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In vitro cytotoxicity against K562 tumor cell line, antibacterial, antioxidant, antifungal and catalytic activities of biosynthesized silver nanoparticles using Sophora pachycarpa extract
⁎Corresponding author. S.mortazavi23@yahoo.com (Sobhan Mortazavi-Derazkola) Sobhan.mortazavi@bums.ac.ir (Sobhan Mortazavi-Derazkola)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
In the present study, we demonstrate the green synthesis of silver nanoparticles using Sophora pachycarpa extract (S. pachycarpa; SPE) as capping, reducing, and stabilizing agents. The biosynthesized silver nanoparticles (SPE-AgNPs) were tested for catalytic, antibacterial, antifungal, antioxidant, and anti-cancer activities. The affecting parameters (the concentration of silver nitrate, the temperature of the reaction, and time of reaction) on the synthesis process were optimized. The biosynthesized SPE-AgNPs were studied by X-Ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDS) and Fourier-transform infrared spectroscopy (FT-IR). The FESEM and TEM results revealed spherical and oval-like morphology with sizes ranging from 30 to 40 nm. Photocatalytic performance experiments of SPE-AgNPs were determined by the rapid degradation of the eriochrome black T (EBT) and methylene blue (MB) under sunlight and UV irradiations. The results showed that SPE-AgNPs degraded more than 90% and 80% of both dyes under UV and sunlight irradiations, respectively. In addition, the SPE-AgNPs exhibited good antibacterial and antifungal properties against S. aureus, S. epidermidis, P. aeruginosa, E. coli, K. pneumoniae, E. faecalis, and C. albicans with MIC values of 6.25, 6.25, 0.78, 0.39, 0.78, 1.56 and 0.78 µg/ml. The green synthesized SPE-AgNPs were found to inhibit the activity of DPPH free radicals efficiently. Eventually, the SPE-AgNPs exhibited significant in vitro cytotoxicity against K562 tumor cell line (IC50 = 19.5 µg/ml). All these studies indicated that AgNPs synthesized using S. pachycarpa extract have applications in the environmental and biomedical fields.
Keywords
Green synthesis
Sophora pachycarpa
Nanoparticle
Anti-cancer
Photocatalytic degradation
1 Introduction
Nanotechnology is an applied science with a wide range of applications in pharmaceutical science (Ardestani et al., 2020; Ghoreishi et al., 2017; Mohammadi-Aghdam et al., 2018), drug design (Yoo et al., 2015), biology (Chandra et al., 2020; Mohammadzadeh et al., 2019; Shirzadi-Ahodashti et al., 2020b), medicine (Guo et al., 2021; Mohammadzadeh et al., 2017), environment (Ahmadi et al., 2020b; Ebrahimzadeh et al., 2019; Ebrahimzadeh et al., 2020b), etc. Nanoparticles with sizes of 1–100 nm and various shapes have unprecedented chemical, physical and optical properties. Among metal nanoparticles, silver is one of the most important and widely used ones in the pharmaceutical, environmental and cosmetic industries (Rafique et al., 2017). Silver nanoparticles offer unique physical and chemical properties such as high electrical and thermal conductivity, suitable size, chemical stability, catalytic activity, and biological properties such as antimicrobial, antioxidant, anticancer effects (Beyene et al., 2017; Naghizadeh et al., 2021). Fouda et al. found that silver nanoparticles can be used to produce bioactive compounds that are applicable in biomedicine with significant antioxidant and antimicrobial properties (Fouda et al., 2020).
Various methods such as chemical reduction, lithography, electrochemistry, lasers, and microwaves are used to synthesize metal nanoparticles (Iravani et al., 2014). One of the disadvantages of chemical approaches is that they remain undecomposed and eventually cause pollution. In addition, the use of high pressure and high temperature during the synthesis of nanoparticles, even in low quantities, consumes lots of resources (Jorge de Souza et al., 2019). To solve this problem, the researchers used a wide range of environmentally friendly solvents. One of the goals of green chemistry is to use environmentally friendly reactants (Rao and Trivedi, 2006). This approach has advantages such as ease of use, biosafety and biocompatibility, lower costs, nontoxicity, and the production of high purity nanoparticles. Garibo et al. showed in 2020 that nanoparticles synthesized through the green synthesis, while less cytotoxic, have more antimicrobial effects than chemically synthesized silver nanoparticles (Garibo et al., 2020).
