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Article

Nanovesicles Loaded with Origanum onites and Satureja thymbra Essential Oils and Their Activity against Food-Borne Pathogens and Spoilage Microorganisms

1
Department of Chemistry “Ugo Schiff”, University of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy
2
Faculty of Pharmacy, Department of Pharmacognosy & Chemistry of Natural Products, National & Kapodistrian University of Athens, Panepistimiopolis, Zografou, 157 71 Athens, Greece
3
Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”—National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Molecules 2021, 26(8), 2124; https://doi.org/10.3390/molecules26082124
Submission received: 15 March 2021 / Revised: 30 March 2021 / Accepted: 3 April 2021 / Published: 7 April 2021

Abstract

:
Food poisoning is a common cause of illness and death in developing countries. Essential oils (EOs) could be effective and safe natural preservatives to prevent and control bacterial contamination of foods. However, their high sensitivity and strong flavor limit their application and biological effectiveness. The aim of this study was firstly the chemical analysis and the antimicrobial evaluation of the EOs of Origanum onites L. and Satureja thymbra L. obtained from Symi island (Greece), and, secondly, the formulation of propylene glycol-nanovesicles loaded with these EOs to improve their antimicrobial properties. The EOs were analyzed by GC-MS and their chemical contents are presented herein. Different nanovesicles were formulated with small average sizes, high homogeneity, and optimal ζ-potential. Microscopic observation confirmed their small and spherical shape. Antibacterial and antifungal activities of the formulated EOs were evaluated against food-borne pathogens and spoilage microorganisms compared to pure EOs. Propylene glycol-nanovesicles loaded with O. onites EO were found to be the most active formulation against all tested strains. Additionally, in vitro studies on the HaCaT cell line showed that nanovesicles encapsulated with EOs had no toxic effect. The present study revealed that both EOs can be used as alternative sanitizers and preservatives in the food industry, and that their formulation in nanovesicles can provide a suitable approach as food-grade delivery system.

Graphical Abstract

1. Introduction

Lamiaceae is a well-known family of aromatic herbs, diffused in many regions of the world. Numerous plants that belong to this family are used for medicinal purposes and in perfumery, and they are extensively applied to impart flavor and aroma to foods. Most of them are rich in essential oils (EOs) [1,2] which consist of complex mixtures of volatile, liquid, odorous, flavorsome and strongly active compounds. Due to their various biological properties, principally antioxidant and antimicrobial, they have been widely used since the Middle Ages and currently, they may have several applications in different fields, from medicine and cosmetics to food. However, their high volatility and low stability to direct exposure to light, oxygen, heat, and humidity can limit their potential.
Nanocarriers represent an innovative challenge to optimize the essential oil formulation, overcoming the main limitations [3]. Liposomes are vesicles containing an aqueous phase entirely surrounded by a bilayer constituted of phospholipids and cholesterol, which can definitely load the essential oils. The resulting nanocarrier is stable and efficient, easily and safely produced, capable of stabilizing the essential oil, modulating its release and optimizing its activity [4]. Recently, a commercial Melissa officinalis L. essential oil was successfully formulated in glycerosomes, being very active in vitro in inhibiting HSV type 1 infection of mammalian cells, without producing cytotoxic effects [5]. In addition, Risaliti and coworkers (2019) demonstrated enhanced antioxidant, anti-inflammatory and antimicrobial activities of the essential oils of Rosmarinus officinalis L. and Salvia triloba L. plants, when formulated in liposomes [6]. The same authors further improved antifungal properties against 10 different drug-resistant Candida strains of Artemia annua L. EO loaded in nanoliposomes [7].
EOs obtained from aromatic and medicinal plants, as well as their components have largely demonstrated antibacterial and antifungal properties against a wide range of microbial pathogens, including numerous food-borne pathogens and key spoilage bacteria [8,9]. Food poisoning is considered as one of the most common causes of illness and death in developing countries, which are associated with bacterial contamination especially from Gram-negative bacteria, mainly represented by Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa [10]. Among Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus are the most causative agents of food-borne illnesses or food spoilage [11]. Over recent years, synthetic preservatives are largely used to successfully prevent and control bacterial contamination of food, but their use can result in unwanted chemical residues in food and feed chains. In addition, there is an increasing resistance of these pathogens to the synthetic preservatives [12]. Hence, the discovery of alternative preservatives, mainly originated by natural sources have been increased during recent years. EOs are reported as potentially effective and safe natural food preservatives by many publications [8]. Although EOs possess good antimicrobial properties, they have potential limitations for commercial applications due to their physical and organoleptic properties such as strong flavor, volatility and chemical instability. Consequently, different types of food-grade delivery systems, mainly nano-emulsions [13], but also innovative nanocochleates [14] have now been evaluated for improving their performance as antimicrobials.
Plants of genus Origanum L. and Satureja L. are well-known aromatic herbs, widely used in traditional and modern medicine, as well as in food and cosmetics. Many of them are referred with the common name “oregano” [15]. Among the oregano plants, O. vulgare L. is one of the most popular aromatic species, including six subspecies; O. vulgare subsp. glandulosum (Desf.) Ietsw., O. vulgare subsp. gracile (K. Koch) Ietsw., O. vulgare subsp. hirtum (Link) Ietsw., O. vulgare subsp. virens (Hoffmanns. and Link) Ietsw., O. vulgare subsp. viridulum (Martrin-Donos) Nyman, and O. vulgare subsp. vulgare [16]. Strikingly, the two most commercially important oregano herbs are considered the O. vulgare subsp. hirtum (known as “Greek oregano”) and O. onites L. (known as “Turkish oregano”, “Island oregano” or “Cretan oregano”) [17,18]. The latter herb is a narrowly distributed East Mediterranean species, occurring mainly in Turkey and Greece. The interest for this species is due to the high essential oil yield and carvacrol content (69.0–92.6%) [18,19]. S thymbra L. is distributed in the Mediterranean region and its smell is close to that of oregano [20]. Its essential oil is rich in monoterpene derivatives such as γ-terpinene, p-cymene and carvacrol [21]. Both EOs exert a broad range of pharmacological properties, especially antimicrobial activity, which are attributed to their chemical constituents (i.e., carvacrol, γ-terpinene and p-cymene) [22,23].
In this study, O. onites and S. thymbra EOs were isolated and analyzed by GC-MS, and for the first time, they were formulated in propylene glycol-nanovesicles, proposed as safe and food-grade delivery systems. Different nanovesicles were developed and optimized in terms of size and essential oil loading and were fully characterized as regards physical and chemical parameters. Successively, they were investigated for the antimicrobial activity against a panel of different bacteria and fungi, comparing the activity with that of pure essential oils. The safety profile was assessed by in vitro test on HaCaT cell line.

