*2.4. Anti-Aging Activity*

The EOs from *P. dioscoridis*, *E. bonariensis*, and the mixture of the two EOs (1:1) have a strong inhibitory activity of the collagenase, elastase, hyaluronidase, and tyrosinase (Figure 5). All the EO treatments exhibited potent inhibition of collagenase enzyme with IC50 of 1.85, 2.90, and 1.73 μg/mL for *P. dioscoridis*, *E. bonariensis,* and the mixture, respectively. Furthermore, the three EO treatments strongly inhibit the elastase enzyme with respective values of IC50 of 14.63, 16.52, and 11.01 μg/mL. Furthermore, strong suppression of hyaluronidase was demonstrated via the three EO treatments based upon the respective observed values of IC50 of 17.18, 15.16, and 13.54 μg/mL. By the same, the three tested displayed strong tyrosinase enzyme inhibition with IC50 values at 19.52, 18.93, and 15.81 μg/mL, respectively. All the results were constructed based upon comparing with the polyphenolic compound, epigallocatechin gallate (EGCG), as a standard ant-aging reference [44] that exhibit inhibition of collagenase, elastase, hyaluronidase, and tyrosinase with IC50 of 1.56, 10.29, 12.71, and 14.37 μg/mL.

**Figure 5.** Anti-aging activities of the EOs extracted from *Pluchea dioscoridis* and *Erigeron bonariensis* against the four enzymes: collagenase, elastase, hyaluronidase, and tyrosinase. Values are IC50 (μg/mL) as an average of three replicates and the bars representing the standard deviation. Different letters (A, B, and C) within each enzyme mean values significant at 0.05 probability level after Duncan's test.

In the matrix of extracellular, the elastin and hyaluronan degradation were principally correlated with the two respective proteolytic enzymes, elastase, and hyaluronidase that cause the main reasons for aging of the skin such as wrinkles, sagging. Moreover, tyrosinase caused the regulation of the synthesis of melanin in human melanocytes that lead to skin ailments.

In the present study, the anti-aging of the 1:1 mixture of the two EOs was evaluated to study the synergetic effects of the combination of the two EOs. Results revealed that the EO of *P. dioscoridis*, *E. bonariensis,* and the mixture of the two EOs (1:1) have strong anti-aging activity. These results might be attributed to the chemical components of these oils. The anti-aging activity was directly correlated with antioxidant potentiality [45]. The main constituents in both EOs, sesquiterpenes, were described to play a significant role as antioxidants, anti-inflammatory agents, and thus anti-aging [45]. Tu and Tawata [45] reported that EO of the leaves of *Alpinia zerumbet* exhibit antioxidant and anti-aging activities due to the high concentration of terpenoids, especially sesquiterpenes. Furthermore, the monoterpenes were documented as active anti-aging agents in EO of *Juniperus communis* [46] and *Origanum vulgare* [47]. These reports concluded that the increase of free radical scavenging constituents in EOs lead to an increase in their anti-aging activity. Based upon this fact, the high concentrations of terpenes especially the oxygenated sesqui- and monoterpenes caused increasing in the anti-aging activity of EOs of these two plants. All these reported data deduced the role of synergetic effects between the components of the EOs. This fact of the role of synergetic effect was very clear in our results in which the mixture of the two EOs (1:1) exhibited better activity than the individual EO of each plant. In the mixture of the two EOs, the raising of concentration of oxygenated terpenes as well the synergetic effects between the components caused increasing of the inhibition potentiality.

#### *2.5. Cytotoxic Activity of EOs of P. dioscoridis and E. bonariensis*

The cytotoxicity of EOs of the above-ground parts of the two plants, *P. dioscoridis* and *E. bonariensis*, as well as a mixture of the two EOs (1:1) against the three cancer cell lines, breast adenocarcinoma cells (MCF-7), lung cancer cells (A-549), and hepatocellular carcinoma cells (HepG2) are shown in Figure 6. The results exhibited that the EO of *P. dioscoridis* have a significant inhibition of the two cancer cells, MCF-7 and A-549, with IC50 of 37.3 and 22.3 μM, respectively (Figure 6A,B), without any activity against HepG2. While, the EO of the *E. bonariensis* showed inhibitory potentiality only against HepG2 with IC50 of 25.6 μM (Figure 6C), with negative results against MCF-7 and A-549. The 1:1 mixture of the two EOs did not exhibit any activity against the three cancer cells.

**Figure 6.** Cytotoxicity of EOs of (**A**) *Pluchea dioscoridis* against MCF-7 cells, (**B**) *P. dioscoridis* against A-549 cells, and (**C**) *Erigeron bonariensis* against HepG2.

The significant activities of the two EOs might be attributed to the chemical composition in which the synergetic effect of the compounds contributes to this activity [48]. The sesquiterpenes in both forms, oxygenated and hydrocarbons, represented very effective compounds as anticancer leaders [49,50]. Several reports deduced that the increasing of sesquiterpene contents in EOs caused increasing in anticancer activity [51,52]. For example, caryophyllene with high concentration in EOs was reported as a known potential cytotoxic agent especially against the growth of breast adenocarcinoma cells (MCF-7) [53,54].

The present data revealed that these two EOs are selective against the tested cancer cells. This selectivity was in full agreement with several documented results of EOs derived from other plants. For example, EO derived from *Sideritis perfoliata*, *Satureia thymbra*, *Salvia officinalis*, *Laurus nobilis,* and *Pistacia palestina* were found to have selective inhibitory effects against, amelanotic melanoma (C32), renal celladenocarcinoma (ACHN), hormonedependent prostatecarcinoma (LNCaP), and breast cancer (MCF-7) [55]. Moreover, EOs extracted from the three plants, *Satureja montana*, *Coriandrum sativum,* and *Ocimum basilicum*, were found to have selective cytotoxic activity against HeLa, MDA-MB-453, K562, and MRC-5 [56]. The disappearance of the mixtures of the two EOs (1:1) might be ascribed to the negative synergetic effects of each EO upon the other and this phenomenon was reported in some reports. Haroun and Al-Kayali [57] found that the different extracts of *Thymbra spicata* showed positive synergetic effects via combination with some references antibiotics against some strains of bacteria and a while negative synergetic effects against other strains.
