The Effect of Cytotoxicity and Antimicrobial of Synthesized CuO NPs from Propolis on HEK-293 Cells and Lactobacillus acidophilus

Background Drug resistance is currently possible anywhere in the world. Due to the discovery of antimicrobials, medicine, and health have made tremendous advances over the past several decades. Aim This research evaluated the antimicrobial and cytotoxicity effects of green synthesis of copper oxide nanoparticles (CuO NPs) on Lactobacillus acidophilus and human embryonic kidney 293 cells (HEK). Method and Materials. Propolis was sampled and extracted. Green synthesis of CuO NPs was synthesized and characterized using SEM, TEM, DLS, BET, and zeta potential methods. L. acidophilus (ATCC 4356) was used, and the antimicrobial tests were carried out at different concentrations (10≥ mg/ml). Moreover, the cytotoxicity was evaluated using an MTT assay on human embryonic kidney 293 cells (HEK). Results Synthesized CuO NPs using propolis extracts from Khalkhal (sample 1) and Gillan (sample 2) showed −13.2 and −14.4 mV, respectively. The hydrodynamic sizes of well-dispersed samples 1 and 2 were 3124.9 nm and 1726.7 nm, respectively. According to BET analysis, samples 1 and 2 had 5.37 and 8.45 m2/g surface area, respectively. The surface area was decreased due to the addition of propolis extract, and the pore size was increased. CuO NPs of samples 1 and 2 were visible on SEM images with diameters ranging from 75 to 145 nm and 120 to 155 nm, respectively. Based on TEM analysis, the size of CuO particles was increased in samples 1 and 2. CuO NPs particles had narrow size distributions with evenly dispersed NPs on all sides. The cell viability of the CuO NPs of samples 1 and 2 after 24, 48, and 72 hours was greater than 50%. As a result of the MIC and MBC tests, it was determined that samples 1 and 2 had the same effect against L. acidophilus (0.0024 mg/ml). Biofilm formation and degradation of sample 1 were more efficient against L. acidophilus. Conclusion There was no evidence of cytotoxicity in the samples. In addition, results showed that the green synthesized CuO NPs from Khalkhal propolis were effective against L. acidophilus. Thus, the green synthesized CuO NPs from Khalkhal propolis were the best candidates for clinical application.


Introduction
Microorganisms in the mouth can destroy teeth. Troughout the world, this disease afects many generations. In recent years, silver metal has been replaced with resin restorations due to the advent of silver amalgam treatments in dentistry in the 19th century [1]. During the last few decades, antibiotics have made considerable advances in the medical feld. An emerging phenomenon called antimicrobial resistance could impede international health and sustainable development goals by compromising therapeutic strategies. As a result of AMR, countries and continents have been afected more rapidly than ever, making it one of the most severe public health crises ever. Antibiotic Stewardship program activities include ensuring appropriate diagnostics and treatment of drug-resistant infections and appropriate use of antibiotics judiciously [2]. Tere is no doubt that nanotheranostics represents an advancement in nanomedicine. Nanomaterials and nanotechnology can be used to improve medicinal Efcacy. A unique physicochemical property, targeted delivery, and reduced chance of developing resistance make NPs a popular alternative to antibiotics. Metallic NPs are most commonly reported as alternative antibacterial agents [2][3][4][5]. Lactobacillus is acidogenic bacteria that cause dental caries. Children and adults sufer from pain, structural damage to their teeth, and eventual tooth loss due to the loss of mineralized tissues. Lactobacillus is the most common bacteria that cause periodontal diseases and cause infammation, and infection [6][7][8][9][10][11]. An arsenal of redox potentials is found in plant extracts. NPs with defned sizes can be produced by biogenic synthesis using phytochemical compounds as stabilizers. Propolis is used in traditional medicine for its biological properties. Te antioxidative, anti-infammatory, and antibacterial properties of propolis are noteworthy. Propolis exhibits antiproliferative and antitumor properties in vitro and in vivo. It depends on the region, climate, and extraction season. Because propolis contains polyphenolic acids, favonoids, and terpenoids, it can produce gold NPs [12][13][14][15][16][17][18]. Te cost of manufacturing metallic NPs can be reduced by capping and reducing metallic NPs with biological components. Since they do not require excessive pressure or energy, they are environmentally friendly and energyefcient [18][19][20][21]. NPs can be manufactured from medicinal plants. Allium sativum, Aleo vera, and Punica granatum have been used to synthesize copper NPs [22,23]. Tis research aimed to synthesize the CuO NPs using propolis extracts and evaluate their antibacterial and cytotoxicity on Lactobacillus acidophilus and HEK-293 cells, respectively.

