Appraisal of the antimicrobial and cytotoxic potentials of nanoparticles biosynthesized from the extracts of Pelargonium quercetorum Agnew

1. Abstract 2. Introduction 3. Material and method 3.1 Plant material, chemicals, and reagents 3.2 Preparation of the extracts 3.3 Instrumentation and characterization 3.4 Formation of AgNP 3.5 Preparation of bacterial strains 3.6 Minimum inhibitory concentration (MIC) 3.7 Cell culture 3.8 Cell viability assay 3.9 Statistical analysis 4. Results and discussion 4.1 Synthesis and characterization of the AgNPs 4.2 Antimicrobial activities of the pq11AgNPs, pq70AgNPs, and pqhAgNPs 4.3 Cytotoxicity of the AgNPs against the MCF-7 cell line 5. Conclusions 6. Author contributions 7. Ethics approval and consent to participate 8. Acknowledgment 9. Funding 10. Conflict of interest 11. References


Introduction
Since the beginning of the 21st century, nanosilver particles (AgNPs) have been used in almost every field, widely used in physical, biological, and pharmaceutical applications [1]. Due to the broad spectrum and high efficiency of AgNPs, their antimicrobial anticancer activities have attracted attention in the field of biomedicine. Besides antimicrobial and anticancer properties, other medical applications of AgNPs include bone healing and wound repair, enhancing the immunogenicity of vaccines, dental applications, antidiabetic agent and biosensing [2]. However, it has been reported in the literature that these nanoparticles may have negative effects on people and the environment [3][4][5][6][7]. Physical and chemical, and biological synthesis methods for AgNPs have been described in the literature [8,9]. Physical synthesis can obtain AgNPs with uniform size distribution and high purity. Chemical synthesis is the most commonly used method to obtain AgNPs due to the size and shape control [2]. The use of hazardous chemicals that may cause potential environmental and biological risks in most of the physical and chemical methods used in the synthesis of AgNPs is the main factor in not selecting these methods [5]. In addition, chemical synthesis methods can absorb certain toxic substances on the surface and prevent their medical use [10]. Biosynthesis of metal nanoparticles is a simpler and less harmful method than a chemical method. Since cheaper and more efficient use of biosynthesized AgNPs than chemical AgNps is a subject that needs to be investigated, appropriate environmental and economic methods should be used for synthesizing these nanoparticles. The search for such a method has led to the need for biomimetic production of AgNPs. Therefore, biological methods using microorganisms and plants are an alternative method applicable for AgNP synthesis. It has been determined by studies that AgNPs can arrest the cell cycle at certain phases, inhibit cell proliferation and lead cancer cells to apoptosis by inducing oxidative stress. Reactive oxygen species damage cellular enzymes (cellular respiratory chain), leading to disruption of the cellular membrane and DNA damage, leading to cell lysis and death. Cellular uptake of smaller nanoparticles may be easier compared to larger particles. Thus, the higher cytotoxicity of smaller particles compared to larger ones may be related to the amount of reactive oxygen species (ROS) produced in the relatively larger surface area of the small nanoparticles. Alternatively, smaller nanoparticles may exhibit or release more silver ions on its surface than larger nanoparticles. The molecular mechanism of AgNPs to show antimicrobial and anticancer properties by penetrating into the cell can be explained in this way.
The family Geraniaceae is represented in Turkey by 4 genera, comprising Biebersteina, Geranium, Erodium, and Pelargonium, and a total of 62 species. Two species of Pelargonium, the most important genus of this family, have been recorded in the vegetation of Turkey [11][12][13]. Pelargonium quercetorum is one of these species and is generally found in Northern Iraq, although it also grows in Hakkari Province in Turkey. This plant, which thrives at high altitudes and humidity, is popular in the spring in the region and is known as Tolk or Tolik. P. quercetorum. It has been given importance by the local people and also has a commercial value in the region. In addition to using this plant for medicinal purposes, the local people also use it for food purposes [14]. The traditional use of P. quercetorum as an antiparasitic is known in the Kurdistan region of Iran [15]. In Turkey, it is used to treat throat disorders and skin wounds, and its seeds and leaves are used to blast boils. It is also used for chronic headaches, neck pains, and migraines [14]. Studies have shown that P. quercetorum has a high content of total phenols and flavonoids, and gallic acid and apocynin have been identified in the root extracts of the plant [16]. Although there are limited studies on this traditionally used plant, it has been shown to cause apoptosis in lung and breast cancer cell lines. In a previous study, it was proved by us that it exhibits antioxidant activity [17][18][19].
It has been shown that silver nanoparticles synthesized by Pelargonium sidoides and Pelargonium. endlicherianum from the Geraniaceae family have strong antimicrobial activity [20,21]. Besides, the strong effects of P. quercetorum and different Pelargonium species on cancer cell lines led us to evaluate the effects of nanoparticles synthesized by P. quercetorum [17,18,22]. For this reason, the biosynthesis of AgNPs was reported for the first time herein using different extracts that were obtained directly from the P. quercetorum plant. The main objectives of this study were (1) the biosynthesis of AgNPs directly using extracts of P. quercetorum; (2) characterization of these AgNPs using zeta potential, scanning electron microscopy (SEM), and UV-vis to its assess quality, morphology, and dimensions; and (3) determination of the antimicrobial and anticancer activity of AgNPs.

