Document Type : Research Paper
Authors
Department of Cell and Molecular Biology, Faculty of Chemistry, University of Kashan, Kashan, Islamic Republic of Iran
Abstract
Keywords
INTRODUCTION
A Japanese scientist, Norio Taniguchi from Tokyo University, introduced nanotechnology [1]. Nanotechnology is a science that researches particles and materials at the nanoscale (1-100 nm) [2,3]. The advantage of nanoscale size is the high surface-to-volume ratio [4,5]. Nanoscale features are often incorporated into bulk materials. They have a large surface-to- volume ratio [6], bio-molecular detection, diagnostics [7], catalysis [8], microelectronics [9], sensing devices and targeting of drugs to cancer cells [10]. The shape, size, and structural properties of NPs strongly depend on synthetic methods. During the last decades, many approaches have been utilized to synthesize NPs classified into three groups: chemical, physical and biological [2,11]. Evaporation-condensation and chemical reduction synthesize metal nanoparticles [12]. Physical and chemical processes can successfully produce nanoparticles. These expensive, high-energy methods are potentially hazardous to humans, animals, and the environment [13].
Nowadays, plant extracts synthesize green nanoparticles [14,15]. The green synthesis of metal particles is environmentally friendly and cost-effective. Moreover, it does not require high temperatures, pressures, energy, and toxic chemicals [16,17]. The plant extract of roots, stem, leaves, and fruit has been utilized to biosynthesize metal and metal oxide nanoparticles [18-20]. The nanoparticles can be synthesized by proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, terpenoids, glycosides, and benzenoids in the plant extract. These compounds may reduce, capping, and stabilize agents in nanoparticle synthesis [21-23].
Metal nanoparticles are important among nanoparticles due to their antimicrobial properties [1,20,24,25]. Medicinal application is one of the important aspects of nanotechnology. Infectious diseases are the major challenges for humans, and multidrug-resistant bacterial are a severe threat to human health worldwide [26,27]. Silver nanoparticles were selected to overcome these problems due to their multiple medical functions. Silver nanoparticles have exhibited different biological activities such as anti-inflammatory, antifungal, anticancer, antiplasmodial, and antimicrobial activity [26]. The antibacterial ability of smaller Ag nanoparticles is higher than that of larger Ag nanoparticles [21,28,29]. This study aimed to synthesize AgNPs through A. elongatum extract and evaluate their biological activity, including antimicrobial, anti-biofilm, and cytotoxicity.
MATERIAL AND METHODS
Silver nitrate (AgNO3, 99.9%) was prepared from Merck. The culture medium includes nutrient agar (NA), nutrient broth (NB), tropic soy broth (TSB), and supplements purchased from Merck. All chemicals and solvents used in this study were purchased from Merck. Brine shrimp eggs used in this study were purchased from Inc., Salt Lake City, Utah 84126.
Collection and preparation of aqueous leaves extract
Arum elongatum leaves were collected from Mount Dena in Kohgiluyeh and Boyer-Ahmad province. At first, the leaves were washed with distilled water and followed shade-dried. Then, the powder (3.5 g) was added to water (200 ml) and heated at 40 °C for 15 min [30]. After filtration, the solution was used for the nanoparticle synthesis reaction.
Synthesis of silver nanoparticle
For the synthesis process, 10 ml of the aqueous extract was added to AgNO3 (1mM) and incubated at 60 °C. The changing color of the solution from colorless to dark brown indicated the synthesis of silver nanoparticles. After 24 h, the nanoparticles were isolated by centrifugation at 12000 rpm for 15 min at 24 °C and washed with double distilled water three times. The clear supernatant solution was discarded, and the pellet was dried and stored until further study.
Characterization of silver nanoparticles
The formation of AgNPs was measured by a Shimadzu UV-1800 spectrophotometer at 300-700 nm. The FT-RI spectrum was determined by the FT-IR spectrophotometer (Shimadzu-8400). The presence of biomolecules in aqueous extracts and silver nanoparticles was selected in the 4000-400 cm-1 range at a resolution of 4 cm-1. The surface morphology and size of synthesized AgNPs were evaluated using a scanning electronic microscope (SEM). The crystal structure of AgNPs was evaluated using a Philips Xpert pro-XRD system. The average crystallite size of the AgNPs was calculated with the Debye-Scherrer equation: D = (kλ)/ (β).
