Chemical Composition, Antioxidant, Insecticidal Activity, and Comparative Analysis of Essential Oils of Leaves and Fruits of Schinus molle and Schinus terebinthifolius

Schinus terebinthifolius Raddi. and Schinus molle L. are perennial woody plants belonging to the Anacardiaceae family, widely distributed in the United States, Europe, Asia, and Africa, and they are broadly used for many applications such as in traditional medicine as an antipyretic, analgesic, depurative, and in the treatment of diseases of the urogenital system as well as culinary and ornamental species. Our work aims to study and compare the chemical composition and the antioxidant and insecticidal activity of the essential oils of the leaves and fruits of the two species of the genus Schinus. The essential oils were characterized by a very spicy aromatic odor, and by the abundance of hydrocarbon monoterpenes in the leaves and fruits of Schinus molle (49.70% and 42.65%), unlike the EOs of the fruits of Schinus terebinthifolius which have a high content in hydrocarbon sesquiterpenes (40.57%). Usually, these oils have shown relatively low antioxidant activity was expressed in IC50; especially, the essential oil of the fruits of S. terebinthifolius revealed a concentration of 3.292 ± 2.82 mg/mL. The evaluation of the insecticidal activity gave good results in the method of exposure of adults of Sitophilus oryzae to EOs by contact; thus, the fruits of Schinus molle are more active against this species of beetle than the other essential oil.


Introduction
e family Anacardiaceae has about 60 to 74 genera and 400 to 600 species. e Anacardiaceae consists of trees, shrubs, lianas, or rarely perennial herbs and tissues of plant organs with resinous conduits or laticifers [1,2]. e genus Schinus has about 30 species, most of which are found in a spontaneous state in the South American region [3]. Schinus molle L. and Schinus terebinthifolius Raddi. are known by Mediterranean cuisine [7]. Leaves composed of 3 to 10 pairs of leaflets, with serrated edges. When it blooms, from September to January, its small aromatic flowers, light yellow in color, attract bees. e flowers are unisexual, hermaphrodite, and arranged in elongated panicles. e fruits are globular drupes of 4 to 6 mm of diameter, round, red, and brilliant and gathered in hanging clusters. Schinus molle and S. terebinthifolius were used to treat urinary tract infections, skin ulcers, gastroduodenal disorders, and ulcers and infections of the respiratory, digestive, and genitourinary systems. Schinus molle is also taken as a diuretic and a digestive tonic for cleansing the system. It presents red and edible pulp [8]. e essential oil (EO) of leaves and fruits of Brazilian S. molle contains as major compounds sabinene (48.63% and 51.74%) and limonene (10.20% and 16.98%), respectively, while bicyclogermacrene (18.12%) was also important in the leaf oil [9]. Previous studies have reported that the EOs of S. molle. are characterized by their antioxidant, antimicrobial [10][11][12][13][14], cytotoxicity, and insecticidal proprieties [3,[14][15][16].
Schinus terebinthifolius Raddi (S. terebinthifolius), called "aroeira-vermelha," "aroeira-pimenteira," "Brazilian pepper," Christmas berry, pink pepper [5], is a dioecious tree of medium size, with a short trunk, generally hidden by interlaced branches. It reaches a height of 5 to 10 meters. Its leaves are dark green and oblong to elliptical. e white or yellow flowers are small and grouped in bouquet-like inflorescences. e bark of the Schinus has an action against fever, hemoptysis, and uterine disorders in general. From the shell, oil is used to treat tumors and diseases of the cornea [17]. S. terebinthifolius was also widely used in Brazilian traditional medicine for many purposes as an anti-inflammatory and healing and to treat some respiratory diseases [6]. In fact, some recent in vitro and in vivo studies [18] have reported numerous biological activities of S. terebinthifolius extracts such as antioxidant, antibacterial, and antifungal [19,20]. e essential oils of the leaves and fruits are rich in limonene (14.21-16.99%), germacrene D (11.45-10.85%), and cadinene (9.21-3.31%), respectively [17].
Sitophilus oryzae L. (S. oryzae) (Coleoptera: Curculionidae), a ubiquitous pest of economic importance, is an internal feeding insect that burrows into the stored grain. Adult weevils feed primarily on endosperm, which reduces carbohydrate content, and the larvae preferentially feed on the germ of the grain, removing much of the protein and vitamins. Insects that selectively attack the germ will cause greater loss of germination than others [21]. e aim of this study was to evaluate the chemical composition of S. molle and S. terebinthifolius leaf and fruit essential oils by GC-MS, antioxidant properties by DPPH free radical scavenging method, and insecticidal activity by contact, repellency, and fumigation against wheat weevil (S. oryzae).

