**2. Results**

#### *2.1. Chemical Composition of Essential Oils*

Thyme and oregano essential oils are, generally, characterized by a large amount of monoterpenes (both hydrocarbons and oxygenated), reaching almost 90% of the whole oil. The two main phenolic monoterpenes, thymol and carvacrol, occur more frequently, often accompanied by their biogenetically correlated compounds *p*-cymene and γ-terpinene [13,24]. Thymus is probably one of the most taxonomically complex genera of its family. Several studies confirmed that the two most common chemotypes are thymol and carvacrol [25],

followed by less common non-phenolic chemotypes [26]. As reported in materials and methods section, the two EO samples of this study were commercial. The chemical composition of our oregano sample has already been published in a previous study with different purposes from this one [27], while the chemical composition of our commercial thyme EO is published here for the first time. The two analytical results are listed together in Table 1 for easier comparison by readers. The samples subjected to this study had chemical profiles quite typical of the *Origanum vulgare* thymol and carvacrol chemotype and the *Thymus capitatus* carvacrol chemotype of essential oils. Gas chromatography (GC) techniques, coupled with a flame ionization detector (FID) and a mass spectrometer (MS), allowed the identification of more than 35 components, covering up to 99% and 98% of the total oil compositions, respectively. Table 1 shows the details of the chemical compositions, listing only the components with a % > 0.05. The most represented class for both samples was that of oxygenated monoterpenes (68.7% and 72.9%, respectively), followed by hydrocarbon monoterpenes (30.0% and 22.3%, respectively). The sum of these classes in the oregano sample reached 96.7%, while in the thyme sample it reached 95.1%. For both samples, sesquiterpenes and other components were below 4%. The common feature of these two oils was to have three main components which alone covered more than 80% of the whole composition. The oregano essential oil profile was characterized by the presence of carvacrol (36%) and thymol (25%) as the main compounds, followed by *p*-cymene (22%). All other compounds were below 5%. The thyme essential oil profile was dominated by a high amount of carvacrol (almost 70%), followed by *p*-cymene (9%) and γ-terpinene (8%). The thymol percentage was negligible (0.5%), as were the percentages of all other compounds, with the exception of β-caryophyllene, which reached 2.6%.

**Table 1.** Chemical composition of commercial *Origanum vulgare* and *Thymus capitatus* essential oils (EOs).



**Table 1.** *Cont.*

a The numbering refers to the elution order. b Literature retention index (RI). c Retention index (RI) relative to the standard mixture of *n*-alkanes on the SPB-5 column. d Identified compounds (those < 0.05% have not been reported). e Data previously reported (see [27]). f Relative peak area percent, representing the averages of three determinations. N.D. = not detected.

#### *2.2. Physicochemical Characterization of Essential Oil-Loaded Nanocapsules (EO-NCs)*

In a previous work, we reported the encapsulation of thyme and oregano essential oils in PCL nanocapsules [28]. Unlike the previous work, here we used commercial essential oils having different compositions of volatile components. This offered numerus advantages, including the lower cost of preparation and the standardization in the compositions of the EOs. In this manner, the obtained nanocapsules could potentially be more attractive for industrial sectors. Table 2 shows the reported values of the physicochemical parameters (z-average diameter, polydispersity index (PDI), zeta potential (*ζ*), encapsulation efficiency (EE) and loading capacity (LC)) which allowed us to characterize the nanocapsules containing thyme and oregano essential oils (TC-NCs and OV-NCs, respectively).

**Table 2.** Physicochemical Characterization of essential oil (EO)-NCs.


The z-average diameters of 198 ± 3 nm and 200 ± 3 nm for the TC-NCs and OV-NCs, respectively, were in agreemen<sup>t</sup> with the nanometric structures of the nanocapsules. Low PDI values for both nanocapsules pointed out a narrow size distribution of nanoparticles and the presence in aqueous solution of monodisperse nanosystems (Figure 1). Negative zeta potentials of −11 and −10 mV for the TC-NCs and OV-NCs, respectively, were very similar to those obtained for other stable PCL nanocapsules [28,29]. The percentages of EE and LC were high, with values of 84 ± 6 and 52 ± 3 for the TC-NCs and 80 ± 9 and 51 ± 4 for the OV-NCs. The prepared TC-NC and OV-NC aqueous nanosuspensions showed a total essential oil content of 5.7 ± 0.3 and 5.8 ± 0.4 mg/mL, respectively.

**Figure 1.** I-weighted distribution of the hydrodynamic diameter (*DH*) of (**a**) OV-NCs and (**b**) TC-NCs.

#### *2.3. Antifungal Activities of Pure EOs and EO-NCs*

The screening of encapsulated essential oils (EO-NCs) against a panel of fungal strains (Table 3) showed marked antifungal activity (Table 4 and Figure 2). The obtained results evidenced that EO-NCs inhibited the growth of assayed fungi at s MIC values ranging from 0.125 to 0.25 mg/mL. These concentrations were two to four times lower than those observed for pure EOs. For both EO-NCs, an MIC of 0.125 mg/mL was effective for inhibiting the growth of *A. fumigatus*, *C. aggregatocicatricatum*, *C. herbarum* and *P. eryngii*. In addition, this concentration for OV-NCs was also enough to inhibit the fungus *Bjerkandera adusta*.

#### **Table 3.** Characteristics of the assayed fungal strains.


IMB-SAS = fungal collection of the Institute of Molecular Biology (Slovak Academy of Sciences). CCBAS = Culture collection of Basidiomycetes, Institute of Microbiology, Academy of Sciences of the Czech Republic.

We also evaluated the fungicidal effect of EO-NCs versus the panel of fourteen fungal strains. In particular, both the TC-NCs and OV-NCs had minimum fungicidal concentrations (MFCs) of 0.25 mg/mL against *A. fumigatus*, *C. aggregatocicatricatum*, *C. herbarum* and *P. eryngii*, while having an MFC of 0.5 mg/mL against *A. flavus*, *P. rubens*, *P. citrinum*, *F. oxysporum*, *G. candidum*, *M. circinelloides*, *E. xenobiotica*, *P. lilacinum* and *P. chrysosporium*. The MFC values of the EOs were two to four times higher than the MFCs of the EO-NCs on the tested strains. The empty NCs revealed no activity on the fungal growth.


**Table 4.** Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of encapsulated essential oils and pure essential oils.

MIC and MFC are expressed in mg/mL. TC-NCs = nanoencapsulated thyme essential oil; OV-NCs = nanoencapsulated oregano essential oil; TC-EO = thyme essential oil; and OV-EO = oregano essential oil.

**Figure 2.** Example of MIC and MFC assays with the isolate *Cladosporium herbarum*. (**A**) Results using a nanoencapsulated oregano EO. (**B**) Results using a nanoencapsulated thyme EO.C=control. The range of nanoencapsulated EO concentrations used was 0.05–0.5 mg/mL.
