*2.3. Allelopathic Effect of the C. amblyocarpa EO*

The EO of *C. amblyocarpa* showed significant allelopathic activity of the seed germination (*p* < 0.05) as well as the shoot and root development of *D. aegyptium* in a dose-dependent manner (Figure 4a). At the highest concentration (100 μg mL−1), the germination was inhibited by 70.18%, whereas the seedling root and shoot were reduced by 75.88% and 61.87%, respectively. Based on the IC50 values, the root was more affected than the shoot, where the roots had an IC50 value of 57.10 μg mL−1, and the root attained 74.07 μg mL−<sup>1</sup> (Figure 4b). Root has been reported to be more affected by allelochemicals than the shoot. This observation was reported for many plant species such as *Deverra tortuosa* [41], *Teucrium polium* [42], *Calotropis procera* [5], *Ficus carica* [43], and *C. plicata* [34]. This could be ascribed to the direct contact of the root with the medium and the high permeability of root cells [34,44].

**Figure 3.** Chemometric analysis of various *Cleome amblyocarpa* ecospecies. (**a**) agglomerative hierarchical clustering (AHC), (**b**) and principal components analysis (PCA). (**F1**) and (**F2**) are factor 1 and 2.

**Figure 4.** Allelopathic activity of the EO from the above-ground parts of *Cleome amblyocarpa* on the germination, root and shoot growth of *Dactyloctenium aegyptium*. (**a**) Various concentration and (**b**) IC50. Different letters within each line mean values significantly different at *p* < 0.05 (Tukey's HSD test).

To the best of our knowledge, the allelopathic activity of the EO from *C. amblyocarpa* has not been studied yet. However, the aqueous, hexane, chloroform, and methanol extracts from *C. amblyocarpa* have been reported to inhibit lettuce germination and growth [45]. At a concentration of6gL−1, ethyl acetate showed a complete inhibition of lettuce growth, whereas *Peganum harmala*, *Raphanus sativus*, and *Silybum marianum* were more resistant. Additionally, Ladhari et al. [46] identified some terpenoids and flavonoids from *C. amblyocarpa*, where dammarane-type triterpenes showed strong allelopathic activity, and flavonoid compounds exhibited <50% inhibition of the targeted species.

In our results, the allelopathic activity of *C. amblyocarpa* EO could be attributed to the activity of a single or combination of the major identified compounds. Caryophyllene oxide is reportedly a major compound of the EOs with substantial allelopathic activity such as *C. plicata* [34], *H. curassavicum* [33], *Acroptilon repens* [47], *Teucrium arduini*, *T. montbretii* [48], and *Nepeta curviflora* [49]. On the other hand, the EO from *Launaea mucronata* and *L. nudicaulis* showed the presence of hexahydrofarnesyl acetone as a major compound, where it revealed significant allelopathic activity against the weed: *Portulaca oleracea* [30]. Additionally, the EO of *H. curassavicum* had hexahydrofarnesyl acetone as the main compound, and showed marked allelopathic activity on *Chenopodium murale* [33]. The major compound, alloaromadendrene epoxide, in the EO of the present *C. amblyocarpa* has also been reported as the main compound (7.32%) of the EOs from *Lactuca serriola* that showed allelopathic activity against the weed *Bidens pilosa* [50]; however, in the EO of *Calamintha nepeta*, it did not show significant allelopathic activity against *Raphanus sativus*, *Lepidium sativum*, *Sinapis arvensis*, *Triticum durum*, and *Phalaris canariensis* [51]. This inconsistency could be attributed to the resistance of the weeds and shows that allelochemicals are species-specific [52].

Generally, the oxygenated terpene compounds have been reported to possess allelopathic activity compared to the non-oxygenated compounds [6]. In the present study, the EO of *C. amblyocarpa* was very rich in oxygenated compounds (81.80%), which could explain the notable allelopathic activity.

#### *2.4. Antioxidant Activity of C. amblyocarpa EO*

The activity of *C. amblyocarpa* EO in the reduction in the DPPH revealed significant antioxidant activity in a dose-dependent manner (Figure 5). At the lowest concentration of the EO (10 mg mL<sup>−</sup>1), the EO showed a 15.28% scavenging activity of the DPPH, whereas at the highest concentration (100 mg mL−1), the antioxidant activity was reduced by 3.7 fold of the lowest concentration of the EO. Based on the IC50, the *C. amblyocarpa* EO had a value of 4.52 mg mL−<sup>1</sup> compared to 2.11 mg mL−<sup>1</sup> of the ascorbic acid as a standard antioxidant. The antioxidant activity of EOs in the present study were lower than those reported from Tunisian ecospecies [37]. This could be ascribed to the variation in the EO chemical compositions (Table 1).