Antibacterial activity
Sixteen strains were collected from Qom Hospital, Iran, including Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. The minimum inhibitory concentration (MIC) was estimated using the broth microdilution procedure [31]. First, the concentration ranges of the AgNPs (1, 0.5, 0.25, 0.125, 0.0625 and 0.0312 mg/ml) were prepared. Afterward, 100 µl of each dilution was added to the wells containing 95 µl of TSB. Then, 5μl of the bacterial suspension was adjusted to 0.5. MacFarland was inoculated to each well. The microplates were incubated at 37 °C for 24 h. Moreover, 195 µl of TSB and 5 µl of the suspension were applied as a control. The MIC value was determined as the lowest concentration of the AgNPs applied for inhibiting the growth of microorganisms. However, minimal bactericidal concentration (MBC) was measured by inoculating 5 μl from the clean well on nutrient agar. MBC was defined as the lowest concentration in which growth was observed.
Anti-Biofilm of AgNPs
The bacterial strains collected from patients with urinary tract infections in different care units of Qom Hospital (Iran) were selected because these strains have the biofilm-forming ability. The anti-biofilm activity of the AgNPs was measured using a microtiter plate assay [32].
The bacterial suspension adjusted to 0.5 MacFarland was diluted 1:100 in TBS medium. The suspension was then added to 96-well microplate (100 μl per well). Also, 100 μl of AgNPs solution (2 mg/ml) was added to each well. After incubation for 24h at 37 ºC, the suspensions were removed from each well and washed twice with phosphate-buffered saline (PBS) to discard the planktonic cells. Then, methanol (200 μl) was added to each well. After 20 min, all wells were washed with PBS; the adherent cells fixed in each well were stained with 1% crystal violet for 5 min and discarded by inversion. Then, the wells were rinsed with PBS. At the last stage, 95% ethanol was added for 5 min, and OD of the samples was recorded at a wavelength of 570 nm using a spectrophotometer.
Cytotoxicity assay
The cytotoxic activity of the AgNPs was evaluated using the brine shrimp lethality test (BST). The different salts were resolved in one liter of distilled water NaCl (23 g), MgCl2 (11 G), NaSO4 (4g), CaCl2 (1.3 g), and KCl (0.7 g). Artemia salina eggs were hatched after 48 h incubation at 37 °C under strong aeration and illuminations. After two days, when A. salina eggs were ready, ten larvae were collected and transferred into each tube containing 5ml of sea salt water and different concentration of the nanoparticles. The negative control contained seawater and ten larvae. Vincristine sulfate was applied as a positive control. The number of survivors larvae was counted after 24 h incubation at 25 ºC. The percentage of the lethality of larvae was calculated.
RESULTS AND DISCUSSION
The present study elucidated the biosynthesized of the AgNPs using A. elongatum leaves extract and evaluated their biological activities.
The antimicrobial activity of nanoparticles is related to properties such as size, shape, and morphology. Active molecules in the extract, such as polyphenols, reduce metal ions to form nanoparticles. This mechanism describes the synthesis of nanoparticles by the plant extract. M, n, and Ar are the metal ion and the number of groups oxidized and the aromatic ring, respectively [33,34].
nM+ n(Ar-OH) n → nM0NPs + n(aromatic ring) + 3nH
UV-Vis spectroscopy
The synthesis of silver nanoparticles was confirmed using the color change of the AgNO3 solution to dark brown (Fig. 1). The changing color was observed when the surface plasmonic resonance phenomena were excited in silver nanoparticles [35]. In the synthesis AgNPs, the intensity of the change in color of the solution increased during the incubation time.
The synthesis of silver nanoparticles was monitored using the UV-Vis Spectrophotometer at 350–700 nm. The results exhibited a single, strong peak around 433 nm after 48 h, whereas the absorption peak was not observed for the extract of A. elongatum and AgNO3 solution used as control (Fig. 1).
Therefore, this result confirmed that the extract could reduce and stabilize the nanoparticles. The synthesized silver nanoparticles covered with biomolecules are well dispersed in the solution [34]. Similar results showed that the absorption peak of silver nanoparticles was 400-500 nm.
The silver nanoparticle synthesized with Aegle marmelos, Berberis lyceum, and Piper nigrum leaves extract revealed 436, 458, and 460 nm [19,36]. The result of UV-Vis Spectroscopy has to overlap with a previous study by Mehmood et al. (2014).