2. Results and Discussion

2.1. Chemical Composition of OOEO and STEO

The yield (v/w) of the EO obtained from O. onites (OOEO) was 3.0%. Overall, thirty-five compounds, representing 100.0% of the OOEO, were identified. In particular, the main components were carvacrol (66.0%), p-cymene (7.9%), γ-terpinene (4.9%) and borneol (2.8%). Furthermore, reasonable levels of β-bisabolene (2.3%), myrcene (2.1%), α-terpinene (2.0%) and terpinen-4-ol (1.7%) were also detected, while α-pinene and thymol were found in low percentages (both 1.0%) (Table 1). Oxygenated monoterpenes (74.1%) comprised the major chemical group of the OOEO, followed by monoterpene hydrocarbons (21.2%) and sesquiterpene hydrocarbons (4.1%), (Table 1). It is well-known that the OOEO is high rich in carvacrol content (up to 90%) [18,19,23,24,25]. Vokou and coworkers (1988) investigated the EOs of O. onites, originated from different parts of Greece, underlying the high yields of EOs from south-eastern islands (Halki, Symi and Tilos) [26]. Given that different OOEO chemotypes were reported based on their main volatile constituents [23], our studied OOEO revealed a carvacrol chemotype. We should point out that linalool was determined in a low amount (0.4%). Previous studies discussed the distinction among the O. vulgare ssp. hirtum and O. onites, mentioning that the EO of the latter species is poor in thymol and/or p-cymene, whereas its borneol content ranges more than 2.0% [18,27]. Our findings comply with this distinction since thymol concentration was low (1.0%) and borneol was found in higher level (2.8%).
In the EO of S. thymbra (STEO; yield 2.8% v/w) were identified twenty-nine chemical constituents, representing 99.9% of the total amount (Table 2). These constituents were grouped into oxygenated monoterpenes (50.0%), monoterpene hydrocarbons (41.9%), sesquiterpene hydrocarbons (7.7%), oxygenated sesquiterpenes (0.2%) and aliphatic alcohols (0.1%) (Table 2). Precisely, the main compounds were carvacrol (46.0%), γ-terpinene (19.7%), p-cymene (7.6%), β-caryophyllene (7.0%) and α-terpinene (5.1%). Other constituents in lower concentrations were myrcene (2.5%), α-thujene (2.4%), α-pinene (1.6%) and linalool (1.3%). Our results are in accordance with previous studies in EO of S. thymbra [20,22,25,28].
Comparing the two investigated EOs, carvacrol was the dominant component, while thymol was detected in very low concentrations (<1.0%) in both samples. We also observed that the high carvacrol (66.0%) content was related to low amount of γ-terpinene (4.9%) in the EO of O. onites, whereas the EO of S. thymbra demonstrated a relatively lower concentration of carvacrol (46.0%) followed by a high content of γ-terpinene (19.7%). This observation is congruous with a previous study [25] and could be attributed to biosynthetic pathways, considering that γ-terpinene and p-cymene are biosynthetic precursors of carvacrol. In addition, the two samples presented similar chemical groups. Both plants were rich in EOs which is directly related to the distinct environmental and geographical conditions of Symi island which belongs to the Dodecanese island complex (SE Aegean region).

2.2. Development and Optimisation of Nanovesicles Loaded with OOEO and STEO

EOs of O. onites (OOEO) and S. thymbra (STEO) were formulated using diverse nanovesicles. Firstly, OOEO was encapsulated in conventional liposomes using PBS as dispersant medium and phosphatidylcholine (P90G) plus cholesterol in different ratios, trying to optimize average diameter and polydispersity of the vesicles. An amount of 10 mg/mL of OOEO was found to be the optimal concentration to obtain stable formulations without OOEO extrusion from the system, but vesicles resulted not homogeneous. Accordingly, other types of vesicles were approached, in particular those obtained by mixing membrane components with a water-soluble, non-volatile organic solvent, such as a polyol. The resulting nanovesicles are physiologically suitable even when administered intravenously into the human body. In particular, both glycerol and propylene glycol are among the most widely used raw materials in food, cosmetic and pharmaceutical industries. Glycerol shows excellent solubility in water, and, due to its GRAS status, it is very safe to use [29]. Propylene glycol is a colorless, odorless and completely water-soluble solvent very similar to glycerol, also having the GRAS status and high safety [30]. Glycerosomes were prepared according to the recent publication by Vanti and coworkers [5]. Different experimental conditions were tested, as reported in the experimental section. Finally, the lipid film was constituted of P90G (600 mg) plus cholesterol (10 mg). It was hydrated using a 5% v/v glycerol/water solution, and OOEO (10 mg/mL) or STEO (10 mg/mL) or OOEO plus STEO (5 mg/mL plus 5 mg/mL), were added in this step, obtaining O. onites essential oil-loaded glycerosomes (OO-GS), S. thymbra essential oil-loaded glycerosomes (ST-GS) and O. onites plus S. thymbra essential oil-loaded glycerosomes (OOST-GS). In parallel, different nanovesicles were prepared hydrating the lipid film with a 1% v/v propylene glycol/water solution and obtaining propylene glycol-nanovesicles (PGV) loaded with: O. onites essential oil (OO-PGV), S. thymbra essential oil (ST-PGV) and O. onites plus S. thymbra essential oil (OOST-PGV). All the samples were analyzed by light scattering techniques and they showed small dimensions, low polydispersity index (PdI) and good ζ-potential (Table 3 and Table 4). The higher standard deviation related to the average sizes of OO-GS and OO-PGV indicates a lower repeatability of sample preparation. However, the application of nanovesicles in food products is not limited by their average dimensions and all the developed formulations have suitable physical characteristics as delivery systems of food preservatives.
The combination of mechanic stirrer and ultrasonic bath during the two hydration processes allowed to obtain homogenous and stable formulations, without any further optimization step. Glycerosomes and PG-nanovesicles loaded with OOEO plus STEO were analyzed by transmission electron microscope (TEM, Figure 1a,b). The obtained micrographs showed vesicles with spherical shape and several lamellae, mainly visible in glycerosomes (Figure 1a), and with dimensions in accordance with those obtained by DLS.
In addition, the encapsulation efficiency (EE) of OOEO and STEO, expressed as percentage of carvacrol, the marker constituent, was evaluated by HPLC-DAD and resulted high in both glycerosomes (between ca. 73% and ca. 77%) and PG-nanovesicles (between ca. 77% and 83%).

2.3. Antibacterial and Antifungal Activities

Results of the evaluation of antibacterial and antifungal activity of pure and formulated OOEO/STEO are reported in Table 5 and Table 6. All tested samples possessed significant antimicrobial effects. The best antimicrobial activities of formulated OOEO/STEO against all tested bacteria and fungi were observed for OO-PGV. The antibacterial minimum inhibitory concentration (MIC) of OO-PGV ranged from 1.00 to 4.00 mg vesicles/mL, while the minimum bactericidal concentrations (MBCs) were within the range of 2.00 to 8.00 mg vesicles/mL. MIC and MBC of essential oils formulated in nanovesicles, reported in Table 5, were calculated taking into account that the amounts of EOs loaded in the nanovesicles were 10 mg EO/g of vesicles. The pure OOEO exhibited stronger antibacterial potential with MICs at 0.0002–0.002 mg/mL and MBCs at 0.0003–0.0025 mg/mL compared to standard antibiotic Streptomycin and formulated OOEO used as reference compounds. The most sensitive bacterial species was S. aureus, while E. coli was the resistant one among all the tested bacteria. MIC and MFC were reported in Table 6: for OOEO, MICs were in the range 0.0002–0.001 mg/mL against all tested fungi, and MFCs varied within the range of 0.0003–0.0012 mg/mL. Antifungal potential of OOEO was higher than that of formulated OOEO and reference drug Ketoconazole. The most sensitive fungus appeared to be P. verrucosum, while both Candida species were found to be resistant. In conclusion, the activity of both pure OOEO and STEO was more prominent when compared to the activity of corresponding amounts of formulated EOs. However, this lower antibacterial and antifungal effectiveness of formulated EOs with respect to pure EOs is mainly due to the prolonged release properties of the EOs loaded in the nanovesicles, as described by other studies on in vitro activity of essential oils formulated in glycerosomes and liposomes [5,31].