Method and Material
2.1. Materials. Propolis was collected from Gillan and Khalkhal ( Figure 1). To synthesize CuO NPs, the following materials need to be used: copper sulfate pentahydrate (CuSO 4 .5H 2 O) (Sigma Aldrich, Co., USA) and absolute ethanol (Merck, Germany). Tis experiment used reagents purchased from Merck, Germany; none of them were purifed before use.

Preparation of Propolis Extract.
Ground samples were prepared and frozen (−20°C) for a while. In this study, raw propolis samples were extracted (by stirring them in a tetrafold amount of ethanol (70%) for three days in a dark environment at room temperature) to quantify their ethanol content. To remove waxes and other less soluble substances from the suspensions, they were fltered (Whatman paper, No.1) to make sure that all waxes and other less soluble substances were removed from the suspensions. It was necessary to repeat this procedure three times to get the desired results. Te freeze-dried extract solution was obtained for the next steps [24].

Green Biosynthesized Copper NPs.
To synthesize CuO NPs, the procedure was to dissolve 1 mg of extract in deionized water to form a solution and then adjust the pH of this solution to 8 by adding NaOH to form a solution. A solution of copper sulfde (6 mM) was slowly added to 100 ml of the extract solution while stirring with a rotation speed of 1,000 rpm. Te samples were then placed in a fask and stirred (24°h, 37°C to 40°C) in the dark at a temperature between 37°C and 40°C in the dark. After the colored mixture (dark brown) had been obtained, it was centrifuged at 13,000 rpm (15 min, 25°C) to obtain the colored mixture (dark brown). A deionized water wash was performed on the pellet twice to ensure that all residues were removed from the extract. A lyophilized precipitate was then stored to be analyzed in the future.

Zeta Potential
Analysis. Zeta Potential analysis was used to detect the hydrodynamic size polydispersity index and the size polydispersity index of the synthesized NPs. Particle sizes were evaluated under an angle of 90 degrees at 25°C.

DLS Analysis.
Tis study used the DLS method to size metal oxide NPs (measured in terms of hydrodynamic radius) according to the range sizing method. An open capillary cell was used in a 37°C water bath, and a disposable folded capillary cell was used to measure the particle sizes of the dispersions after they had been prepared using a disposable folded capillary cell.
2.6. BET Analysis. As a part of the investigation, the surface area of NPs under vacuum was determined using the Brunauer-Emmett-Teller (BET). As part of the surface area measurement, a 2-hour vacuum degassing of the sample was carried out at 120°C.

SEM Analysis.
To determine the structure and morphology structure of the NPs, the SEM examination was carried out. As part of the SEM studies, approximately 25 μl of NPs were applied to the CuO stub, and the results were evaluated. Using a scanning electron microscope, photographs of the samples were taken [25]. Approximately 10 kV of accelerating voltage was used with a working distance of approximately 3 mm. Te brightness and contrast correction also resulted in clear and distinguishable images that could be displayed. Te lengths of the measurement scales, 500 nm and 20 μm, were calibrated using the NISTRM for calibration purposes.

TEM Analysis.
Te shapes of the CuO NPs were observed using TEM imaging based on suspensions induced by propolis-mediated suspensions accelerated at 200 KV and tilted at X-tilt ± 60°. A copper grid coated with carbon was used as a platform for the droplet of NPs solutions to achieve this goal. A specimen holder was used to mount the grid once it had dried, and a few minutes later, it was mounted on the specimen holder for drying. Te TEM image of the selected area of the sample was overlaid with the selected area's difraction pattern to clarify the sample's lattice pattern and crystallite size [26].
2.9. Cytotoxicity Assay. Biosynthesized NP solutions were sonicated before using in any experiments. Te NPs were sterilized using UV light. In the culture of the human embryonic kidney 293 cells (HEK), 100 U/mL penicillin, 10% fetal bovine serum (Himedia), 100 U/mL penicillin, and 2 mM L-glutamine were used. Te cells were maintained at 70-80% confuency by passage every 2-4 days in a 25 cm 2 fask [27]. Human embryonic kidney 293 cells (HEK) were used to test the green biosynthesized NPs [28][29][30][31]. In 96-well plates, 100 μl of cell suspension was flled into each well, containing 10000 cells. A set of parameters was followed during incubation. Te concentrations of CuO NPs were 2.5, 5, 10, 25, 50, 75, and 100 μg/mL, and the control group was cell culture. Once the initial model had been dissolved in DMSO, the NPs were dispersed in PBS. After adding the samples, the plates were incubated for 48 hours. A control medium was used without test samples. Each well was incubated for four hours at 37°C with 15 μl of MTT in PBS. We dissolved the formazan crystals in 100 μl of DMSO after removing the MTT medium and the medium containing MTT. 570 nm measurements were taken to determine the absorbance. Te cell viability percentage was calculated using the following equation:

Determination of Minimal Inhibitory/Bactericidal Concentrations (MIC/MBC).
Tis experiment used Lactobacillus acidophilus (ATCC 4356) as the bacteria. 1.5 × 10 8 CFU/mL was the fnal concentration [32]. MICs and MBCs of green synthesized CuO NPs from Khalkhal (sample 1) and Gillan (sample 2) propolis were determined following the guidelines of the NCCLS. To prepare the nutrient broth medium for dilution, a concentration of 10 mg/mL of CuO NPs was added to the nutrient broth at 10 mg/mL. To create solid nutrient agar plates, solid nutrient agar was used for plating the bacteria. It was determined that an inhibitory concentration was reached when the lowest concentration inhibited bacterial growth. Te MBC needs to be determined in a concentration that allows bacterial growth to be inhibited entirely. Our experiments were repeated in equal numbers for each sample. Te average growth rate of the bacteria on each plate was calculated based on the average growth rate on each plate [33].

Bioflm Formation Analysis.
In the process of conducting this test, microdilutions were used as a dilution method. It was observed that bacteria were cultured for 24 hours using TSB. TSB containing 1% sucrose was used as the dilution solution, and the suspension was diluted 1 : 100 in the solution. Te extracts containing 10× MBC were used for this test. To transfer 100 μl of a mixture of bacterial suspension and extract solution to each of the microplate wells, 200 μl of 100 μl of a suspension of pathogenic bacteria was transferred, 100 μl of extract solution, and 100 μl of mouthwash. As a positive control, 0.2% chlorhexidine was added as a solution to 200 μl of bacterial suspension and physiological saline in which bacterial suspension and physiological saline were mixed. During the incubation period of 24 hours, the plate surface was incubated at 37°C in an incubator. Using a bufer containing phosphate and saline, the wells were washed three times with the solution after the contents had been removed from them. It was necessary to do this to remove disconnected cells. In the following step, 200 μl of 33% glycolic acetic acid was added to the wells containing cells that had adhered to the bottom to remove them. After 15 minutes, the optical density at 570 nm of each well of each sample was measured using an ELISA reader, and bioflm formation rates (%) were calculated using the mentioned formula based on the optical density at 570 nm of each sample.
The biofilm formation rate � Samples(OD) Control(OD) × 100. (2) OD treatments and OD controls are defned as the absorbance at 570 nm in each well after the dissolving solution was added to the well with and without the sample, respectively.

Te Bioflm Degradation Analysis.
Tis test was also conducted to investigate whether microdilution has any destructive efects on bioflms. Microplates were inoculated with bacteria using TSB medium, 3% glucose, and synthetic saliva (McDougall solution) for growth. A bioflm was formed as soon as the remaining culture medium of the culture was discarded. Te MBC solution was diluted ten times with the sample solution. To remove the bioflms on the walls of the wells, phosphate bufer was used. After Evidence-Based Complementary and Alternative Medicine applying it to them, it took 15 minutes for the walls to become saturated with 1% violet crystal. Sterile solutions of water mixed with 95% alcohol were used to clean the wells, and three rinses of sterile water were used after each. After the suspension was transferred from one microplate to another, it was incubated for 45 minutes before the new microplate was used. Using a microplate reader at a wavelength of 570 nm, we measured the optical density of the suspension in each well to determine the extent of degradation of the bioflm. Te positive control was chlorhexidine 0.2%. Trough the use of the equation mentioned, we were able to calculate the percentage rate of bioflm degradation using the following equation [34]: OD treatments and OD controls are defned as the absorbance at 570 nm in each well after the dissolving solution was added to the well with and without the sample, respectively.

Statistical Analysis.
It was performed independently in triplicates for each of the tests. A one-way ANOVA test (SPSS statistics model 20) was used to compare the means among the groups, and Tukey's post hoc test allowed further comparisons. Te signifcance level was P value <0.05.

