Sesquiterpenes-Rich Essential Oil from Above Ground Parts of Pulicaria somalensis Exhibited Antioxidant Activity and Allelopathic Effect on Weeds

Pulicaria genus (fleabane) is characterized by its fragrant odor due to the presence of essential oil (EO). According to the literature reviews, the EO of Pulicaria somalensis O.Hoffm. (Shie) is still unexplored. For the first time, 71 compounds were characterized in EO derived from above-ground parts of P. somalensis collected from Saudi Arabia. Sesquiterpenes represented the main components (91.8%), along with minor amounts of mono-, diterpenes, and hydrocarbons. Juniper camphor (24.7%), α-sinensal (7.7%), 6-epi-shyobunol (6.6%), α-zingiberene (5.8%), α-bisabolol (5.3%), and T-muurolol (4.7%) were characterized as main constituents. The correlation analysis between different Pulicaria species showed that P. somalensis has a specific chemical pattern of the EO, thereby no correlation was observed with other reported Pulicaria species. The EO showed significant allelopathic activity against the weeds of Dactyloctenium aegyptium (L.) Willd. (crowfoot grass) and Bidens pilosa L. (hairy beggarticks). The IC50 value on the germination of D. aegyptium was double that of B. pilosa. The IC50 values on the root growth of B. pilosa and D. aegyptium were 0.6 mg mL−1 each, while the shoot growths were 1.0 and 0.7 mg mL−1, respectively. This variation in the activity could be attributed to the genetic characteristics of the weeds. Moreover, the EO exhibited significant antioxidant effects compared to ascorbic acid. Further studies are necessary to verify if these biological activities of the EO could be attributable to its major compounds.


Introduction
Since their early presence on Earth, humans have depended largely on plants for food, energy, and medicine [1]. Nowadays, even with highly scientific and technological developments, aromatic and medicinal plants are still the main source of food and medicinal products. Most of the scientists focused on finding and developing new products derived from plants, plant extracts, and constituent choices for the treatment of different diseases and illnesses [2].

Plant Materials
We collected the above-ground parts of P. somalensis from three populations at Alwashla, Riyadh region, Saudi Arabia (24 • 25'36.1" N 46 • 39'07.3" E). Within each population, we collected samples from five individuals and mixed them as one composite sample. At the laboratory, we cleaned the samples from dust, dried them in a shaded place at room temperature, and ground them into powder using a grinder (IKA®MF 10 Basic Microfine Grinder Drive, Breisgau, Germany). We identified the plant according to Chaudhary [21] and deposited a voucher specimen (RIY-15647) in the National Herbarium and Genebank, Riyadh, Saudi Arabia.

Extraction of EO
Hydro-distillation of the prepared plant materials from above-ground parts of P. somalensis (400 g) was achieved using a Clevenger-type apparatus for three hours. The dark yellow oil (0.5% w/w) was separated and then dried with anhydrous NaSO 4 . EOs from all the three samples of P. somalensis populations were extracted in the same way and stored at 4 • C until further gas chromatography-mass spectrometry (GC-MS) analysis was performed.

GC-MS Analysis and Identification of Components of EO
The chemical composition of the extracted EO samples was analyzed separately by GC-MS according to our published protocol [22].

Allelopathic Activity of the EO
To assess the allelopathic activity of the extracted EO from the above-ground parts of P. somalensis, we targeted two weeds from different families: D. aegyptium (Poaceae) and B. pilosa (Asteraceae). The seeds of D. aegyptium were collected from newly reclaimed fields near New Mansoura City, northern Nile delta, Egypt (31 • 29 57.3" N 31 • 21 59.3" E), while the seeds of B. pilosa were collected from the gardens of Mansoura University campus, Mansoura, Egypt (31 • 02 38.1" N 31 • 21 01.7" E). We selected Agronomy 2020, 10, 399 3 of 14 seeds of both weeds with homogenous size and color. We surface sterilized the seeds with sodium hypochlorite (0.3%), rinsed them with water (distilled and sterilized), and then dried them over a sterilized Whatman ® cellulose filter paper (Sigma-Aldrich, Taufkirchen, Germany) [23].
We prepared different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg mL −1 ) of the extracted EO using 1% Tween ® 80 (Sigma-Aldrich, Darmstadt, Germany). For bioassay, we placed 20 seeds of each weed in sterilized Petri plates (Ø: 9 cm) lined with sterilized Whatman No. 1 filter paper and immediately added 4 mL of each concentration. The plates were sealed with Parafilm ® and incubated at 27 ± 2 • C in a growth chamber with a light cycle of 8 h dark and 16 h light. Tween (1%) was used as a negative control. After seven days for B. pilosa and ten days for D. aegyptium, we counted the number of germinated seeds and measured the length of the seedling root and shoot for both weeds. The inhibition of seed germination, root growth, and shoot growth was calculated according to the following equation: The bioassay experiment was repeated three times with three replications (three plates), and the IC 50 (the concentration of EO required for 50% inhibition)was calculated graphically as the amount of the EO necessary for 50% inhibition.

