**2. Results and Discussion**

#### *2.1. Chemical Compositions of EO of P. dioscoridis*

The chemical characterization of *P. dioscoridis* EO was extracted via hydro-distillation afforded golden yellow (0.037%). The chemical profiles of the extracted EO were assigned depending upon the GC-MS analysis. The GC-MS chromatogram of the EO is presented in Figure 1 exhibiting the main peaks from all identified components. Twenty-nine compounds were identified from the EO of *P. dioscoridis* represented 96.91% of the total oil. All the identified compounds along with their chemical and physical properties were summarized in Table 1.


**Table 1.** Components of essential oils of *Pluchea dioscoridis* and *Erigeron bonariensis*.

[a] Rt: retention time, [b] Literature Kovats retention index on DB-5 column with reference to *n*-alkanes [36], [c] experimental Kovats retention index; values of each compound are average ± SD from duplicates. The identification of essential oil (EO) components was performed based on the (a) mass spectral data of compounds (MS) and (b) Kovats indices with those of Wiley spectral library collection and NIST (National Institute of Standards and Technology) library database.

**Figure 1.** Gas chromatography-mass spectroscopy (GC-MS) chromatograme of the essential oils (EO) of *Pluchea dioscoridis*. The main peaks were numbered (1–7).

The constituents of EO of *P. dioscoridis* were characterized by the presence of four classes of compounds including sesqui- (93.40%), and monoterpenes (0.19%), carotenoid derived compounds (0.28%) in addition to other acyclic compounds (1.33%). The terpenoids were found as abundant compounds with a relative concentration of 93.59% in addition to traces of carotenoids and acyclic compounds with a complete absence of diterpenoids. GC-MS analysis of EO derived from *E. bonariensis*, revealed the presence of four categories of compounds comprising sesqui- (81.06%), and monoterpenes (14.16%), diterpenes (2.44%) in addition to other acyclic compounds (0.55%). Furthermore, the terpenoids were characterized as the main components by a relative concentration of 95.22% with traces of diterpenoids and other compounds. These results deduced the fact of the preponderance of the terpenoids in the different species of *Conyza* genus [32,34,37].

Sesquiterpenoids were found as the main compounds of the EO of *P. dioscoridis* with mixtures of oxygenated and non-oxygenated compounds. The abundance of sesquiterpenes was found in full agreement with previous data of EOs of this plant [32,38]. From all identified sesquiterpenes, α-maaliene (18.84%), berkheyaradulen (13.99%), dehydrocyclolongifolene oxide (10.35%), aromadendrene oxide-2 (8.81%), *β*-muurolene (8.09%), α-eudesmol (6.79%), *β*-caryophyllene (4.95%), t-muurolol (3.88%), represented the major compounds.

Berkheyaradulen, muurolene, eudesmol, tau-muurolol, and caryophyllene, were found as marker compounds for this plant in the previous study [32] and this data is in the same line with our results. While the reported data of EO of the leaves of this plant [38] exhibited variations in chemical constituents than those data described previously by our team [32] and also than our results herein. Elshamy, et al. [32] documented that *α*-cadinol is the main sesquiterpene and this data is different than our results in which *γ*-cadinol is present as a minor compound. Additionally, eudesmol and tau-muurolol were reported as major sesquiterpenes in EO of the leaves of this plant, and this data agreed with our results.

The results of GC-MS of EO of *P. dioscoridis* revealed that the monoterpenes are traces with only one compound, α-pinene (0.19%). The scarcity of monoterpenes is consistent with the results of Elshamy, et al. [32] and El-Seedi, et al. [38].

In EO derived from *P. dioscoridis*, diterpenes were completely absent and this result is inconsistent with the published data [32,37], while El-Seedi, et al. [38] characterized only one diterpene, phytol, from the leaves of this plant. *α*-ionone was the only identified carotenoid-derived compound from EO of *P. dioscoridis* that was not reported before from this plant [32].

The other compounds (1.33%) including hydrocarbons were characterized as traces in EO of *P. dioscoridis* that was in agreement with the previous data [32,39]. In contrast, El-Seedi, et al. [38] documented that the monoterpenoid compounds represented a high concentration (26.6%) of the total mass of EO of the leaves of *P. dioscoridis*.

#### *2.2. Chemical Compositions of EO of E. bonariensis*

The hydro-distillation of the above-ground parts of *E. bonariensis* afforded golden yellow EO (0.049%). The chemical characterization of the extracted EO was performed based on the GC-MS analysis. Figure 2 represented the GC-MS chromatogram including the major peaks. Thirty-five components were assigned representing 98.21% of the total oil mass. The characterized constituents as well as retentions times (RIs), molecular formulas (MFs), and literature and calculated Kovats indexes (KIs) were compacted in Table 1.