XRD
The XRD technique determined the structure of crystal AgNPs in the range of 5 - 90° at 2ϴ angle. Fig. 2b showed several diffraction peaks characteristic of the crystalline nanoparticle at 2ϴ values of ~ 28°, 33°, 38°, 45°, 47°, 65°, and 78°. The XRD peaks at ~28° and ~33° were attributed to the Cl of AgCl. Also, at 2ϴ =38.36, it has the highest peak, related to Ag. The average crystalline size of the synthesized AgNPs was 23 nm, estimated by the Scherrer equation.
FT-IR
Phenolic compounds such as gallic acid, benzoic acid, and flavonoids are plant metabolites. The FT-IR spectrum is evaluated to compare the capped biomolecules on the AgNPs, and the organic compounds and functional groups of the leaves extract [37,38].
The absorption peaks of the leaves extract were determined at 3049 cm-1 (stretch O-H), 2925 cm-1(stretch C=H), 1595cm-1 (stretch), 1403cm-1 (stretch C-H), 1076 cm-1(stretch C-O), and 830cm-1, 766 cm-1, 695 cm-1, 617 cm-1 compounds aromatic ring in the phenolic [38-40].
In contrast, these six peaks are absent in the corresponding silver nanoparticle spectra. However, the picks around 3430 cm-,1 and 1634 cm,-1 and 1383cm-1 were observed in the nanoparticles. The absorption pikes at 3430 cm-1 indicate hydroxyl groups assigned to O-H stretching frequency. Also, the bands at 1634cm-1 are commonly related to alkenes [41], and 1383cm-1 ascribed to C-N stretching vibrations of aromatic [42]. The other bands in the FTIR spectrum occurred due to the biomaterials of the extract. They provided stable nanoparticles with the functional groups in this region, responsible for reducing/stabilizing the AgNPs.
Elinkages in C-O and C=O functional groups are related to being groups of flavones, terpenoids, and polysaccharides in the plant extract. The biomolecules of the extract have been identified on the surface of AgNPs synthesized from the plant extract [43].
SEM
The SEM micrograph demonstrated that the morphology of the nanoparticles was an almost spherical shape. Furthermore, the size of the nanoparticles was in the range of 32–38 nm, and the average diameter of 6.45 nm (Fig. 3A and Fig. 3 B).
In similar study, Sahoo et al. reported spherical silver nanoparticles synthesized using Punica granatum L. with a size of 40-80 nm [43].
The histogram of the size distribution of nanoparticles was obtained by counting 100 particles in different regions and is shown in Fig. 3D.
EDX analysis determined the presence of the silver element. Moreover, other signals were detected in the EDX spectrum, confirming the present elements like Cl, O, N, Mg, and C. It suggested that the element peaks such as Cl, O, N, and C were obtained from plant extract [44].
Antibacterial activity of AgNPs
Silver nanoparticles could be applied against various bacterial pathogens that are antibiotics resistant. The antimicrobial properties of silver nanoparticles depend on size, pH, ionic strength, and capping agent [45]. In this study, the antimicrobial effect of AgNPs was investigated against the sixteen human pathogenic strains using the broth microdilution method. The MIC values are summarized in Table 1.
The nanoparticles exhibited an antibacterial effect on all the tested strains, with MIC and MBC values in the range of >2 ±0.00 to 0.0625±0.00 and 2>±0.00 to 0.125 ±0.00, respectively.
Kim et al. reported the antibacterial activity of silver nanoparticles against E.coli and S.aureus, which confirmed our results [46]. Moodley et al. synthesized AgNPs from moringa aleifera leaf extract and evaluated their antibacterial potential against different bacteria. The MIC values against K.pneumoniae, P.aeroginosa, and S.aureus obtained 25 μg /ml, and MIC against E. coli and E. faecalis was 12.5 μg/ml. This study recorded lower antibacterial activity than ours [5]. In another study, AgNPs showed antibacterial effects against E.coli and methicillin-resistant S.aureus with MIC 1.2 µg/ml and 0.25 µg/ml, respectively [47]. However, silver nanoparticles join the cell membrane, penetrate the bacteria, and lead to protein denaturation [48]. Antibacterial activity depends on the different concentrations of silver nanoparticles. Further, it has been shown that smaller nanoparticles have more antibacterial activity [49]. Yousefzadi et al. investigated biosynthesis AgNPs using green alga Enteromorpha flexuosa and the antibacterial potential of AgNPs. Their results indicated that in MBC, the value of S. epidermidis was 12.5 μg/ml and MBC of S. cerevisiae, S.aureus, and B. subtilis was 50 μg/ml [50]. A previous study showed that AgNPs synthesized using Uritica diocia ethyl acetate extract have high antimicrobial activity and can be used as an alternative to antibiotics (Binsalah et al. 2022).
