3.1. Color change
The reduction of silver ions into silver nanoparticles can be followed by color change. The fresh extract of M. officinalis was pale yellow in color. After addition of AgNO3 and stirring at room temperature, the solution color changed into pale pink (within 30 minutes for aqueous extract) and light red (within 15 minutes for methanolic extract). The emulsion turned dark brown after 24 and 36 h for methanolic and aqueous extracts respectively.
In this study different volumes of aqueous extract (7, 10, 15 and 20 ml) was used. Change in color in the aqueous extract of lemon balm during chemical reaction of biosynthesis of silver nanoparticles was initiated within 30 minutes. At first, color change was occurred in the sample of 7-ml aqueous extract. This process was completed in this sample earlier than the other volumes of aqueous extract (Fig. 1).
The methanolic extract was used at 0.1, 0.5, 1, and 5 mg /50 ml of the reaction volume. The concentration of 0.1 mg showed no color variation and silver nanoparticles were not produced at this concentration. Change in color in the methanolic extract of lemon balm was initiated within 15 minutes. Our obtained results showed that the speed of chemical reaction was increased by increasing the concentration of methanolic extract. So in the used concentrations, 5 milligrams responded best (Fig. 2). As the concentration of lemon balm extract varies, the nanoparticle synthesis also varies.
The size of colloidal particles is between 1 to 1000 nm. The formation of colloidal solutions from the reduction of silver ions occurs in two steps (nucleation and growth) (Fig. 3). According to the researchers, metal precursor, reducing and stabilizing agents are major components in the formation of metal nanoparticles [28, 29]. In the biological process plant metabolites such as sugars, proteins, terpenoids, polyphenols, alkaloids, flavonoids and phenolic acids are responsible for the reduction and stabilization of nanoparticles [30]. It has been reported that the OH groups present in flavonoids play a role in the reduction of silver ions to AgNPs [31]. Based on the results obtained till this step, two experiments (DPPH and SEM) were performed only on the samples of 7-ml aqueous and 5-mg /50 ml methanolic extracts.
3.2. Antioxidant capacity (DPPH radical scavenging assay)
The DPPH free radical scavenging assay was determined by measuring the ability of plant extracts to capture the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) [25, 32]. The primary DPPH solution is violet which changes to yellow. The violet color disappears as soon as adding synthesized silver nanoparticles because of the presence of antioxidant in the medium. However, the process of yellowing may slow down and the target substance exhibits poor antioxidant activity or no color change. The inhibitory concentration of the material is investigated based on the absorbance of 2,2-diphenyl-1-picrylhydrazyl radiation at a wavelength of 517 nm. The antioxidant effect of lemon balm extracts and silver nanoparticles showed that the methanolic extract had the highest antioxidant activity among the tested specimens. Subsequently, the aqueous extract and the nanoparticle showed antioxidant activity. The color change rate in the methanolic extract was also higher than that of the aqueous and silver nanoparticles (Fig. 4).
Several studies with enhanced DPPH scavenging activity by AgNPs from plant extracts have been investigated [33–35]. The production of iron nanoparticles using aqueous extract of 26 tree species and antioxidant properties of their extract have been reported. These researchers interestingly found dried leaves produce extracts with higher antioxidant capacities than non-dried leaves. This is probably due to evaporation of water present in the leaves and increasing of antioxidants concentration [36].
3.3. Scanning electron microscope
To determine the morphological characters of silver nanoparticles synthesized by lemon balm extracts, scanning electron microscope (SEM) was used. The SEM images showed rod-shaped nanoparticles formed with diameter in the range of 19–40 nm from the aqueous extract. Also the nanoparticles derived from the methanolic extract were spherical with diameter of 13–35 nm (Fig. 5).
It has been reported that the nanoparticles deformation as different shapes and diameters is the result of altering the plant extract and/or the reaction conditions depending on the application [30]. The surface areas and the shapes of nanoparticles affect on their antimicrobial activity [37, 38] due to different effective surface areas and active facets [39, 40]. It was suggested that Melissa officinalis scavenged DPPH radical in a concentration-dependent manner [21] that is in agreement with the result of our study. DPPH assay revealed the sample of 5-mg /50 ml methanolic extract had the highest antioxidant activity compared to the 7-ml aqueous extract.
Based on the SEM results, the particles produced by methanolic extract were spherical and smaller in size in comparison to the rod-shaped particles derived from the aqueous extract. Therefore, further experiments were conducted using the methanolic sample.
3.4. Ultraviolet-visible spectroscopy
Color change from yellow to brownish-red and dark brown is the first sign of nanoparticle production and is due to excitation of the surface Plasmon resonance [41]. The biosynthesis of nanoparticle should be confirmed via physical methods like UV-Vis spectrophotometer, SEM, XRD and FTIR [34, 42].
The biosynthesis of SNPs and the reduction of Ag+ ions to Ag atoms were recorded by UV-Vis spectroscopy. The reaction was conducted for a duration of 2 h. The UV-Vis absorption spectra of colloidal solutions of SNPs using M. officinalis methanolic extract had absorbance near 450 nm. The broadening peak is a sign of the poly-dispersed particles formation (Fig. 6). Hafez et al. (2017) produced AgNPs that showed UV-Vis absorbance at 425 nm [43] and Keshari et al. (2018) reported the absorption band of AgNPs at 442 nm [44]. Also, the absorption of the SNPs was observed near 430 nm in the UV–Vis spectrum [45]. The absorbance wavelength depends on the concentration of plant extract [46, 47], different times [47, 48], fresh and freeze-dried samples [14] and particle size [40, 49].
