Ecofriendly Synthesis of Silver Nanoparticles Using Ananas comosus Fruit Peels: Anticancer and Antimicrobial Activities

Metallic nanoparticles are valuable materials and have a range of uses. Nanoparticles synthesized from plant wastes by environment-friendly methods have attracted the attention of researchers in recent years. Also, the advantages of biological resources and synthesis methods are attracting attention. In this study, silver nanoparticles were synthesized from Ananas comosus fruit peels using ecofriendly method steps. The characterization of the particles obtained was determined by using a UV-visible spectrophotometer (UV-Vis.), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction diffractometer (XRD), Fourier scanning electron microscope (FESEM), and transmission electron microscopy (TEM). The nanoparticles showed maximum absorbance at 463 nm, measuring 11.61 in crystal nanosize, and presented spherical in appearance. An antimicrobial activity test was determined with the minimum inhibition concentration (MIC) method. The nanoparticles showed promising inhibitory activity on the Gram-positive and Gram-negative pathogen microorganisms (Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, Bacillus subtilis ATCC11774, Pseudomonas aeruginosa ATCC27833 bacteria, and Candida albicans yeast) at low concentrations. The cytotoxic and growth inhibitory effects of silver nanoparticles on different cancer cell lines were examined via the MTT assay.


Introduction
Nanomaterials synthesized by different methods, produced with metallic nanoparticles, are very valuable products that can be used in many areas. Nanoparticles show biological, optical, magnetic, and catalysis properties depending on their shape and size [1]. Some features of nanoparticles (NPs) make them superior. Having a large surface area and being resistant to high temperatures are some of these. With these features, they can be used in many fields such as material science, pharmaceutics, and electronics [1].
Physical, chemical, and biological methods are used to synthesize nanoparticles [2]. Physical and chemical methods have certain disadvantages compared to biological methods. e presence of toxic chemicals in the synthesis stages and the difficulty of controlling these steps also bring along problems such as high energy requirements and increased costs. In that aspect, biological methods are more advantageous [3].
For nearly 5000 years, ancient civilizations used silver materials while consuming foods and beverages or storing them for a long time [4]. Especially for their antimicrobial effects [5], silver nanoparticles (AgNPs) are widely used in anticancer agents [6], cosmetics [7], the food industry [8], electronics, catalysis [9], and bioremediation [10] applications such as dye removal. ere are many biological sources to synthesize these particles. Bacteria [11], algae [12], fungi [13], yeasts [14], and plants [2] are among these. Using plants offers several advantages over other biological resources. Factors such as the ease of application, no risk of pathogenicity, and lack of problems including production conditions are some of these. In the synthesis of AgNPs with plant sources, the root [15], peel [16], fruit [17], leaf [18], and flower [19] parts or the whole plant can be used [20].
Cancer, with its different types and difficult treatment process that is often insufficient, is a tough disease to cope with, in today's world [21]. A lot of research is being carried out to develop new treatment methods every day [22][23][24]. AgNPs can contribute to this process, in finding new and effective methods. ere are some studies on the use of AgNPs as anticancer agents [22,23]. e use of AgNPs as antimicrobial and anticancer agents has been revealed as a result of many studies [23,24]. AgNPs obtained from plant sources exhibit a biocompatible structure which provides great convenience for medical applications [6,12].
Antibiotic resistance of microorganisms poses a serious problem. ousands of people succumb to these microorganisms and die due to hospital pathogens being resistant to the antibiotics used [11]. Studies have shown that AgNPs can, at this point, contribute to the search for antimicrobial agents against pathogenic microorganisms [5,11].
is study aimed to synthesize AgNPs from waste Ananas comosus fruit peels economically and easily via an ecofriendly method that does not involve toxic chemicals, characterize them, and examine their anticancer and antibacterial activities.

Preparation of Plant Extract and Stock Solution.
e peels of Ananas comosus fruits were cut and removed. After washing with tap water several times followed by distilled water, the peels were dried at room temperature. ese dried peels were cut into smaller pieces and prepared to weigh. 200 grams of the dried fruit peels was weighed and left to boil in 500 mL of distilled water, half covered. From the moment it started boiling, we waited for about 15 minutes and left it to cool fully covered. First, coarse filter paper and, subsequently, a filtering process with Whatman 0.1 mm filter paper were used. e extract was stored at + 4°C for use in the synthesis phase. A solution of 10 mM (millimolar) concentration was prepared from the solid AgNO 3 .

