Generation and Characterization of Silver Nanoparticles in Mentha pulegium Extract and Evaluation of Biological Activities of the Prepared Extract
The aim of the present study was to synthesize silver nanoparticles (AgNPs) using Saudi Mentha pulegium leaves, to characterize the physicochemical properties of the resulting AgNPs, and to evaluate the biological activities of the resulting AgNP-containing extract. The formation of AgNPs in M. pulegium extract was indicated by a change in color following the addition of silver nitrate and was confirmed using UV-visible spectroscopy with a maximum absorbance at 425 nm. Energy dispersive X-ray spectroscopy (EDX) indicated that the anisotropic AgNPs were spherical, and Fourier transform infrared spectroscopy (FTIR) spectral analysis indicated that the aqueous M. pulegium extracts were responsible for reducing Ag+ to Ag0. The secondary metabolite contents of the methanolic M. pulegium extract corresponded to 17 mg GAE/g DW. DPPH and ABT radical-scavenging assays indicated that the M. pulegium extracts possessed antioxidant activity ( and 3 μg/mL, respectively). Disc and broth dilution assays revealed that the extracts exerted significant antibacterial activity, with the inhibition zone diameters and minimal inhibition concentrations of 17–24 mm and 0.08–0.62 mg/mL, respectively. These findings clearly indicate that modified plant extracts have high biological importance and potential use as preservatives in the pharmaceutical and food industries.
The biosynthesis of nanoparticles from plants, bacteria, and fungi is an environmentally safe strategy for generating small particles (10–100 nm) with unique physicochemical and optical properties [1, 2]. In fact, the green synthesis of nanomaterials and nanoparticles, for the purpose of protecting the environment from hazardous wastes, has been implemented in the textile, food packaging, and cosmetics industries, as well as in labeling experiments, biosensors, and cell imaging [3–9].
Silver nanoparticles (AgNPs) have several characteristics, including important thermal and electrical conductivities, surface-enhanced Raman scattering, chemical stability, and catalytic and biological activities (antioxidant, antimicrobial, antiviral, and antitumor) [8–13]. Because the typical chemical and physical techniques used to produce nanoparticles involved hazardous wastes and/or high energy costs, the use of biological methods is an attractive alternative.
The biological activities and chemical compositions of many plant species have been well studied. However, few studies have investigated either the biosynthesis of AgNPs using Mentha species or the antibacterial, antifungal, or antioxidant activities of AgNP-containing extracts of Saudi Mentha pulegium.
The flora of Saudi Arabia is very diverse and contains over 1200 medicinal plant species, of which many exhibit pronounced endemism . The genus Mentha (Lamiaceae) includes ~13 species and is distributed throughout the temperate regions of Eurasia, Australia, and South Africa . One of the important Mentha species in Saudi Arabia is M. pulegium, which is locally known as Al-Medina mint [15, 16].
In general, M. pulegium is known by ancient popular as an important remedy for treating diseases (e.g., bronchitis, flatulence, anorexia, and ulcerative colitis), owing to its antibacterial, antifungal, and antioxidant activities [17, 18]. Recently, Mentha spp. have been used to synthesize ferric chloride, silver, and iron oxide nanoparticles [12, 13, 19, 20].
To valorize the Mentha species, the aim of the present study was to characterize AgNPs synthesized from Saudi M. pulegium using a variety of techniques, including transmission electron microscopy, UV-visible spectrophotometry, and Fourier transform infrared spectroscopy (FTIR).
2. Materials and Methods
2.1. Plant Material and Extraction Methods
Mentha pulegium plants were identified in an agricultural region in Al-Kharj, Saudi Arabia, and their identities were confirmed using the “Flora of the Kingdom of Saudi” . Leaves were harvested, shade-dried, and ground into fine powders. Then, for extraction, 2 g samples were each combined with 20 mL ultrapure water, incubated for 2 h at 37°C, and then filtered using Whatman filter paper no. 1.