The free radical scavenging activities of plant extracts and/or EOs were directly correlated with the concentration of the oxygenation of their constituents due to the increase in the free electrons [53]. The present data revealed 81.08% of oxygenated compounds, which means that a wealth of free electrons can act to diminish free radicals in the evaluation reaction. More specifically, the activity of this EO may be attributed to the activity of major compounds, either singularly or in synergy. Caryophyllene oxide has been reported to possess antioxidant activity [34]. EOs rich in hexahydrofarnesyl, such as *B. muricata* [10] and *H. curassavicum* [33], showed substantial antioxidant activity. The EO extracted from *L. serriola* has been reported to be rich in alloaromadendrene epoxide and isoshyobunone, where it expressed strong antioxidant activity [50].

**Figure 5.** Antioxidant activity of the EO from the above-ground parts of *Cleome amblyocarpa*. Different letters of the line mean values significant at *p* < 0.05 (Tukey's HSD test).

## *2.5. Anti-Inflammatory effect of C. amblyocarpa EO*

For the first time, the anti-inflammatory of EO of *C. amblyocarpa* has been evaluated via inhibition of the enzymes, lipoxygenase (LOX), cyclooxygenases (COX1, and COX2), as well as membrane stabilization. The results presented in Figure 6 revealed that the EO exhibited a significant anti-inflammatory action via the inhibition of LOX, COX1, and COX2 with respective IC50 values of 1.67, 12.77, 13.43 μg mL<sup>−</sup>1, whereas ibuprofen showed inhibition with IC50 values of 1.53, 10.26, and 12.71μg mL<sup>−</sup>1, respectively. Additionally, the EO significantly inhibited the lysis of the hypotonic solution of the RBCs at an IC50 value of 15.25 μg mL<sup>−</sup>1, compared with indomethacin which presented a result of 14.34 μg mL<sup>−</sup>1.

**Figure 6.** The anti-inflammatory activity of the EO of *Cleome amblyocarpa*, based on membrane stabilizing, and the inhibition of lipoxygenase (LOX) and cyclooxygenase (COX1 and COX 2). (ns) non-significant, \* *p* < 0.05, \*\* *p* < 0.01 (two-tailed *t*-test). Data are mean values ± standard error (*n* = 4).

These data revealed that this EO has potent anti-inflammatory potentialities comparable with the two reference drugs, ibuprofen, and indomethacin, especially via the inhibition of lipoxygenase (LOX) and cyclooxygenase (COX1). This capability of EO for the inhibition of inflammations might be directly ascribed to the terpenoid contents as the main components (84.88%) [54]. Terpenoids represented major components of many of the documented plants with significant anti-inflammatory potentialities such as *Araucaria heterophylla* [8], *Ocimum basilicum* [55], and *Limnophila indica* [56].

Many studies have deduced that volatile sesquiterpene compounds have a potent anti-inflammation role in in vivo and in vitro models, especially caryophyllene and its oxide form [57]. Chavan et al. [58] described that the caryophyllene oxide, isolated from *Annona squamosa*, has significant in vivo anti-inflammatory activity. EO derived from *Cordia verbenacea* as well as its active constituent, caryophyllene, were demonstrated to exhibit strong anti-inflammatory activity was discussed with regard to their interfering with the production of TNF-*α* [59]. Moreover, Medeiros et al. [60] described that *trans*-caryophyllene showed potent anti-inflammatory in rats by significantly decreasing the migration of neutrophils as well as increasing the NF-κB-induced stimulation by lipopolysaccharides.

The EO of Sardinian *Santolina corsica* has been reported to contain a remarkable content of aromadendrene derivatives that exhibit a significant anti-inflammatory [61]. The enriched EO of Algerian *Myrtus communis* with myrtenyl acetate (38.7%) was shown to reduce the mice's inflammation and paw edema at a concentration of 100 mg/kg [62]. In addition to these significant roles of the main components, the other compounds were expected to have an important contribution via synergetic effects [8]. Based upon all these reported data, it is very clear that the present data agree with the previously documented. Additionally, the present results could be attributed to the prevalence of sesquiterpenes, especially *trans*-caryophyllene and caryophyllene oxide.