Characterization Results.
A UV-visible spectrophotometer was used in a previous study and performed surface plasmon resonance measurements on CuO NPs. In UV-Vis spectra of CuO NPs prepared from propolis (Khalkhal) extract, a characteristic peak at 385 nm can be seen on the spectrum. As a result of further UV-Vis spectrophotometry investigation into CuO NPs using propolis extract (Gillan), peaks are observed at 243, 292, and 350 nm. CuO NPs were analyzed using XRD techniques using extracts of propolis (Khalkhal), resulting in crystallographic planes of facecentered cubic (FCC) with peaks of difraction around 2θ � 35.74°, 39.04°, and 49.04°. Te difraction peaks of CuO NPs using extracts of propolis (Gillan) were observed around 2θ � 25.54°, 26.69°, 38.79°, and 48.84°. Te Khalkhal propolis extract FTIR spectrum showed a sharp peak at 3422 cm −1 due to free hydroxyl groups and their intramolecular and intermolecular hydrogen bonds. Sharp peaks at 2925, 1637, and 1515 to 1076 cm −1 were associated with C�O and C�C aromatic stretching frequencies. CuO NP monoclinic phase exhibits an absorption band of 602 cm −1 . As a result of free hydroxyl groups and their intramolecular and intermolecular H-bonds, CuO NPs of the Gillan propolis extract spectrum also peak at 3410 cm −1 . CSp3-H and aromatic stretching frequencies of C�O and C�C were related to the peaks at 2920, 1614, and 1515 to 1057 cm −1 . An absorption band of 603 cm −1 was observed in the monoclinic phase of CuO NPs [35].

Zeta Potential DLS Analysis. CuO NPs produced from
Khalkhal propolis extract (sample 1) showed a zeta potential of −13.2 mV. In contrast, the produced CuO NPs from Gillan propolis extract (sample 2) had a zeta potential of −14.4 mV (Figure 2). As a result of using the sonication method provided by the National Institute of Standards and Technology (NIST), the CuO NPs were separated from CuO NPs synthesized from Khalkhal (sample 1) and Gillan propolis extracts (sample 2); however, there was no noticeable diference between the well-synthesized CuO NPs synthesized using propolis extract and those synthesized using propolis extracts. For well-dispersed samples 1 and 2, it was found that the hydrodynamic sizes were 3124.9 nm and 1726.7 nm, respectively, based on DLS data (Figure 3). Table 1 shows that the amount of precursor used in the Brunauer-Emmett-Teller (BET) process determines the system's surface area. As a result of the synthesized CuO NPs using diferent extracts of propolis, it was found that they had a surface area of 5.37 to 8.45 m 2 /g for samples 1 and 2, respectively. Propolis extract increased the pore size and decreased the surface area.

SEM and TEM Evaluation.
Images obtained from SEM micrographs of the synthesized CuO NPs using propolis extract showed that the crystallization structure of CuO NPs synthesized from propolis extract was quite polydisperse and similar to anatase phase CuO crystallites. As a result of the experiments in sample 1 (Khalkhal), NPs with diameters ranging from 75 to 145 nm were observed. Tere were also NPs ranging in diameter between 120 and 155 nanometers in sample 2. Based on the diameter of the propolis extracts from Gillan and Khalkhal, it was possible to compare the extracts based on their diameters ( Figure 4). As can be seen in Figure 4, green CuO NPs were biosynthesized with propolis extracts and taken from Khalkhal and Gillan. In part, the increase in CuO particle size can be attributed to the green biosynthesis among samples 1 and 2. It is important to note that CuO NP particles had narrow sizes and were uniformly dispersed throughout all sides of the particle.

Cell Viability Evaluation.
Various concentrations of samples 1 and 2 NPs were incubated with HEK cells to determine their efect on the cell-cultured cells (CuO NPs concentration: 2.5, 5, 10, 25, 50, 75, and 100 μg/mL and control: cell culture) for 24, 48, and 72 h. To determine the viability of cells, the cytotoxicity test must be performed. As a result of both the dose and time dependence, the number of viable cells was decreased in samples 1 and 2 NPs. It was determined that the cell viability percentages for the HEK cells and the control groups were calculated for 24, 48, and 72 hours. As a result of MTT data analyses, the cell survival rate of both samples was approximately more than 50% during 24, 48, and 72 hours ( Figure 5).  (Table 2). As seen in Table 2, levels of MBC were within the range of 0.0024 for L. acidophilus.