Preparation of Plant.
e leaves and fruits of both species of Schinus were collected from the region of Rabat (34°01′53.34″) in the month of February-March. e confirmation of both species was carried out by Prof. El Aboudi Ahmed, a botanist at the department of Biology, Faculty of Sciences Mohammed V University. e two parts of the plants were separated and dried away from humidity after two weeks and then crushed in order to perform the extraction.

Extraction of Essential
Oil. e extraction of essential oils by hydrodistillation of S. molle and S. terebinthifolius was achieved using a Clevenger-type instrument [22]. First, 100 g of crushed leaves or fruits of S. molle and S. Terebinthifolius was placed in a 1000 ml flask at a plant material/water ratio of 1 : 3. e flask was heated for 6 h. e extraction was continued until no essential oil was collected. e resulting mixture of water and essential oils was then dehydrated and stored in dark bottles.

Chemical Analysis of Essential Oils by GC-MS.
Analyses were carried out using a Perkin-Elmer Clarus 690 (Waltham, MA, USA) equipped with dual flame ionisation detection (FID) system and fused-silica capillary columns, namely, Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethyleneglycol) (60 m × 0.25 mm i. d; film thickness 0.25 μm). e oven temperature was programmed from 60 to 230°C at 2°C/min and then held isothermally at 230°C for 30 min: hydrogen was employed as carrier gas (0.7 mL/min). e injector and detector temperatures were maintained at 280°C, and samples were injected (0.5 μL HE diluted in ethanol grade FID) in the split mode (1/80). Retention indices (RI) of compounds were determined relative to the retention times of a series of n-alkanes (C5C30) by linear interpolation using the Van den Dool and Kratz equation with the aid of software from Perkin-Elmer (Total Chrom navigator). e relative percentages of the oil constituents were calculated from the GC peak areas, without the application of correction factors.
Samples were also analysed with a gas chromatograph of the Hewlett-Packard type (HP 6890 series) coupled with a mass spectrometer (HP 5973 series). Fragmentation is carried out by electronic impact under a field of 70 eV. e column used is an HP-5 MS capillary column (30 m x 0.25 mm), and the film thickness is 0.25 μm. e temperature of the column is programmed from 50 to 250°C at a rate of 4°C/min. e carrier gas is helium, the flow rate of which is set at 1.5 mL/ min. e injection mode is split (leakage ratio: 1/70, flow rate 112 mL/min). e device is connected to a computer system managing a NIST 11 mass spectrum library.

Evaluation of Antioxidant Activity.
e antioxidant power of the essential oils of the four samples was tested by the DPPH (2,2-diphenyl picryl-hydrazyl) as a relatively stable free radical method [23]. e reaction is achieved in a total volume of 2.5 ml containing 0.5 ml of DPPH at 0.2 mM solubilized in methanol. e essential oil samples were prepared by dissolving in absolute methanol at 20 mg/ml, and these solutions will then be diluted to obtain the final concentrations of 15%, 17

Assessment of Insecticidal Activity by
Contact. e contact toxicity of extracted EOs for adults of S. Oryzae was evaluated in acetone to obtain the required concentrations. About 25 to 200 μL of EO was applied to the surface. e oil + acetone solution is uniformly distributed on a disc of filter paper, and a control receives 200 μL of acetone only. Four adults of S. Oryzae, individuals are placed on the filter paper in the middle of the Petri dishes (5.5 cm diameter × 1.5 cm). Mortality control was carried out by counting dead insects from the first day of treatment until the death of all individuals [25].

Assessment of Insecticidal Activity by Fumigation.
e fumigant effect of EOs was determined according to the method of Khani and Asghari [26], with some modifications. Four adults of S. Oryzae were placed in 50 mL plastic bottles. 5, 25, 50, 100, and 150 μL of EO were added to individual 3 cm diameter filter paper. e filter paper was then attached to the undersurface of the cap firmly attached to the vial to produce concentrations of 0.1, 0.5, 1, 2, and 3 μL/L air, respectively. e control vial did not receive any trace of oil. Insect mortality was determined after 24, 48, 72, 96, 120, 144, 168, and 192 hours of exposure. Insects were considered dead when they could no longer move parts of their bodies.

