3.1. Essential Oil Content
Our results showed different results for the EO depending on the elicitor, its dosage, and the plant species.
Figure 2 represents the EO content expressed by mL/100 g of dry material.
In marjoram, higher dosages both of MeJa and SA were significantly more effective than lower dosages, but none of them differed statistically from the control, except for the 2 mM of MeJa. The latter and 2 mM of SA increased the EO content by 23% and 15% respectively.
In peppermint, similarly, the higher dosage of SA resulted in a slight increase of the EO content, while the lower (0.1 mM) dosage was not effective. However, treatments with MeJa seem to stop the accumulation of volatiles, interestingly. Especially the 0.1 mM treatment caused a significant decrease.
Hyssop also showed greater sensitivity to SA than to MeJa. The highest EO content (0.35 mL/100 g d.w.) was measured in the samples sprayed with 0.1 mM of SA. The higher dosage of it also elevated EO content but was not significant. Spraying with MeJa in either concentration had no significant elicitor effect on the volatiles of this species.
Basil showed a distinct reaction to the treatments compared to the previously mentioned species, as the EO content dropped in consequence of each treatment, except for the 2 mM of MeJa. Especially the higher dosage of SA resulted in a significant decrease of the volatile accumulation, by approximately 20%.
Our findings partly agree with a few studies where the application of different dosages of SA or MeJa/JA did not have a significant difference on the accumulation of EO in other
Lamiaceae species, such as summer savory (
Satureja hortensis) and thyme (
Thymus daenensis Celak) [
23,
24].
Yadegari et al. [
25] showed that a low dosage of SA (0.1 mM) failed to enhance the EO production of sage (
Salvia officinalis) in contrast to higher dosages. At the same time, elicitation with 0.1 mM of MeJa was effective for increasing the EO percentage in anise hyssop (
Agastache foeniculum) [
26].
3.2. Essential Oil Composition
The results of GC-MS analysis of marjoram in
Table 4 revealed the presence of 15 compounds reaching 97.4–98.6% of the total area percentages were identified in the EO. in each sample,
cis-sabinene hydrate and terpinen-4-ol were detected as major components, however, their ratios changed according to the treatments.
The concentration of the sabinene hydrate isomers decreased as a consequence of all treatments, among which significant differences of 22% and 16% were detected in the ratio of cis-sabinene hydrate due to the spraying with 0.1 mM and 2 mM of SA, respectively. At the same time, the ratio of terpinen-4-ol was increased by all treatments and significant (30%) elevation was registered at the higher dosage of SA. Furthermore, the two sesquiterpenes identified in the samples both increased due to the spraying of both dosages of MeJa and 0.1 mM of SA. The highest concentration of beta-caryophyllene was obtained by 0.1 mM SA, while the same dosage increased bicyclogermacrene by approximately 45%. As a result, the ratio of total sesquiterpenes in the oil was elevated, too.
The findings about the elicitation of marjoram with SA in Egypt were partly in accordance with ours: the treatment of 0.1 mM of SA increased the ratio of both
cis-sabinene hydrate and terpinen-4-ol in the EO [
27]. Interestingly, elicitation by the application of compost (Nitrogen-fixers +
Bacillus circulans) had the same results as those of our study: ratio of the sabinene hydrate decreased, but that of terpinen-4-ol was elevated [
28].
The statistical analysis of the EO composition of peppermint in
Table 5 revealed that in the ratio of the main compounds menthol and menthone, there are no significant differences in the treated samples compared with the control (
Table 5). Only the ratio of pulegone was reduced after spraying 2 mM of SA by approximately 43%, which seems to be advantageous considering the EU regulation on limits for pulegone and menthofuran [
29]. Yet, in another experiment, 0.1 mM of SA was able to increase the menthol and the menthyl acetate concentration significantly, when applied exogenously to peppermint [
30]. On the other hand, it appears that a physical elicitor, such as UV-B may have more effect on the peppermint EO quality, where UV radiation greatly increased methofuran, menthyl acetate, and menthone but significantly decreased the menthol level. This can be a serious drawback due to the quality requirements and importance of the menthol compound in the industry [
31]. In our experiment, the treatments likewise did not cause significant changes in the total mono and sesquiterpene ratio.
The EO analysis of hyssop in
Table 6 reveals that the ratio of β-phellandrene decreased significantly, by 46% with 0.1 mM SA, however, the other treatments had no significant effects. Pentylbenzen and the main component isopinocamphone changed significantly due to the SA treatments. Their concentrations were elevated by 65% and 60% respectively when 0.1 mM was applied, and by 39 and 28% with 2 mM of SA. Interestingly, MeJA in 2 mM concentration also elevated the ratios of the two compounds mentioned, by the same rate. The highest percentage of isopinocamphone was detected by 0.1 mM SA, around 46% which may be advantageous considering the biological activities and wide use of this compound in perfumery and cosmetics [
32,
33]. SA was proven in other studies as well to be an important elicitor in increasing the main EO components in
Achillea millefolium L. [
34],
Citrus aurantium L. [
35] and
Melissa officinalis [
36]. Among sesquiterpenes, β-bisabolol was reduced after spraying 2 mM of MeJa and 0.1 mM of SA by 66% and 65%, respectively. Overall, our results demonstrated a significant decrease in total sesquiterpenes with both dosages of MeJa and 0.1 mM of SA, which contradicts a previous report that SA was able to stimulate the production of sesquiterpenes of hyssop [
37].
