Antibacterial Activities of Phytofabricated ZnO and CuO NPs by Mentha pulegium Leaf/Flower Mixture Extract against Antibiotic Resistant Bacteria

Purpose: In this study, leaf/flower aqueous extract of medicinal plant species Mentha pulegium was used to synthesize ZnO and CuO nanoparticles (NPs) as a cost-effective, one-step, and eco-friendly method. Methods: Physicochemical properties of both metal oxide NPs (MONPs) were determined by UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscope (SEM) and energy dispersive X-ray (EDX) techniques. Results: Phytofabricated ZnONPs and CuNPs illustrated 65.02±7.55 and 26.92±4.7 nm with antibacterial activities against antibiotic-resistant Escherichia coli and Staphylococcus aureus. Higher antibacterial activities were observed for CuONPs compared with ZnONPs. Conclusion: Large surface area and more reactivity resulted from smaller size as well as higher production of reactive oxygen species (ROS) were considered to antibacterial efficiency of CuONPs against antibiotic-resistant E. coli and S. aureus.


Introduction
Infectious diseases resulted from antibiotic-resistant microorganisms specifically multidrug-resistant (MDR) bacteria are increasing owing to the inefficiency of conventional antibiotics. 1 Several mechanisms including higher expression of efflux pump and antibiotic degradable enzymes are recognized for this ability in bacteria. 2 In recent years, nanotechnology has illustrated the appropriate antibacterial capacities of metal nanoparticles (MNPs)/metal oxide NPs (MONPs) compared to their bulk materials and common antibiotics. 3 These abilities can be resulted from unique properties such as higher aspect ratio and surface area to volume ratio of these nanomaterials (NMs) compared with bulk materials. In this case, antibacterial activities of MNPs/MOPNs against MDR bacteria have been reported by various investigations. 4 ZnO and CuO NPs with less cytotoxicity compared to other MNPs/MONPs such as Ag NPs may be a suitable option to obtain efficient antibacterial formulation. 5,6 Ions released by these MONPs are defined as a major antibacterial mechanism which can disrupt the integrity of cell envelope of bacteria. 7 In addition, the antibacterial and biocompatibility of these NPs can be modified by several approaches. For instance, green synthesis of ZnO and CuO NPs by medicinal plant extracts can decrease cytotoxicity and increase therapeutic applications of these MONPs. 8,9 For this purpose, there are many studies related to phytosynthesis of Ag, Cu, CuO, ZnO, and Fe 3 O 4 NPs with antibacterial effects on sensitive and MDR bacteria. 3 In this regard, green synthesized TiO 2 , Fe 3 O 4 , ZnO, Cu, and Ag NPs by Artemisia haussknechtii plant species demonstrated average diameter size of 92.58, 83.4, 60, 35.36 and 10.69 nm with antibacterial activities against Gram-positive and Gram-negative bacteria. [10][11][12] In this study, we used Mentha pulegium plant species to synthesize ZnO and CuO NPs. M. pulegium is flowering plant in Mentha genus of Lamiaceae family which can be found widely in Middle East, North Africa and Europe regions as native species. Stems, leaves and flowers of this plant species have antiseptic activities against wide range of microorganisms. 13,14 Result of disc diffusion assay for essential oils of M. pulegium against E. coli and S. aureus showed inhibition zone diameters (IZDs) by 12.6±0.5 and 21.4±0.8 mm respectively. 15 In previous study, leaves extracts of M. pulegium were used to prepare AgNPs with size distribution in the range 5-50 nm. These MNPs demonstrated antibacterial effects on E. coli, S. aureus and Streptococcus pyogenes bacteria. 16 In fact, both flower and leaf extracts of M. pulegium have shown antimicrobial properties. 17,18 These abilities can be resulted from various oxygenated monoterpenes in secondary metabolites of this plant species. 15 According to above argument, we used leaf/flower aqueous extract to fabricate ZnO and CuO NPs. After characterization of physicochemical properties of these MONPs by standard techniques, antibacterial activities of each MONP against antibiotic-resistant E. coli and S. aureus were determined by disc diffusion, agar well diffusion, minimum inhibition and bactericidal concentrations (MIC/MBC) assays. In the case of the bacterial loading on the glass surface, ATP-bioluminometer instrument was applied to measure cell number upon MONPs stress.

Materials
All materials obtained from commercial sources were utilized without further purification.

Preparation of Leaf/flower extract
Healthy leaves of M. pulegium were sampled from the Amrooleh mountainous region, in the Kermanshah province, west of Iran during July 2019 followed by identification and authentication by an expert of Kurdistan agriculture and resource research center (Sanandaj, Kurdistan). Aqueous leaf/flower extract of M. pulegium was obtained by freshly amassed leaf/flower (10 g). The leaves and flower surface were cleaned with running tap water, followed using distilled water, and air dried on a paper towel for 2 weeks. Dry leaves and flowers were grounded in a tissue grinder to get fine powder, and boiled with 150 mL of double distilled water at 60°C for 1 hour. The filtered suspension was collected and stored at 4°C till further use.