The rapid growth of industries such as textiles, dyes, and large amounts of plastic waste that end up in water, lead to serious environmental pollution. Dye contaminants, being one of the most essential contaminations, affects the transparency of water and causes the abnormal color of water (Azeez et al., 2018). Various methods, such as adsorption or coagulation, have been used to remove organic matter. One of the efficient methods of removing organic dyes developed over the last few decades is photocatalysts (Mekasuwandumrong et al., 2010; Shirzadi-Ahodashti et al., 2020a). Various studies have been carried out to date on the photocatalytic properties of silver nanoparticles. Kadam et al. introduced silver nanoparticles as an effective multifunctional tool for dye degradation (methylene blue) in industrial effluents and the detection of mercury-based contaminants (Kadam et al., 2020). Rajkumar et al. also reported that green silver nanoparticles synthesized with Chlorella vulgaris degraded 96.51% of methylene blue dye (100 ppm) within 3 h of incubation (Rajkumar et al., 2021). In previous research, the destruction of pollutants has not been significant. In this study, we obtained the high degradation efficiency of contaminants using biosynthesized nanoparticles.
Over the last five decades and the decline in the death rate from infectious diseases, cancer has become the leading cause of death in developed countries. The cancer cell line K562 is a myeloid blood leukemia cell that was first observed in a 53-year-old woman with chronic leukemia. It has about 1.5 times as many chromosomes as normal cells (Klein et al., 1976). In recent years, the effects of synthesized nanoparticles on different types of cancer cells were investigated (Ahmed et al., 2019; Anandan et al., 2019). Hashemi et al., by studying the antitumor property of silver nanoparticles, found that these nanoparticles have potential therapeutic effects and could be valuable in the production of cancer drugs (Hashemi et al., 2020). Ahmadi et al. also assessed the cytotoxic effects of silver nanoparticles on K562 and MCF-7 cell lines (Ahmadi et al., 2020a).
Sophora is a perennial herb with 7–12 pairs of leaves, cream-colored flowers, and fruits of the genus Fabaceae, widespread in East and Southwest Asia, Greece and southern Russia. The biologically active compounds of this genus include quinolizidine alkaloids, flavonoids, and steroidal glucosides (Emami et al., 2007). The antimicrobial, sedative, antipyretic, analgesic, anti-inflammatory, and antitumor effects of quinolizidine alkaloids, and the antioxidant, antibacterial, anti-inflammatory, anticancer, and antiviral effects flavonoids have already been proven (Kumar and Pandey, 2013).
This study aimed to green synthesize silver nanoparticles using hydroalcoholic Sophora pachycarpa extract (S. pachycarpa) and use it as an antibacterial and antifungal agents against Gram-positive bacteria (S. aureus, S. epidermidis, and E. faecalis) and Gram-negative bacteria (P. aeruginosa, E. coli, and K. pneumoniae) and C. albicans as fungal. The potential anticancer effect of the synthesized nanoparticles on K562 cell line was also assessed through the MTT assay. In addition, the photocatalytic activity was investigated by the degradation of eriochrome black T and methylene blue dyes under UV and sunlight irradiations.
2 Materials and methods
2.1 Materials and characterization techniques
All chemical reagents were at least reagent grade. Silver nitrate (AgNO3, 99.99%), sodium hydroxide (NaOH), and methanol solution were obtained from Sigma-Aldrich Company. All water used in the experiment was triply deionized water. The fresh parts of S. pachycarpa were collected from the Southern Khorasan province of Iran and washed with deionized water. Characterization of biosynthesized silver nanoparticles was determined by using various analytical techniques such as field emission scanning electron microscopy (FE-SEM; TESCAN BRNO-Mira3 LMU), Fourier transform infrared spectroscopy (FT-IR; PerkinElmer Spectrum TwoTM IR spectrometer; Model L160000U), UV-Visible spectroscopy (NanoDrop, BioTek model Epoch, USA), dynamic light scattering (DLS; NanoBrook 90Plus-Brookhaven Instruments, model 18051; USA; DLS), X-ray diffraction (Philips PW 1800 using Cu Kα radiation), transmission electron microscopy (TEM; Zeiss-EM10C-100 KV) and energy-dispersive X-ray spectroscopy (EDS).