2.4. Cytotoxicity on HaCaT Cell Line

The cytotoxic effect of pure and formulated OOEO/STEO (Table 7) was assessed on the HaCaT cell line, a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin, a very sensible cell line used as an effective in vitro alternative for an initial orientating screening of safety issues of substances. Blank-GS and Blank-PGV samples showed no toxicity towards HaCaT up to 500 µg/mL, which is indicative of carrier low cytotoxicity. All the investigated samples showed to be weakly cytotoxic towards this cell line. The results showed that OO-GS sample showed the highest cytotoxic effect on the human immortalized keratinocytes, followed by OOST-GS (combination of essential oils loaded in glycerosomes). OO-PGV (propylene glycol-nanovesicles loaded with O. onites essential oil) was the only sample with a cytotoxic effect below 500 µg/mL. Although weak cytotoxicity could be acknowledged for some samples, further application of propylene glycol-nanovesicles should be considered when formulating antimicrobial preparations, since this carrier expressed no toxicity to immortalized cell line. These safety data are encouraging for further safety studies to demonstrate the safe use of the developed nanovesicles.

3. Materials and Methods

3.1. Plant Materials

The fresh aerial parts of the wild plants Origanum onites L. and Satureja thymbra L. were collected from Symi island (SE Aegean, Greece) in summer 2018. The plant materials were authenticated by Associate Prof. Th. Constantinidis; Voucher specimens were deposited to a personal Herbarium of the Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, NKUA (Voucher Specimen Numbers: Tomou and Skaltsa 004/005).

3.2. Chemicals

Phosphatidylcholine (Phospholipon 90G, P90G) was purchased from Lipoid AG (Cologne, Germany) with the support of the Italian agency AVG srl. Cholesterol 95%, dichloromethane, methanol and acetonitrile were purchased from Sigma-Aldrich (Milan, Italy); vegetable glycerol Eur Ph. and propylene glycol Eur Ph. were purchased from Galeno srl (Prato, Italy). Ultrapure water was produced by a synergy UV Simplicity water purification system provided by Merck KGaA (Molsheim, France). Phosphotungstic acid (PTA) was purchased from Electron Microscopy Sciences (Hatfield, PA, USA).

3.3. Hydrodistillation and Identification of OOEO and STEO by Gas Chromatography–Mass Spectrometry (GC-MS)

Air-dried parts (40.0 g) of each plant were cut into small pieces and subjected separately to hydrodistillation for 2 h, using a modified Clevenger type apparatus with a water-cooled oil receiver to reduce artifacts produced during distillation by over-heating according to Hellenic Pharmacopoeia [32]. The EOs were obtained by gas chromatography (GC) grade n-pentane and dried over anhydrous sodium sulfate and stored at −20 °C. The compositions of the volatile constituents were established by GC/MS analyses, performed on a Hewlett-Packard 7820A-5977B MSD system operating in EI mode (70 eV) equipped with a split/splitless injector, using a fused silica HP-5 MS capillary column (30 m × 0.25 mm I.D., film thickness: 0.25 μm). Helium was used as a carrier gas at a flow rate of 2.0 mL/min. The oven temperature was increased from 60 to 300 °C at a rate of 3 °C/min, and subsequently held at 300 °C for 10 min. Injection was at 220 °C in a split ratio 1:5. Injection volumes of each sample were 2 μL. Retention indices for all compounds were determined according to the Van den Dool approach [33], using n-alkanes as standards. The identification of the components was based on comparison of their mass spectra with those of Wiley and NBS/NIST Libraries and those described by Adams (2017) [34], as well as by comparison of their retention indices with literature data. In many cases, the essential oils were subjected to co-chromatography with authentic compounds (Fluka, Sigma). Semi-quantification through peak area integration from GC peaks was applied to obtain the component percentages. The analyses were carried out twice for each sample.

3.4. HPLC-DAD Analysis

Quantitative analyses of OOEO and STEO in nanovesicles was based on the determination of carvacrol, the most abundant and marker constituent of both essential oils. Analyses were carried out using a 1100 High Performance Liquid Chromatograph (HPLC) equipped with a diode array detector (DAD), by Agilent Technologies Italia Spa (Rome, Italy). The chromatographic analyses were performed using a reverse-phase column Luna C-18 100 Å (250 × 4.6) mm, 5 µm particle size, maintained at 25 °C; and the chromatograms were acquired at 276 nm [35]. A gradient elution method, with 1 mL/min flow rate, was applied, using (A) acetonitrile and (B) formic acid/water (pH 3.2) as mobile phases. The analytical method was: 0–3 min 30–30% (B), 3–10 min 30–80% (B), 10–15 min 80–80% (B), 15–20 min 80–95% (B), 20–22 min 95–95% (B), 22–27 min 95–30%. The calibration curve was prepared using a 0.001 μL/μL standard solution of carvacrol in methanol and successive dilutions. The coefficient of determination (R2) of carvacrol calibration curve was 0.9999.

3.5. Preparation of Vesicles Loaded with OOEO and STEO

O. onites and S. thymbra essential oils (OOEO and STEO) were formulated in lipid nanovesicles by the lipid film hydration method [36], in two steps [5,37]. Different amounts of phosphatidylcholine (P90G) and cholesterol were tested for the development of the formulation, and the experimental conditions of the preparation were optimized varying media and time of hydration, as well as the use of ultrasonic bath and/or mechanic stirrer, as reported in Table 8. The selected formulations were prepared with 600 mg of phosphatidylcholine and 10 mg of cholesterol dissolved in dichloromethane, using the ultrasonic bath for 1 min in order to improve the dissolution. Subsequently, evaporation of dichloromethane was carried out using the rotavapor for 20 min at 30 °C, in order to obtain a homogenous lipid film on the internal surface of the flask. At this stage, OOEO (100 μL) or STEO (100 μL) or OOEO plus STEO (50 μL + 50 μL) were added to the flask and the lipid film was hydrated with 5 mL of 5% v/v glycerol/water solution (glycerosomes, GS) or 1% v/v propylene glycol/water solution (propylene glycol-nanovesicles, PG-nanovesicles, PGV), by using the mechanic stirrer for 30 min at 25 °C and immersing the flask in the ultrasonic bath, as shown in Table 8, Table 9 and Table 10. Then, a further 5 mL of the selected dispersant medium was added, and the dispersion was mechanically shaken for additional 30 min, at 25 °C, in the ultrasonic bath.

3.6. Physical Characterization of Nanovesicles Loaded with OOEO and STEO

Average hydrodynamic diameter (nm), polydispersity index (PdI) and ζ-potential (mV) of the developed nanovesicles were measured by Dynamic and Electrophoretic Light Scattering, DLS/ELS (Zetasizer Nanoseries ZS90) by Malvern instrument (Worcestershire, UK), at 25 °C, with a scattering angle of 90 °C [38,39]. Glycerosomes and PG-nanovesicles loaded with the essential oils were diluted with ultrapure water before measurements, in order to achieve a suitable scattering intensity. Successively, the two systems loaded with OOEO plus STEO were observed by transmission electron microscope, TEM (CM12 TEM, PHILIPS, Eindhoven, The Netherlands) equipped with an OLYMPUS Megaview G2 camera, with an accelerating voltage of 80 kV. A drop of sample, 5-folds diluted in water, was applied and dried by desiccation on a carbon film copper grid and it was counterstained with 1% w/v of phosphotungstic acid solution for 3 min [40]. Then, the sample was examined at different amplifications.

