Comparison of antibacterial and antioxidant potentials of pure and nanoemulsified Nepeta pogonosperma essential oil

Abstract The current study aimed to investigate the antiradical and antibacterial potential of pure and its nanoemulsified (NNE) Nepeta pogonosperma essential oil (PNE). Antimicrobial activity of the essential oil against two Gram‐positive (E. faecalis and B. cereus) and two Gram‐negative (M. catarrhalis and K. pneumonia) food‐related pathogens during 60‐day storage was investigated based on disc diffusion, minimum inhibition concentration (MIC), and minimum bactericidal concentration (MBC). The chemical compounds of Nepeta essential oil were estimated by GC/MS. The physical properties of the nanoemulsion including polydispersity index (PDI), mean particle diameter, and viscosity were also determined. 4aα,7α,7aβ‐Nepetalactone (46.31%), 1,8‐cineole (23.13%), and (Z)‐α‐bisabolene (4.01%) were the main compounds of this essential oil. The Nepeta nanoemulsion had a mean droplet diameter of 254.07 nm, PDI of 0.281, and viscosity of 0.887 cP. NNE had stability for up to 60 days. The PNE showed a higher IC50 value than NNE (p < .05). During storage, the antiradical performance of both PNE and NNE was decreased (p < .05). However, emulsification was successful to control this decreasing trend. E. faecalis was the most susceptible bacteria to PNE and NNE, while the lowest inhibition zone was obtained for K. pneumoniae. At the first time, the antibacterial effect of PNE was more than NNE. However, over time nanoemulsion became more successful in maintaining its antibacterial effect. Overall, the incorporation of Nepeta pogonosperma essential oil into a nanoemulsion system can be a promising system to maintain the bioactivity of the essential oil for a longer time.

Emulsion-based technology is the most attractive approach for the delivery of essential oils due to its easy and inexpensive preparation. In this method, hydrophobic material (e.g., essential oil) is encapsulated within colloidal-based delivery systems by various techniques such as ultrasonication, microfludization, high pressure, and homogenization stabilized by surfactant molecules (McClements, 2011;Nishad et al., 2021;Yazgan, 2020). Among these techniques, the ultrasonication method as a green technology can obtain nanoemulsions with lower polydispersity index (PDI), higher stability, and smaller droplet size, using less amount of surfactant and energy consumption compared to microfludization and high-pressure homogenization. Ultrasonic power produces oil droplets by generating intensive disruptive forces and breaking up the water and oil phases (Nirmal et al., 2018;Pongsumpun et al., 2020). Therefore, the ultrasonication method was used in the current study to prepare emulsion of Nepeta essential oil.
Nanoemulsion is an oil-in-water (o/w) or water-in-oil (w/o) emulsion with a mean droplet size of 20-200 nm. Nanoemulsions exhibit optical transparency, controlled releasing, suitable solubility, high physical stability, and bioactivity even at lower concentrations (Flores et al., 2011;Mahdi & Maraie, 2019;Rao & McClements, 2013). Several studies have compared the bioactivity of various pure essential oils (e.g., Verbenaceae, lemon myrtle and anise myrtle, grapefruit peel, lemongrass, and basil) and their nanoemulsions, and investigated the promising potential of nanoemulsions as an effective treatment for improving bioactivity of essential oils (Balasubramani et al., 2017;Kumar & Kumar, 2018;Nirmal et al., 2018;Prakash et al., 2018;Seibert et al., 2019;Sundararajan et al., 2018;Yazgan, 2020). However, the antibacterial and antioxidant potential of N. pogonosperma and essential oil nanoemulsion have not been studied. To the best of our knowledge, this is the first study addressing the antibacterial and antioxidant potential of Nepeta pogonosperma and nanoemulsions. Therefore, the current study aimed to investigate the antiradical potential of pure Nepeta pogonosperma and essential oil and its nanoemulsion. The antimicrobial activity of essential oils against two Gram-positive (E. faecalis and B. cereus) and two Gramnegative (M. catarrhalis and K. pneumonia) food-related pathogens during 60 days of storage was studied to investigate the benefits of nanoemulsion compared to the pure form. Moreover, the chemical compounds of Nepeta pogonosperma were estimated by GC/ MS. The properties of nanoemulsion such as polydispersity index (PDI), mean particle diameter, viscosity, and stability were also determined.