2.2. Silver Nanoparticle Biosynthesis
The filtered extract of M. pulegium leaves was utilized to produce the silver nanoparticle. In fact, the preparation of silver nitrate solution 0.1 M was carried out by adding 0.169 gr of to 10 mL of distilled water. After dilution of 1 mL for 20 times, the extract was added to 0.4 mL of 0.1 M of prepared silver solution and incubated for 24 h at 37°C .
2.3. Silver Nanoparticle Characterization
Previous studies have reported that the development of brown coloration by prepared extracts indicates successful AgNP biosynthesis [10, 22]. Three analytical methods were used to characterize the synthesized nanoparticles. More specifically, T60 UV-visible spectrophotometry (resolution of 2 nm at 280 and 720 nm) was used to estimate the optical properties of the biosynthesized AgNPs. Fourier transform infrared spectroscopy was used to estimate the rate of AgNP reduction by other plant extract components, and scanning electron microscopy (SEM: TS Quanta 250), in conjunction with energy dispersive X-ray spectroscopy (EDX), was used to evaluate the size, shape, and elemental composition of AgNPs in room-temperature-dried extract droplets.
2.4. Secondary Metabolite Analysis
The phenolic content of the prepared extract, expressed as mg gallic acid equivalent per g dry weight (mg GAE/g DW), was evaluated using Folin-Ciocalteu reagent (Sigma-Aldrich) as described previously . Meanwhile, total flavonoid content, expressed as rutin equivalent per mL extract (RE/mL extract), was evaluated using the aluminum chloride colorimetric method . Briefly, 1 mL of the evaluated extract was added to 1 mL 2% AlCl3 (in methanol) and incubated for 15 min at 25°C, and absorbance at 430 nm was measured using a 160-UV spectrophotometer (Shimadzu, Tokyo, Japan).
2.5. Antioxidant Activity Evaluation
The antioxidant ability of the prepared extract was estimated using two assays. First, the method reported by Hatano et al.  was used to estimate DPPH radical-scavenging activity. Briefly, 1 mL extract was mixed with 0.5 mL DPPH solution (0.2 mM), and the resulting mixture was incubated in the dark for 30 min, after which the absorbance of the solution at 517 nm was estimated. DPPH radical-scavenging activity was then estimated using the following equation: , where and represent the absorbance of the control and sample, respectively.
To estimate ABTS radical-scavenging activity, ABTS stock solution and potassium persulfate (2.45 mM) were mixed, and the resulting mixture was incubated at room temperature in the dark for 24 h to produce ABTS cation radical (ABTS+) . ABTS radical-scavenging activity was then estimated as the percent reduction in ABTS+ content, using the following formula: , where and represent the absorbance of the blank and sample, respectively.
2.6. Antimicrobial Activity Evaluation
The antimicrobial activity of the prepared extract against six pathogenic bacteria (Salmonella typhimurium NCTC 6017, Listeria monocytogenes ATCC 7644, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, and Bacillus cereus ATCC 1247) was evaluated using two methods.
The disc diffusion method was used to evaluate the qualitative antibacterial activity of the prepared extract . Briefly, samples of each bacteria strain (100 μl, 107 CFU/mL) were spread on separate Muller-Hinton agar plates. Sterile filter paper discs (6 mm) were impregnated with 15 μl of the prepared extract and placed onto the inoculated plates, using gentamicin (10 μg)-impregnated and untreated filter paper as positive and negative controls, respectively. To determine the solvent activity, solvent control disc was employed. Then, the plates were incubated at 37°C for 24 h, and the antimicrobial activity of the prepared extract was estimated using inhibition zone (IZ) diameter.
Meanwhile, the broth dilution method was used to evaluate the quantitative antibacterial activity of the extract, as described by Cosentino et al.  and modified by Aouadhi et al. . More specifically, the method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which are defined as the lowest concentrations that prevent visible bacterial growth and kill 99.9% of the initial bacteria, respectively .