3.5. Antimicrobial property of AgNPs
The antimicrobial effect of spherical silver nanoparticles was investigated by disc diffusion and agar well diffusion methods. Based on our findings, green synthesis of the silver nanoparticles using this herb showed an effective antimicrobial activity against B. subtilis and S. aureus gram-positive and E. coli gram-negative bacteria and S. cerevisiae (Figs. 7 and 8).
The inhibition zone of bacteria and fungus was measured in millimeter. In disc method the size of inhibition zone for B. subtilis, S. aureus, E. coli and S. cerevisiae was measured 5.7, 5.6, 7 and 4 respectively (Fig. 7). The size of inhibition zone of B. subtilis, S. aureus, E. coli and S. cerevisiae was calculated in agar well diffusion method 10, 10, 11.3 and 9.25 mm, respectively (Fig. 8). In both methods gram-negative bacterium of E. coli showed the highest sensitivity to the silver nanoparticles. The bactericidal effect of silver nanoparticles is size and shape dependent. Smaller particles have higher percentage of the surface area than bigger particles [50]
In a previous study, antibacterial property of SNPs derived from aqueous extract of lemon balm leaves against S. aureus and E. coli was confirmed [19]. Our results elucidated that silver nanoparticles from methanolic extract of lemon balm inhibited bacterial and fungal growth. As expected, increasing the amount of nanoparticles showed more deterioration and increased the growth halo. In the present study it was revealed that both gram-negative and gram-positive bacterial strains were more susceptible to AgNPs than the fungus strain. Although AgNPs exert antifungal activity due to interaction with the fungal cell wall and membrane which leads to cell death through disruption of cell membrane structure [51]. These differences in bactericidal and fungicidal effects of the AgNPs are due to the differences in organization of the bacterial and fungal cells. The bacteria are evolutionarily prokaryotic types and are less complex. Therefore, they are unable to fight the toxic effects of AgNPs as effectively as the eukaryotic fungi. The eukaryotic organisms have superior detoxification system that makes them resistance to higher concentrations of AgNPs [52].
Attack on the surface of the bacteria membrane through interaction with sulfur-containing proteins [53], disruption of cell permeability and respiration, form the pits on the cell surface and induce the proton leakage as a consequence of cell death [54, 55], inhibition of respiratory enzymes of bacterial cells by combining with the group thiol [50] as well as cell retention of DNA replication and preventing reproduction [56, 57] are among the reasons given for the antimicrobial properties of silver nanoparticles. A possible mechanism was depicted for AgNPs formation and their antimicrobial activities (Fig. 9).
3.6. X-ray diffraction analysis
X-ray diffraction (XRD) is one of the most widely used techniques to characterize the structural properties of NPs. To gain structural information, the resulting diffraction pattern obtained from the penetration of X-rays into the nanomaterials is compared with standards [58, 59]. Figure 10 shows the XRD pattern of the synthesized AgNPs using the methanolic extract of lemon balm. The AgNPs diffractogram displayed several sharp intense peaks at 2 theta angles, which indicated towards the crystallinity of the AgNPs and confirmed the formation of the silver nanoparticles. Four distinct reflections at 37.5° (111), 44.37° (200), 64.56° (220) and 76.58° (311) evidently indicated the formation of the face-centered cubic structure of the AgNPs in the prepared sample (Fig. 10).
The XRD outline accordingly obviously displayed that the silver nanoparticles formed by the reduction of Ag+ ions by Melissa officinalis extract are crystal-like in nature. This result is consistent with XRD analysis of Shaik et al. (2018) [46]. Additional peaks at 32.25° and 54.62° were observed on the preparation of AgNPs using M. officinalis methanolic extract (Fig. 10). These peaks are attributed to the existence of some bioorganic compounds in M. officinalis leaf broth [60] or related to unreduced and remained AgNO3 in the sample [61]. It has been suggested that magnesium chlorophyll is the center of X-ray diffraction in the bioorganic crystalline phase [62].
3.7. Fourier transform infrared spectroscopy
Here, Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical composition of lemon balm responsible for reduction of Ag ions (Fig. 11). FTIR is useful for characterizing the surface chemistry. Using this technique organic functional groups (COO−, OH, …) attached to the Ag and other chemical residues surface are detected.
The methanolic extract of aerial parts of lemon balm displayed a number of absorption peaks, reflecting its complex nature. The results of FTIR analysis showed different stretches of bonds shown at different peaks including 3446.51 cm− 1 could be assigned to O-H stretch, H-bonded corresponded to alcohols and phenols, 2357.56 cm− 1 assigned for single aldehyde, 1151.92 cm− 1 indicates the fingerprint region of C-O stretching, 674.99 cm− 1 could be attributed to the presence of C-H bend alkynes and 674.99 cm− 1 and 599.95 cm− 1 correspond to halo compound. Figure 11 shows the peaks near 3000 cm− 1 assigned to O-H stretching and aldehydic C-H stretching. The absorption peaks between 1500 to 2000 cm− 1 can be attributed to the presence of C-O stretching in carboxyl coupled to the amide linkage in amide I which is characteristic of the presence of protein and enzymes in the supernatant and confirms the extracellular formation of AgNPs [34, 63–67]. Consequently, the occurrence of these peaks in the FTIR spectrum evidently indicates the dual role of the M. officinalis extract, both as a green reducing and stabilizing agents.
Interactions between metabolites in the extract and metal ions cause the bioreduction of metal salts and synthesis of nanoparticles. The functional groups in the plant extract act as reducing, capping, and stabilizing agents [58, 68]. Negatively charged (COO−) and polar (OH and CO) groups presented in the plant extract attach on the Ag surface with high tendency and contribute in both reduction and stabilization of AgNPs [69].