3.7. Chemical Characterization of Nanovesicles Loaded with OOEO and STEO

Encapsulation efficiency (EE) and total recovery (R) of OOEO and STEO loaded inside nanovesicles were evaluated in terms of carvacrol content, the marker constituent of both the essential oils. EE was calculated according to the following equation:
E E = ( e n c a p s u l a t e d   c a r v a c r o l i n i t i a l   c a r v a c r o l ) × 100
where encapsulated carvacrol is the concentration of the single component after the purification step. In fact, vesicles were purified from free EOs by the dialysis bag method [41], using Spectra/Por® regenerated cellulose membranes with 3.5 KDa molecular weight cut-off, by Repligen Europe B.V. (Breda, The Netherlands). The dialysis bag was stirred in 1 L of ultrapure water, at room temperature for 1 h. After that, the purified formulations were diluted in methanol, in order to break vesicles and release the encapsulated EOs. Samples were centrifuged at 14,000 rpm for 10 min and supernatants were analyzed by HPLC-DAD. OOEO and STEO total recovery (R) was determined using the same procedure without the purification step by dialysis, and it was calculated according to the following equation:
R = ( t o t a l   r e c o v e r e d   c a r v a c r o l i n i t i a l   c a r v a c r o l ) × 100
where total recovered carvacrol is the concentration of the single component after the preparation of the formulation, determined by chromatographic analysis.

3.8. Determination of Antibacterial and Antifungal Activity

The Gram-positive bacteria Bacillus cereus (food isolate), Staphylococcus aureus ATCC 11632 and Listeria monocytogenes NCTC 7973, and the Gram-negative bacteria Escherichia coli ATCC 35210, Pseudomonas aeruginosa ATCC 27853 and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 were used in order to determine the potential antibacterial activity. For determination antifungal activity 6 strains of fungi were used: Aspergillus fumigatus ATCC 1022, Aspergillus niger ATCC 6275, Trichoderma viride IAM 5061, Penicillium verrucosum var. cyclopium (food isolate), Candida albicans (oral isolate) and Candida krusei (oral isolate) were tested for their susceptibility. The bacterial strains were cultured on solid Tryptic Soy agar (TSA), while micromycetes were cultured on solid malt agar (MA) and yeast were sustained on Sabouraud dextrose agar (SDA) medium. The cultures were sub-cultured once a month and stored at 4 °C for further utilization. All the tested microorganisms are deposited at the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Siniša Stankovic”—National Institute of Republic of Serbia, University of Belgrade, Serbia. The antimicrobial activity of samples was determined by the modified microdilution method [42,43]. The results were presented as minimum inhibitory concentrations (MICs) and minimum bactericidal/fungicidal concentrations (MBCs/MFCs). Streptomycin (Sigma-Aldrich S6501, St. Louis, MO, USA) and ketoconazole (Zorkapharma, Šabac, Serbia) were used as positive controls, and blank-glycerosome (Blank-GS) and blank-propylene glycol-nanovesicles (Blank-PGV) were used as negative control. All the experiments on antimicrobial activity were repeated in triplicate.

3.9. Evaluation of Cytotoxicity in HaCaT Cell Line

Crystal violet assay was used for determination of the antiproliferative effect, according to the previous protocol [44] with modifications. Antiproliferative effect of pure and formulated OOEO/STEO was analyzed on spontaneously immortalized human skin keratinocytes (HaCaT) cell line. Cell line was grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin and streptomycin (Invitrogen) at 37 °C in 5% CO2. Twenty-four hours before treatment with the extract 1 × 104 cells/well were seeded in a 96-well plate. After, the medium was removed, fresh medium supplemented with different concentrations of the extract and compound (6.25–400 μg/mL) dissolved in phosphate buffered saline was added to the cells. Control cells were grown in medium. Potassium dichromate (K2Cr2O7) was used as a positive control and PBS as negative control. The experiment was performed in triplicate for each condition and cells were incubated with the extract for 24 h. After that period, the medium was removed and the cells were washed twice with phosphate buffered saline (PBS), stained with 0.5% crystal violet staining solution and incubated for 15 min at room temperature. Afterwards, crystal violet was removed, the cells were washed in a stream of tap water and left to air-dry at room temperature for 24 h. The absorbance of dye dissolved in methanol was measured in a microplate reader at 590 nm (OD590). The results were expressed as IC50 (%) value in μg/mL. The criterion used to categorize the cytotoxic activity of pure and formulated OOEO/STEO to cancer cell lines was as follows: IC50 ≤ 20 µg/mL = highly cytotoxic, IC50 ranged between 21 and 250 µg/mL = moderately cytotoxic, IC50 ranged between 201 and 500 µg/mL = weakly cytotoxic, and IC50 > 501 µg/mL = no cytotoxicity. All analyses were performed in triplicate; each replicate was quantified also three times. Data were expressed as mean standard deviation, where applicable. In the cases where statistical significance differences were identified, the dependent variables were compared using Tukey’s honestly significant difference (HSD) test.

4. Conclusions

During recent years, synthetic preservatives are generally used to protect food against microorganisms. However, there is an urgent need to search new antimicrobials because of the increasing resistance against these microorganisms. EOs represent a valid alternative to synthetic preservatives in the food industry, and Origanum essential oil has been largely investigated as antimicrobial and antioxidant additive in food products [45]. However, in many cases their organoleptic impact in foodstuffs limits their usage. Techniques such as nanoencapsulation can address this problem. Thus, this study was designed in order to develop propylene glycol-nanovesicles loaded with EOs from O. onites and S. thymbra for the first time and to evaluate them as safe and food-grade delivery systems.
In this study, the chemical profiles of the EOs of O. onites and S. thymbra were identified. Oxygenated monoterpenes comprised the major chemical class in both EOs. In particular, the main components of O. onites EO were carvacrol (66.0%), p-cymene (7.9%), γ-terpinene (4.9%) and borneol (2.8%). Whereas, the principal compounds of S. thymbra EO were carvacrol (46.0%), γ-terpinene (19.7%), p-cymene (7.6%), β-caryophyllene (7.0%) and α-terpinene (5.1%). Afterwards, we succeeded to encapsulate the EOs in nanovesicles, which presented high homogeneity and optimal encapsulation efficiency either for O. onites or S. thymbra. Both pure EOs and formulated EOs were evaluated against different food-borne pathogens. The high antimicrobial activity of both EOs could be attributed to their chemical constituents, not only to the high concentration of carvacrol but also to the potential synergy of all the compounds. Our results showed that pure EOs were more active compared to the corresponding amounts of formulated EOs. We should point out that the lower antibacterial and anti-fungal effectiveness of formulated EOs with respect to pure EOs is assigned mainly to the prolonged release properties of the EOs loaded in the nanovesicles. Cytotoxicity was also tested in HaCaT cells.
In conclusion, the present study unveiled that the tested nanovesicles could represent potential biocontrol agents against fungal and bacterial food pathogens with promising GRAS status in mammalian systems, besides being an innovative and completely biodegradable approach for the prolonged and sustained release of the EO, preserving functional properties.

Author Contributions

Conceptualization, H.S.; methodology, H.S., A.R.B. and A.Ć.; investigation, E.-M.T., G.V. and D.S.; writing—original draft preparation, E.-M.T., G.V. and D.S.; writing—review and editing, H.S., A.R.B. and A.Ć.; supervision, H.S., A.R.B. and A.Ć. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful to the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-9/2021-14/200007).