2.7. Statistical Analysis
All experiments were performed three times, and data are presented as values. Differences were considered significant at . The AgNP size distribution was evaluated using SEM images, ImageJ, and Origin 8.0.
3. Results and Discussion
3.1. Silver Nanoparticle Properties
The variation of the color of the prepared extract after addition of AgNO3 solution was used as indicator of the biosynthesis of silver nanoparticles. In the present study, the formation of AgNPs was indicated by a change in extract color (to yellowish-brown) following the addition of AgNO3 (Figure 1). Yousaf et al.  reported that the color change is the result of Ag+ reduction. AgNP formation was also confirmed by wavelength scan (280–720 nm) using a T60 UV VIS spectrophotometer. The prepared extract yielded an absorbance peak at 420 nm, which indicated the synthesis of spherical and uniform AgNPs (Figure 2) [22, 31].
FTIR spectral analysis (510–4000 cm) was used to identify the functional groups responsible for reducing Ag+ ions into AgNPs in the prepared extract [22, 32]. The analysis was achieved by comparing the prepared extract to another extract with a half-reduced nitrate solution volume (Figure 3). The results of the FTIR spectral analysis indicated that the AgNPs were associated with four distinct peaks (Figure 3). To identify the functional groups responsible for reducing Ag+ ions into AgNPs, the observed bands were compared to standards. The peaks at 675.17/cm corresponded to C-O groups, C-C groups, and/or aromatic C-H bending, whereas the peaks around 1640/cm corresponded to stretching vibrations of C=O groups in carboxylic acid, flavonoids, proteins, the aromatic groups, and ester bonds of polyphenols [22, 31, 33]. In addition, the peaks at 3400/cm could be related to the stretching vibration associated with NH stretching in amides, the hydroxyl groups of phenolic compounds, alcohols, and carboxylic acid [3, 22, 31], and the peak at 403.9/cm can be attributed to the presence of more important quantity of AgNPs [22, 34, 35]. Rizwana et al.  in a recent study characterized AgNPs synthesized from M. pulegium leaf extract and showed peaks at different positions on the IR spectrum. The peaks at 3,446, 1,622, 1,384, and 1,041 cm−1 shown in the FTIR spectrum of M. pulegium-AgNP indicate the presence of an OH group of alcohol or phenols, –NH group of amide or proteins, and carbonyl group of esters .
The FTIR spectral analysis indicated that M. pulegium aqueous extract reduces Ag+ to Ag0, and that agglomeration of silver particles is inhibited by a variety of functional groups, which function as capping agents on the surfaces of the biosynthesized nanoparticles .
SEM analysis revealed that the AgNPs were spherical, with diameters of ≤1 nm (Figure 4). However, this finding differs from previous reports, namely, the production of spherical and face-centered cubic AgNPs with an average diameter of 29 nm using an aqueous extract of Iranian M. pulegium leaves  and between 4 and 60 nm using an aqueous extract of Saudi M. pulegium .
In addition, EDX spectrum analysis indicated that the major constituents of the AgNPs were silicon, oxygen, and carbon. The registered Ag signals at 3 keV were related to the metallic AgNPs . Other element (C, O, and Na) signals were recorded. It is possible that these elements were derived from the organic compounds and proteins functioning as AgNP-capping agents .
3.2. Extract Biological Activities
The colorimetric dosage was used to estimate the secondary metabolite contents in the produced silver nanoparticle by M. pulegium leaf extract. Obtained data showed that the prepared extract was richer in secondary metabolites ( GAE/g DW and Eq catechin/g DW of total phenolic and flavonoids, respectively). These contents are important to these observed in M. pulegium leaf methanolic extract. In fact, Alharbi et al.  found that the total phenolic and flavonoid contents in methanolic extracts of Saudia M. pulegium were 48.4 GAE mg/g DW and 28.87 RE mg/g DW, respectively.