Table 7 shows the result of the CG-MS analysis of the basil EO. It can be established that there was no significant difference for the major component linalool after the application of either of the elicitors. Among monoterpenes, only 1.8-cineole and iso-bornyl acetate changed significantly.
The first compound fell by 48% due to the 0.1 mM SA treatment, while the second one rose with 2.0 mM MeJa but dropped with 2.0 mM SA. Sesquiterpenes were characteristically more strongly affected. Both α-guaiene and bicyclogermacrene levels were enhanced with the lower dosages of both elicitors; 0.1 mM MeJa and 0.1 mM SA increased α-guaiene by 42% and 44%, respectively; and bicyclogermacrene was 70% and 72%, respectively. Besides, 0.1 mM SA was able to increase β-elemene and bicyclogermacrene by 61% and 72%, respectively. In parallel, the same lower dosage of MeJa reduced the ratio of
trans-α-bergamotene compound by 38%, while 0.1 mM SA increased it approximately by 24%. Based on the above, the present result could not support the large increase (113%) in the ratio of linalool following application of 0.1 mM of SA, a result which was registered in a previous experiment under hydroponic conditions [
38].
Some other reports also indicate different results with basil, depending on the elicitor and the cultivar. A concentration of 0.5 mM MeJa was proven to increase linalool level in the Genovese cultivar under salinity stress, while it decreased the same component in the Rubi cultivar [
39]. Elicitation with copper sulfate in vitro significantly increased eugenol while inducing some compounds that were absent in the untreated samples [
40]. In our study, no qualitative differences were detected in the spectrum. The total ratio was shifted significantly in the direction of monoterpenes due to the higher dosage of MeJa; and was shifted in the opposite direction by increasing the sesquiterpenes after the lower dosage of SA.
3.3. Total Phenolic Content
The effect of treatments on the total phenolic content (TPC) of the four species is demonstrated in the bar graph below (
Figure 3). TPC is expressed by mg GAE/g d.w. of the. After the treatments, significant differences were observed. The TPC of marjoram ranged between 200 and 360 mg GAE/g d.w., the lowest one with foliar application of 2 mM of MeJa and the highest one with 0.1 mM of SA. This latter treatment increased the TPC by approximately 36% compared to the control. Various studies have mentioned the elicitation effect of MeJa and JA in enhancing bioactive compounds including phenolic compounds and flavonoids in different plant species, such as butter lettuce (
Lactuca sativa L.), melon (
Cucumis melo), and Saint John’s wort (
Hypericum perforatum), whether applied in vivo or in vitro [
41,
42,
43]. However, Zlotek observed the failure of MeJa to change the TPC especially in marjoram [
44]. In our study, the 0.1 mM and 2 mM of MeJa decreased the content by approximately 27% and 31%, respectively.
In the case of peppermint, both SA dosages elevated the TPC, while treatment with MeJa did not result in any significant changes. The effect of SA on the phenolic accumulation in peppermint was also previously observed, where increasing the TPC by 65% and 31% was reached after treatment with the dosages of 0.5 and 2 mM, respectively [
45].
As for hyssop and basil, the applied elicitors had contradictory results. All the treatments decreased the TPC in hyssop significantly, and the sample treated with 2 mM of MeJa was lower than the control by 28%. However, in basil, the treatments increased the TPC and the concentration 0.1 mM of MeJa was the most effective. The latter findings are partly in agreement with Kim et al. [
46] where the 0.1 mM of MeJa could not increase the TPC in basil, but a higher dosage (0.5 mM of MeJa) was effective.
3.4. Antioxidant Capacity
The effect of treatments on the AC of the four species (expressed by mg AAE/g d.w.) is demonstrated in
Figure 4. The results closely correlate with the TPC data in all species and for all treatments. For both marjoram and hyssop, the treatments decreased the AC except for the lower dosage of SA treatment in marjoram, which showed a 50% stronger activity than the control. In hyssop, each treatment decreased the AC except for the lower dosage of MeJa. This is in contradiction to several reports about the effect of MeJa in elevating the AC; not only in healthy plants but also in plants subjected to water deficit stress, where this phytohormone presumably enhances the protection mechanisms [
42,
47,
48].
Similarly, to these publications, in the case of peppermint, both dosages of SA were able to increase the AC in our experiment also, where the 2 mM dosage resulted in the highest activity. This result is similar to that of Figueroa Pérez et al. [
45] where different dosages of SA ranging between 0.5 and 2 mM increased the AC. As for MeJa treatments, no significant differences were registered.
SA is a plant hormone that plays a pivotal role in regulating physiological and biosynthetic processes. If applied exogenously, SA triggers a hypersensitive response by causing a temporary increase of reactive oxygen species (ROS) followed by phenolic compounds, biosynthesis and elevated antioxidant activity [
49,
50]. It was found that MeJa is effective only in basil for stimulating the accumulation of phenolic compounds and increasing AC. A considerable amount of literature supports the contribution of polyphenols in lowering the risk of health disorders, such as cancer, cardiovascular diseases, chronic inflammations, degenerative diseases, and diabetes. This is because of their strong antioxidant activity and ability to scavenge free radicals, thus reducing oxidative damage [
51,
52].
For basil, all treatments increased the AC but only 0.1 mM of MeJa and 2 mM of SA were significant, where they raised AC by 40% and 35%, respectively. The results of Wang et al. [
53] and Blanch et al. [
54] support these findings, as they found that both SA and MeJa increased the AC in blackberries (
Rubus sp.) and table grapes (
Vitis vinifera).