DPPH Radical Scavenging Activity
The EO capability to react with the free DPPH radical (Sigma-Aldrich, Darmstadt, Germany) and reduce its color was determined according to the method of Miguel [24]. In brief, we prepared different concentrations (10,20,40,60,80, 100 µg mL −1 ) of the EO in methanol (70%). A reaction mixture of 2 mL of each concentration and 2 mL of DPPH (0.3 mM) was prepared in screwcap test tubes, shaken well, and incubated in dark conditions at 25 • C for 15 min. Negative control was performed using 2 mL of 1% Tween instead of the EO. We measured by a spectrophotometer (Milton Roy Spectronic 21D UV-Visible Spectrophotometer, California, USA) at 512 nm. In addition, positive control with ascorbic acid (as a standard antioxidant) was prepared in a range of 1-25 mg mL −1 and treated as previously mentioned for the EO treatments. We calculated the scavenging activity according to the following equation: Also, the IC 50 was calculated as the concentration of the EO required for 50% scavenging of the DPPH.

ABTS-Free Radical Scavenging Activity
To confirm the antioxidant activity of the extracted EO, we determined the scavenging of the ABTS radical (Sigma-Aldrich, Darmstadt, Germany) according to the method of Re et al. [25]. The free radical was prepared using 7 mM of ABTS and 2.45 mM of K 2 S 2 O 8 . The mixture (1/1, v/v) was kept at room temperature (25 ± 2 • C) in dark conditions. We then diluted the radical by MeOH until it reached the absorbance of 0.700 ± 0.02 at 734 nm. A reaction mixture of 2 mL of each concentration of the EO and 2 mL of the freshly prepared ABTS was prepared, mixed well, and incubated at room temperature (25 • C) for 6 min. We then measured the absorbance at 734 nm using a spectrophotometer (Milton Roy Spectronic 21D UV-Visible Spectrophotometer, California, USA). Ascorbic acid was also used as a positive control. We calculated the scavenging activity and the IC 50 as mentioned in DPPH method.

Treatment of Data
We repeated the experiments of allelopathic and antioxidant activity three times with three replications for each. We subjected the data of antioxidant experiments, as a percentage of scavenging activity in triplicates, to a one-way analysis of variance (ANOVA) test followed by Duncan's test, where the significant differences among the various tested concentrations were assessed at p ≤ 0.05 using CoStat software program, version 6.311 (CoHort Software, Monterey, CA, USA). However, the data of allelopathic activity, as a percentage of inhibition in triplates, were subjected to two-way ANOVA at p ≤ 0.05 using the CoStat program, version 6.311 (CoHort Software, Monterey, CA, USA), which afforded the concentration of the EO and the types of weed as two factors.

