**2. Results**

*2.1. GC-MS and Gravimetric Analyses*

The chromatographic analysis of EOs shows phytocomplexes that are quite different (Table 1). *Lavandula angustifolia* has linalyl acetate and β-linalool at respective concentrations of 33.35% and 28.36%, while *L. intermedia* EO has the same components at concentrations of 36.47% and 27.99%, respectively. The EO of *Origanum hirtum* is mainly characterized by thymol, γ-terpinene and p-cymene at 36.3%, 23.81% and 18.83%, respectively, while the EO of *Satureja montana* has carvacrol as a major compound (concentration of 63.1%), followed by γ-terpinene (concentration of 13.44%). Both *Monarda didyma* and *M. fistulosa* EOs show carvacrol (20.59% and 35.18%, respectively) and γ-terpinene (13.07% and 16.85%, respectively) as major compounds, while thymol and *p*-cymene are the third most concentrated components in the respective *M. didyma* and *M. fistulosa*. The rest of the components present in EOs show concentrations lower than 10%.

The analysis of Hy (Table 2) shows β-linalool, α-terpinen-4-ol and α-terpineol (42.5%, 20.33 and 19.1%, respectively) as major chemical compounds of *L. angustifolia* Hy. *L. intermedia* Hy is characterized by β-linalool, camphor and 1,8-cineol (34.17%, 22.12% and 19.08%, respectively) as major compounds, while *S. montana* has carvacrol and thymol as the major compounds (85.79% and 13.88%, respectively). *O. hirtum* Hy has only one component, thymol (100% concentration).

*M. didyma* has carvacrol and thymol (48.44% and 34.03%, respectively) as major compounds, while *M. fistulosa* has only carvacrol (84.68%) at a concentration above 10%. All the other components show a concentration lower than 10%. It is important to remember that the concentrations of chemicals identified in the Hys are referred at most to 1 g/L, which is the maximum terpenes concentration present in Hy. Results of the gravimetric analyses are shown in Table 2. The qualitative and quantitative analyses of the extract obtained for the gravimetric analysis are not shown because they are redundant and perfectly superimposable to those obtained from the gas-chromatographic analysis.


**Table 1.** Chemical composition of EOs.


**Table 1.** *Cont.*

Note. RI = Retention Indices. SD < 5%, RI-E = RI experimentally determined, RI-L = RI determined through Libraries.


Note. RI = Retention indices. a Values are expressed as % (*w*/*w*). SD < 5%, RI-E = RI experimentally determined, RI-L = RI determined through Libraries.

#### *2.2. Broth Microdilution Susceptibility Test*

Table 3 shows the Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC) of the tested EOs. The table also displays the values of Inhibition Rate or Lethal Rate of 90% (IR90 and LR90, respectively) of strains. The EOs of *S. montana* and *O. hirtum* are the most active, showing IR90 values of 0.25% and 1 % *<sup>v</sup>*/*<sup>v</sup>*, respectively, and LR90 values of 0.25% *v*/*v* and 1% *<sup>v</sup>*/*<sup>v</sup>*, respectively. All the other EOs have IR90 and LR90 values greater than or equal to 2% *<sup>v</sup>*/*<sup>v</sup>*, except *M. didyma* EO showing IR90 and LR90 values equal to 1% *v*/*v* and > 2% *<sup>v</sup>*/*<sup>v</sup>*, respectively. Specifically, while the EO of *S. montana* acts in equal measure on all three microbial types (bacteria, yeasts, and dermatophytes), the EO of *O. hirtum* acts primarily on bacteria and yeasts, while that of *M. fistulosa* on dermatophytes.


**Table 3.** Inhibitory and lethal activities of EOs.

Note. D = Designation, IR90= Inhibition Rate of 90% of strains, LR90 = Lethal Rate of 90% of strains, IR50 = Inhibition Rate of 50% of strains, LR50 = Lethal Rate of 50% of strains, LA = *Lavandula angustifolia,* LI=*Lavandula intermedia,* OH = *Origanum hirtum,* SM = *Satureja montana,* MD = *Monarda didyma,* MF = *Monarda fistulosa.*

> As shown in Table 4, values obtained from the analysis of the antimicrobial effectiveness of the Hys indicate the Hys of *O. hirtum* and *M. didyma* (IR90 value 50% *v*/*v*) as more active than the others against bacteria, yeas<sup>t</sup> and dermatophytes. However, it was not possible to study Hys concentrations greater than 50% *<sup>v</sup>*/*<sup>v</sup>*, as this would have introduced a significant methodological bias by reducing the amount of nutrient broth necessary for microbial growth.

