A new strategy to achieve high antimicrobial activity: green synthesised silver nanoparticle formulations with Galium aparine and Helichrysum arenarium

Silver nitrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Müller Hinton broth, Müller Hinton agar, Sabouraud dextrose broth, and Sabouraud dextrose agar were purchased from Merck (Darmstadt, Germany). The plants H. arenarium and G. aparine were purchased commercially in dry form (Arpas Arifoglu Marketing Distribution and Trade Inc.). E. coli (ATCC number: 25922), S. aureus (ATCC number: 25923), C. albicans (ATCC number: 10231), and A. flavus (ATCC number: 204304) were obtained from the Yıldız Technical University Molecular Biology and Genetics Department, Laboratory of Microbiology (Istanbul, Turkey). Ultra-pure water was obtained from the Millipore MilliQ Gradient system (Millipore Corporation, Bedford, MA, USA).

We opted for a fast, efficient, and environmentally friendly MAE method using water, a non-toxic and reliable solvent, instead of chemical solvents (39). To prepare the aqueous extract, 4.5 g of powdered H. arenarium and G. aparine leaves were mixed with 100 mL of distilled 18.2 MΩ/cm water (4.5% w/v) and boiled for 5 min. To remove plant residues, the extracts were first centrifuged (NF 800R, Nuve, Istanbul, Turkey) at 4025 g and 25 °C for 20 min and then filtered through Whatman filter paper No. 1 and were kept at 4 °C for further tests.

The synthesis of G-AgNPs and H-AgNPs was optimised by trial and error, using different parameters such as pH (6–10), temperature (25–80 °C), AgNO₃ concentration (0.1–2 mmol/L), and reaction time (0–240 min). The optimum green synthesis parameters for G-AgNPs were pH 8, temperature 70 °C, AgNO₃ concentration 1 mmol/L, and reaction time 4 h. Interestingly, the optimum synthesis parameters for H-AgNPs were different: pH 9, temperature 60 °C, AgNO₃ concentration 1 mmol/L (as in G-AgNPs), and reaction time 3 h. G-AgNPs and H-AgNPs analysed in this study were synthesised under these optimal conditions for biological activity.

To synthesise H-AgNPs 1 mL of aqueous H. arenarium extract was added dropwise to 20 mL of AgNO₃ solution in the 1:20 ratio (v:v) at 60 °C to obtain a concentration of 1 mmol/L. The pH of the mixture was then adjusted to 9 with 1 mol/L NaOH using a pH-meter (HANNA instruments HI 2211 pH/ORP Meter). The mixture was stirred on a magnetic stirrer at 60 °C for 3 h. To synthesise G-AgNPs, 1 mL of aqueous G. aparine extract was added dropwise to 20 mL of AgNO₃ solution in the 1:20 ratio (v:v) at 70 °C to reach a concentration of 1 mmol/L, and the pH of the mixture was then adjusted to 8. The mixture was stirred on a magnetic stirrer at 70 °C for 4 h.

The reaction was followed with a UV-Vis spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan). The produced particles were collected by centrifugation at 9056 g and 25 °C for 30 min (Nüve NF 800R), washed three times with ultrapure water to remove unreacted plant residues and silver nitrate, and then lyophilised. All lyophilised nanoparticles were stored at -80 °C until further analysis. NF-1, NF-2, and NF-3 nanoformulations (NF) were prepared by combining G-AgNPs and H-AgNPs as follows: 1000 µg/mL G-AgNP + 1000 µg/mL H-AgNP (NF-1), 500 µg/mL G-AgNP + 1500 µg/mL H-AgNP (NF-2), and 1500 µg/mL G-AgNP + 500 µg/mL H-AgNP (NF-3).

The formation of nanoparticles was first confirmed visually based on the change in the colour of the reaction solution from yellow to brown. Analytically it was confirmed from the intensity of surface plasmon band absorption of nanoparticle solutions measured in the wavelength range of 200–800 nm at 25±0.1 °C using a UV-Vis spectrophotometer (UV-1700, Shimadzu). All measurements were made at a 1:10 dilution of the samples. For the blank we used ultrapure water.

