Phyto-mediated silver nanoparticles via Mellisa ocinalis aqueous and methanolic extracts: synthesis, characterization and biological properties

The aim of the study was to examine the inuence of extraction method on the size, shape and morphology of synthesized silver nanoparticles (AgNPs). Silver nanoparticles were prepared by the aqueous and methanolic extracts of lemon balm. The properties of obtained nanoparticles were characterized by UV-Vis, SEM, XRD and FTIR techniques. The UV-Vis spectroscopy conrmed the formation of AgNPs by observing a distinct surface Plasmon resonance band around 450 nm. SEM images showed different shape, size and morphology of AgNPs using two different extracts types. AgNPs derived from the aqueous extract were rod-shaped with a diameter of 19 to 40 nm whereas spherical particles were synthesized by the methanolic extract found smaller with size distribution ranging from 13 to 35 nm. The XRD pattern indicated that AgNPs formed by the reduction of Ag + ions using methanolic extract of M. ocinalis were crystal-like in nature. The functional groups of M. ocinalis methanolic extract involved in synthesis and stabilization of AgNPs were investigated by FTIR. In addition, AgNPs-containing methanolic extract showed higher antioxidant activity. These particles exhibited remarkable antimicrobial activity against gram positive and negative bacteria and a fungus. The nanoparticles produced by the methanolic extract of the lemon balm showed antioxidant and antimicrobial activity. The production of silver nanoparticles using plant extract is rapid, low cost and eco-friendly. Therefore, green chemistry is a good alternative to industrial production of nanoparticles. Silver nanoparticles can be used as an antiseptic to sterilize the surrounding area and the hospital wastes.


Introduction
Utilization of biocompatible and ecofriendly processes for the production of nanoparticles is of great importance in current nanotechnology research [1]. Nanomaterials have been synthesized by different methods including hard template [2], bacteria [3], fungi [4] and plants [5]. During the synthesis of metallic nanoparticles by chemical methods toxic compounds are often produced which remains adsorbed on the particle surface and has adverse effects on human health. However, the green synthesis is based on nontoxic and environmentally friendly ingredients. It is cost-effective, low energy consumption, less complex and less time-consuming process [6]. Among all types of nanoparticles, silver nanoparticles (AgNPs) have been used for a variety of applications in everyday life [7]. AgNPs have more application in the medical eld as antimicrobials and sterilizers [8]. The antimicrobial property of silver nanoparticles is well known and mediated by the released silver cations from silver nitrate (AgNO 3 ) [9].
Because of advantages of biological processes in synthesis of nanoparticles [10], various plant resources have been explored by researchers to synthesize nanoparticles such as Coriandrum sativum [11], Berberis vulgaris [12], Cassia toral [13], Moringa oleifera [14], Malva sylvestris [15], Gossypium hirsutum [16], Gelidium amansii [17], Aloe vera [18] and Melissa o cinalis [19]. Melissa o cinalis L. is a perennial and medicinal herb from the Labiatae (Lamiaceae) family. Because of its lemon-like fragrance known as lemon balm [20]. Different organic compounds present in lemon balm extract including protein, essential oils, avonoids, rosmarinic, caffeic and gallic acids and phenolic contents are responsible for bio-reduction of Ag + ions [19,21]. M. o cinalis as a valuable source of natural antioxidants has strong antimicrobial activity [22,23]. In the present study, silver nanoparticles (AgNPs) were synthesized using aqueous and methanolic extracts of Melissa o cinalis. Silver nitrate was used as a precursor and M. o cinalis extracts as reducing and stabilizing agents. To investigate the in uence of extraction method on the size, shape and morphology of synthesized AgNPs, aqueous and methanolic extracts of lemon balm were examined. To survey the effect of extract concentration on the rate of nanoparticle synthesis, the AgNPs were prepared using various concentrations of aqueous (7, 10, 15 and 20 ml) and methanolic (0.1, 0.5, 1 and 5 mg) extracts of lemon balm. The properties of obtained nanoparticles were characterized by ultraviolet-visible spectroscopy (UV-Vis), Scanning Electron Microscope (SEM), X-Ray Diffraction (XRD) and Fourier Transform Infrared (FTIR) techniques. The antioxidant ability of the AgNPs was assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging as well. Moreover, the bactericidal and fungicidal effects of AgNPs on Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae were con rmed.