Cytotoxicity activity of AgNPs
The cytotoxicity effect of the silver nanoparticles was determined using the BSL assay. The cytotoxicity potential of the AgNPs was evaluated at concentrations 300, 250, 200, 150, 100, and 50µg/ml and compared to vincristine sulfate (VS) as a positive control. All the samples showed good brine shrimp larvicidal effect. The AgNPs have killed (90%) brine shrimp larvae at the highest concentration (300 μg/ml). Furthermore, minimum mortalities (20%) were indicated at 50 µg/ml concentration. The results showed that the LC50 of Ag nanoparticles and vincristine sulfate were 120±0.00 and 0.751±0.008 μg/ml, respectively (Fig. 4).
The previous research determined that toxicity against A. salina included inactive (LC50>1000µg/ml), weakly toxic (LC50<500-1000 µg/ml), moderately toxic (LC50 100-500 µg/ml), and highly toxic (LC50<µg/ml) [51]. According to these reports, the AgNPs synthesized using A. elongatum have moderately brine shrimp larvicidal activity (LC50120±0.00). This study is comparable to studies carried out by other researchers. Patil Shriniwas et al. reported the cytotoxicity activity of AgNPs synthesized from Lantana camara L. leaves using brine shrimp lethality assay with LC50 514.50 μg/ml [52]. According to the findings of Vijayan et al., AgNPs synthesized from seaweed had a cytotoxicity effect (LC50 88.914±5.04) using assay [53]. Moreover, according to the results of the BSL test, synthesized nanoparticles using extracts of Agave Americana, Mentha spicata, and Mangifera indica have good cytotoxicity.
Anti-biofilm
One of the major causes of nosocomial infections is biofilm formation by E.coli. In this study, biofilm inhibition potentials of the AgNPs were evaluated against sixteen strains of the human uropathogenic E.coli, S.aureus, and K.pneumoniae. These strains were able to form biofilms. As result, the AgNPs (1 μg/ml) inhibited over 90% biofilm formation.
The effect of AgNPs against the formation of biofilm was reported in some studies [53-55]. AgNPs synthesized using leaves extract of Convolvulus arvensis exhibited biofilm degrading activity against both S.aureus and P.aeruginosa [56]. In another study, silver nanoparticles synthesized from garlic extract showed a robust anti-biofilm effect against S.aureus [57]. A similar study has been previously reported that AgNPs could inactivate the biofilm formation of S. aureus strains [58]. Sahoo et al. reported that AgNP synthesized using Punica granatum L. was a biofilm inhibitor against S. aureus [59]. Further, another researcher has reported that AgNP made from fruit extract of Chrysophyllum albidum has a high biofilm effect on methicillin-resistant S.aureus [60].
The finding of this study showed that the silver nanoparticles were successfully synthesized by A.elongatum leaves extract. The formation of AgNPs was confirmed by UV-visible spectroscopy, XRD, FTIR, SEM, and EDX analysis. The analysis showed that the AgNPs had spherical morphology. The biological activity of the AgNPs was evaluated using anti-biofilm, anticancer, and antimicrobial activity. Brine shrimp lethality (BSL) assay was applied to investigate the anticancer activity. The nanoparticles exhibited good cytotoxicity activity. Furthermore, the synthesized AgNPs indicated powerful antibacterial and anti-biofilm effects against human uropathogenic. These results suggested that the silver nanoparticles could be used as antimicrobial, anticancer, and anti-biofilm against pathogenic bacteria. However, the antibacterial effect of the AgNPs could be due to the interaction between the cell membrane bacteria and AgNPs.
CONCLUSION
In this study, a robust antimicrobial and cytotoxic silver nanoparticle was produced using a novel, simple and environmentally friendly pathway with leaves extract of A.elongatum collected from Kohgiluyeh and Boyer-Ahmad province, Iran. The results showed that the nanoparticles have an antibacterial effect on human pathogenic strains, including E.coli, S. aureus, and K. pneumonia, in Qom Hospital, Iran. Moreover, the silver nanoparticles inhibit the biofilm of uropathogenic bacteria. Based on the results, the silver nanoparticles can be a good candidate for treating urinary infections caused by a biofilm of uropathogenic bacteria.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.
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