2.2 Preparation of Sophora pachycarpa extract (SPE)
A herbarium specimen of Sophora pachycarpa was identified by a botanist and examined and approved by a specialist in the Herbarium of Birjand University of Medical Sciences in May 2020 in South Khorasan, Iran. The plant roots were pulverized by a grinder and the alcoholic extract was stirred by soaking the powder in a 1:10 solution of 80% methanol at room temperature for 24 h. The mixture was then filtered using a paper filter (Blue Ribbon, Grade 589, Germany), and its solvent was concentrated using a rotary evaporator (Heidolph, Germany), then was dried by a freeze dryer (FD-5005-BT, Dena Vacuum Industry, Iran), and powder was stored at −20 °C until use.
2.3 Biosynthesis of silver nanoparticles using SPE (SPE-AgNPs)
The SPE capped AgNPs (SPE-AgNPs) were synthesized using our previous protocol (Shirzadi-Ahodashti et al., 2021a). Typically, 0.3 g of S. pachycarpa extract was dissolved in 20 ml of distilled water, and the pH was raised to 12. The preparation of silver nanoparticles was observed by a color change from yellow to blood red according to the surface plasmon resonance (SPR). After that, the silver solution was further centrifuged and washed three times at room temperature. Finally, the purified AgNPs were stored for further study. Furthermore, a description of the samples prepared under different conditions is exhibited in Table. 1.
2.4 Antibacterial and antifungal assay
Staphylococcus aureus (ATCC 16538; S. aureus), Staphylococcus epidermidis (ATCC 12228; S. epidermidis), Pseudomonas aeruginosa (ATCC 27853; P. aeruginosa), Escherichia coli (ATCC 25922; E. coli), Klebsiella pneumoniae (ATCC 9997; K. pneumoniae), Enterococcus faecalis (ATCC 15753; E. faecalis), and Candida albicans fungi (C. albicans), were obtained from the Pasteur Institute of Iran. To obtain pure colonies from the nutrient broth culture medium containing pathogenic bacteria, an isolated culture was performed on the nutrient and blood agar media to obtain a 0.5 McFarland solution (1.5 × 108 CFU/ml) from the colonies that appeared on the surface of the culture medium. C. albicans fungi colonies were used to make suspension after revitalization and purification in the Sabouraud dextrose agar medium. For minimum inhibitory concentration (MIC) experiment: 100 μl TSB (Tryptic Soy Broth) was added to the culture medium in each micro-well, and 100 μl of the nanoparticle solution produced was added to the first micro-well of each row and mixed. The MIC was considered as the lowest concentration of bacterial growth inhibitor (Riesenberg et al., 2016). In this study, ceftriaxone as an antibiotic and amphotericin B as an antifungal was used to compare the antibacterial and antifungal effects of the nanoparticles and the extract, respectively. In order to do determine the minimum bactericidal concentration (MBC), 10 µl of turbidity-free micro-wells (at MIC concentrations and above) was taken under completely sterile conditions and inoculated and cultured on a blood agar medium. After 24 h of incubation at 37 °C, the most minor dilution that killed 99.9% of the bacteria indicated the MBC. For the reliability of the result, the experiment was repeated three times and an average was calculated (Ebrahimzadeh et al., 2020a).