Phytosynthesis of CuO and ZnO NPs
In order to phytosynthesize CuO and ZnO NPs, the Erlenmeyer flask with 100 mL volume of CuSO 4 and [Zn(NO₃)₂ •6H2O] salts by 0.1M concentrations were stirred for 3 hours at 25 ○ C. The aqueous leaf/flower extract of M. pulegium was filtered via Whatman No. 1 filter paper followed by centrifugation at 5000 rpm for 1h. Twenty mL of resulted leaf/flower aqueous extract samples were separately added to 80 mL of CuSO 4 and [Zn(NO₃)₂ •6H2O] at room temperatures under stirred condition for 24 hours. In order to purify NPs, mixture solutions were centrifuged at 4000 rpm for 30 minutes. After drying the colloidal solution, the resulting powder was crushed and subsequently the obtained sediment was washed repeatedly with absolute ethanol and deionized water. In the case of ZnO NPs synthesis, the samples were calcined at 400°C to obtain the powder for subsequent analyses. 19 Physicochemical characterization UV-Vis spectroscopy (Tomas, UV 331), X-ray diffraction (XRD) analysis (model PW1730, PHILIPS, Netherlands), Fourier-transform infrared (FTIR) spectroscopy (AVATAR, Thermo, United States), and FE-SEM (MIRA III, TESCAN, Czechia) technique were applied to determine physicochemical properties of ZnO and CuONPs. Zeta potentials of each NP were indicated by DLS (model ZEN3600, MALVERN, United Kingdom). The intensities related to absorption peaks of ZnO and CuONPs were examined by UV-Vis spectroscopy in the wavelength range of 200 to 600 nm. XRD was applied in the scanning range of 10°-80°(2θ) using Cu Ka radiations of wavelength 1.5406 Å for identification of the crystal phases and determination of the average crystal size of NPs.

Antibacterial activities
Bacteria species Escherichia coli and S. aureus, as respectively gramnegative and gram-positive bacteria species were obtained from Kowsar hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran. In order to determine the sensitivity of these species, after culturing of bacteria on NA medium and incubation for 24 hours at 37 ○ C, antibiotics including amoxicillin, azithromycin, ciprofloxacin, cefixime, doxycycline, gentamycin, and sulfamethoxazole as the amount of 10 µg/disc were tested on these bacteria (Table 1).

Agar well diffusion and MIC/MBC assays
Broth cultures of S. aureus and E. coli as cell density ≈ 1.5 × 10 8 CFU/mL (standardized by 0.5 McFarland standard) were prepared in the PW medium with the concentration of 0.1%. The bacteria species were swabbed on the MHA plate. Wells (6 mm) were made with a sterile metal punch on the surface of the agar plates. Wells were filled by 50 µL of ZnO and CuONPs with serial concentrations of 0.625, 1.25, 2.5, 5, and 10 mg/mL followed by incubation at 37 ○ C for 24 hours. IZDs were measured by calipers as averages of three independent analyses plus standard deviation. 20 Minimum bacteriostatic and bactericidal concentrations

ATP-bioluminometer
ATP-bioluminometer (UltraSnap TM -Surface ATP) is used to determine bacterial loading on the environmental surfaces and medical equipment. 21,22 For this purpose, NP with higher antibacterial activities was selected to measure bacterial removal ability from the infected surface. It should be considered that E. coli bacteria were more resistance to antibiotic compared to S. aureus. Four glass slides were sterilized in 150 ○ C for 120 minutes followed by an addition of 100 µL of E. coli to each slide. The initial cell number of bacteria was measured as amount as 3673 by ATP-bioluminometer. Afterwards, volumes of 25, 50, and 100 µL of NPs with 5 mg/mL value were incubated on the surface of slides containing bacteria. One slide without NPs was considered as the control sample and cell number was obtained after 15 minutes.

UV-Vis spectroscopy and zeta potential
The formation of MONPs can be determined by their surface plasmon absorption band. UV-Vis spectra for ZnO and CuONPs showed peaks at 255 and 278 nm wavelengths respectively (Figure 1). In a similar study, there was a peak at 370 nm for green synthesized ZnO NPs by Artemisia haussknechtii, medicinal plant species. 19 The maximum absorbance for biosynthesized CuONPs via Malus domestica leaf extract was at 335 nm wavelength. 23 The absorption band at 366 nm was found for green prepared ZnONPs via Catharanthus roseus leaf extract. 24 Electrokinetic potentials of both NPs in colloidal dispersion were indicated by zeta potential results. ZnONP demonstrated -7.14 mV and 4.96 mS/cm for zeta potential and conductivity respectively. Also, the values of zeta potential and conductivity were respectively -3.45 mV and 3.43 mS/cm for CuONPs. In contrast, the positive charge of zeta potential as the amount of 2.61 mV was found for phytosynthesized ZnONPs using Solanum torvum leaf extract. 25 Similarly, there was a negative charge of zeta potential around -30 mV at pH≈7 for green synthesized CuONPs by leaf extract of Punica granatum plant species. 26