Chemical Composition of the EO
The EO with a dark yellow color from the above-ground parts of P. somalensis, collected from Saudi Arabia, was extracted by the hydrodistillation method and yielded 0.5% (v/w) oil. The EO was analyzed via GC-MS. The chromatogram, including the main components, is indexed in Figure 1. All the identified constituents of EO comprising 71 compounds are listed in Table 1, representing 100% of the total mass. Mono-, di-, and sesquiterpenes as well as hydrocarbons and aromatic phenolic compounds were characterized as components of the EO.
Sesquiterpenes represented the main characterized class (91.8%) of compounds, including both oxygenated (72.4%) and non-oxygenated (19.3%) sesquiterpenes. Oxygenated monoterpenes were one of the identified compounds with a concentration of 3.7% from overall identified monoterpenes (4.8%) in addition to minor monoterpenes hydrocarbons (1.0%). Diterpenoids were the usual minor compounds in EOs derived from aromatic and medicinal plants [22]. Herein, diterpenes are characterized as minor constituents with a concentration of 2.5%, including the concentration of 1.8% of oxygenated and 0.7% of non-oxygenated diterpenes. A concentration of 2.0% from overall mass represented the other compounds, including oxygenated and non-oxygenated hydrocarbons (0.8% and 0.2%) as well as 0.94% of volatile aromatic compounds.
In our findings, sesquiterpenes were the backbone of the characterized compounds in the EO. From 36 identified oxygenated sesquiterpenes, juniper camphor (24.7%), α-sinensal (7.6%), 6-epi-shyobunol (6.6%), α-bisabolol (5.3%), and T-muurolol (4.7%) represented the main components, while isoaromadendrene epoxide was a minor one with a concentration of 0.1%. By comparing our results with the literature survey of EOs of other Pulicaria species, it was clear that the chemical Agronomy 2020, 10, 399 5 of 14 composition EO of P. somalensis is comparable to some other Pulicaria species, with a preponderance of sesquiterpenes such as P. dysenterica [13] and P. gnaphalodes [14]. In our findings, sesquiterpenes were the backbone of the characterized compounds in the EO. From 36 identified oxygenated sesquiterpenes, juniper camphor (24.7%), α-sinensal (7.6%), 6-epishyobunol (6.6%), α-bisabolol (5.3%), and T-muurolol (4.7%) represented the main components, while isoaromadendrene epoxide was a minor one with a concentration of 0.1%. By comparing our results with the literature survey of EOs of other Pulicaria species, it was clear that the chemical composition EO of P. somalensis is comparable to some other Pulicaria species, with a preponderance of sesquiterpenes such as P. dysenterica [13] and P. gnaphalodes [14].
In most of the cases, the EOs derived from the aromatic and the medicinal plants were poor resources of diterpenoids with some exceptions, such as Lactuca serriola L. (prickly lettuce), where the diterpene isocembrol was determined in high concentration (17.4%) [22]. This fact was achieved in our study by minor diterpene constituents. Only two diterpenoid components were identified, which includes the oxygenated one, geranyl linalool (1.8%) and the non-oxygenated one, geranyl-α-terpinene (0.7%). The previous studies of EOs of Pulicaria species deduced that these plants almost do not have diterpene components [16,18]. For example, the EO of P. mauritanica was described to have only one diterpenoid with a concentration of 0.2 of the total mass [15], while the EOs of P. dysenterica [13], P. gnaphalodes [14], P. undulata [17], and P. jaubertii [16] had no diterpenes.
Volatile aromatic and phenolic terpenoid compounds are very common in the EOs of Pulicaria species, especially cymene derivatives and isomers such as p-cymene, m-cymene, and p-cymen-8-ol [15,16,18]. In the same line, the EO of P. somalensis contained only one aromatic compound, p-cymene, with a low concentration (1.0%).

Correlation Between P. somalensis and other Pulicaria Species
The application of PCA on the concentration of various classes of the EO from different Pulicaria species revealed that P. somalensis, P. stephanocarpa, and P. vulgaris from Italy, P. vulgaris from Tunisia, P. dysenterica from Greece, P. gnaphalodes from Mashhad, Iran, and P. glutinosa were correlated with each other due to the similarity in the content of sesquiterpenes (Figure 2a). However, P. dysenterica from Iran was not correlated with other Pulicaria species, as it was characterized by the presence of diterpene. On the other hand, P. undulata from Egypt, P. undulata from Sudan, P. vulgaris from Iran, P. odora, P. mauritanica from Morocco, and P. mauritanica from Algeria, and P. jaubertii were correlated with each other, since these species have monoterpenes as the major class.
The application of AHC on the data of the major compounds (>5%) of the EO from different Pulicaria species showed that the P. somalensis was separated from other Pulicaria species (Figure 2b). These results reflected the characteristic pattern of the chemical composition of P. somalensis. Similarly, P. odora, P. vulgaris from Iran, P. vulgaris from Italy, P. gnaphalodes from Mashhad, Iran, P. dysenterica from Iran, P. vulgaris from Tunisia, P. glutinosa, and P. incisa from Algeria were also separated alone.
However, P. undulata from Sudan and Egypt were grouped together into one group, while P. undulata from Algeria, P. jaubertii, P. incisa from Egypt, P. mauritanica from Morocco, and P. mauritanica from Algeria were grouped together. P. sicula, P. dysenterica from Greece, and P. stephanocarpa were separated together, while P. gnaphalodes from Tehran, Iran and P. vulgaris from Iran were grouped together.
The obtained data from AHC showed that the same species varied in the chemical composition according to their geographical region. Abd El-Gawad et al. [41] reported that the chemical composition of the EO varied among different plant ecotypes due to variation in climate, soil, environmental variables, and the genetic pool. Our previous work on Xanthium strumarium L. (cocklebur), Symphyotrichum squamatum (Spreng.) Nesom (bushy starwort), and Launaea (Morrar) species indicated that a variation of the chemical composition of the EO was found to be strongly correlated with variation in the habitats [42,43]. The overall correlation analysis showed that P. somalensis has a specific chemical pattern of the EO, where it could be related to the genetic characteristics. These data of the EO chemical composition could be helpful for the chemotaxonomy of Pulicaria genus.
The overall correlation analysis showed that P. somalensis has a specific chemical pattern of the EO, where it could be related to the genetic characteristics. These data of the EO chemical composition could be helpful for the chemotaxonomy of Pulicaria genus.

