Chemicals and reagents

Copper acetate Cu(CH3COO)2 was purchased from Sigma, Aldrich, Germany and was used for copper solution preparation. Standard antibiotics (Ampicillin, Streptomycin and Fluconazole, 1000 µg/ml) were purchased from Amoun pharmaceutical company, Cairo, Egypt. All chemicals were analytical grade.

Stevia rebaudiana leaf aqueous extract

Stevia leaves were collected and rinsed utilizing distilled water, then dried in a dark place. Five grams of grounded leaves was soaked in 100 ml of distilled water, then heated for 2 min at 85 °C in order to prepare aqueous extract. The boiled extract was then filtered utilizing a Whatman No. 1 filter paper and used for the biosynthesis of CuNPs (Srihasam et al. 2020).

Microbial pathogens cultures collection

Thirteen pathogenic microbial strains (8 bacteria & 5 fungi) were used to assess the antimicrobial action of stevia-CuNPs. The pathogenic bacterial strains were Klebsiella quasipneumoniae ATCC 700603, Bacillus cereus ATCC 11778, Shigella sonnei DSM 5570, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi DSM 17058, Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 8379. The fungal pathogens were Aspergillus flavus ATCC 9643, Fusarium oxysporum ATCC 62506, Alternaria solani ATCC 62102, Rhizopus oryazae ATCC 96382, and Candida albicans DSM 1386. The pathogens were acquired from the Faculty of Agriculture, Agricultural Microbiology Department at Ain Shams University in Egypt.

Media used

Medium (1): Nutrient agar medium was utilized to maintain bacterial cultures. It comprised (g/l): Beef extract, 3; peptone, 5; agar 15 g and pH 7.2. Medium (2): MGYP agar medium was utilized to maintain fungal cultures. It comprised (g/l): malt extract, 3; peptone, 10 and dextrose, 10 and pH 5.2 (Difco 1984). *Broth media was the exact composition of the same agar medium without adding agar.

Standard inoculum preparation

Making standard inoculums for pathogens necessitated employing the Sen and Batra (Sen and Batra 2012) method. For fresh bacterial inocula, a loop that consisted of the alive bacterial growth in 50 ml med. (1) broth was employed, which was agitated at 150rpm for 24 h at 37 °C using a rotary shaker incubator (Shin Saeng, South Korea). For fungal inocula, active spore suspension was inoculated in med. (2) broth at 28 °C for 72 h at 150rpm. One ml of standard culture containing 4.5 × 105 colony forming unit (CFU/ml) was used as standard inoculum for shake flasks experiments. Scrape the fungi agar to obtain a pathogen fungus spore suspension in 10.0 ml sterilized saline solution. The obtained spore suspensions (1.1 × 108/ml) were used as standard inoculum for shake flasks experiments.

Biosynthesis and recovery of stevia-CuNPs

According to Das et al. (2020), 0.8 g of copper acetate was dissolved in 20 ml of demineralized water (2  mM Copper solution) and introduced dropwise to 80 ml of stevia extract solution. The reaction combination was agitated for 3 h at 65 °C until a reddish-brown color emerged, suggesting that CuNPs were successfully synthesized. The nanoparticles were recovered by centrifugation, which caused particle settling, and rinsed repeatedly with demineralized water. The resulting nanoparticles were dried for one hour at 100 °C.

Stevia-CuNPs characterization

Visual color shift was the initial indication that stevia-CuNPs were forming. An aliquot of primary examination for stevia-CuNPs construction utilizing UV-Visible spectroscopy (JASCO Corp., V-570 USA) by spectral analysis at the wavelength of 200–800 or 900 nm to prove the reduction of Cu2+. Zeta potential and size determination for stevia-CuNPs suspension was examined by Zeta potential and Dynamic Light Scattering (DLS) techniques utilizing a Zeta seizer (Malvern Zeta sizer Nano ZS90, UK) instrument. The morphology and size of stevia-CuNPs were studied after the suspension of CuNPs was dried and exposed to the TEM technique. For this, a sample of CuNPs suspension was placed onto a copper grid coated with amorphous carbon, dried, and then examined with HR-TEM (Joel JEM 2100, Japan) at 80 kV. The complementary investigations include estimating concentration via an atomic absorption spectrophotometer, determining the crystalline structure of CuNPs powder via XRD, and identifying chemical residues involving amine, carbonyl, and hydroxyl functional groups in a molecule via FTIR approach (Shimadzu FTIR Tracer-100, United Kingdom) in the 500–4000 cm− 1 spectrum. All previously mentioned tests were conducted in the Nawah Scientific, Cairo, Egypt.