Aqueous extract
The aerial parts of Melissa o cinalis were washed with distilled water and shade dried for 7 days at room temperature. The dried plants were ground into a ne powder. For preparation of the aqueous extract 150 ml of sterile distilled water was added to 6 gr of powdered samples and boiled for 10 min.
The suspension was left for 3 h and then ltered through Whatman No. 1 lter paper. The samples were centrifuged at a high speed (20000 rpm) for 15 min and kept at 4 °C until further analysis.

Methanolic extract
About 6 gr of powdered samples was uniformly packed into a thimble and placed in the Soxhlet extractor.
It was exhaustible extracted with methanol for the period the solvent in siphon tube of extractor becomes colorless. The extracts were ltered using lter paper and the solvent evaporated from the extract in a rotary evaporator to have a syrupy consistency. The extract was stored at 4 °C for further experiments.

Biosynthesis of silver nanoparticles
The procedure for the preparation of silver nanoparticles has been adopted from Savithramma et al. [24] with slight modi cations. 1 mM AgNO 3 (silver nitrate) solution was prepared and stored in amber colored bottle. 50 ml of AgNO 3 solution (1 mM) was added into 5 ml of lemon balm extracts with constant stirring at room temperature until the color change was observed. The color change of the solution is a sign for the formation of silver nanoparticles. The extract content was then centrifuged at 14000 rpm for 30 min. To monitor the reduction of Ag + ions and the formation of AgNPs ultraviolet-visible spectroscopy was used.

Characterization of synthesized silver nanoparticles
The biosynthesized AgNPs were analyzed using UVD-3200 spectrophotometer. Their spectral analysis was done in the range of 300 to 800 nm. The size and shape of AgNPs were determined by SEM using MIRA-3 SEM machine. The lyophilized powdered sample was used for XRD and FTIR (Bruker Tensor 27) spectroscopy analysis. The XRD patterns were recorded using Bruker D8 Advance X-ray diffractometer with a Cu K α radiation. FTIR was used to characterize the nanoparticles using the lyophilized samples by potassium bromide pellet technique in the range of 500 to 4000 cm − 1 .
2.4. DPPH free radical scavenging assay Antioxidant activity of AgNPs was quantitatively studied using DPPH method [25]. Brie y, 150 µl of 1 mM nanoparticle solution and ascorbic acid (positive control) were taken separately and mixed with 2850 µl of a methanolic solution of DPPH. The reaction mixture was shaken and kept in a dark place for 24 h. After incubation, the absorbance of the samples (reduction of DPPH radical) was measured by UV-Vis spectrophotometer at 517 nm against methanol as blank. The methanolic solution of DPPH without the sample served as control. The DPPH free radical scavenging activity (percentage of inhibition) was calculated using the following formula:

Antimicrobial assays of the biosynthesized AgNPs
The antimicrobial e cacy of synthesized silver nanoparticles was observed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae. This property was assayed by both the standard Kirby-Bauer disc diffusion [26] and agar well diffusion methods [27]. Brie y, the agar plates were inoculated with 100 µl of 1⊆10 6 bacterial and fungal suspension using spread-plating. After drying the plates, lter paper discs of 6-mm diameter were soaked in 30 and 40 µl of biosynthesized AgNPs solution (1 mM), plant extract and distilled water as control and placed on LB agar plates followed by incubation at 37 °C (Bacteria) and 28 °C (Saccharomyces cerevisiae) for 24 h. Susceptibility of organisms were determined by measuring diameter of inhibition zone (mm) with a ruler.
For agar well diffusion test, the agar plates were inoculated with 100 µl of 1⊆10 6 bacterial and fungal suspension using spread-plating. After drying the plates, wells of 6-mm diameter were punched into the agar medium, aseptically lled with 20, 30 and 40 µl of biosynthesized nanoparticle solution (1 mM nanoparticle) and plant extract and allowed to diffuse for 2 h at room temperature. The plates were subsequently incubated at 37 °C (Bacteria) and 28 °C (Saccharomyces cerevisiae) for 24 h. The antimicrobial activity was analyzed by measuring the diameter of inhibition zone (mm) around the wells.

Color change
The reduction of silver ions into silver nanoparticles can be followed by color change. The fresh extract of M. o cinalis was pale yellow in color. After addition of AgNO 3 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 rst, 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, avonoids and phenolic acids are responsible for the reduction and stabilization of nanoparticles [30]. It has been reported that the OH groups present in avonoids 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.

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-1picrylhydrazyl 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][34][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].

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 o cinalis 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.

Ultraviolet-visible spectroscopy
Color change from yellow to brownish-red and dark brown is the rst sign of nanoparticle production and is due to excitation of the surface Plasmon resonance [41]. The biosynthesis of nanoparticle should be con rmed 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. o cinalis 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].

Antimicrobial property of AgNPs
The antimicrobial effect of spherical silver nanoparticles was investigated by disc diffusion and agar well diffusion methods. Based on our ndings, 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 con rmed [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 ght the toxic effects of AgNPs as effectively as the eukaryotic fungi. The eukaryotic organisms have superior detoxi cation 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 con rmed the formation of the silver nanoparticles. Four distinct re ections 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 o cinalis 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. o cinalis methanolic extract (Fig. 10). These peaks are attributed to the existence of some bioorganic compounds in M. o cinalis leaf broth [60] or related to unreduced and remained AgNO 3 in the sample [61]. It has been suggested that magnesium chlorophyll is the center of Xray diffraction in the bioorganic crystalline phase [62].

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  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 con rms the extracellular formation of AgNPs [34,[63][64][65][66][67]. Consequently, the occurrence of these peaks in the FTIR spectrum evidently indicates the dual role of the M. o cinalis 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].

Conclusion
Here, biosynthesis of silver nanoparticles using lemon balm extracts and their antibacterial, antifungal and antioxidant activities were studied. According to the results, the nanoparticles produced by the methanolic extract of the lemon balm showed antioxidant and antimicrobial activities. The production of AgNPs using plant extract is rapid, eco-friendly and low-cost. In this method, there is no need to use expensive raw and hazardous materials [70]. Therefore, green chemistry is a good alternative to industrial production of nanoparticles. Due to the antimicrobial properties of these particles on the tested materials, they can be used as an antiseptic to sterilize the surrounding area and the hospital wastes. Also, antioxidant properties of nanoparticles can be exploited in food industry. The syntheses of spherical and rod-shaped silver nanoparticles with different dimensions were observed using M. o cinalis methanolic and aqueous extracts respectively. Therefore, deformation of resulting nanoparticles indicated that by changing the plant extract type and the reaction conditions, nanoparticles with different shapes and diameters formed.    The reducing agents (in plant extract) donate electrons to Ag+ ions lead them as neutral Ag atoms (Ag0).

Abbreviations
These atoms due to van der waal's forces of attractions come closer and combine to form cluster of Ag atoms of diameter 1 to 100 nm, called Ag nanoparticle. A silver nanoparticle in nano scale may contain about hundreds atoms of silver. If these clusters (nanoparticles) come closer they agglomerate rst and then aggregate to form bulky particles, which is not Ag nanoparticles. Plant extract has ability to stabilize silver colloids in water and prevent agglomeration and aggregation of the nanoparticles [28,[71][72][73].