*2.1. Yields and Chemical Constituents of P. undulata EOs*

The aerial parts of Saudi and Egyptian *P. undulata* (150 g each) were subjected separately to the hydrodistillation for 3 h in Clevenger-type apparatus, provided pale yellow oil with an average yield of 0.43% and 0.36% (*v*/*w*), respectively. The yields of EOs in our study were comparable to those reported from other Egyptian ecospecies (0.23–0.60%) [9,16,20]. However, the yield of the present *P. undulata* ecospecies was lower than that reported in previous studies for other ecospecies such as Yemeni (2.10%) [13], Iranian (0.50–1.34%) [15,19,24], Sudanian (1.40–2.50%) [6,17], and Algerian ecospecies (1.20%) [14]. These variations in the yield of the EOs might be attributed to the difference in the geographical region, in addition to the environmental conditions such as soil, climate, as well as genetic pool [22,25–27].

In total, 80 compounds were characterized depending upon GC–MS analysis of the two EOs of *P. undulata* including 61 and 34 compounds from Saudi and Egyptian ecospecies, respectively. The identified constituents were classified into eight classes namely, (i) monoterpene hydrocarbons, (ii) oxygenated monoterpenes, (iii) sesquiterpene hydrocarbons, (iv) oxygenated sesquiterpenes, (v) carotenoid-derived compounds, (vi) apocarotenoid-derived compounds, (vii) nonoxygenated hydrocarbons, and (viii) oxygenated hydrocarbons, (Figure 1a). Oxygenated sesquiterpenes were the most represented class—they represented 55.03% and 40.34% of the total oil of the Egyptian and Saudi ecospecies, respectively. Monoterpenes were determined with a high content (39.46%) of the EO of Saudi ecospecies, while it represented a minor class in Egyptian ecospecies (6.70%). Additionally, hydrocarbons represented 13.32% of the total EO content of the Egyptian eco-sample, while completely absent in the Saudi plant sample. Overall, the Egyptian ecospecies had oxygenated compounds as the main elements, while non-oxygenated compounds were represented as the main constituents of Saudi ecospecies (Figure 1b). The identified compounds, accounting for 97.22% and 97.61%, respectively, of overall EO mass, in addition to their retention times, and literature and experimental Kovats indices are presented in Table 1.

**Figure 1.** Classification of the chemical compounds of *Pulicaria undulata* EOs of Saudi and Egyptian ecospecies. (**a**) various classes and (**b**) oxygenated and non-oxygenated compounds.

**Table 1.** Chemical constituents of the EOs of the aerial parts of Saudi and Egyptian ecospecies of *Pulicaria undulata*.



**Table 1.** *Cont.*


**Table 1.** *Cont.*

<sup>a</sup> Rt: retention time; <sup>b</sup> values are mean (*<sup>n</sup>* = 2) <sup>±</sup> SD of Saudi ecospecies; <sup>c</sup> Egyptian ecospecies; <sup>d</sup> literature Kovats retention index; <sup>e</sup> experimental Kovats retention index; MS: mass spectral data of compounds; KI: Kovats indices with those of Wiley Spectral Library collection and National 104 Institute of Standards and Technology (NIST) Library database.

> The analysis of the data revealed that the EOs of the two plant samples were very rich with terpenoids, with respective concentrations of 95.78% and 66.17% in addition to carotenoids (2.77% and 20.15%, respectively). The variations in the quantitative and qualitative analysis of EOs of the two plant samples were attributed directly to the environmental and climate variations between the Saudi and Egyptian environments [28,29].

> More in-depth data indicated that the EO of the Saudi *P. undulata* contained mainly terpenoids, including almost equal concentrations of mono (46.27%) and sesquiterpenes (49.51%) with traces of carotenoids and a complete absence of diterpenoids and hydrocarbons. In comparison, the chemical characterization of the EO of the Egyptian plant showed that terpenoids were the major compounds, including minor elements of monoterpenes (6.40%) and abundance of sesquiterpenes (59.77%), as well as a high concentration of carotenoids. Similarly, the EO of the Egyptian plant was characterized by the complete absence of diterpenes and the presence of a remarkable concentration of hydrocarbons. The sesquiterpenes were found as major constituents of the EOs of both ecospecies (Saudi and Egyptian); this result was different than those reported for Yemeni leaves (2.1%) [13], Iranian aerial parts (0.5%) [15], and Egyptian aerial parts (0.6%) [20] of *P. undulata*. The sesquiterpenes in the EOs of Saudi and Egyptian ecospecies were categorized as sesquiterpene hydrocarbons (9.17% and 4.74%), and oxygenated sesquiterpenes (40.34% and 55.03%). Isoshyobunone (7.67%), 6-epi-Shyobunol (6.51%), spathulenol (3.43%), and *trans*-nerolidol (3.33%) represented the main oxygenated sesquiterpene of EO of the Saudi plant. In comparison, spathulenol (30.86%), *α*-bisabolol (6.34%), 4,4-dimethyltetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9-ol (4.68%), and τ–cadinol (3.65%) were found to be the abundant oxygenated sesquiterpenes of EO of Egyptian ecospecies. Most of the studied ecospecies of *P. undulata* have been described as non-rich of sesquiterpene [13,15,20]. However, EOs of other *Pulicaria* species such as *P. somalensis* [1], *P. dysenterica* [30], and *P. gnaphalodes* [31] were reported as rich in sesquiterpene.