| Material
The aerial parts of N. pogonosperma were collected from the Alamut region (Qazvin province, Iran) in July 2020. A voucher specimen (MPIH-527) has been deposited at the central herbarium of Tehran, Iran. The aerial parts of Nepeta were dried at shade (23 ± 3°C). Then, the air-dried plant was milled (model 320P; Pars-Khazar) and kept in a dark bottle. All chemicals in analytical grades were bought from Merck (Darmstadt, Germany) and Dr. Mojalali (Tehran, Iran). The bacterial strain cultures were purchased from the Iranian Research Organization for Science and Technology (IROST).

| Nepeta pogonosperma essential oil extraction
The pure Nepeta essential oil (PNE) was extracted from the air-dried plant (100 g) based on the hydrodistillation technique for 4 h using a Clevenger apparatus (Azmiran). The oil was dried with anhydrous Na 2 SO 4 and kept in a sealed dark glass bottle at 4 ± 1°C until analysis (Sefidkon & Akbari-Nia, 2003).

| Chemical composition of Nepeta essential oil
The volatile compound of essential oil was analyzed by gas chromatography-mass spectrometry (GC/MS) system Agilent 6890N/5973 inert (Agilent Technologies, USA) fitted with a DB-1-fused silica capillary column (60 m × 0.25 mm and 0.25 μm thickness). The injected volume was 1 μl and the split ratio was 1/50. The oven's initial temperature was increased (ramp rate: 5°C min −1 ) from 60 to 250°C and for 10 min held at the final temperature. The injector temperature was 220°C. Diluted Nepeta essential oil (1 μl) in hexane was injected into the column by using a split-less injec-

| Physical properties of nanoemulsion
The polydispersity index (PDI) and mean particle size of the emulsified droplets were measured by a dynamic light scattering (DLS) (Nanophox Sympatec GmbH, Germany) system based on a laser diffraction particle size analyzer at 25°C. Each sample was diluted with deionized water at a 1:20 ratio to avoid interparticulate interaction and multiple scatterings (Kumar & Kumar, 2018

| Free radical scavenging capacity
The scavenging activity of Nepeta essential oil and its nanoemulsion were estimated on the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical. First, the essential oil or its nanoemulsion (0.1 ml) at various concentrations was mixed with 5-ml methanolic solution of DPPH (4.5 ml of methanol and 0.5 ml of DPPH solution). After 1 h incubation at room temperature, the absorbance of the solutions was read at 517 nm (Shimadzu 2501UV spectrophotometer). The percentage of radical scavenging capacity (RSC) was calculated using the following equation: where A blank and A sample are control (methanolic solution of DPPH) and extract absorbance at 517 nm, respectively.
The IC 50 value (50% inhibitory concentration) was evaluated using linear regression analysis from the obtained RSC values (Koleva et al., 2002).

| Bacterial culture
The food-related pathogens were used to evaluate the antimicrobial effect of the pure and emulsified Nepeta essential oil as follow: Enterococcus faecalis ATCC29212, Klebsiella pneumoniae ATCC700603, Bacillus cereus PTCC1247, and Moraxella catarrhalis ATCC700603.

| Bacterial inhibition assay
The bacterial inhibition effect of the Nepeta essential oil and its nanoemulsion was evaluated by agar disc diffusion assay (Murray et al., 1982) with some modifications. Bacterial suspension (1 ml; 10 8 CFU ml −1 ) was spread on nutrient agar plates, and then, paper disc (diameter: 6 mm) was impregnated with 50 μl of Nepeta essential oil or its nanoemulsion. The discs were placed over plates of Muller Hinton agar (MHA, Difco) seeded with each bacterium.
Turbidity was set to 0.5 Mc Farland standard. The plates were incubated at 37 ± 1°C for 24 h. The antibacterial effect was evaluated by measuring the zones of inhibition (mm) around each of the discs (Domig et al., 2007).