Repellent Activity.
e study of the repulsive power of the EOs was achieved by calculating the percentage of repulsion of the studied EOs towards S. Oryzae by the preferential area method on filter paper described by Jilani and Saxena [27]. Filter paper discs of 11 cm diameter are cut into two equal parts; one half of the paper is treated with EOs plus acetone, and the other half is treated with acetone only. 25, 50, 100, 150, and 200 μL of essential oil are diluted, respectively, in 200 μL of acetone so that the distribution is homogeneous on the filter paper. Four insects were introduced in the middle of the Petri dishes. e solution (oil + acetone) is evenly distributed on one half of the filter paper disc, and the other half of the disc receives acetone only. e two half discs of filter paper are air-dried, and the disc is reconstituted and put in a Petri dish. After half an hour of treatment of the insects under laboratory conditions, the count of the insects on the half disks is achieved. e percentage of repulsion (PR) is thus calculated according to the formula used by Nerio et al. [28] as follows: (i) N ac : Number of insects present on the half disc treated with acetone, (ii) N EO: Number of insects present on the half disc treated with the oil solution.

Essential Oil Yield.
e essential oil obtained from S. molle is pale yellow color contrary to the oil of S. terebinthifolius which is of greenish yellow color, with a very spicy peppery aromatic smell in both species. e yield of essential oil from the leaves and fruits of each of the two species is expressed in percentage and is presented in Table 1. e determination of the yield according to the used parts of the plant detected a difference in yield between the leaves and fruits of S. terebinthifolius. Otherwise, the determination of the yield according to the selected plant revealed that S. molle is richer in essential oil than S. terebinthifolius.
A study carried out by Santos et al. [9] on the extraction of Brazilian S. molle leaves and fruits, and S. terebinthifolius leaves and fruits by a Clevenger apparatus and during 1 h of distillation, gave a yield of 1.24% and 0.49% for the leaves and fruits, respectively, for S.M., while for S.T., the yield is 0.74% and 0.16%, respectively.
In 2012, Santana et al. [29] found that the EO content of S. terebinthifolius leaves is around 0.17%. In Costa Rica, Martins et al. [30] reported that S. molle leaves are very rich in EO than fruits, with a content of 1.09% and 0.91%, respectively. Dos Santos Cavalcanti et al. [31] achieved the extraction of 80 g each of the leaves and fruits of S. molle and S. terebinthifolius by hydrodistillation for 6 h and found that the EO content is very low in the leaves of S. terebinthifolius of about 0.10%, while the content of fruits is 1.74% and the Evidence-Based Complementary and Alternative Medicine

Chemical
Composition. GC-MS analysis showed a difference in the chemical composition of the EOs of the two species studied. A total of 46 and 44 components were identified in S. molle and S. terebinthifolius representing, respectively, for leaves-Fruits (91%-92%) and (89%-84%) of the total content given in Table 2. e EO of S. molle is characterized by the presence of c-muurolene, c-gurjunene, c-cadinene, 10-epi-elemol, guaiol, and α-acorenol, while that of S. terebinthifolius can be dis-  Previous studies that have been achieved on the EOs of both species stated that these oils are rich in monoterpene compounds, which is compatible with our results, but the difference lies in the dominant monoterpene compounds which differ from one study to another. However, Bendaoud et al. [32] stated that the main difference between the essential oils of the genus Schinus is the high content of sesquiterpenes in the fruits of S. terebinthifolius which is similar to our results. e chemical composition of essential oils of S. molle marks a difference with that collected in Tunisia, and this fruit oil is characterized by a high percentage of hydrocarbon monoterpenes 79.69% and 11.75% of oxygenated monoterpenes with only 3.69% of hydrocarbon sesquiterpenes and 1.83% oxygenated sesquiterpenes. Specifically, this oil showed high content of α-phellandrene (46.52%) and β-phellandrene (20.81%) [32]. Eryigit et al. [33] found that S. molle fruits grown in Turkey yield EO rich in α and β-phellandrene (31.74 and 16.49%) and p-cymene (11.36%). Also, Aboalhaija et al. [34] compared the EOs of leaves and fruits from different regions of Jordan and marked that stereoisomers α-and β-phellandrene represent the major components of the EOs studied, while the essential oil of S. molle fruits grown in Mexico gave a high percentage of myrcene (39.7), p-cymene (19.5%), cadinene (7.8%), and phellandrene (7.1%) [35].