> Numerous *Pulicaria* plants were described to have spathulenol as minor and/or major compounds of their EOs such as *P. somalensis* [1] and *P. stephanocarpa* [32]. *α*-Bisabolol was detected as the main sesquiterpene in EOs derived from some *Pulicaria* species such as *P. somalensis* [1], *P. dysenterica* [30], and *P. gnaphalodes* [31]. Moreover, the major sesquiterpene, cadinol, in this study has been described as a major component in EO derived from aerial

parts of the *P. undulata* collected from the Algerian Sahara [14], while it was reported as minor or trace element in other ecospecies.

Monoterpenes were reported as the main constituents of several *Pulicaria* ecospecies [13,33]. Saudi *P. undulata* was found to be in harmony with the reported documents where the monoterpenes represented around half of the total oil (46.27%) including hydrocarbons (39.46%) and oxygenated (6.81%) forms of monoterpene. However, the monoterpenes were identified as trace elements (6.40%) in the EO of Egyptian plants, including traces of non-oxygenated and oxygenated forms, with respective relative concentrations of 3.04% and 3.36%. In the EO from the Saudi sample, *β*-pinene (21.14%), *α*-pinene (5.08%), and *α*terpinolene (3.84%) were assigned as the main monoterpene hydrocarbons, while myrtenyl acetate (2.50%) and terpinen-4-ol (1.20%) were characterized as main oxygenated monoterpenes. Only four monoterpenes were identified from overall compounds of EO of Egyptian ecospecies. *β*-Pinene (1.32%) was assigned as the main monoterpene hydrocarbons, and carvotanacetone (3.36%) was the only identified oxygenated one. Carvotanacetone was stated as the main monoterpene of *P. undulata* collected from Yemen [13] and from the Egyptian Western Desert region [8,20]. The present data revealed that the variations in the components in EO of Egyptian and Saudi samples might be attributed to the variations in collection areas, in addition to the environmental conditions such as soil, climate, as well as their genetic pool [23]. The abundance of pinene and myrtenyl derivatives, *α*-terpinolene, terpinen-4-ol was in complete harmony with the data reported from the Iranian *P. undulata* [15,18].

Carotenoid-derived compounds were represented as trace constituents in the EO of the Saudi ecospecies, with a concentration of 2.77%, comprising carotenoids (1.26%) and apocarotenoid-derived compounds (1.51%). Hexahydrofarnesyl acetone was found as the main component in all characterized carotenoid-derived compounds. By contrast, carotenoid-derived compounds derived from the Egyptian EO sample were characterized by high concentration (20.12%), representing carotenoid-derived compounds (2.03%) and apocarotenoid-derived compounds (18.12%). Additionally, hexahydrofarnesyl acetone represented the predominated compound in all overall carotenoid-derived compounds. Hexahydrofarnesyl acetone is a common apocarotenoid-derived compound in EOs derived from the plant kingdom such as *Hildegardia barteri* [34], *Stachys tmolea* [35], and *Bassia muricata* [36].

The hydrocarbons represented 13.32% of the total identified oil of the Egyptian plant involved non-oxygenated (8.98%) and oxygenated (4.34%) forms. *n*-nonadecane (1.57%) and *n*-nonacosane (1.49%) were identified as the majors of non-oxygenated hydrocarbons, while *cis*-9-hexadecenoic acid (3.73%) represented the main oxygenated hydrocarbons. Hydrocarbons were completely absent from the EO of the Saudi plant, and this result was found in agreement with Iranian *P. undulata* [15,18].

#### *2.2. Chemometric Analysis of the EOs of Pulicaria Ecospecies*

The application of the EOs profiles of the 2 ecotypes of *P. undulata* and the other 11 ecotypes were subjected to principal component multivariate data analysis (PCA) and agglomerative hierarchical clustering (AHC). The cluster analysis revealed that the EOs could be categorized into four clusters. Cluster-I consisted of the Iran–Baluchestan ecotype, while the EOs of the presently studied ecospecies (Saudi and Egyptian) were grouped as cluster-II. Further, the Egypt–Elba Mountain-2 and Egypt–Sinai ecospecies showed a close correlation, and therefore, they were grouped as cluster-III. Finally, cluster-IV contained Iranian (Iran–Baluchestan, Iran–Fars, and Iran–Hormozgan samples), Algerian, Sudanian, Yemeni, Egyptian (Elba Mountain-2, and Sadat) ecospecies (Figure 2a).

**Figure 2.** Chemometric analysis of the essential oil of different *Pulicaria undulata* ecospecies: (**a**) agglomerative hierarchical clustering (AHC) and (**b**) principal components analysis (PCA). SA: Suadi, EG: Egyptian, IR: Iranian, AL: Algerian, SU: Sudanian, and YE: Yemeni. The blue color represents the present samples.

The PCA score plot showed the distant separation of Egypt–Elba Mountain-2 and Egypt–Sinai ecospecies in the PC2, while Egypt–Elba Mountain-2, Egypt Sadat, Yemeni, Algerian, and Sudanian ecospecies were distantly distributed along the right side of the PC1 (Figure 2b). Conversely, the present samples (Saudi and Egyptian) as well as Iranian and Algerian were clustered together in the center of the PCA and had positive score values. In addition, the examination of the loading plot showed that piperitone was the most correlated/abundant compound in Egypt–Elba Mountain-2 and Egypt–Sinai ecospecies. However, carvotanacetone showed an abundance in Egypt–Elba Mountain-2, Egypt Sadat, Yemeni, Algerian, and Sudanian ecospecies. The detected variation among different ecospecies could be ascribed to the effect of climatic and environmental conditions, as well as the genetic characteristics [22,25,26,37].