Bioflm Formation and Degradation Evaluation.
It was determined that the samples could prevent bioflm formation using microdilution tests as a method of determining their efectiveness. Table 3 shows the bioflm formation results, and the OD (570 nm) was determined to compare the ODs of treated groups (samples 1 and 2). Sample 2 of the NPs showed the highest levels of bioflm growth compared to sample 1 which showed the highest efect against L. acidophilus. Bioflms that had already been formed were also treated with the same method to test the treatment's efects. As a result of the degradation of bioflm, a percentage degradation was calculated (Table 4). Tere was a signifcant efect of sample 1 NPs against L. acidophilus. Figure 6 shows the bioflm formation and degradation tests of both NPs against L. acidophilus.

Discussion
Health risks arise from bioflms containing pathogenic microorganisms. Lactobacillus species can colonize tooth surfaces due to bacterial adhesion. Along with mechanical plaque removal, natural antimicrobial mouthwashes enhance it. Chlorhexidine mouthwashes promote tooth decay more than herbal mouthwashes [13]. Global public health  has been afected by AMR in recent years. MDR strains of pathogenic bacteria are becoming more resistant to antibiotics due to the emerging trend of AMR. Recently, metallic nanoscale materials have been used more frequently in nanotechnology. Te NPs show promising therapeutic effects due to their unique physicochemical properties [36,37]. In addition, NPs require a thorough understanding of their physicochemical characteristics and appropriate synthesis methods [36,38]. A new method for synthesizing metal NPs has been developed by utilizing biological cells' highly structured physical and biosynthetic activities. As nanotechnology continues to improve, it is now used in nearly all biomedical applications, from laboratory to large-scale manufacturing. In this study, CuO NPs were synthesized using phase-pure, green propolis extract. Due to phenolic compounds, antimicrobial properties are present in natural materials [39]. In Barbosa et al., silver NPs (AgNP-P) were synthesized from Brazilian propolis and studied for their antimicrobial properties. A factorial design was used to optimize the synthesis conditions for smaller particles. UVvisible spectra revealed that AgNP-P was formed in a spherical structure, with maximum absorbance at 412 nm.
Tis new material shows a good size distribution and a low polydispersity index resulting from dynamic light scattering. Silver NPs were found to contain propolis after centrifugation, and microscopy analysis confrmed it. Te principal    planes of the metallic silver crystalline structure were identifed by X-ray difraction, while infrared spectroscopy indicated that 22% of the AgNP-P in the sample was reduced to silver. Silver NPs and propolis synergistically demonstrated antimicrobial activity and had signifcant antimicrobial efects [40]. Te CuO NPs were synthesized by Hajizadeh et al. and characterized by FTIR, XRD, and UV-Vis absorption spectra. According to FTIR analysis, propolis extract compounds were found to modify the surface of synthesized NPs. Tere was a sharp peak at 3422 cm − Manikandan et al. report that green-fabricated CuO NPs with a −17.2 mV zeta potential could have more excellent colloidal stability when fabricated from a higher negative charge. NP's catalytic activity is primarily infuenced by its surface area, crystal size, and crystallinity. It may be possible for NPs to be highly photocatalytic if the BET value is high, the crystal size is small, and the crystallinity is high. Using propolis increased the rate of photodegradation and specifc surface area while decreasing crystal size [41].
According to Moustakas et al., acute CuO NPs exposure caused photosynthetic activity, oxidative stress, and CuO bioaccumulation in seagrasses (Cymodocea nodosa). Te CuO NPs were characterized with SEM and DLS measurements at 4, 12, 24, 48, and 72 hours (Optimum CuO NPs exposure). In terms of size distribution, CuO NPs synthesized with Pdi 0.35 had an average size of 233 nanometers based on their results [42]. Te present study demonstrated that propolis extract could be used to synthesize CuO NPs, but no smaller particles were found. Khalkhal and Gillan propolis extracts were found to interact with CuO NPs in terms of physical interactions.
Green tea (Camellia sinensis L.) and lavender (Lavandula angustifolia) have been found to synthesize green and pure CuO NPs, respectively. Based on SEM images of lavenderproduced samples, it appears that NPs tightly adhered to each other, and lavender was more efcient in generating pure and uniform CuO NPs (50 nm) [43]. A compact distribution of NPs was observed with narrow dispersion. According to the average particle size measurement, NPs were spherical and ranged from 75 to 145 nm for sample 1 (extracted from Khalkhal) and 120 to 155 nm for sample 2 (extracted from Gillan). It has been possible to detect aggregates and NPs of synthetic CuO NPs with spherical shapes on a micrograph.