Preparation of the extracts
First, the standardized extract of P. quercetorum (EPs 7630) was prepared with 11% ethanol, and then the dried root parts of the plant were roughly powdered and extracted 3 times for 8 h at 37 • C in a shaking water bath with the 11% ethanol (pq11) and 70% methanol (pq70). The obtained extracts were combined and concentrated in a rotavapor (37-38 • C) under vacuum. The aerial part of the vegetable material was extracted in the same way as the 70% methanol (pqh) and then concentrated in the rotavapor. All of the extracts were lyophilized and stored at -20 • C until analysis.

Instrumentation and characterization
Formation of the AgNPs was characterized using SEM, UV-Vis spectrometry, dynamic light scattering (DLS) and Zeta potential. SEM images were obtained using a ZEISS EVO LS10 SEM (Oberkochen, Germany) with a working voltage of 25 kV. The effective diameter and surface charge of AgNPs were measured using Zetasizer (Malvern Panalytical Ltd., Malvern, UK).

Formation of AgNP
For this, 1 mg/mL concentrations of pqhAgNP, pq11AgNP, and Pq70AgNP were prepared as stock solutions. The AgNPs were synthesized from the P. quercetorum extracts, according to the method of a previous study [20].

Preparation of bacterial strains
Bacterial cultures comprising gram-negative: Escherichia coli American Type Culture Collection (ATCC) 25922, gram-positive: Staphylococcus aureus ATCC 25923, and yeast: Candida albicans ATCC 14053 were used for the microdilution method. The bacteria were grown in NA, while the yeast was grown in DSA. The bacterial suspensions equivalent to the density of 0.5 McFarland (for bacteria 108 CFU/mL, for yeast 106 CFU/mL) were prepared by comparing the density standard, using a PhoenixSpec Nephelometer (Becton Dickinson, NJ, USA), of the fresh subcultures in broth.

Minimum inhibitory concentration (MIC)
Antibacterial activity of the AgNPs was tested using the microdilution technique of Clinical and Laboratory Standards Institute [23]. The MIC values were determined for the antimicrobial activity method of Altinsoy [24].

Cell culture
MCF-7 ATCC HTB 22 cells (human breast adenocarcinoma cell line) were obtained from the ATCC. For the MCF-7 cells, DMEM, inactivated fetal bovine serum, antibiotic mixture, and L -glutamine were used.

Cell viability assay
After the MCF-7 cells were developed under suitable conditions, they were seeded at a ratio of 10,000 cells per well into a 96-well microplate. The grown cells were incubated with various concentrations of AgNPs (3.125-100 µg/mL) for 24 h. The cytotoxic properties of AgNPs were investigated using the using MTT method [25]. Finally, the absorbance was read using an ELISA Synergy HT microplate reader (BioTek, Winooski, VT, USA) at 570 nm. The experiments were repeated in triplicate and results were given as the mean ± SD.

Statistical analysis
The experimental data were shown as the mean ± SD. Statistical analysis was performed using ANOVA. Statistical significance was accepted as p < 0.05 using the Tukey pairwise comparison test.