| Minimum inhibition/bactericidal concentration (MIC/MBC)
The MIC and MBC values of Nepeta essential oil and its nanoemulsion against food-related pathogens were measured. Briefly, 1 ml of pure or emulsified essential oil (stock solution: 50 mg ml −1 ) was added to the first tube in each series and diluted with sterile Muller-Hinton Broth (MHB, Merck, Germany). Then, 1 ml of each bacterial suspension (10 6 CFU ml −1 ) was added to each tube. The final concentrations of the sample were 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.19 mg ml −1 . All samples were incubated at 35°C for 24 h. The tubes were investigated for turbidity of the medium as an indicator of microbial growth. The MIC values were defined as the lowest Nepeta essential oil concentration inhibiting visible growth of the tested microorganism. MBC was evaluated by subculturing the contents of tubes of MIC into Mueller-Hinton Agar (MHA), which showed no growth (CLSI, 2008).

| Statistical analysis
All analyses were performed in three replicates and the results were reported as mean values and standard division (mean ± SD).

| Chemical composition of Nepeta essential oil
It was reported that some environmental and geographical factors such as soil properties, ozone concentration, temperature, wind exposure, relative humidity, photoperiod, light intensity, and partial CO 2 pressure as well as the genetic structure could significantly affect the secondary metabolites synthesis, and consequently, the chemical composition and yield of Lamiaceae essential oil (Kofidis & Bosabalidis, 2008;Layeghhaghighi et al., 2017;Talebi et al., 2019).
For example, Layeghhaghighi et al. (2017) showed the significant effect of altitude on the quality and quantity of Nepeta pogonesperma essential oil collected from the Alamut region. The maximum percentage of 1,8-cineole, ρ-cymene, and nepetalactone was observed in 2400, 2600, and 2800 m, respectively.

| Physical properties of Nepeta pogonosperma nanoemulsion
The mean droplet size (Z-average) and PDI of Nepeta pogonosperma nanoemulsion are presented in Figure 2. The Nepeta nanoemulsions had a relatively narrow size distribution with a single peak at around TA B L E 1 Chemical composition of Nepeta pogonosperma essential oils  In the current study, the mean particle diameter of the nanoemul-
The pure essential oil showed higher IC 50 value than nanoemulsion (53.66 vs. 49.90; p < .05). During storage, the antiradical performance of pure and emulsified essential oil was decreased (p < .05).
Investigation of various nanoformulations indicates that emulsification increased the antiradical potential of essential oils. In this regard, Seibert et al. (2019) showed that the nanoemulsion of Cymbopogon densiflorus essential oil has a more antiradical activity than its pure essential oil (IC 50 values of 14.689 and 3.692 mg ml −1 ), respectively (Seibert et al., 2019). The IC 50 value for the DPPH scavenging activity of pure and emulsified Ocimum basilicum essential oil was 13.21 and 10.47 μg ml −1 (Sundararajan et al., 2018). Moreover, the IC 50 value of Vitex negundo essential oil and its nanoemulsion was 28.87 and 23.26 μg ml −1 (Balasubramani et al., 2017). It was demonstrated that nanostructures enhance the activity of biocompounds.
Therefore, the nanoemulsified essential oil was able to reproduce bioactivity compared with pure essential oil even in lower concentrations (Balasubramani et al., 2017;Seibert et al., 2019).

| Antibacterial activity
The antibacterial property of Nepeta essential oil and its nanoemulsion against Gram-positive bacteria (E. faecalis and B. cereus) and Gram-negative bacteria (M. catarrhalis and K. pneumonia) during 60 days of storage are given in  by the previous author (Sonboli et al., 2009). In this regard, Sonboli et al. (2017) suggested that 1,8-cineole and nepetalactone might be involved in the antimicrobial response of Nepeta hormozganica essential oil (Sonboli et al., 2017).
As seen in Table 2, on the first day of storage, the antibacterial effect of PNE (inhibition zone = 8.55-25.67 mm) was more than NNE (inhibition zone = 6.60-17.40 mm). Moghimi et al. (2017) reported that the antimicrobial potential of Salvia officinalis essential oil against Haemophilus influenza was higher than that recorded for the nanoemulsified one. However, for Streptococcus pneumonia and Moraxella catarrhalis, the nanoemulsion was much more effective than the pure essential oil (Moghimi et al., 2017). In addition, the superiority of the antibacterial capability of nanoemulsion as compared to essential oils has already been reported (Anwer et al., 2014;Maté et al., 2016;Yazgan et al., 2019). For example, Yazgan et al. (2019) reported that E. faecalis was more sensitive to lemon nanoemulsion (24.25 mm) than lemon essential oil (19.00 mm) (Yazgan et al., 2019). It can be related to the difference in bioactive compounds of essential oil, formulation, and particle size of emulsion as well as the type of bacterial strain (Donsì & Ferrari, 2016). Over time, nanoemulsion became more successful in maintaining its antibacterial effect (Table 2). This effect could be related to the increased surface area per mass of hydrophobic compounds formulated in nanoemulsions. These small oily droplets penetrate faster in the microbial membranes and improve the ability of bioactive compounds to interact with the microorganisms. It reduces the concentration of the essential oil needed to create a certain antibacterial effect (Donsì & Ferrari, 2016). Moreover, the restructuring of the bioactive materials within the nanoemulsion droplets and a higher local concentration of them could be a reason for the increased activity of nanoemulsion as compared to its essential oil (Garzoli et al., 2020).