Determination of stevia-CuNPs biocompatibility

To establish a complete monolayer sheet, the 96-well tissue culture (TC) plate was initially inoculated with 1 × 105 Vero cells/ml (100 µl/well) and then incubated at 37 °C and 5% CO2 for 24 h. After confluency, the growth medium was carefully discarded from the 96-well TC plates. The cell monolayer was subsequently washed twice with phosphate buffer solution (PBS) pH 7.0. To evaluate the effects of the stevia-CuNPs, six concentrations (31.25, 62.5, 125, 250, 500, and 1000 µg/ml) were prepared in Roswell Park Memorial Institute (RPMI) medium supplemented with 2% serum. In each well, 0.1 ml of each concentration was added, meanwhile three wells were assigned as controls and received only the maintenance medium (RPMI medium supplemented with 2% serum). The plate was then incubated at 37 °C with 5% CO2 and visually examined for any physical signs of toxicity, such as partial or complete loss of the monolayer, cell rounding, shrinkage, or granulation. Following the incubation, a 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution in PBS (prepared by BIO BASIC CANADA INC) was introduced. Then, 20 µl of the MTT solution was added to each well and mixed thoroughly on a shaking plate at 150 rpm for 5 min. The plate was incubated at 37 °C with 5% CO2 for 4 h to allow for the metabolic conversion of MTT. Subsequently, the medium was discarded, and if necessary, the plate was dried on paper towels to remove any residue. The formazan, a metabolic product of MTT, was resuspended in 200 µl of dimethyl sulfoxide (DMSO) and mixed thoroughly on a shaking plate at 150 rpm for 5 min. Finally, the optical density of the resulting solution was measured at 560 nm, and the background absorbance at 620 nm was subtracted. The observed optical density directly correlates with the quantity of cells present (van de Loosdrecht et al. 1994).

Application of stevia-CuNPs as antimicrobial agents

The antimicrobial application of CuNPs was applied using the well agar diffusion test technique (Guzman et al. 2012). The gram-positive pathogenic bacteria of B. cereus, E. faecalis and S. aureus also gram-negative pathogenic bacteria of K. quasipneumoniae, P. aeruginosa, E. coli, S. typhi, and S. sonnei were maintained on med. (1) agar slants. The tested fungal cultures A. flavus, A. solani, F. oxysporum, C. albicans, and R. oryazae. The fungal cultures were maintained on the med. (2) agar slants. The tested fungal spores and bacterial suspensions (100 µl) were inoculated on med. (1) and med. (2) agar, respectively. The wells were filled with freshly prepared stevia-CuNPs at concentrations of 10, 50, 100, 500, and 1000 µg/ml using distilled water. The specimens were primarily incubated for 15 min at 4 °C (to enable diffusion) and then consecutively for the fungal and bacterial cultures at 28 °C for 96 h and 37 °C for 24 h. The test was considered positive when an inhibition zone was shown around the well after incubation. Ampicillin (G− ve bacteria), Fluconazole (fungi), and Streptomycin (G+ve bacteria) of 1000 µg/ml were used as controls.

Calculation of stevia-CuNPs antimicrobial activity index

By Singariya et al. (2012), the IZDs of CuNPs were compared with the standard reference antibiotic using the subsequent formula to determine the activity index:

$$\begin \text\,\text\,(AI)\, = \text\,\text\,\text/ \hfill \\\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\text\,\text\,\text\,\text \hfill \\ \end$$

(1)

Minimum inhibitory concentration (MIC) for stevia-CuNPs

Serial dilutions of the stevia-CuNPs with final concentrations of 12.5, 25, 50, 75, 125, 250, 500, 1000 µg/ml were carried out following the clinical and laboratory standard Institute (CLSI) guidelines (Humphries et al. 2018). These dilutions were poured into the previously prepared wells in the inoculated plates as mentioned before on med. (1) and med. (2) for bacteria and fungi, respectively. Then incubated at 37 ℃ for 24 h and 28 °C for 96 h in the case of bacteria and fungi, respectively, IZD was recorded and calculated according to Eq. (1). The MIC was determined as the lowest concentration of stevia-CuNPs inhibited microbial growth (Valdez-Salas et al. 2021).

Minimum lethal concentration (MLC) of stevia-CuNPs

According to Rabe et al. (2002), the MLC value is the lowest concentration that showed no growth on the appropriate nutritional medium for microbes. Following the results of MIC, MLC was determined by reinoculation of the inhibition zones results of MIC on med. (1) and med. (2) incubated at 37 ℃ for 24 h and 96 h for bacteria and fungi, respectively. The microbial growth was observed and the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were defined as the lowest concentration of stevia-CuNPs inhibited microbial growth (Abd-Elhalim et al. 2023).

Assessment of stevia-CuNPs mode of action

Stevia-CuNPs mode of action was assessed according to the following equation:

$$\begin \text\,\operatorname\,\text\,\text = \text\,\text\,\text\,\text\,\text/ \hfill \\\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\text\,\text\,\text \hfill \\ \end$$

(2)

Stevia-CuNPs is considered a bactericidal or fungicidal agent if MBC or MFC /MIC value is ≥ 4. On the other hand, it is considered bacteriostatic or fungistatic if the value is ≤ 2 (Galal et al. 2021).

Statistical analysis

Using IBM® SPSS® Statistics software (2017), all specimens and gathered data were statistically examined and reported as means. Duncan’s test was used with a 0.05 P-value (Duncan 1955; Bryman and Cramer 2011). The cytotoxicity results calculated as IC50 were reported as mean ± SD and the difference between the groups was tested using two-way ANOVA, using Graph Pad Prism 8.4.1 (GraphPad Software, San Diego, CA, www.graphpad.com) and the interaction was found significant as P < 0.05. All experiments were carried out as n = 3.