Acknowledgments

The authors thank MIUR-Italy (“Progetto dipartimenti di eccellenza 2018–2022” allocated to Department of Chemistry “Ugo Schiff”, University of Florence, Italy). The authors express thanks to Maria Cristina Salvatici, Electron Microscopy Centre (Ce.M.E.), ICCOM, CNR, Sesto Fiorentino, Florence, Italy for data curation of TEM. E.-M.T. would like to thank the program Erasmus+ Studies for the research mobility. The authors thank Tsakiris Philippe for the collection of the plant materials.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

References

  1. Nieto, G. Biological Activities of Three Essential Oils of the Lamiaceae Family. Medicines 2017, 4, 63. [Google Scholar] [CrossRef] [Green Version]
  2. Karpiński, T.M. Essential Oils of Lamiaceae Family Plants as Antifungals. Biomolecules 2020, 10, 103. [Google Scholar] [CrossRef] [Green Version]
  3. Bilia, A.R.; Guccione, C.; Isacchi, B.; Righeschi, C.; Firenzuoli, F.; Bergonzi, M.C. Essential oils loaded in nanosystems: A developing strategy for a successful therapeutic approach. Evid. Based Complement. Altern. Med. 2014, 2014, 651593. [Google Scholar] [CrossRef] [Green Version]
  4. Bilia, A.R.; Piazzini, V.; Risaliti, L.; Vanti, G.; Casamonti, M.; Wang, M.; Bergonzi, M.C. Nanocarriers: A successful tool to increase solubility, stability and optimise bioefficacy of natural constituents. Curr. Med. Chem. 2019, 26, 4631–4656. [Google Scholar] [CrossRef]
  5. Vanti, G.; Ntallis, S.G.; Panagiotidis, C.A.; Dourdouni, V.; Patsoura, C.; Bergonzi, M.C.; Lazari, D.; Bilia, A.R. Glycerosome of Melissa officinalis L. Essential Oil for Effective Anti-HSV Type 1. Molecules 2020, 25, 3111. [Google Scholar] [CrossRef]
  6. Risaliti, L.; Kehagia, A.; Daoultzi, E.; Lazari, D.; Bergonzi, M.C.; Vergkizi-Nikolakaki, S.; Hadjipavlou-Litina, D.; Bilia, A.R. Liposomes loaded with Salvia triloba and Rosmarinus officinalis essential oils: In vitro assessment of antioxidant, antiinflammatory and antibacterial activities. J. Drug Deliv. Sci. Technol. 2019, 51, 493–498. [Google Scholar] [CrossRef]
  7. Risaliti, L.; Pini, G.; Ascrizzi, R.; Donato, R.; Sacco, C.; Bergonzi, M.C.; Salvatici, M.C.; Bilia, A.R. Artemisia annua essential oil extraction, characterization, and incorporation in nanoliposomes, smart drug delivery systems against Candida species. J. Drug Deliv. Sci. Technol. 2020, 59, 101849. [Google Scholar] [CrossRef]
  8. Kerekes, E.B.; Vidács, A.; Török Jenei, J.; Gömöri, C.; Takó, M.; Chandrasekaran, M.; Kadaikunnan, S.; Alharbi, N.S.; Krisch, J.; Vágvölgyi, C. Essential oils against bacterial biofilm formation and quorum sensing of food-borne pathogens and spoilage microorganisms. In The Battle against Microbial Pathogens: Basic Science; Technological Advances and Educational Programs, Ed.; A Méndez-Vilas: Bajadoz, Spain, 2015; Volume 1, pp. 429–437. [Google Scholar]
  9. Vergis, J.; Gokulakrishnan, P.; Agarwal, R.K.; Kumar, A. Essential Oils as Natural Food Antimicrobial Agents: A Review. Crit. Rev. Food Sci. Nutr. 2015, 55, 1320–1323. [Google Scholar] [CrossRef] [PubMed]
  10. Zhou, Y.; Hartemink, A.E.; Shi, Z.; Liang, Z.; Lu, Y. Land use and climate change effects on soil organic carbon in North and Northeast China. Sci. Total Environ. 2019, 647, 1230–1238. [Google Scholar]
  11. Wei, S.; Daliri, E.B.M.; Chelliah, R.; Park, B.J.; Lim, J.S.; Baek, M.A.; Nam, Y.-S.; Seo, K.-H.; Jin, Y.-G.; Oh, D.H. Development of a multiplex real-time PCR for simultaneous detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in food samples. J. Food Saf. 2019, 39, e12558. [Google Scholar] [CrossRef] [Green Version]
  12. King, M. Spoilage and preservation of food. Food Qual. Stand. 2009, III, 41–59. [Google Scholar]
  13. Amaral, D.M.F.; Bhargava, K. Essential oil nanoemulsions and food applications. Adv. Food Technol. Nutr. Sci. Open J. 2015, 1, 84–87. [Google Scholar] [CrossRef]
  14. Asprea, M.; Leto, I.; Bergonzi, M.C.; Bilia, A.R. Thyme essential oil loaded in nanocochleates: Encapsulation efficiency, in vitro release study and antioxidant activity. LWT 2017, 77, 497–502. [Google Scholar] [CrossRef]
  15. Lombrea, A.; Antal, D.; Ardelean, F.; Avram, S.; Pavel, I.Z.; Vlaia, L.; Mut, A.-M.; Diaconeasa, Z.; Dehelean, C.A.; Soica, C.; et al. A Recent Insight Regarding the Phytochemistry and Bioactivity of Origanum vulgare L. Essential Oil. Int. J. Mol. Sci. 2020, 21, 9653. [Google Scholar] [CrossRef]
  16. Euro+Med PlantBase. 2011. Available online: http://ww2.bgbm.org/EuroPlusMed/query.asp (accessed on 24 March 2021).
  17. Drabova, L.; Alvarez-Rivera, G.; Suchanova, M.; Schusterova, D.; Pulkrabova, J.; Tomaniova, M.; Kocourek, V.; Chevallier, O.; Elliott, C.; Hajslova, J. Food fraud in oregano: Pesticide residues as adulteration markers. Food Chem. 2019, 276, 726–734. [Google Scholar] [CrossRef] [PubMed]
  18. Stefanaki, A.; Cook, C.M.; Lanaras, T.; Kokkini, S. The Oregano plants of Chios Island (Greece): Essential oils of Origanum onites L. growing wild in different habitats. Ind. Crops Prod. 2016, 82, 107–113. [Google Scholar] [CrossRef]
  19. Tasdemir, K.; Kaiser, M.; Demirci, B.; Demirci, F.; Baser, K.H.C. Antiprotozoal Activity of Turkish Origanum onites Essential Oil and Its Components. Molecules 2019, 24, 4421. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Skoula, M.; Grayer, R.J. Volatile oils of Coridothymus capitatus, Satureja thymbra, Satureja spinosa and Thymbra calostachya (Lamiaceae) from Crete. Flavour Fragr. J. 2005, 20, 573–576. [Google Scholar] [CrossRef]
  21. Tepe, B.; Cilkiz, M. A pharmacological and phytochemical overview on Satureja. Pharm. Biol. 2015, 54, 375–412. [Google Scholar] [CrossRef] [Green Version]
  22. Glamočlija, J.; Soković, M.; Vukojević, J.; Milenković, I.; Van Griensven, L.J.L.D. Chemical Composition and Antifungal Activities of Essential Oils of Satureja thymbra L. and Salvia pomifera ssp. calycina (Sm.) Hayek. JEOR 2006, 18, 115–117. [Google Scholar]