Allelopathic Activity of the EO
The extracted EO from P. somalensis above-ground parts exhibited a significant allelopathic inhibitory activity on the germination and the seedling growth of the tested weeds (B. pilosa and D. aegyptium) in a dose-dependent manner (Figure 3). At a concentration of 1 mg mL −1 of the EO, germination, root growth, and shoot growth of B. pilosa were inhibited by 61.4%, 73.6%, and 55.4%, respectively.

Allelopathic Activity of the EO
The extracted EO from P. somalensis above-ground parts exhibited a significant allelopathic inhibitory activity on the germination and the seedling growth of the tested weeds (B. pilosa and D. aegyptium) in a dose-dependent manner (Figure 3). At a concentration of 1 mg mL −1 of the EO, germination, root growth, and shoot growth of B. pilosa were inhibited by 61.4%, 73.6%, and 55.4%, respectively.   On the other hand, germination, root, and shoot growth of D. aegyptium were inhibited by 75.0%, 72.1%, and 66.2%, respectively. A highly significant difference in seed germination and shoot growth was observed between the two test weeds (p < 0.0001), while no significant difference (p < 0.0883) was observed based on the root growth ( Figure 3). Usually, the root is more affected, as it is the first sprout organ and because it has direct contact with allelochemicals, as described in many studies [23,[43][44][45][46].
According to the IC 50 , the EO showed a more inhibitory effect against D. aegyptium (Figure 4). The IC 50 value on the germination of D. aegyptium was doubled compared to B. pilosa. The IC 50 values on the root growth of B. pilosa and D. aegyptium were comparable (0.6 mg mL −1 , each), while the IC 50 values on the shoot growth were 1.0 and 0.7 mg mL −1 , respectively ( Figure 4). Overall, the EO of P. somalensis showed more inhibitory activity against D. aegyptium than B. pilosa. This variation in the activity could be attributed to the genetic characteristics of the weeds [47].
Agronomy 2020, 10, x FOR PEER REVIEW 11 of 15 On the other hand, germination, root, and shoot growth of D. aegyptium were inhibited by 75.0%, 72.1%, and 66.2%, respectively. A highly significant difference in seed germination and shoot growth was observed between the two test weeds (p < 0.0001), while no significant difference (p < 0.0883) was observed based on the root growth ( Figure 3). Usually, the root is more affected, as it is the first sprout organ and because it has direct contact with allelochemicals, as described in many studies [23,[43][44][45][46].
According to the IC50, the EO showed a more inhibitory effect against D. aegyptium (Figure 4). The IC50 value on the germination of D. aegyptium was doubled compared to B. pilosa. The IC50 values on the root growth of B. pilosa and D. aegyptium were comparable (0.6 mg mL −1 , each), while the IC50 values on the shoot growth were 1.0 and 0.7 mg mL −1 , respectively ( Figure 4). Overall, the EO of P. somalensis showed more inhibitory activity against D. aegyptium than B. pilosa. This variation in the activity could be attributed to the genetic characteristics of the weeds [47]. The inhibitory activity of P. somalensis EO could be attributed to the high content of oxygenated terpenoid compounds, particularly sesquiterpenes (Table 1). Major compounds such as juniper camphor, α-sinensal, 6-epi-shyobunol, α-zingiberene, α-bisabolol, and T-muurolol could act either individually or synergistically as inhibitors for the germination and the growth of the B. pilosa weed. The oxygenated terpenoids usually have a significant role in biological activity compared to nonoxygenated compounds due to the reactivity of oxygen [41]. The EOs from S. squamatum and L. serriola have been reported to inhibit the germination and the seedling growth of the B. pilosa Abd-ElGawadm et al. [22] and Abd-ElGawad, et al. [42] due to the presence of sesquiterpenes as major components.
Juniper camphor has been reported as the main compound (15.5%) of antibacterial, antioxidant, and phytotoxic active EO from Syzygium samarangense Merr. & Berry (rose apple) Lawal, et al. [48]. Additionally, the EO from Artemisia argyi Levl et Vant (mugwort) has been reported to possess antifungal activity due to the high content of juniper camphor [49].

