing 50 durum wheat seeds mixed with the ethanolic and aqueous extracts of
the aerial part of P. graveolens at five selected doses (0; Dn/2; Dn; 2Dn; and 4Dn), either an
extract weight of 0 g, 0.0078 g, 0.0156 g, 0.0321 g, and 0.0624 g, respectively, for the ethanolic
extract or an extract weight of 0 g, 0.0127 g, 0.0254 g, 0.0508 g, and 0.1017 g, respectively,
for the aqueous extract. The tests were carried out under the conditions of breeding for
S. oryzae. Mortality control was done by enumerating dead insects from the first day of
treatment to the death of all individuals. For each dose, the experiments were repeated
three times.

3.4.4. Data Analysis

The LC50 and LC99 were determined using the Finney probit method [67]. Mortalities
were corrected using Abbott’s formula [68]. The lethal times required for the death of 50%
(TL50 ) and 99% (TL99 ) of adults exposed to different concentrations of the essential oil and
extracts were estimated.

3.5. Statistical Analysis

Statistical analysis of data regarding the antioxidant activity was carried out by using
one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple compar-
isons tests; conversely, the t-test was employed for total phenolic content data handling
(GraphPad Prism Software for Science or Statistica 13.3 (TIBCO Software Co., Palo Alto,
CA, USA)). In all the selected tests, p-values lower than 0.0001 were considered statistically
significant. To compare the effects of the essential oil and the extracts tested on insecticidal
activity, analysis of variance (ANOVA) followed by the 5% Scheffé test was performed
using Excel version 2010 software.

4. Conclusions
In this contribution, the essential oil and the extracts (aqueous and ethanolic) obtained
from the aerial parts of Pelargonium graveolens grown in Er-Rachidia, Morocco, have been
assayed for their in vitro antioxidant activity and insecticidal properties against the rice
weevil (Sitophilus oryzae), one of the most destructive pests of stored cereals and processed
cereal products worldwide.
The results of the antioxidant tests showed the best activity for the ethanolic extract,
followed by the aqueous one, whereas EO exhibited weak antioxidant properties, indicating
that phenolic compounds play a major role in the observed effects. A thorough characteriza-
tion of the phenolic profile of the aqueous and ethanolic extracts has been performed, which
revealed quite a complex and different pattern, including phenolic acids and flavonoids.
Differently, the essential oil displayed the strongest toxicity against S. oryzae, which could
depend mainly on the presence of some monoterpenoids in high amounts. Notably, even
Molecules 2024, 29, 4036 14 of 17

the ethanolic and the aqueous extracts exhibited toxicity against rice weevils, with the
former being more active than the latter, which could be related to phenolic compounds.
Based on the remarkable results achieved for antioxidant and insecticidal activity,
the aerial parts of P. graveolens could be considered as an alternative source of bioactive
compounds to be advantageously employed as botanical insecticides against several stored
and processed product insect pests.

Author Contributions: Conceptualization, Z.M. and N.M.; Data curation, Z.M., F.D., M.E., A.A.,
F.C., R.L.V., L.M., N.M. and M.F.T.; Investigation, Z.M., F.D., F.M., F.C. and R.L.V.; Supervision, N.M.;
Writing—original draft, Z.M., F.D., F.M., F.C., R.L.V. and N.M.; Writing—review & editing, L.M., N.M.
and M.F.T. 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: The data presented in this study are available on request from
the authors.
Acknowledgments: Federica Davì thanks the “Prof. Antonio Imbesi” Foundation for the fellowship.
The authors are grateful to Shimadzu and Merck Life Science Corporations for the continuous support.
The authors are grateful to Benkhnigue Ouafae of the Botanics and Plant Ecology Department of the
Scientific Institute of Rabat, Morocco for the identification of the Plant.
Conflicts of Interest: The authors declare no conflicts of interest.

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