  23. Tepe, B.; Cakir, A.; Sihoglu Tepe, A. Medicinal Uses, Phytochemistry, and Pharmacology of Origanum onites (L.): A Review. Chem. Biodivers. 2016, 13, 504–520. [Google Scholar] [CrossRef] [PubMed]
  24. Kokkini, S.; Vokkou, D. Carvacrol-rich Plants in Greece. Flavour Fragr. J. 1989, 4, 1–7. [Google Scholar] [CrossRef]
  25. Economou, G.; Panagopoulos, G.; Tarantilis, P.; Kalivas, D.; Kotoulas, V.; Travlos, I.S.; Polysiou, M.; Karamanos, A. Variability in essential oil content and composition of Origanum hirtum L., Origanum onites L., Coridothymus capitatus (L.) and Satureja thymbra L. populations from the Greek island Ikaria. Ind. Crops Prod. 2011, 33, 236–241. [Google Scholar] [CrossRef]
  26. Vokou, D.; Kokkini, S.; Bessière, J.-M. Origanum onites (Lamiaceae) in Greece: Distribution, Volatile Oil Yield, and Composition. Econ. Bot. 1988, 42, 407–412. [Google Scholar] [CrossRef]
  27. Kokkini, S.; Karousou, R.; Hanlidou, E.; Lanaras, T. Essential Oil Composition of Greek (Origanum vulgare ssp. hirtum) and Turkish (O. onites) Oregano: A Tool for Their Distinction. JEOR 2004, 16, 334–338. [Google Scholar]
  28. Azaz, A.D.; Kürkcüoglu, M.; Satil, F.; Can Baser, K.H.; Tümen, G. In vitro antimicrobial activity and chemical composition of some Satureja essential oils. Flavour Fragr. J. 2005, 20, 587–591. [Google Scholar] [CrossRef]
  29. Becker, L.C.; Bergfeld, W.F.; Belsito, D.V.; Hill, R.A.; Klaassen, C.D.; Liebler, D.C.; Marks, J.G., Jr.; Shank, R.C.; Slaga, T.J.; Snyder, P.W.; et al. Safety Assessment of Glycerin as Used in Cosmetics. Int. J. Toxicol. 2019, 38 (Suppl. 3), 6S–22S. [Google Scholar] [CrossRef]
  30. Fiume, M.M.; Bergfeld, W.F.; Belsito, D.V.; Hill, R.A.; Klaassen, C.D.; Liebler, D.; Marks, J.G., Jr.; Shank, R.C.; Slaga, T.J.; Snyder, P.W.; et al. Safety assessment of propylene glycol, tripropylene glycol, and PPGs as used in cosmetics. Int. J. Toxicol. 2012, 31 (Suppl. 5), 245S–260S. [Google Scholar] [CrossRef]
  31. Valenti, D.; De Logu, A.; Loy, G.; Sinico, C.; Bonsignore, L.; Cottiglia, F.; Garau, D.; Fadda, A.M. Liposome-incorporated Santolina insularis essential oil: Preparation, characterization and in vitro antiviral activity. J. Liposome Res. 2001, 11, 73–90. [Google Scholar] [CrossRef]
  32. Hellenic Pharmacopoeia, 5th ed.; Chapter 28.12; National Organization for Medicines: Greece, Athens, 2002.
  33. Van den Dool, H.; Kratz, P.D. A generalization of the Retention Index System including linear temperature programmed Gas Liquid partition Chromatography. J. Chromatogr. 1963, 11, 463–471. [Google Scholar] [CrossRef]
  34. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing Corp.: Carol Stream, IL, USA, 2017. [Google Scholar]
  35. Hajimehdipoor, H.; Shekarchi, M.; Khanavi, M.; Adib, N.; Amri, M. A validated high performance liquid chromatography method for the analysis of thymol and carvacrol in Thymus vulgaris L. volatile oil. Pharmacogn. Mag. 2010, 6, 154. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Risaliti, L.; Yu, X.; Vanti, G.; Bergonzi, M.C.; Wang, M.; Bilia, A.R. Hydroxyethyl cellulose hydrogel for skin delivery of khellin loaded in ascosomes: Characterization, in vitro/in vivo performance and acute toxicity. Int. J. Biol. Macromol. 2021, 179, 217–229. [Google Scholar] [CrossRef] [PubMed]
  37. Manca, M.L.; Zaru, M.; Manconi, M.; Lai, F.; Valenti, D.; Sinico, C.; Fadda, A.M. Glycerosomes: A new tool for effective dermal and transdermal drug delivery. Int. J. Pharm. 2013, 455, 66–74. [Google Scholar] [CrossRef]
  38. Vanti, G.; Wang, M.; Bergonzi, M.C.; Zhidong, L.; Bilia, A.R. Hydroxypropyl methylcellulose hydrogel of berberine chloride-loaded escinosomes: Dermal absorption and biocompatibility. Int. J. Biol. Macromol. 2020, 164, 232–241. [Google Scholar] [CrossRef]
  39. Manaia, E.B.; Abuçafy, M.P.; Chiari-Andréo, B.G.; Silva, B.L.; Junior, J.A.O.; Chiavacci, L.A. Physicochemical characterization of drug nanocarriers. Int. J. Nanomed. 2017, 12, 4991. [Google Scholar] [CrossRef] [Green Version]
  40. Vanti, G.; Bani, D.; Salvatici, M.C.; Bergonzi, M.C.; Bilia, A.R. Development and Percutaneous Permeation Study of Escinosomes, Escin-Based Nanovesicles Loaded with Berberine Chloride. Pharmaceutics 2019, 11, 682. [Google Scholar] [CrossRef] [Green Version]
  41. Vanti, G.; Coronnello, M.; Bani, D.; Mannini, A.; Bergonzi, M.C.; Bilia, A.R. Co-Delivery of Berberine Chloride and Tariquidar in Nanoliposomes Enhanced Intracellular Berberine Chloride in a Doxorubicin-Resistant K562 Cell Line Due to P-gp Overexpression. Pharmaceutics 2021, 13, 306. [Google Scholar] [CrossRef]
  42. CLC-Pred Web-Service. Available online: http://www.way2drug.com/Cell-line/ (accessed on 3 March 2021).
  43. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard, 8th ed.; CLSI Publication M07-A8; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2009. [Google Scholar]
  44. Stojković, D.; Drakulić, D.; Gašić, U.; Zengin, G.; Stevanović, M.; Rajčević, N.; Soković, M. Ononis spinosa L., an edible and medicinal plant: UHPLC-LTQ-Orbitrap/MS chemical profiling and biological activities of the herbal extract. Food Funct. 2020, 11, 7138–7151. [Google Scholar] [CrossRef] [PubMed]
  45. Rodriguez-Garcia, I.; Silva-Espinoza, B.A.; Ortega-Ramirez, L.A.; Leyva, J.M.; Siddiqui, M.W.; Cruz-Valenzuela, M.R.; Gonzalez-Aguilar, G.A.; Ayala-Zavala, J.F. Oregano essential oil as an antimicrobial and antioxidant additive in food products. Crit. Rev. Food Sci. Nutr. 2016, 56, 1717–1727. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Pictures of (a) glycerosomes loaded with O. onites plus S. thymbra essential oils (OOST-GS) and (b) propylene glycol-nanovesicles loaded with O. onites plus S. thymbra essential oils (OOST-PGV), obtained by transmission electron microscope (TEM) analysis.