XRD results
As shown in Figure 2a and 27 As illustrated in Figure 2b, in the case of CuONPs, there was a crystallite structure of face-centered cubic structure (FCC) by the peaks at 35.20 ○ , 38.66 ○ , 47.50 ○ and 53.99 ○ for the planes of (002), (111), (-202) and (020) respectively. 28 These planes were indicated for prepared CuONPs by aqueous extract of oak fruit hull. 29 The obtained crystal size of ZnONPs and CuONPs were respectively 18.09 and 18 nm. Hexagonal morphology of ZnO NPs with 36.83 nm grain size was observed in the case of phytofabricated ZnONPs via C. roseus plant leaf extract. 24 Phytosynthesized ZnONPs by A. haussknechtii leaf aqueous extract showed an average crystal size of 53 nm. 19 In addition, CuONPs biosynthesized by Halomonas elongata IBRC-M 10214 demonstrated grain size in the range of 57-79 nm. 30 According to the present results,  grain sizes of our NPs were smaller compared to similar previous investigations.

FTIR spectra
FTIR spectra of ZnO and CuONPs are presented in Figure 3a and 2b. Prominent peaks at 3435.74 and 3423.06 cm -1 were related to -OH stretching vibration, which can be associated to water adsorption on NPs surface. In the case of ZnONPs, peaks at 1382.34, 1116.70 and 519.94 cm -1 wavenumber were corresponded to C-H bending (aldehyde and alkane), C-O stretching (tertiary alcohol and aliphatic ether) and C-I stretching (halo compound) respectively (Figure 3a). For CuONPs, sharp and intense peaks were at 1627.64, 1101.71 and 600.41 cm -1 respectively related to C=C stretching (alkene), C-O stretching (secondary alcohol and aliphatic ether) and C-Br stretching (halo compound) (Figure 3b). Similar peaks also were indicated to green synthesized ZnO and CuONPs by Abelmoschus esculentus mucilage and algal extract respectively. 31,32 These functional groups may be resulted from the interaction of MONPs with primary and secondary metabolites of leaf extract. 33 C-O stretching (secondary alcohol and aliphatic ether) and C-O stretching (tertiary alcohol and aliphatic ether) were a common functional group for both CuO and ZnONPs. This functional group can be resulted from the presence of secondary metabolites containing alcohol and aliphatic ether in leaf/flower extract. 34 In addition, these peaks may be responsible for the stabilization of ZnO and CuONPs. 35

SEM and EDX analyses
According to the results of SEM images, spherical shape was a common shape of ZnO and CuO NPs (Figure 4a and 4b). Average diameter sizes for ZnO and CuO NPs were respectively 65.02±7.55 and 26.92±4.7 nm ( Figure  4c). As shown in Figure 5, EDX analysis of ZnONPs showed respectively 77.98%, 12.25%, 4.85%, and 3.05% for Zn, O, C and K elements ( Figure 5a). As illustrated in Figure 5b, elemental weights of CuONPs were 51.39%, 21.49%, 13.91%, 9.91% and 3.31% for O, C, Cu, S and K respectively. Oxygen, carbon and potassium elements were common in green synthesis of these MONPs by M. pulegium. Contribution of carbon, phosphor and sulfur indicated for green synthesized CuONPs using Malus domestica leaf extract. 23 In addition to zinc and oxygen, lichen synthesized ZnONPs by P. muralis showed sulfur and chlorine elements in the EDX spectrum. 36

Agar well diffusion and MIC/MBC assays
Agar well diffusion showed antibacterial activities of CuONPs contrast to ZnONPs with any antibacterial effects in all concentrations (    ) are common ROS, which may be resulted in damage of bacterial envelope and biological macromolecules. 28,42,43

Conclusion
This study reported a one-pot, cost-effective, and ecofriendly method to synthesize ZnONPs and CuONPs by leaf/flower extract of M. pulegium medicinal plant. The contribution of various metabolites in the stabilizing of MONPs was confirmed by FTIR spectra. C-O stretching (tertiary alcohol and aliphatic ether) and C-O stretching (secondary alcohol and aliphatic ether) were a common functional group for both ZnO and CuONPs. This functional group can be related to secondary metabolites containing alcohol and aliphatic ether with MONPs stabilization property. Based on the results of XRD spectra, the hexagonal wurtzite and FCC crystallite structures were determined for ZnO and CuO NPs respectively. Despite the similar shape (spherical) for both MONPs, there was a smaller size of CuONPs compared to ZnONPs. Antibacterial effects on antibiotic-resistant E. coli and S. aureus were higher for CuONPs in the face of ZnONPs. A large surface area as well as more reactivity of CuONPs rather than ZnONPs may be resulted in more ROS formation in the bacterial medium. The structural