Synthesis and characterization of the AgNPs
The SEM images of AgNPs formed using pq11, pqh, and pq70 are shown in Fig. 1A-C. While the pq11, pqh, and pq70 mediated AgNPs were all spherical, they exhibited different sizes and size distributions, comprising ∼47 nm for pq11AgNP, ∼96 nm for pqhAgNP, and ∼35 nm for pq70AgNP, respectively. The UV-vis spectrum of pq11AgNP, pqhAgNP, and pq70AgNP showed absorption peaks at ∼434, ∼460, and ∼417 nm, respectively, which are all characteristic peaks of AgNPs (Fig. 1D). DLS was used to measure the effective diameter and size distribution of pq11AgNP, pqhAgNP, and pq70AgNP, as shown in Fig. 1E-G, respectively. The maximum density was found to be 43-68 nm for pq11AgNP, 78-190 nm for pqhAgNP, and 28-50 nm for pq70AgNP, respectively. The sizes of pq11AgNP (112 nm), pqhAgNP (122 nm), and pq70AgNP (61 nm) were effective diameters measured using DLS. As in the AgNPs synthesized from microorganisms in a previous study, the large dimensions can be attributed to the presence of plant phytochemicals adhering to the surface of the AgNPs and to the partial accumulation of the Ag-NPs. Karatoprak et al. [19] reported that while the total amount of flavonoids in 70% methanolic P. quercetorum extract was (pq70) 64.95 ± 2.93 mgCA/g, 11% ethanol extract (pq11) was found as 46.63 ± 1.93 mgCA/g, and herba extract (pqh) was found as 30.89 ± 3.58 mgCA/g [20]. It was assumed that during the pq70 formation, the presence of total phenolic, flavonoids, and flavonol contents may have had an effect, since these molecules tend to easily react with metal ions to form complexes, they can participate in the formation process of AgNp to form small-sized nanoparticles.

Antimicrobial activities of the pq11AgNPs, pq70AgNPs, and pqhAgNPs
The antimicrobial effects of AgNPs against various microorganisms are widely used. Their size, morphology, surface charge, surface coating, and synthesis procedures have been reported to affect the antimicrobial performance of AgNPs [26][27][28][29]. Due to the various plant phytochemicals with antimicrobial effects, many plant extracts have widespread use as antimicrobial agents [30,31]. In addition, essential oils rich in terpenic composition have gained importance as effective antimicrobial agents against commercial bacterial strains that cause infections [32]. In the last decade, several studies have reported the biosynthesis of Ag nanoparticles obtained from plant extracts, microorganisms, and fungi, and green synthesized AgNPs are considered as good sources of antimicrobial molecules [33]. AgNPs obtained from some microorganisms have been efficiently used against a wide range of microorgan-isms [34]. The antibacterial activities of the synthesized AgNPs at different concentrations (500-3.91 µg/mL) were tested against E. coli, S. aureus, and C. albicans. The percentage of inhibition was compared with a positive control (Meropenem and Fluconazole) and a negative control (AgNO 3 ). Antimicrobial activity results were detected using the microdilution method and the results are shown in Fig. 2 and Table 1. According to the results, the AgNPs exhibited approximately 90% inhibition against the studied gram-positive and gram-negative bacteria at a concentration of 500 µg/mL. It can be understood from Fig. 2 that Ag-NPs exhibited inhibition in a dose-dependent manner. The AgNPs demonstrated 70% antimicrobial activity against C. albicans at a concentration 500 µg/mL. It was seen that the antimicrobial effect of pq70AgNP tended to be higher than those of pqhAgNP and pq11AgNP based on the microdilution test. It was determined that 125 µg/mL of concentrated pq11AgNP reduced the proliferation of E. coli, S. aureus, and C. albicans by 55%, 63%, and ∼43%, respectively. At the same concentration, pq70AgNP and pqhAgNP caused a decrease in viability of 68%, ∼73%, and ∼58%, and 58%, 60%, and 30% for E. coli, S. aureus, and C. albicans, respectively. Similarly, 31.25 and 15.62 µg/mL of pq70AgNP, respectively, showed much effective inhibition on S. aureus (∼53% and ∼27%), and E. coli (∼35% and ∼30%) when compared to C. albicans (∼27% and ∼20%).
When the IC50 (µg/mL) values of the pq11Ag NPs, pq70AgNPs, and pqhAgNPs on E. coli were examined, all of the nanoparticles were statistically different. No sample had the same significance as Meropenem. On S. aureus, while the pq11AgNPs and pq70AgNPs were statistically the same (p > 0.05), no sample could reach the activity of Meropenem. The results are summarized in Table 1.
The highest inhibition activity on C. albicans was found with the pq70AgNPs, while it was statistically different (p < 0.05) than that of the pqhAgNPs and pq11Ag NPs. Given the overall effect on all of the pathogens, it was clear that the pq70AgNPs had lower IC50 values.
The minimum inhibitory concentration (MIC) values of the pqhAgNPs and pq70AgNPs were determined as 7.81 µg/mL for S. aureus (Table 2). Overall, the activity showed by the Pq70AgNPs was significantly higher than that of the Pq11AgNPs. In a previous study, AgNPs synthesized from P. endlicherianum showed a higher inhibitory effect against gram-positive S. epidermidis [20]. The cell membrane interactions of S. aureus and AgNPs predicted a very strong interaction when compared to E. coli and C. albicans. S. aureus, a gram-positive bacterium, has a very thick cell wall due to proteoglycan layers, which may strengthen the interactions between S. aureus cells and Ag-NPs, where the cells are effectively and rapidly deactivated. In contrast to that, gram-negative E. coli has thin cell walls, which may weaken the interaction between, E. coli cells and the AgNPs.  Values given as the mean ± SD are within the ±95% confidence interval. pq11, 11% ethanol root extract; pq70, 70% methanol root extract; pqh, 70% methanol herba extract.