Figure 1. Pictures of (a) glycerosomes loaded with O. onites plus S. thymbra essential oils (OOST-GS) and (b) propylene glycol-nanovesicles loaded with O. onites plus S. thymbra essential oils (OOST-PGV), obtained by transmission electron microscope (TEM) analysis.
Molecules 26 02124 g001
Table 1. Chemical composition (% v/v) of O. onites essential oil.
Table 1. Chemical composition (% v/v) of O. onites essential oil.
NoCompoundsRI aComposition (%)
1α-thujene9200.8
2α-pinene9281.0
3camphene9410.6
41-octen-3-ol9700.4
5myrcene9852.1
6α-phellandrene10000.4
7δ-3-carene10040.2
8α-terpinene10102.0
9p-cymene10177.9
10β-phellandrene10210.8
11(E)-β-ocimene10400.1
12γ-terpinene10514.9
13trans-sabinene hydrate10720.3
14α-terpinolene10830.4
15linalool10910.4
16α-campholenal11200.1
17trans-pinocarveol11320.1
18borneol11632.8
19terpinen-4-ol11711.7
20α-terpineol11830.6
21carvone12350.1
22carvacrol methyl ether12410.4
23linalyl acetate12500.2
24carvenone12530.2
25thymol12851.0
26carvacrol130066.0
27carvacrol acetate13660.2
28β-cubebene13830.7
29β-caryophyllene14140.7
30aromadendrene14350.2
31ledene14950.1
32β-bisabolene15012.3
33δ-cadinene15180.1
34spathulenol15750.1
35caryophyllene oxide15800.1
Total identification 100.0
Grouped components (% v/v) of O. onites essential oil
Classes of EO Constituents%
Monoterpene hydrocarbons21.2
Oxygenated monoterpenes74.1
Sesquiterpene hydrocarbons4.1
Oxygenated sesquiterpenes0.2
Aliphatic alcohols0.4
a RI: Retention Index, calculated against C9–C24 n-alkanes on the HP 5MS column capillary column.
Table 2. Chemical composition (% v/v) of S. thymbra essential oil.
Table 2. Chemical composition (% v/v) of S. thymbra essential oil.
NoCompoundsRI aComposition (%)
1α-thujene9202.4
2α-pinene9281.6
3camphene9410.3
4β-pinene9700.6
5myrcene9852.5
63-octanol9870.1
7α-phellandrene10000.6
8δ-3-carene10040.2
9α-terpinene10105.1
10p-cymene10177.6
11β-phellandrene10210.9
12(E)-β-ocimene10400.1
13γ-terpinene105119.7
14trans-sabinene hydrate10720.3
15α-terpinolene10830.3
16linalool10911.3
17borneol11630.3
18terpinen-4-ol11710.9
19thymol methyl ether12320.7
20carvone12350.1
21thymol12850.3
22carvacrol130046.0
23carvacrol acetate13660.1
24β-caryophyllene14147.0
25aromadendrene14350.1
26α-humulene14500.4
27ledene14950.1
28δ-cadinene15180.1
29caryophyllene oxide15800.2
Total identification 99.9
Grouped components (% v/v) of S. thymbra essential oil
Classes of EO Constituents%
Monoterpene hydrocarbons41.9
Oxygenated monoterpenes50.0
Sesquiterpene hydrocarbons7.7
Oxygenated sesquiterpenes0.2
Aliphatic alcohols0.1
a RI: Retention Index, calculated against C9–C24 n-alkanes on the HP 5MS column capillary column.
Table 3. Physical and chemical parameters of glycerosomes loaded with: O. onites essential oil (OO-GS), S. thymbra essential oil (ST-GS), O. onites plus S. thymbra essential oils (OOST-GS). From left: Size, polydispersity index (PdI), ζ-potential, recovery (R) and encapsulation efficiency (EE); mean ± SD (n = 3).
Table 3. Physical and chemical parameters of glycerosomes loaded with: O. onites essential oil (OO-GS), S. thymbra essential oil (ST-GS), O. onites plus S. thymbra essential oils (OOST-GS). From left: Size, polydispersity index (PdI), ζ-potential, recovery (R) and encapsulation efficiency (EE); mean ± SD (n = 3).
FormulationSize (nm)PdIζ-Potential (mV)R (%)EE (%)
OO-GS148.40 ± 48.340.20 ± 0.04−41.68 ± 5.7887.02 ± 6.1972.97 ± 6.46
ST-GS105.51 ± 13.920.22 ± 0.05−34.03 ± 3.5791.38 ± 4.7777.35 ± 7.63
OOST-GS105.43 ± 15.190.17 ± 0.02−26.50 ± 1.4889.48 ± 5.9373.26 ± 6.44
Table 4. Physical and chemical parameters of propylene glycol-nanovesicles loaded with: O. onites essential oil (OO-PGV), S. thymbra essential oil (ST-PGV), O. onites plus S. thymbra essential oils (OOST-PGV). From left: Size, polydispersity index (PdI), ζ-potential, recovery (R) and encapsulation efficiency (EE); mean ± SD (n = 3).
Table 4. Physical and chemical parameters of propylene glycol-nanovesicles loaded with: O. onites essential oil (OO-PGV), S. thymbra essential oil (ST-PGV), O. onites plus S. thymbra essential oils (OOST-PGV). From left: Size, polydispersity index (PdI), ζ-potential, recovery (R) and encapsulation efficiency (EE); mean ± SD (n = 3).
FormulationSize (nm)PdIζ-Potential (mV)R (%)EE (%)
OO-PGV138.23 ± 29.170.17 ± 0.03−34.93 ± 8.8187.92 ± 8.2783.12 ± 5.16
ST-PGV73.95 ± 5.710.21 ± 0.01−30.68 ± 6.6984.64 ± 12.0279.04 ± 7.34
OOST-PGV101.09 ± 8.240.22 ± 0.06−28.80 ± 1.1585.79 ± 9.2776.73 ± 8.27
Table 5. Antibacterial activity (mg EO/mL medium) of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO).
Table 5. Antibacterial activity (mg EO/mL medium) of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO).
B. cereusS. aureusL. monocytogenesE. coliP. aeruginosaS. enterica Serovar
Typhimurium
Blank-GSMICn.a.n.a.n.a.n.a.n.a.n.a.
MBCn.a.n.a.n.a.n.a.n.a.n.a.
OO-GSMIC0.0150.0150.0100.0400.0200.010
MBC0.0200.0200.0400.0800.0400.020
ST-GSMIC0.0200.0100.0200.0400.0400.020
MBC0.0400.0200.0400.0800.0800.040
OOST-GSMIC0.0200.0150.0200.0400.0200.020
MBC0.0100.0300.0400.0800.0400.040
Blank-PGVMICn.a.n.a.n.a.n.a.n.a.n.a.
MBCn.a.n.a.n.a.n.a.n.a.n.a.
OO-PGVMIC0.0100.0100.0100.0400.0200.015
MBC0.0200.0200.0200.0800.0300.040
ST-PGVMIC0.0150.0100.0100.0400.0300.020
MBC0.0400.0200.0200.0800.0400.040
OOST-PGVMIC0.0100.0200.0150.0400.0200.020
MBC0.0200.0400.0200.0800.0400.040
OOEOMIC0.00060.00120.00200.00100.00020.0005
MBC0.00120.00250.00250.00200.00030.0006
STEOMIC0.00030.00120.00060.00100.00030.0010
MBC0.00120.00250.00120.00120.00060.0012
StreptomycinMIC0.100.050.200.200.100.20
MBC0.200.100.400.400.200.30
MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; n.a.: not active at tested concentration of 8 mg formulation/mL medium, corresponding to 0.080 mg EO/mL medium; Blank-GS: blank-glycerosomes; OO-GS: glycerosomes loaded with O. onites essential oil; ST-GS: glycerosomes loaded with S. thymbra essential oil; OOST-GS: glycerosomes loaded with O. onites plus S. thymbra essential oils; Blank-PGV: blank-propylene glycol-nanovesicles; OO-PGV: propylene glycol-nanovesicles loaded with O. onites essential oil; ST-PGV: propylene glycol-nanovesicles loaded with S. thymbra essential oil; OOST-PGV: propylene glycol-nanovesicles loaded with O. onites plus S. thymbra essential oils.
Table 6. Antifungal activity (mg EO/mL of medium) of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO).
Table 6. Antifungal activity (mg EO/mL of medium) of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO).
A. fumigatusA. nigerT. virideP. verrucosumC. albicansC. krusei
Blank-GSMICn.a.n.a.n.a.n.a.n.a.n.a.
MFCn.a.n.a.n.a.n.a.n.a.n.a.
OO-GSMIC0.0150.0100.0100.010-0.080
MFC0.0400.0200.0200.020->0.080
ST-GSMIC0.0100.0200.0150.0200.0400.060
MFC0.0400.0400.0300.0400.0800.080
OOST-GSMIC0.0200.0150.0200.0150.0400.060
MFC0.0400.0300.0400.0300.0800.080
Blank-PGVMICn.a.n.a.n.a.n.a.n.a.n.a.
MFCn.a.n.a.n.a.n.a.n.a.n.a.
OO-PGVMIC0.0100.0200.0200.0200.0600.080
MFC0.0200.0400.0400.0400.080>0.080
ST-PGVMIC0.0050.0100.0200.0050.0800.080
MFC0.0100.0400.0400.010>0.080>0.080
OOST-PGVMIC0.0200.0100.0200.0150.0800.060
MFC0.0400.0200.0400.030>0.0800.080
OOEOMIC0.00020.00040.00030.00030.00100.0006
MFC0.00030.00060.00060.00060.00120.0012
STEOMIC0.00040.00060.00030.00030.00060.0003
MFC0.00060.00120.00060.00060.00120.0006
KetoconazoleMIC0.200.201.000.200.500.50
MFC0.500.501.500.501.001.00
MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration; n.a.: not active at tested concentration of 8 mg formulation/mL medium, corresponding to 0.080 mg EO/mL medium; Blank-GS: blank-glycerosomes; OO-GS: glycerosomes loaded with O. onites essential oil; ST-GS: glycerosomes loaded with S. thymbra essential oil; OOST-GS: glycerosomes loaded with O. onites plus S. thymbra essential oils; Blank-PGV: blank-propylene glycol-nanovesicles; OO-PGV: propylene glycol-nanovesicles loaded with O. onites essential oil; ST-PGV: propylene glycol-nanovesicles loaded with S. thymbra essential oil; OOST-PGV: propylene glycol-nanovesicles loaded with O. onites plus S. thymbra essential oils.
Table 7. Cytotoxic properties of pure and formulated of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO) on human immortalized keratinocyte cell line.
Table 7. Cytotoxic properties of pure and formulated of pure and formulated EOs of O. onites (OOEO) and S. thymbra (STEO) on human immortalized keratinocyte cell line.
SamplesIC50% (µg/mL) HaCaT Cell Line
Blank-GS>500
OO-GS311.24 ± 8.22 b
ST-GS487.34 ± 4.46 d
OOST-GS385.83 ± 1.51 c
Blank-PGV>500
OO-PGV492.14 ± 11.74 d
ST-PGV>500
OOST-PGV>500
OOEO>500
STEO>500
K2Cr2O716.29 ± 1.42 a
Blank-GS: blank-glycerosomes; OO-GS: glycerosomes loaded with O. onites essential oil; ST-GS: glycerosomes loaded with S. thymbra essential oil; OOST-GS: glycerosomes loaded with O. onites plus S. thymbra essential oils; Blank-PGV: blank-propylene glycol-nanovesicles; OO-PGV: propylene glycol-nanovesicles loaded with O. onites essential oil; ST-PGV: propylene glycol-nanovesicles loaded with S. thymbra essential oil; OOST-PGV: propylene glycol-nanovesicles loaded with O. onites plus S. thymbra essential oils. Different letters mean significant difference between IC50 values of samples (p > 0.01).
Table 8. Preparation of OOEO-loaded vesicles.
Table 8. Preparation of OOEO-loaded vesicles.
P90G:Chol Ratio (mg/mL)OOEO Conc (mg/mL)Hydration Time (min)Hydration Volume (mL)Ultrasonic BathMechanic Stirrer
15:0.510305 (PBS)noyes
30:110305 (PBS)noyes
30:61030/605 (PBS)noyes
60:11030 + 302.5 + 2.5 (1%G/W)yesyes
60:11030 + 302.5 + 2.5 (5%G/W)yesyes
60:11030 + 302.5 + 2.5 (10%G/W)yesyes
60:11060 + 602.5 + 2.5 (5%G/W)yesyes
10:11030 + 302.5 + 2.5 (5%G/W)yesyes
60:11030 + 302.5 + 2.5 (1%PG/W)yesyes
60:11030 + 302.5 + 2.5 (5%PG/W)yesyes
G/W = glycerol/water solution; PG/W = propylene glycol/water solution.
Table 9. Preparation of STEO-loaded vesicles.
Table 9. Preparation of STEO-loaded vesicles.
P90G:Chol Ratio (mg/mL)STEO Conc (mg/mL)Hydration Time (min)Hydration Volume (mL)Ultrasonic BathMechanic Stirrer
60:11030 + 302.5 + 2.5 (5%G/W)yesyes
60:11030 + 302.5 + 2.5 (1%PG/W)yesyes
G/W = glycerol/water solution; PG/W = propylene glycol/water solution.
Table 10. Preparation of OOEO plus STEO-loaded vesicles.
Table 10. Preparation of OOEO plus STEO-loaded vesicles.
P90G:Chol Ratio (mg/mL)OOEO + STEO Conc (mg/mL)Hydration Time (min)Hydration Volume (mL)Ultrasonic BathMechanic Stirrer
60:15 + 530 + 302.5 + 2.5 (5%G/W)yesyes
60:15 + 530 + 302.5 + 2.5 (1%PG/W)yesyes
G/W = glycerol/water solution; PG/W = propylene glycol/water solution.
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Vanti, G.; Tomou, E.-M.; Stojković, D.; Ćirić, A.; Bilia, A.R.; Skaltsa, H. Nanovesicles Loaded with Origanum onites and Satureja thymbra Essential Oils and Their Activity against Food-Borne Pathogens and Spoilage Microorganisms. Molecules 2021, 26, 2124. https://doi.org/10.3390/molecules26082124

AMA Style

Vanti G, Tomou E-M, Stojković D, Ćirić A, Bilia AR, Skaltsa H. Nanovesicles Loaded with Origanum onites and Satureja thymbra Essential Oils and Their Activity against Food-Borne Pathogens and Spoilage Microorganisms. Molecules. 2021; 26(8):2124. https://doi.org/10.3390/molecules26082124

Chicago/Turabian Style

Vanti, Giulia, Ekaterina-Michaela Tomou, Dejan Stojković, Ana Ćirić, Anna Rita Bilia, and Helen Skaltsa. 2021. "Nanovesicles Loaded with Origanum onites and Satureja thymbra Essential Oils and Their Activity against Food-Borne Pathogens and Spoilage Microorganisms" Molecules 26, no. 8: 2124. https://doi.org/10.3390/molecules26082124

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