**Table 4.** Inhibitory and lethal activities of Hys.



**Table 4.** *Cont.*

Note: D = Designation, IR90 = Inhibition Rate of 90% of strains, LR90 = Lethal Rate of 90% of strains, IR50 = Inhibition Rate of 50% of strains, LR50 = Lethal Rate of 50% of strains, LA = *Lavandula angustifolia,* LI = *Lavandula intermedia,* OH = *Origanum hirtum,* SM = *Satureja montana,* MD = *Monarda didyma,* MF = *Monarda fistulosa.*

> In particular, the *O. hirtum* Hy at a concentration of 50% *v*/*v* is the only one that can inhibit all bacteria growth but is unable to exert cytocidal effect at the same concentration, while fungi (yeast and dermatophytes) show greater sensitivity to Hys (Table 3). Specifically, the Hys of *S. montana*, *O. hirtum* and *M. didyma* have inhibitory and cytocidal effect against most dermatophytes at a concentration equal to 50% *<sup>v</sup>*/*<sup>v</sup>*, and only *M. fistulosa* is able to inhibit all strains at a concentration of 25% *v*/*v,* but it is not capable of having cytocidal effects for values <50% *<sup>v</sup>*/*<sup>v</sup>*.

#### *2.3. Comparison Between EOs and Hys*

Table 5 shows the values of the peaks' total areas of the chemicals of both EOs (EOTA) and Hys (HYTA), the volatiles' Conversion Factor (CF) obtained as EOTA/ HYTA, and the value of the IR50Hy/CF ratio. This last parameter indicates the value that the IR50Hy would have if the Hy were concentrated as the EO. As shown in Table 5, the value of the IR50Hy/CF ratio is lower than that of IR50Eo for all the EOs.

**Table 5.** Volatile concentrations in EOs and HYs, their relationships, and IR50 comparison at equivalent volatile concentrations.


Note: EOTA = Essential Oil Total volatiles Area, HYTA = Hydrolate Total volatiles Area, CF= volatiles' Conversion Factor.

This means that, to have the same antimicrobial activity in the EO, a relative concentration of volatiles between 1.11 (*S. montana*) and 71.43 (*M. fistulosa*) times as high as that contained in the Hy is required.

The same difference is evidenced in the activity of EO and Hy against each microbial strain. Table 6 shows the concentration of EOs and Hys necessary to obtain the Inhibitory concentration of the 50% (IC50) of the initial inoculum, and the IC50Hy/CF ratio that is the IC50Hy value normalized according to the volatiles' concentration. IC50 values were obtained, starting from the inhibition curve calculated using OD450 values obtained from the micro-broth dilution test. In Table 6, values of dermatophytes are not reported. In fact, due to the inhomogeneity of their growth, they were only evaluated by visual reading, as specified in "Material and Methods section". Additionally, in this case, the visual exam points out that IC50Hy/CF ratios are significantly lower than the respective IC50EO values.



#### *Antibiotics* **2021**, *10*, 88

More generally, the average values of IC50Hy/CF and IC50EO, calculated on four bacterial strains (excluding 01SA(R) and 0.6EF strains) and four yeasts, indicate that the two distillation products (EO and Hy) from *S. montana* show the smallest differences in terms of effectiveness related to volatiles concentrations: in the average of the eight cases, an amount of the EO 8.2 times as concentrated as that of the Hy is needed to attain the same inhibition of microbial growth. However, products obtained from the *O. hirtum* and *Monarda* genus illustrate the greatest difference in terms of the biological activity related to the volatiles' concentration. In fact, a quantity of *O. hirtum*, *M. didyma* and *M. fistulosa* EOs, respectively, 5.7, 16 and 42.3 times as concentrated as the corresponding Hys is necessary. In this respect, the IC50 comparison between EOs and Hys outlines the same ranking as the IR50 comparison between EOs and Hys (Table 5), strengthening the differences in efficacy between the two distillation products.