Veisi et al. studied CuO NPs synthesis with the help of aqueous extracts of Stachys lavandulifolia fowers, and they demonstrated that TEM analysis provides morphological information about CuO NPs shapes and dimensions. According to the authors, NPs are synthesized with moderately good monodispersity, with sizes ranging between 15 and 25 nm without agglomeration [44]. As the surface-to-volume ratio decreases, the particle size decreases inversely, facilitating easy penetration of the cell wall and rapid destruction of microorganisms; Khalkhal and Gillan (Khalkhal and Gillan) propolis extract enhanced the antimicrobial activity of green synthesized CuO NPs. Botteon et al. (2021) described the biosynthesis of AuNPs and evaluated their structural properties. An SPR band was observed at 535 nm in AuNPs. Te Brazilian red propolis (BRP) sample used in the reaction afected the sizes and morphologies. All strains tested showed antimicrobial activity against AuNPdichloromethane and AuNPhexane. In both T24 and PC-3 cells, AuNPs showed dose-dependent cytotoxicity. ANPdichloromethane and AuNPextract were the most cytotoxic. Apoptosis-related mechanisms are also responsible for biogenic nanoparticle cytotoxicity [12]. Rao and colleagues employed the coprecipitation method to stabilize and cap Zinc oxide NPs (2018). NPs (PZnO) were synthesized from P betel. PZnO was tested for its antibacterial activity against dental pathogens using a well-difusion method. Both tested microbes were inhibited by PZnO at concentrations of as low as 3.25 μg/mL, indicating higher antimicrobial activity than DZnO. PZnO and DZnO inhibited cellular growth by 40% using Balb 3T3 mouse fbroblast cell lines [45]. Tere were no signifcant cytotoxicity results during the incubation of samples 1 and 2 NPs during the 24, 48, and 72 hours of incubation, which showed that the viability of the samples was approximately more signifcant than 50%.
NPs synthesized from biosynthetic materials were subjected to MTT assays to assess their impact, and they signifcantly reduced the survival of cancer cells. NPs synthesized using green methods have been shown to possess antiproliferative properties against cancer cell lines [46,47]. Silver NPs were green synthesized using leaf extract from Justicia glauca by Emmanuel et al. AgNO 3 solution was mainly reduced to AgNPs by water-soluble organics in leaf extracts. TEM images showed that AgNPs were 10-20 nm in diameter. S. mutans, S. aureus, L. acidophilus, and C. albicans were used, and the antibacterial and antifungal activities of AgNPs were demonstrated. AgNPs demonstrated MICs between 25 and 75 g/mL [6]. Based on extracts of A. javanica leaf, CuO NPs' antibacterial properties were evaluated against P. aeruginosa, E. coli, S. aureus, and A. baumannii. Tere was greater efectiveness of the CuO NPs against S. aureus, P. aeruginosa, A. baumannii, and E. coli. According to the sample, S. aureus had a maximum inhibition zone of 9 ± 1 mm in the sample. As a result of the extractions with A. javanica, P. aeruginosa displayed an even more signifcant inhibition zone. Te pathogens that were most active against CuO NPs were S. aureus, A. baumannii, P. aeruginosa, and E. coli, while those with minor activity were E. coli and P. aeruginosa. It has been demonstrated previously that S. aureus was more susceptible to CuO NPs than E. coli, based on a comparison between the two samples [48]. Tere was no diference between samples 1 and 2 in terms of their efect on L. acidophilus (0.0024 mg/ml) in the MIC and MBC tests in this study. It was found that the efect 8 Evidence-Based Complementary and Alternative Medicine of green synthesized CuO NPs from Khalkhal (sample 1) on bioflm formation was more efective than green synthesized CuO NPs from Gillan (sample 2).

Conclusion
Te use of nontoxic, cost-efective, ecofriendly, and easyto-use materials have proven successful and expanded. All samples were tested for cytotoxicity, and it showed that all samples were free of any notable cytotoxicity. In addition, the results showed that the green synthesized CuO NPs from propolis had antibacterial efects against L. acidophilus. It can be concluded that green synthesized CuO NPs derived from propolis may be the best candidate for clinical application due to their high antibacterial properties.

Data Availability
Te data generated or analyzed during this study are included in this published article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.