Antioxidant Activity.
In vitro antioxidant activity test was carried out by varying the concentration of the methanolic extracts of essential oils studied by calculating the inhibitor percentage for each concentration from the absorbance obtained by spectrometry at 517 nm. e results are grouped into two graphs (Figures 3(a) and 3(b)), one to compare the percentages of inhibition of EOs of the fruits and the other of the leaves of both species. e evaluation of the antioxidant activity of the EO of leaves and fruits (5 mg/mL) revealed a percentage of radical inhibition of 17.99% and 53.30% in S. molle and 49.31% and 77.82% in S. terebinthifolius, respectively, which shows that the fruits exhibited higher antioxidant activity than the leaves. e antioxidant effect of the different EOs was expressed in IC 50 , which represents the 50% inhibitory concentration of the radicals with reference to ascorbic acid (Table 3). e calculation of the IC 50 of the 4 oils studied showed that they have a rather low antioxidant activity compared to the reference antioxidant. However, comparing the IC 50 of the essential oils of leaves and fruits, we find that fruits have a higher activity than leaves, and this may be due to the richness of the berries by sesquiterpene compounds  (hydrocarbon and oxygenated). However, the essential oil of the fruits of S. terebinthifolius revealed a concentration of 3.292 ± 2.82 mg/mL higher than that of ascorbic acid (1.91 μg/mL) and lower than those of other EOs, which proves that S. terebinthifolius fruits have a stronger antioxidant activity than S. molle fruits, and this can be attributed to the presence of globulol characterizing the fruits of S. terebinthifolius.
In the literature, the essential oils of S. molle leaves and fruits have reported low antioxidant activity [30,38,39]. ese authors correlated their results with the small percentage of oxygenated terpene.
In summary, the chemical composition of EOs is responsible for this low antioxidant activity due to the fact that monoterpenes present the majority of the components of these EOs. is has been reported by several authors [40][41][42][43], who stated that essential oils rich in monoterpenes such as α-pinene, β-pinene, limonene, β-myrcene, sabinene, and terpinolene did not show strong antioxidant activities.

Assessment of Insecticidal Activity by Contact.
Evaluation of contact insecticidal activity showed that insect mortality increased with increasing concentrations of  ). e lowest mortality values are recorded in the control boxes; they are 25% after 7 days of exposure. e EOs used at doses of 200, 150, and 100 μL/cm 2 showed higher mortality rates and even from the first day in the fruit EOs of the two species studied. e essential oil of S. molle fruits reported 100% mortality against S. oryzae at 25 μL/cm 2 concentration after 3 days of exposure, while for leaves, this mortality value was marked after 7 days of exposure for the same concentration.
us, Petri dishes of 200 μL/cm 2 concentration recorded a mortality rate of 100% after the first and second day of exposure for fruits and leaves, respectively.
For the essential oil of S. terebinthifolius, a mortality rate of 100% was reached at a concentration of 25 μL/cm 2 after 8 days of exposure for fruits and leaves. While at a concentration of 200 μL/cm 2 , the boxes record the total mortality of the insects after 1 and 2 days of exposure to the essential oils of fruits and leaves, respectively. e evaluation of the insecticidal activity of the EO of the leaves and fruits of the two species showed a significant difference based on the calculation of the lethal dose 50 (LD 50 ) which represents the concentration marking 50% of the mortality of insects (Table 4). e lethal dose 50 (LD 50 ) was determined graphically from the plots representing the mortality rates after 24 h of exposure of insects to essential oils as a function of concentrations and is presented in Table 4.
In the light of these results, the EO of the fruits of S. molle has a higher insecticidal activity due to the fact that its lethal dose (50 μL/cm 2 ) is lower than that of the leaves contrary to S. terebinthifolius which showed a lower lethal dose in leaves (83.33 μL/cm 2 ) than in fruits, while Mohamed and Abdelgaleil [44] found that the essential oil of S. terebinthifolius leaves used for contact treatment on S. oryzae marks an LD 50 � 0.42 mg/cm 2 . e analysis of the insecticidal effect of the EOs of the false pepper tree revealed significant differences for the factors oils, doses, and duration of exposure, which made it possible to classify the oils used according to their toxicities. e DL 50 decreases with increasing duration of EO exposure, and we find that SMF is the most toxic ( Figure 5). e essential oil of S. molle fruits represents 50% mortality at a concentration of 50 μL/cm 2 , and this may be due to its special chemical composition characterized by the presence of β-linalool (0.23%), thujone (0.34%), and ledol (0.36%). However, Mohamed and Abdelgaleil [44] stated that alcoholic monoterpenes such as geraniol, linalool, and menthol have a pronounced activity on S. oryzae. Park et al. [45] stated that the essential oil of Chamaecyparis obtusa, in a toxicity study against adults of S. oryzae exposed to direct contact, gave 80% mortality at 0.26 mg/cm 2 due to the presence of bornyl acetate and terpinolene.