2.5 Antioxidant activity
To examine the antioxidant properties, we used Zantox Total Antioxidant Capacity (TAC) Assay Kit (Kavosh Arian Azma Co., Iran) based on DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. In this standard method, 10 µl of S. pachycarpa extract samples, SPE-AgNPs (sample no. 11), control sample (available in the kit), distilled water (blank sample), and various standards (in concentrations of 62.5, 125, 250, 500, and 100 µM) were poured into micro-wells. Then 250 μl of the prepared DPPH solution was added to each of the micro-wells according to the kit protocol, and the plate was shaken for 30 s. Finally, samples were incubated in the dark at room temperature for 15 min and their absorption was measured at 517 nm using a spectrophotometer. The radical scavenging of DPPH was calculated as follow (Morabbi Najafabad and Jamei, 2014):
2.6 MTT assay of antitumor properties of SPE-AgNPs
The K562 (Chronic Myeloid Leukemia) Cells were cultured in a complete RPMI 1640 medium containing 10% FBS and 1% Pen/Strep at 37 °C in a humidified atmosphere of 5% CO2. The cytostatic effect of SPE-AgNPs on K562 cells was investigated by MTT methods. The Cells in exponential phase and the number of passage three were centrifuged, and plate was collected. Then, 20
103 cells/ml was added to each well of 96-well cell culture micro-plate in triplicate and treated with concentrations of 6.25, 12.5, 25, and 50 µg/ml of SPE-AgNPs. After 24 h of incubation (5% CO2 and 37 °C), 20 μl of the MTT solution (5 mg MTT dissolved in 1 ml PBS) was added to each micro-well and further incubated for 3 h. 100 μl of DMSO solution was then added to each micro-well. After 10–15 min and upon dissolution of the formazone particles, the absorption was measured at a wavelength of 570 nm with an ELISA reader (Mosmann, 1983). The survival rate was calculated as follow:
2.7 Photocatalytic activity
In order to investigate the photocatalytic degradation properties of eriochrome black T (as an anionic contaminant) and methylene blue (as a cationic contaminant), the synthesized SPE-AgNPs (sample no. 11; optimized sample) was applied to degrade the pollutants in a reactor under sunlight and UV irradiations. The photocatalytic experiment was performed by applying 5 ppm of methylene blue (main absorbance band at λ = 668 nm) and 10 ppm of eriochrome black T (main absorbance band at λ = 574 nm). 30 mg of SPE-AgNPs was added into 50 ml of each contaminants solution and stirred for 30 min without irradiation to reach the absorption equilibrium. Next, at the specified time intervals, 10 ml of suspension was withdrawn, centrifuged (6000 rpm and 10 min) to separate the photocatalyst, and determined by UV-Vis spectrophotometer. The contaminants degradation percentage was calculated as follow (Ghoreishi, 2017): D (%) = (A0-At)/A0 * 100 (3); where D is the photocatalytic efficiency (after t min), A0 is the initial absorbance quantity of pollutants solution and At is the absorbance of anionic and cationic contaminants after irradiation (after t min).
3 Results and discussion
3.1 UV-Vis spectrographic analysis
The best formation of AgNPs (in terms of size and morphology) depends on some factors (such as concentration, temperature, and time). The surface plasmon resonance (SPR) of synthesized biogenic silver nanoparticles exhibited a peak centered about 420 nm related to the absorbance of AgNPs (Shirzadi-Ahodashti et al., 2021b). So, in this research, evaluation of various critical experimental parameters, including the concentration of silver ion (5, 10, 15, 20, and 40 mM), reaction time (5, 15, 30, 60, and 90 min), and temperature (room temp., 60, and 85 °C) were studied. Each of these experimental parameters was confirmed by UV-Vis spectral study.
3.1.1 Effect of silver ion concentration
Fig. 1a shows the UV-Vis spectra of the AgNPs synthesized using 5 ml of S. pachycarpa extract in the presence of AgNO3 concentrations varying from 5 mM to 40 mM. As can be seen, 40 mM concentration of silver nitrate with 5 ml of S. pachycarpa extract illustrates more absorbance than the other four concentrations (5, 10, 15, and 20 mM). Past research showed that increasing the AgNO3 concentrations enhanced the absorption intensity implying the increase in the formation of nanoparticles (Hebeish et al., 2013). Therefore, the optimum value of the AgNO3 concentration is 40 mM, which was applied for the next experiments.
UV-Vis absorption spectra of AgNPs at various conditions: (a) concentration of AgNO3 (5, 10, 15, 20, and 40 mM), (b) time (5, 15, 30, 60, and 90 min) and (c) temperature (room tempt., 60, and 85 °C.
3.1.2 Effect of contact time
Fig. 1b shows the UV-Vis spectra recorded at different reaction times. As can be seen, increasing the contact time increased the intensity of the SPR band (5–60 min). However, by increasing the reaction time from 60 to 90 min, there was a decrease in the intensity of the SPR band. This decrease is related to the increased agglomeration of the obtained products. So, the optimal contact time to obtain silver nanoparticles of suitable size and morphology is 60 min.