| Determination of minimum inhibition/ bactericidal concentration (MIC and MBC)
Results of the MIC and MBC of the pure and emulsified nepeta essential oil on the food-related pathogen microorganisms are summarized in Table 3. Both PNE and NNE were active against E. faecalis proved by previous studies (Badawy et al., 2019). The antibacterial ability of essential oil can be attributed to the -OH groups of bioactive compounds located at the meta and ortho positions. These OHgroups can interact with the cytoplasmic membrane of bacterial cells which leads to cell destruction and death (Shahbazi, 2019 TA B L E 3 Minimum inhibition concentration (MIC: mg ml −1 ) and minimum bactericidal concentration (MBC: mg ml −1 ) against food-related pathogens were of >50 mg ml −1 , which was in agreement with previous studies (Ashrafi et al., 2020;Gharenaghadeh et al., 2017;Yazgan, 2020). Sonboli et al. (2017) showed that Nepeta hormozganica essential oil exhibited a moderate-to-strong antibacterial activity against S. epidermidis, S. aureus, and E. coli, and the effectiveness of this essential oil against Gram-positive bacteria (e.g., Staphylococcus epidermidis) was higher than Gram-negative bacteria (e.g., Escherichia coli).
Moreover, Gharenaghadeh et al. (2017) also reported that MIC of Salvia multicaulis essential oil was 6.25 and 50 μg ml −1 for E. faecalis and K. pneumoniae, respectively. Yazgan (2020) reported that the bactericidal effect of sage essential oil against E. faecalis was low.
However, Nepeta has a suitable bactericidal effect on Gram positive. The presence of the lipopolysaccharide layer in the structure of Gram-negative bacteria is a barrier against the penetration of hydrophilic compounds (Youseftabar-Miri et al., 2021;Zheng et al., 2017).
The comparison of the antibacterial potential of Nepeta essential oil and its nanoemulsion showed that there was no significant difference between those at the first time. Over time, although the effectiveness of both essential oil and nanoemulsion significantly reduced, the decreasing trend was more pronounced in essential oil as compared to the nanoemulsion ( Table 3). Emulsification of essential oil at the nanoscale increased the bioactivity through activation of the cell absorption mechanism (Nirmal et al., 2018). In 1.87 v/v% and MIC for B. cereus: 0.94 v/v%) (Garzoli et al., 2020).
Therefore, nanoemulsions can be considered an effective delivery system for Nepeta essential oil.

| CON CLUS ION
The results show that on the first day of storage, the Nepeta pogonosperma essential oil has slightly more antioxidant and antibacterial potential than its nanoemulsion. However, the nanoemulsion of Nepeta essential oil was more successful in maintaining its bioactivity over time. The outcomes confirmed that the conversion of Nepeta pogonosperma essential oil into nanoemulsion improved the bioactivity of the essential oil. Decreasing the mean size of essential oil droplets within the nanoemulsion increases the local concentration of bioactive compounds and antioxidant and antimicrobial potentials of the nanoemulsion as compared to pure essential oil. It seems that NNE has great potential as a natural preservative to enhance the shelf-life of food products, especially, in food products that Gram positive are dominant microorganisms. Further studies are needed to determine the mechanism action of PNE and NNE against foodborne pathogens and spoilage bacteria and confirm the preservative effects of Nepeta nanoemulsion on food products.

ACK N O WLE D G E M ENTS
The authors gratefully acknowledge the support of Islamic Azad University, Qazvin, Iran.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
This article does not contain any studies with human participants or animals performed by any of the authors.