*2.1. EO Composition of C. amblyocarpa*

The hydrodistillation of the air-dried powder of the above-ground parts of *C. amblyocarpa* yielded 0.38% (*v/w*) of golden-yellow oil. Depending upon the GC-MS analysis (Figure 1), 48 compounds were characterized, representing 97.17% of the total mass (Table 1). The Egyptian ecospecies of the present study yielded higher EO yield compared to Iranian [27] and Saudi [28] ecospecies, which had 0.20 and 0.21%, respectively. This variation in oil production could be ascribed to environmental, climatic, or genetic factors [5,6,29–31]. The term "ecospecies" means that species of the plant can be divided into several ecotypes (a genetically distinct population of plants that is growing in a particular habitat).


**Table 1.** Chemical components of the essential oil of above-ground parts of *Cleome amblyocarpa*.

**<sup>a</sup>** RT: Retention time. **<sup>b</sup>** RC: Relative concentration. **<sup>c</sup>** MF: Molecular formula. **<sup>d</sup>** KILit: Kovats retention index according to Adams (2017) on a DB−5 column in reference to *<sup>n</sup>*-alkanes. **<sup>e</sup>** KIExp: Experimental calculated Kovats retention index. **<sup>f</sup>** EO constituent identification was constructed via compound mass spectra (MS) and Kovats retention indices (KI) with those of Wiley spectral library collection and NIST library databases.

**Figure 1.** Chromatogram of the chemical compounds identified via GC-MS in the EO of *Cleome amblyocarpa* above-ground parts. The major compound peaks are numbered (1–6).

In the EO of *C. amblyocarpa*, six classes of components were determined, comprising oxygenated sesquiterpenes, sesquiterpenes hydrocarbons, oxygenated monoterpenes, diterpenes hydrocarbons, oxygenated diterpenes, apocarotenoid-derived compounds, carotenoid-derived compounds, and other compounds (Figure 2). These compounds pooled as 81.80% oxygenated compounds and 15.37% as non-oxygenated compounds. From overall mass, terpenoids represented the main constituents with a relative 84.88% with a preponderance of sesquiterpenes (75.77%), a remarkable concentration of monoterpenes (8.72%), and traces of diterpenes (0.39%). The abundance of terpenoids in the EO of *C. amblyocarpa* was in agreement with the data reported for samples collected from Iran [27] and the United Arab Emirates [26]. In contrast, the plurality of sesquiterpenes was inconsistent with the Iranian *C. amblyocarpa*, in which diterpenoids were determined as the major class [27], and *C. amblyocarpa* collected from the United Arab Emirates, in which monoterpenes were the main constituents [26]. These significant variations of chemical composition might be ascribed to environmental circumstances (such as temperature, rainfall, soil factors, altitude, etc.) and genetic characteristics [11,30,32,33].

**Figure 2.** Concentrations of various identified classes of the chemical compounds of the *Cleome amblyocarpa* EO (**a**) and the percentage of oxygenated and non-oxygenated compounds (**b**).

Sesquiterpenes were assigned as the main components involving the oxygenated compounds as majors (60.62%) in addition to a relative concentration of 15.15% of sesquiterpene hydrocarbons. Out of the 16 identified oxygenated sesquiterpenes, caryophyllene oxide (36.01%), alloaromadendrene epoxide (6.17%), isoshyobunone (4.52%), and shyobunol (4.19%) represented the major compounds, whereas *β*-cubebene (0.13%) was the minor compound. Caryophyllene oxide is a common major compound in several EOs derived from plants such as *Cullen plicata* [34], *Schinus polygamus* [35], *Curcuma sahuynhensis* [36]. Caryophyllene oxide was documented as a minor compound in the EO of Iranian *C. amblyocarpa* [27], and totally absent from the EO of *C. amblyocarpa* collected from the United Arab Emirates [26], Tunisia [37], and Saudi Arabia [28]. On the other hand, *trans*-caryophyllene (3.45%), and *α*-muurolene (2.30%) were found to be the main sesquiterpene hydrocarbons, whereas silphiperfol-5,7(14)-diene (0.13%) was determined as a minor component. *trans*caryophyllene has been reported in trace amounts of the EO of the Iranian ecospecies of *C. amblyocarpa* [27], whereas it is completely absent from the EO of Saudi [28], Emirati [26], and Tunisian [37] ecospecies.

The oxygenated monoterpenes were represented by 8.72%, which contained seven compounds, with myrtenyl acetate (5.73%) and borneol (1.12%) as major compounds. These two compounds are totally absent from the other ecospecies of *C. amblyocarpa* [26–28,37]. On the other hand, low diterpene contents were determined and represented by two compounds, geranyl-*α*-terpinene (0.22%) and phytol (0.17%). However, diterpenes were absent from other ecospecies of *C. amblyocarpa*. In other species of *Cleome* genus, phytol was reported in a high concentration such as *C. monophylla* [23], *C. serrata* [24], and *C. serrata* [38].

Carotenoid-derived compounds were determined in a concentration of 3.03%, that represented only two compounds, dihydroedulan II (1.64%) and theaspirane A (1.39%). Only one apocarotenoid-derived compound, hexahydrofarnesyl acetone, was identified with a high relative concentration (7.92%), whereas it was completely absent from the other reported ecospecies of *C. amblyocarpa* [26–28,37]. Hexahydrofarnesyl acetone is a widely distributed major compound in the EOs of several plants such as *Bassia muricata* [10], *Heliotropium curassavicum* [33], *Hildegardia barteri* [39], *Trianthema portulacastrum* [40].

Finally, traces of other non-terpenoid components were characterized including only two compounds, *p*-isopropy-l-benzaldehyde (1.16%) and 9,12-octadecadienoic acid (0.18%).