Synthesis and Characterization of AgNPs.
e prepared 500 mL plant extract and 10 mM solution were mixed in a 1000 ml glass flask at room temperature by using a magnetic stirrer. e solution was observed for color changes.
Related to color changes, samples were periodically taken (30,45, 60, 90, 120 min) and measurements were made with a UV-Vis spectrophotometer to determine the formation and presence of AgNPs. FTIR device frequencies were used for determining the functional groups responsible for the reduction. After the reaction, the dark liquid sample was centrifuged at 10.000 rpm and the precipitate was dried for other characterization processes. Crystal nanosize and structure were evaluated with XRD data. e FESEM, TEM, and EDX data were used to determine morphological structure and element composition. Muller Hinton broth for bacteria, Roswell Park Memorial Institute (RPMI) broth for yeast, and solutions containing different concentrations of AgNPs were added to 96-well microplates. Firstly, wells were designed and then a series of dilutions were performed. After the microorganism suspension prepared for each strain was added to wells, the same procedures were repeated for vancomycin (used for Gram-positive strains), colistin (used for Gram-negative strains), and fluconazole (used for C. Albicans yeast) antibiotics to compare the inhibitory effects of AgNPs. e microplates were incubated to propagate at 37°C for 24 hours. At the end of this period, the well before the well where propagation started was determined as the minimum inhibition concentration (MIC). e cultured flasks were incubated at 37°C, under 5% CO 2 , 95% air, and humidity conditions. After the cells reached approximately 80% confluency, the cell was counted by using a hemocytometer and 10 4 cells were seeded per well of 96-well plates and subjected to overnight incubation. After the incubation period, the cells were treated with nanoparticles in concentrations of 200 μg/mL, 100 μg/mL, 50 μg/mL, and 25 μg/ mL and incubated for 48 hours. After this period of waiting, the MTT solution was added to the plate wells, and 3 hours after incubation with MTT reagent, the medium was aspirated gently and 100 μl of DMSO was added to each well and incubated for 15 min at RT with gentle shaking. e absorbance of the microplates at 540 nm wavelength was measured using the Multi ScanGo, ermo device.

Examining the Cytotoxic
Utilizing these absorbance values, the concentration in which the percentage of the viability of AgNPs is inhibited on cells was calculated.
% viability � U/C * 100 [25,26], where U is the absorbance of cells treated with AgNPs and C defines the absorbance values of control cells.

Statistical Analysis.
e experiments were performed in triplicate by means of the t-test and ANOVA. P < 0.05 was regarded as significant.

UV-Vis Spectrophotometer Data.
Colour transformation from yellow to dark brown was observed one hour after mixing the plant extract and the 10 mM AgNO 3 solution [8].
is color change is caused by the reduction of silver ions to AgNPs and the occurrence of vibrations (SPR) on the plasma surface [6]. e maximum absorbance was found to be at 463 nm after analyzing the samples taken periodically via the UV-Vis device (Figure 1). Colour transformation and maximum absorbance data show that AgNPs formed in the reaction liquid [27]. In the synthesis study with Holoptelea integrifolia plant extract, 460 nm maximum absorbance data were evaluated with the presence of AgNPs [18]. In another synthesis study of plant origin, 460 nm was associated with the formation and presence of AgNPs [28].

FTIR Data.
FTIR data were evaluated to examine the functional groups involved in the formation of AgNPs. e frequency shifts that occurred between 3334.96 and 3338.80 cm −1 and 1635.35 and 1634.97 cm −1 occurred, suggesting that the functional groups of-OH (hydroxyl) [29] and C�O (I amide) [30], respectively, play a role in the reduction (Figure 2).

FESEM, TEM, and EDX Data. FESEM, TEM, and EDX
analysis data were used to determine the morphological structures and element compositions of AgNPs. FESEM and TEM images showed that the AgNPs obtained were spherical, and the presence of substantially strong silver peaks [18] were detected in the EDX profile. Weak peaks in the EDX profile such as Cl, O, and C were due to phytochemicals in the extract [35] (Figure 4).

