Morus alba L. Leaves-Based Silver Nanoparticles for Aging-Related Infectious Diseases

,


Introduction
2][3][4] Metallic NPs, which are in the NP class, are becoming more popular with their wide usage areas, and the interest in them is increasing day by day.Silver nanoparticles (AgNPs), which are included in metallic NPs, are used quite frequently, especially in biomedical applications due to their antimicrobial effect. 5ynthesis studies using plant sources for these methods attracted attention with advantages such as being cheap, being easy, not requiring special conditions to obtain the source, and showing biocompatible properties. 6,7lant parts used as supplies for synthesis include leaves, flowers, fruits, seeds, and roots.Extracts from plant sources are a rich source of phytochemicals.These phytochemicals have functional groups such as alcohol/ phenol, amine, and carboxylic acids which show bioactive properties. 8,9][12] The Moraceae family includes the species Morus alba L., which is indigenous to China, Korea, and Japan.It is well known that this plant, which can be found growing all over the world, has had medicinal uses for thousands of years.This plant with a rich content of bioactive components has many effects such as antimicrobial, antidiabetic, and anticancer. 13his study aimed to analyze the eco-friendly and low-cost economic synthesis, characterization, and antibacterial effect of AgNPs using M. alba L. (Mulberry) tree leaves grown in Orduzu, Malatya region (Figure 1).

Morus alba L. Leaf Extract and Metal Solution Preparation
Mulberry leaves were harvested in the Orduzu Malatya region by the end of August and were repeatedly cleaned

Original Article
2][3] The liquid extract was prepared for synthesis after cooling and filtering. 4ilver nitrate (AgNO3), a solid compound salt from Sigma Aldrich, was dissolved in water at a concentration of 2.5 mM for use in green synthesis. 4

Synthesis of Silver Nanoparticles with Extract Prepared from Mulberry Leaves
The leaf extract and 2.5 mM AgNO 3 solution were mixed in a suitable container as one unit of the extract and two units of metal solution and kept at room temperature at 25 °C.The color change was observed by observation against time. 4By taking samples from the synthesis medium at regular intervals, 300-800 nm wavelength scans were performed through a Perkin Elmer One, ultraviolet-visible spectrophotometer (UV-Vis). 5The presence of Ma-AgNPs was analyzed with their maximum wavelength absorbance. 3

Characterization of Synthesized Silver Nanoparticles
Morphological appearances of synthesized Ma-AgNPs are presented in a Jeol Jem.It was evaluated using micrographs taken via the 1010 transmissive electron microscope (TEM). 2 The topographic structures and distributions of Ma-AgNPs were determined using a Park System XE-100 atomic force microscopy (AFM). 3The crystal patterns and sizes of Ma-AgNPs reflected on the plane between 20-80 at 2θ were determined using a Rigaku Miniflex 600 model X-ray diffraction (XRD) instrument.][12][13][14]

Antibacterial Effects of Synthesized Silver Nanoparticles
Using the microdilution approach, the minimum inhibition concentrations (MICs) of created Ma-AgNPs have been determined on the development of pathogenic bacteria. 15,16The microorganisms utilized in the experiment came from the microbiology lab of Mardin Artuklu University in Mardin, Turkey.Gram-positive and gram-negative strains of the bacteria Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used to test the antibacterial action.In order to prepare the bacteria for antibacterial research, the bacteria were cultured in acceptable media and adjusted according to the McFarland standard 0.5 [17][18][19] turbidity requirements.Then, 96-well microplates were filled with the appropriate medium.The sterilization process and growth control procedures involved the use of some wells.Furthermore, transferring Ma-AgNPs made at various concentrations to the wells of the microplate served as the microdilution process.The prepared bacteria (McFarland 0.5) were then delivered into the wells, and they (McFarland 0.5) were then transferred into the wells after being prepared.To allow bacteria to interact with Ma-AgNPs, antibacterial microplates were placed in a hot oven at 37 °C for 24 hours.Following incubation, growth control was carried out, and MIC was calculated.