Assessment of Insecticidal Activity by Fumigation.
Assessment of the insecticidal activity of S. molle and S. terebinthifolius essential oils by fumigation on S. oryzae adults showed that these oils are less effective against these insects by comparing their contact activities ( Figure 6). In the treated batches, Weevil death ranged from 2 to 7 days, whereas in the control batch, this parameter ranged over 7 days. us, the toxicity of EOs depends on the dose and duration of exposure. e death of insects increases as the dose and the exposure time increase. e batches of 5 μL/L of air recorded a mortality rate of 75% and 50% after 8 days of exposure to the essential oils of the fruits and leaves, respectively, of S. molle, while the batches of 150 μL/L of air marked a mortality rate of 100% after 5 and 4 days of exposure to the EOs of the fruits and leaves of SM (Figures 6(a) and 6(b)). e batches of 5 μL/L of air did not record any mortality after 8 days of inhalation of essential oils of the leaves and fruits of S.T. which explains the low activity of this plant on S. oryzae, while the batches of 150 μL/L of air marked a mortality rate of 100% and 75% after 7 days of inhalation with essential oils of fruits and leaves, respectively, of Aroeira (Figures 6(c) and 6(d)).
Mohamed and Abdelgaleil [44] tested the fumigant effect of S. terebinthifolius and revealed LC 50 � 56, 48 μL/L air after 72 h of exposure, which reveals that it has a moderate fumigant activity compared to Mentha microphylla which is very strong. Sahaf et al. [46] found that the highest concentration of Vitex pseudo-negundo oil (925.9 μL/L air) induces more than 50% mortality after 6 h and reached a level of 100% at 12 h after treatment. e lowest concentration was found to be capable of killing 50% of S. oryzae within 24 h. At 740.7 μL/L of air, complete mortality of beetles was achieved after 15 h of exposure. Moreover, Negahban et al. [47] found that the essential oil of Artemisia siberi has a good fumigant activity against S. oryzae by registering 50% mortality at a concentration of 3.86 μL/L air. Alternatively, Benzi et al. [48] tested the insecticidal activity of EOs from the leaves and fruits of S. molle on S. oryzae and reported that these did not produce a fumigant effect on S. oryzae adults during the 4 days tested, and this may have been due to the fact that the entry of these compounds is through the cuticle and has higher concentrations to achieve mortality or to a longer exposure time. According to Pérez-López et al. [35], rice weevils are sensitive to coriander essential oil with a percentage of mortality equal to 56% in 24 h.

Repellency activity.
e repellent activity of the studied EOs, after an exposure time of 30 min, is dependent on the dose deposited in the Petri dishes. is activity increases as the dose increases, and it also depends on the used part of the plant (Figure 7).
For berries, it can be seen that S. molle is highly repellent to adults of S. oryzae than S. terebinthifolius which is moderately repellent even at the highest dose. us, at the dose of 150 μL/cm 2 , both plants are moderately repellent, whereas at 100 μL/cm 2 and 50 μL/cm 2 , S. molle alone has a repellent activity.
For leaves, S. molle is more repellent than S. terebinthifolius at 200 μL/cm 2 and 150 μL/cm 2 doses, while at 100 μL/cm 2 , they have the same percentage of repellency. e calculation of the average percentage of repulsion of different doses by the method of McDonald et al. [49] shows that the EO of fruits S. molle and S. terebinthifolius and S. molle leaves is moderately repulsive and belongs to class II with percentages of 28.18%, 26.66%, and 21.52%, respectively, Evidence-Based Complementary and Alternative Medicine while that of S. terebinthifolius leaves is weakly repellent (8.56%) and belongs to class I. e probit analysis showed that S. oryzae was more sensitive (RD 50 � 0.084 μL/cm 2 ) to coriander EO than linalool standards (RD 50 � 0.206 μL/cm 2 ) according to Sriti Eljazi et al. [50]. erefore, it is important to explore the uses of medicinal plants because they are used for the treatment of different infections. World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population [51][52][53][54].

Conclusion
Loss of cereals caused by insect pests remains a problem of concern to farmers through reduction in commercial value and viability of seeds. However, the use of plants to fight against these beetles has become a famous objective. In our study, the insecticidal and antioxidant activity of EOs of the two species of the genus Schinus against S. oryzae was explored. e comparative study of these species allowed noticing the insecticidal effect of the fruits against S. oryzae, so the chemical analysis shows the presence of different constituents responsible for this insecticidal activity. However, the abundance of monoterpenes is accompanied by a weak antioxidant activity of essential oils. Finally, we can conclude that the fruits of S. molle can be used for the preservation of stored foodstuffs.

Data Availability
All the data are cited in the main text of this document.