**Figure 2.** GC-MS chromatogrames of the EO of *Erigeron bonariensis*. The main peaks were numbered (1–7).

In EO of *E. bonariensis,* sesquiterpenes represented also the main constituents including several oxygenated and non-oxygenated metabolites. With a relative concentration of 81.06% of sesquiterpenes, our results are completely agreed with the previous data by Harraz, et al. [34] that reported a relative concentration of 92.50%; trans-α-Farnesene (25.03%), isolongifolene-5-ol (5.53%), *α*-maaliene (6.64%), berkheyaradulen (4.82%), and *α*-muurolene (3.99%) were found the main sesquiterpenoid contents. The main sesquiterpene, trans-α-farnesene, was widely distributed in the EOs of *Conyza* species such as *C. bonariensis* (≈*E. bonariensis*) collected from Venezuela and Vietnam [40], *C. canadensis* [39], and *C. sumatrensis* [41]. However, in the only stated study of EO of *E. bonariensis* [34], α-bergamotene was described as the main compound in addition to some farnesene derivatives such as, *β*-farnesene, and (*E*)-farnesene epoxide. The abundance of α-maaliene (6.64%), berkheyaradulen (4.82%), and α-muurolene (3.99%) were found in perfect harmony with our results of EO of *P. dioscoridis*. The variations of secondary metabolites comprising EOs might be attributed to the plant age and development, plant organs, as well as the environmental factors including such as altitude, seasonality, atmospheric composition and temperature, and water availability [11,42,43].

Monoterpenes represented a remarkable concentration of the EO of *E. bonariensis* with a wealth of *O*-ocimene (12.58%). *O*-Ocimene was reported here for the first time in EO of this plant, contrariwise, Harraz, et al. [34] reported the complete absence of it from EO of the aerial parts of this plant collected from Alexandria, Egypt.

The diterpenoids were represented by a relative concentration of 2.44% from over all mass of the oil of *E. bonariensis*. The total relative concentration of diterpenes was determined in the EO of *E. bonariensis* with only one compound, neophytadiene, which is not reported before from the EO of this plant [34].

Carotenoid derived compounds were not identified from the EO of *E. bonariensis* and this result is in harmony with Mabrouk, et al. [37]; also, hydrocarbons and the other components were represented by traces in EO of *E. bonariensis* (0.55%) that agreed the previous described studies [32,39].

#### *2.3. Chemometric Analysis*

The EOs chemical compositions of the major compounds (>3%), reported from different ecospecies of *P. dioscoridis* and *E. bonariensis* were constructed in a matrix. These collected data were subjected to agglomerative hierarchical clustering (AHC) and principal component analysis (PCA). The cluster analysis of *P. dioscoridis* EOs showed that the present studied sample of *P. dioscoridis* is closely correlated to the Egyptian ecospecies collected from El-Sadat City, and little correlated to that collected from Cairo–Suez desert road, Egypt (Figure 3a). However, the present sample was different than those purchased from a commercial source in Cairo, Egypt. This means that the commercial samples are not in pure form or may be mixed with other plants.

**Figure 3.** Chemometric analysis of the EOs from the present studied *Pluchea dioscoridis* ecospecies and other reported ecospecies. (**a**) agglomerative hierarchical clustering (AHC) and (**b**) principal component analysis (PCA).

The PCA of the *P. dioscoridis* ecospecies showed that the sample collected from Cairo-Suez desert road, Egypt is mainly characterized by farnesol, germacene d-4-ol, and longifolene (Figure 3b). However, the purchased sample from a commercial source in Cairo, Egypt is characterized by hexadecanoic acid and *α*-cadinol.

On the other side, the cluster analysis of *E. bonariensis* EOs revealed that the present Egyptian sample is closely related to the Venezuelan ecospecies, while it was different than other ecospecies (Figure 4a).

**Figure 4.** Chemometric analysis of the EOs from the present studied *Erigeron bonariensis* ecospecies and other reported ecospecies. (**a**) agglomerative hierarchical clustering (AHC) and (**b**) principal component analysis (PCA).

While the Indian and Tunisian ecospecies showed a close relation in the composition of the EO. The PCA showed that the present sample of *E. bonariensis* is characterized by *trans-α*-Farnesene, O-ocimene, and *trans-β*-Farnesene (Figure 4b). The sample collected from Alexandria, Egypt, showed a close correlation with *α*-bergamotene, limonene, and *α*-curcumene, while the Indian ecospecies is characterized by *β*-eudesmol, caryophyllene oxide, allo-aromadendrene, and carvacrol.

The observed variation among the present samples and other reported ones revealed the profitable information derived from the EOs analysis, which could be a useful tool in chemotaxonomy [27].