Evaluation of the Properties of Synthesized Silver Nanoparticles
A color change from yellow to brown was observed 20 minutes after mixing the extract and AgNO3 solution for synthesis.][23][24][25] The determination of the morphological appearance and distribution of the synthesized AgNP was made with the images obtained from the TEM and AFM micrographs given in Figures 3 and 4. TEM pictures illustrated that Ma-AgNPs have a morphological appearance that was predominantly spherical, and a small number of them seemed triangular.][28][29] The crystal structures and sizes of the particles synthesized using mulberry leaves were determined by the data received at 2θ via XRD.Bragg's expansions at 111o, 200o, 220o, and 311o showed that Ma-AgNPs mostly have a central faceted cubic pattern (fcc), [30][31][32][33] and the values obtained in the data were measured as 38.68, 44.54, 64.76, and 77.38, respectively (Figure 5).The crystal nano dimensions of Ma-AgNPs were calculated using the FWHM value of Debye-Sherer's high peak, and the crystal size of Ma-AgNPs was 63.56 nm.In a green synthesis study in which different extraction processes were applied, it was calculated that the extracts have crystal sizes of 56 nm and 67 nm, respectively. 34

Findings of Antibacterial Effect of Synthesized Silver Nanoparticles
Ma-AgNPs synthesized from mulberry leaves were determined on pathogenic Gram-negative and Grampositive bacteria by MIC microdilution method.MIC values of Ma-AgNPs suppressing the growth of the strains are presented in Table 1.As can be seen, 0.25-0.50µg mL -1 concentrations were found to have a suppressive effect on E. coli and S. aureus.These concentrations were lower than the commercial antibiotics used in practice (Table 1).
Ma-AgNPs were ionized and exhibited considerable reactivity in liquid media.6][37][38][39] By causing damage to microorganisms' membrane structures, they enhanced the production of reactive oxygen species and altered the biomolecules'   structures, which caused a decline in their functions and the extinction of microorganisms. 18,40In this study, Ma-AgNPs were synthesized easily and quickly with the extract obtained by collecting the leaves of the M. alba L. plant in Orduzu, Malatya region.The outcomes of several microscopic and spectroscopic analyses were used to evaluate the characteristics of Ma-AgNPs.Ma-AgNPs can be produced as a powerful substitute, particularly in the struggle against microorganisms with multiple drug resistance.

Discussion
][23][24][25] It was observed that Ma-AgNPs were mostly spherical in morphological appearance in TEM images, and they also showed triangular appearance, although a small number of them.In AFM findings, it was observed that they showed a distribution below 100 nm. 28,291][32][33] The values obtained in the data were measured as 38.68, 44.54, 64.76, and 77.38, respectively (Figure 4).The crystal nano dimensions of Ma-AgNPs were calculated using the FWHM value of Debye-Sherer's high peak, and the crystal size of Ma-AgNPs was 63.56 nm.In a green synthesis study in which different extraction processes were applied, it was calculated that the extracts had crystal sizes of 56 nm and 67 nm, respectively. 34As seen in the findings, 0.25-0.50µg mL-1 concentrations were found to have a suppressive effect on E. coli and S. aureus.These concentrations were lower than the commercial antibiotics used in practice (Table 1).Ma-AgNPs, In liquid medium are ionized and show high reactivity.6][37][38][39] They cause an increase in reactive oxygen species (ROS) by disrupting the membrane structure of microorganisms.They disrupt the structure and functions of RNA, DNA, and enzymes with high affinity for these species.The structures of biomolecules damaged by these species and as a result their functions deteriorate, resulting in the death of microorganisms. 8,18,40

Conclusions
Green synthesis methods are a very popular area that aims to design safer and more sustainable processes without the use of toxic chemicals.The Ma-AgNPs had size distributions below 100 nm, an ultimate absorbance of 457.92 nm, and a mainly spherical shape.By defining the MIC, the antibacterial properties of pathogen gram-negative and positive bacteria have been identified by microdilution.Furthermore, Ma-AgNPs were discovered to be more successful than generic antibiotics, according to the data.

Figure 1 .
Figure 1.Schematic Illustration of Nanoparticle Synthesis Steps in Detail

Table 1 .
Concentrations of Ma-AgNPs, Antibiotics, and Silver Nitrate Solution Where They Exert an Antimicrobial Effect on the Growth of Microorganisms