Cytotoxicity of the AgNPs against the MCF-7 cell line
Exposure of cells to AgNP can induce changes in the form of the cells, decrease cell viability, increase lactate dehydrogenase release, and ultimately result in cell apoptosis and necrosis [35]. According to the results of this study, the AgNPs, synthesized by using a plant extract, were determined to have anticancer potential against the breast cancer cell line MCF-7. The nanoparticles, pq11AgNP, pq70AgNP, and pqhAgNP, at concentrations between 25 and 100 µg/mL were found to be statistically different from the control group (p < 0.001). At a concentration of 100 µg/mL, pq11AgNP and pq70AgNP showed 89.47% and 91.66% lethal activity against the MCF-7 cells, respectively. It was noticed that the cytotoxic effect of the pqhAgNPs was lower than that of the pq11AgNPs and pq70AgNPs. The pq70AgNPs at a concentration of 3.125 µg/mL inhibited 52% of the MCF-7 cell growth. Size, shape and surface charge are factors that affect the anticancer activity of AgNPs [2]. In our study, pq70AgNP with the smallest particle size showed the highest anticancer activity. The cytotoxic effect against the MCF-7 cells is shown in Fig. 3. The AgNPs, synthesized from various microorganisms, confirmed a cytotoxic effect on the MCF-7 breast cancer cell line, confirming that the nanoparticle with the smallest particle size had a higher inhibitory effect [24].
The antibacterial and cytotoxic activity of the nanoparticle depends on the shapes and sizes of the nanoparticles; this can be confirmed by examining the inhibition of bacterial growth and cancer cells by different shaped nanoparticles. Nanoparticles of various sizes and shapes were synthesized according to the plant extracts used in the plant-mediated synthesis of AgNPs. Nanoparticles synthesized from different plants were detected in size from 1 to 100 nm. Silver nanoparticles synthesized by green synthesis were obtained in various shapes such as spherical, crystal, polydispersed, circular, cubic, smooth edges, triangular, cuboidal and irregular [36]. In our previous study and this study, the nanoparticles obtained from Pelargonium species were found to be spherical and smaller than 100 nm in size [20]. Pal et al. [26], reported that triangular nanoparticles complete inhibition of bacterial growth was observed even at a total silver content of 1 µg, spherical nanoparticles, a total silver content of 12.5 µg reduced the number of colonies, bacteria grew in rod-shaped particles even at 100 silver content. Apart from the size of the silver nanoparticle, being spherical, rod or triangular causes it to show different antibacterial effects. It has been shown that the most active silver nanoparticles among them are those with a triangular shape, while those with a rod shape have the lowest activity [26].

Conclusions
It can be concluded that the P. quercetorum components function as both reducing and stabilizing agents for the synthesis of pq11, pqh, and pq70 AgNPs and induce AgNP formation with different size and size distributions. The synthesized AgNPs exhibited dramatically enhanced inhibitory properties against the pathogenic strains. In addition, these molecules had intrinsic anticancer properties and also showed promising toxicity against the MCF-7 cell line; hence, these results will shed light on studies regarding their use as potential antimicrobial and anticancer agents. Herein, the use of AgNPs synthesized in conjunction with physicochemical interactions of AgNP and plant phenolic compounds from P. quercetorum extracts offered a promising alternative to reduce the use of chemical agents in the treatment of infection and cancer. In future work, focus will be aimed at in vivo biological activity and toxicity studies.

Ethics approval and consent to participate
Not applicable.

Acknowledgment
Thanks to all the peer reviewers for their comments and suggestions on earlier drafts of this manuscript.

Funding
This research received no external funding.

Conflict of interest
The authors declare no conflict of interest.