The average size (hydrodynamic diameter) and polydispersity index (PDI) of nanoparticles were measured with dynamic light scattering (DLS), and the zeta potential with electrophoretic light scattering (ELS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 4.0 mV He–Ne laser (633 nm) (7). Measurements were carried out in triplicate, at 25±0.1 °C, using the 0.8872 cP viscosity and 1.330 refractive indexes for the solutions and dielectric constant of 79 f(ka) 1.50 (Smoluchowski value). The number of runs and run durations were chosen automatically. All samples were diluted to a 1:10 ratio with distilled water before measurement.

The functional groups involved in both plant powders (G. aparine and H. arenarium) and the synthesised AgNPs (G-AgNPs and H-AgNPs) were determined using a Fourier transform-infrared (FT-IR) spectrophotometer (Shimadzu) (8). The analysis was performed in universal attenuation total reflectance (ATR) mode at 25±0.1 °C with a scanning spectrum of 800–3500 cm^-1 wavenumber with 32 scans per sample and resolution of 4 cm^-1.

The surface morphology, topography, size, and size distribution of AgNPs were determined with both atomic force microscopy (AFM) (Shimadzu SPM 9600) and scanning electron microscopy (SEM) (FEI QUANTA 450 FEG, Thermo Fisher Scientific, Waltham, MA, USA). AFM images were taken with the force constant of 0.02–0.77 N/m, tip height 10–15 nm, and silicon lever in contact mode (40). Freeze-dried powdered nanoparticles were fixed on metallic studs with double-sided conductive tape, then coated with gold under vacuum and analysed with SEM (7).

The antimicrobial activity of G-AgNPs and H-AgNPs alone and in nanoformulations (NF-1, NF-2, and NF-3) was tested against E. coli (ATCC 25922), S. aureus (ATCC 25923), C. albicans (ATCC 10231), and A. flavus (ATCC 204304) in vitro using the broth microdilution method, which is a quantitative method. The results were obtained by spectrophotometric measurements (OD₆₀₀ nm) and the standard plate counting method, as described by the Clinical & Laboratory Standards Institute (CLSI) standard (41, 42).

We prepared stock solutions of AgNPs and nanoformulations at a concentration of 2 mg/mL and serial dilutions in the range of 1–0.03125 mg/mL. 100 µL of the prepared samples were added to six wells each of the 96-well microplate. Microorganism suspension (10⁶ CFU/mL) was added to each well to adjust the final volume in the wells to 200 µL. Microplates containing bacteria were incubated at 37 °C and microplates containing fungi at 28 °C for 16–18 h. After incubation, we determined minimum inhibition concentrations (MIC) and minimum bactericidal or fungicidal concentrations (MBC or MFC, respectively) by spectrophotometry (OD₆₀₀ nm) and standard plate counting.

For statistical analysis we used the SPPS software version 22 (SPPS Inc., Chicago, IL, USA). All data are expressed as means ± standard deviations (SD) of three independent measurements. Statistical differences in MIC and colony counts between control and treated groups were analysed with the two-way analysis of variance (ANOVA) and significance set to p<0.05.

Table 2 shows the minimum inhibitory, bactericidal, and fungicidal concentrations of G-AgNPs, H-AgNPs, and their three combinations against E. coli, S. aureus, C. albicans, and A. flavus. G-AgNPs were effective against E. coli and C. albicans, and H-AgNPs against S. aureus and A. flavus, which is evident from the Petri dish images (Table 3).

Table 3

In turn, all nanoformulations combining H-AgNPs and G-AgNPs were significantly more effective against E. coli than their components alone, with the MIC lowered to 31.25 µg/mL for all three formulations, which points to a synergistic effect. In addition, NF-2 and NF-3 nearly halved the MIC and MBC of H-AgNPs alone against S. aureus, which points to an additive effect. Neither G-AgNPs or H-AgNPs alone or in combination showed antifungal effect, and their bactericidal effects against E. coli did not improve when combined.