Evaluation of Antimicrobial Activities of AgNPs.
Metallic silver ions are inert in their dry state. ey show highly reactive properties when they are ionized in water. Ionized silver contacts microorganisms via its electrostatic attraction force. is causes an increase in reactive oxygen species (ROS). e structure of the cell wall is disrupted by the increase in ROS. e structure of the cell membrane and nucleus membrane also deteriorates. Structures such as DNA and RNA have a high affinity for these species. By affecting the activities of these structures, they disrupt their functions and cause cell destruction and death [3,36].
Antimicrobial effects of AgNPs were tested on pathogens such as Gram-positive and Gram-negative bacteria and yeast. e inhibitive effects of silver nitrate, AgNPs, and standard antibiotics on tested microorganisms were compared. Based on these results, the AgNPs were found to be effective at least 2 more times than the antibiotics used in the treatment (Table 1).
AgNPs obtained with Pistacia terebinthus extract were said to be effective on S. aureus, E. coli, and C. albicans microorganisms at concentrations of 0.04, 0.66, and 0.16 μg/ mL, respectively [31,33]. In a study where different sizes were synthesized, the MIC concentrations of AgNPs that were 5 nm in size for B. subtilis, S. aureus, and E. coli were reported in size 0.8, 6, and 6 μg/mL, respectively [37]. In another similar study, a concentration of 30 μg/mL was   effective for Pseudomonas aeruginosa ATCC 27853 [35]. In another study, it was reported that silver nanoparticles showed effective inhibition of the growth of Gram-negative and Gram-positive bacteria [24,38].

Investigation of Cytotoxic Activities of AgNPs.
e cytotoxic activities of AgNPs synthesized using Ananas comosus peel extract on U118, CaCo-2, and Skov-3, cancer cells were examined via the MTT method (Table 2). According to the data obtained, on CaCo-2 cell lines at a concentration of 25 μg/mL, it showed an inhibitory effect of 81% depending on the % viability of 18.20 on the cell line. Increases in the percentage of viability in some cell lines during transitions to high concentrations were due to the proliferative features of cancer cells [39].
AgNPs show a strong oxidative structure. Ag + release can induce cytotoxic and genotoxic formations in biological structures, and it is important to evaluate these effects [40].
AgNPs interact with the cell surface and cause the cellular composition to deteriorate [12]. AgNPs settle in structures such as the cell membranes, nucleus, and mitochondria. AgNPs cause an increase in ROS; besides, they show toxic effects by stimulating apoptosis [39,41].   In a study conducted to examine the toxic effects of AgNPs, a concentration of 3.75 μg/mL showed toxic effects on Caco-2 cells [42]. In another study, the concentration at which it showed toxic effects on Skov-3 cells was determined to be 9.4 μg/mL [43].
Some features can have a significant effect on the toxicity of nanoparticles. Concentration, exposure time, burden, the chemistry of surface composition, degree of accumulation, shape, and size are some of these [3,23]. e different cytotoxic concentrations of AgNPs in the studies may be due to the synthesis of AgNPs from different sources or their different sizes and morphological structures.

Conclusions
Antibiotic resistance is a serious problem. AgNPs will significantly contribute to the research for antimicrobial agents to solve this situation. Studies have demonstrated that AgNPs synthesized with biological sources show biocompatible properties.
AgNPs are effective on pathogenic species in many studies. However, the toxic effects of their use should also be noted. Cytotoxic activity studies are significant data that will help eliminate this concern.
Waste is among the ever-growing problems in our world. A wide variety of methods are being developed to use such waste in the fields and areas that will benefit people.
In the study, AgNPs were synthesized in an easy, economical, and ecofriendly method by using the bioactive components in pineapple fruit peels to reevaluate waste. e obtained AgNPs were characterized via UV-Vis, FTIR, XRD, TEM, FESEM, and EDX data in which they exhibited at 463 nm maximum absorbance, 11.61 nm in crystal nanosize, and spherical appearance. e AgNPs showed antimicrobial effects at low concentrations. To examine the usability of these particles as an anticancer agent, their cytotoxic effects were examined, and it was determined that 25 μg/mL concentration showed 25-81% inhibition in different cancer cell lines. In addition, the anticancer effect of AgNPs obtained by green synthesis on the U118 cancer cell line was investigated for the first time in this study. e resulting AgNPs can be developed and used in the medical and pharmaceutical industries.

Data Availability
All data used to support the findings of this study are included in the article.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this paper.