Concerning the antioxidant activity, two in vitro methods (DPPH and ABTS radical scavenging activities) were utilized to evaluate the antioxidant power of prepared extract. It showed an important antioxidant activity (butylated hydroxytoluene (BHT)) with (DPPH); 3 μg/mL (ABTS) and (DPPH); 20 μg/mL (ABTS), respectively). As for the secondary metabolites, the antioxidant activity of the AgNPs biosynthesized by the M. pulegium extract was greater than that of the methanolic M. pulegium extract evaluated by Alharbi et al. . Based on the obtained data, it can be signaled that the high levels of total phenolics and total flavonoids correlated with the antioxidant activities. These findings are in agreement with previous studies that have suggested that the radical-scavenging abilities of plant extracts are correlated with the hydroxyl groups of phenolic compounds .
3.3. Extract Antimicrobial Activity
In order to demonstrate the value of the AgNPs biosynthesized using M. pulegium, the antimicrobial activity of the prepared extract against six indicator bacteria (E. coli, S. typhimurium, P. aeruginosa, S. aureus, L. monocytogenes, and B. cereus) was evaluated using two methods.
The qualitative method, which involved IZ measurement, indicated that the prepared extract exhibited significant antibacterial activity (IZ diameters of 17–26 mm), which was comparable to that of gentamicin (IZ diameters of 18–25 mm). In addition, the extract was the most effective against E. coli (diameter: 26 mm) and least effective against L. monocytogenes (diameter: 15 mm), and the extract was more effective against E. coli than standard antibiotics.
Meanwhile, the quantitative broth dilution assay yielded MIC and MBC values of 0.08–0.62 and 0.16–1.25 mg/mL, respectively (Table 1), with the strength of antimicrobial activity varying between among the bacteria species. Similarly, the assays indicated that the extract was least effective against Gram-positive bacteria (e.g., B. cereus and L. monocytogenes; MIC: 0.62 g/mL) and most effective against the Gram-negative bacterium E. coli (MIC: 0.08 mg/mL), followed by the Gram-negative bacterium S. typhimurium (MIC: 0.16 mg/mL). The significantly lower susceptibility of the Gram-positive bacteria, when compared to Gram-negative bacteria, may be due to differences in the bacterial structures.
The antimicrobial activity of the prepared extract with biosynthesized AgNPs was similar to that reported by previous studies (Table 2). For example, AgNPs prepared using Iranian M. pulegium exerted antimicrobial activity against both Gram-positive bacteria (B. cereus and S. aureus) and Gram-negative bacteria (E. coli and K. pneumoniae), with MICs of 61.5–125 μg/mL .
Similarly, the silver sulfide nanoparticles biosynthesized by rosemary leaf extract have been reported to exert moderate antibacterial activity against E. coli, S. aureus, and both Shigella and Listeria bacteria, with IZ diameters of 8–17 mm , and AgNPs produced using Ulva fasciata etyle acetat have been reported to exert antibacterial activity against Xanthomonas malvacearum, with a mean IZ diameter of . Furthermore, Gnanadesigan et al.  reported that AgNPs synthesized using Avicennia marina extract were found to have an important IZ diameter (30 mm) against both Gram-positive and Gram-negative bacteria, with IZ diameters ranging from (S. aureus) to (E. coli), and AgNPs produced using Ocimum tenuiflorum extracts were reported to be efficient against S. aureus and E. coli () .
The findings of the present study use to accomplish that the syntheses of AgNPs from M. pulegium can be used for the development of antimicrobial preservatives.
In the present study, M. pulegium leaf extract was used in an ecofriendly method for producing silver nanoparticles. Nanoparticle biosynthesis was confirmed using several techniques. The prepared extract, which contained the synthesized AgNPs, was richer in secondary metabolites and exhibited measurable antioxidant and antimicrobial abilities. These findings demonstrate that M. pulegium extract-mediated biosynthesis can be used as an alternative technique for producing AgNPs.
No data were used to support this study.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
This research was supported by the Princess Nourah bint Abdulrahman University Researchers supporting Project number (PNURSP2022R84), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
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