Antioxidant Activity of the EO
The antioxidant capacity of the EO from the above-ground parts of P. somalensis was tested by the ability to scavenge the DPPH and the ABTS. The results revealed that the scavenging activity was significantly increased by the increase of EO concentration ( Table 2). The EO attained IC50 values of 81.2 mg mL −1 and 64.4 mg mL −1 based on DPPH and ABTS assays, compared to ascorbic acid with IC50 values of 21.7 mg mL −1 and 18.4 mg mL −1 , respectively. These data showed that the EO of P.  The inhibitory activity of P. somalensis EO could be attributed to the high content of oxygenated terpenoid compounds, particularly sesquiterpenes (Table 1). Major compounds such as juniper camphor, α-sinensal, 6-epi-shyobunol, α-zingiberene, α-bisabolol, and T-muurolol could act either individually or synergistically as inhibitors for the germination and the growth of the B. pilosa weed. The oxygenated terpenoids usually have a significant role in biological activity compared to non-oxygenated compounds due to the reactivity of oxygen [41]. The EOs from S. squamatum and L. serriola have been reported to inhibit the germination and the seedling growth of the B. pilosa Abd-ElGawadm et al. [22] and Abd-ElGawad, et al. [42] due to the presence of sesquiterpenes as major components.
Juniper camphor has been reported as the main compound (15.5%) of antibacterial, antioxidant, and phytotoxic active EO from Syzygium samarangense Merr. & Berry (rose apple) Lawal, et al. [48]. Additionally, the EO from Artemisia argyi Levl et Vant (mugwort) has been reported to possess antifungal activity due to the high content of juniper camphor [49].

Antioxidant Activity of the EO
The antioxidant capacity of the EO from the above-ground parts of P. somalensis was tested by the ability to scavenge the DPPH and the ABTS. The results revealed that the scavenging activity was significantly increased by the increase of EO concentration ( Table 2). The EO attained IC 50 values of 81.2 mg mL −1 and 64.4 mg mL −1 based on DPPH and ABTS assays, compared to ascorbic acid with IC 50 values of 21.7 mg mL −1 and 18.4 mg mL −1 , respectively. These data showed that the EO of P. somalensis has meaningful antioxidant activity. This antioxidant activity could be ascribed to the major constituents of the EO, such as juniper camphor, α-sinensal, and 6-epi-shyobunol. These oxygenated sesquiterpenes might act individually or synergistically as antioxidants. The antioxidant role of the oxygenated compounds might be attributed to the free electrons due to the high oxygenation [42,43]. Although juniper camphor has been reported to have antifungal [49] and antibacterial activity and cytotoxicity [50], its antioxidant activity has still not been studied. Therefore, we recommend further studies to determine the biological activity, particularly the antioxidant activity, of the pure form of major compounds, especially juniper camphor. Table 2.

Conclusions
For the first time, the present study showed that the EO from P. somalensis has 71 compounds. Juniper camphor, α-sinensal, 6-epi-shyobunol, α-zingiberene, α-bisabolol, and T-muurolol were found as main constituents. The correlation analysis revealed that it has a specific EO chemical pattern via the absence of the correlation with other Pulicaria ecospecies. Biologically, EO showed significant allelopathic activity on the weeds (B. pilosa and D. aegyptium). Therefore, this EO could be integrated into the methods of the management of these weeds as an eco-friendly way, but after further study on the assessment of its activity, durability, and safety as bioherbicide at the field scale. Moreover, the EO reflected meaningful antioxidant activity compared to ascorbic acid. Because the biological activities of the pure form of the identified major compounds are still undetermined, a further study is recommended for the characterization of the pure major compounds, particularly juniper camphor.