3.1.3 Effect of temperature
The influence of the reaction temperature on the preparation of silver nanoparticles is exhibited in Fig. 1c. The UV results showed that with increasing temperature, the intensity of adsorption increased. Moreover, decreasing wavelength (blue shift) from 423 to 411 cm−1 can be observed with temperature increasing (from room temp. to 85 °C). From this reduction, it can be concluded that AgNPs with smaller sizes can be obtained at a higher temperature. So, the optimum value of the temperature is 85 °C.
3.2 X-ray diffraction
The crystallinity and nature of the synthesized silver particles using S. pachycarpa extract (under the best conditions) was evaluated by X-ray diffraction (XRD). The XRD pattern of AgNPs, which confirmed the crystalline nature of silver nanoparticles, was shown in Fig. 2. The four distinct diffraction peaks at 38.37°, 44.34°, 64.62°, and 77.59° in the range 10–80°, can be related to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) Bragg reflections from the fcc structure of nanoparticles (JSDS 01-087-0717). Moreover, no impurities were observed in XRD pattern that indicating that the crystal was a single phase. Our results were matched with the findings of Awwad et al. (Awwad et al., 2013). In addition, the grain size of biosynthesized metallic silver nanoparticles was determined using the Debye-Scherrer equation: D = nλ/βcosθ (4). Where λ is the X-ray wavelength, D is the crystallite size, θ is the diffraction angle and β is full-width half maximum (FWHM). Based on the above equation, the size of the synthesized nanoparticles was determined about 32 nm.
XRD pattern of biosynthesized AgNPs using S. pachycarpa extract (40 mM, 60 min and 85 °C).
3.3 Fourier transforms infrared (FTIR) analysis
The investigation of the functional groups in the silver ion (Ag+) reduction using S. pachycarpa extract for the green synthesis of AgNPs is shown in Fig. 3. The strong and broad absorption band at around 3400 cm−1 is related to the hydroxyl stretching band (O-H) of the phenolic compounds in the plant extract (Judith Vijaya et al., 2017). The band at approximately 2920 cm−1 can be related to methylene or methyl stretching vibration (C-H). The peaks at around 1600 and 1300 cm−1 corresponds to phenolic group and stretching aromatic ring (-C = C), respectively. So, water-soluble plant metabolites like quinones, flavonoids, and polyphenols were possibly responsible for reducing metal ions (Ag+) into silver nanoparticles (Ag0). As seen in the FTIR spectra of SPE-AgNPs, the absorption peaks of SPE-AgNPs were similar to the S. pachycarpa extract absorption peaks with a minor amount of displacement and intensity than absorption peaks of S. pachycarpa extract, which was consistent with the results of previous studies (Li et al., 2021).
FT-IR spectrum of the S. pachycarpa extract and SPE-AgNPs (40 mM, 60 min and 85 °C).
3.4 FESEM, TEM, and EDX analysis
The size and morphology of biosynthesized silver nanoparticles were investigated by field emission scanning electron microscopy (FESEM) images with different magnifications. Fig. 4 shows the FESEM micrographs of as-synthesized silver nanoparticles in the presence of S. pachycarpa extract with different magnifications (SPE-AgNPs). The results showed that the produced nanoparticles are spherical-like, and agglomerated in some areas. This agglomeration confirms the presence of stabilizing and capping agents in the process of formation AgNPs. Furthermore, regular and uniform structures with sizes in the range of 30–40 nm are another characteristic of synthesized nanoparticles. Fig. 5 shows TEM images of the synthesized silver nanoparticles in the presence of S. pachycarpa extract (optimized sample; SPE-AgNPs). Uniform nanoparticles and suitable distribution extract on the surface of silver nanoparticles are illustrated in Fig. 5. Moreover, the TEM images verify the synthesis of spherical and oval-like nanoparticles with even distribution and an average size of approximately 36 nm. Fig. 6 illustrates the EDS microanalysis of the obtained SPE-AgNPs. A strong Ag absorption peak appeared at 3 KeV. The Ag, O, and C elements in the chemical composition of SPE-AgNPs were detected. These results are directly related to XRD results.
FESEM images of the biosynthesized AgNPs using S. pachycarpa extract (40 mM, 60 min and 85 °C).
TEM images of SPE-AgNPs (40 mM, 60 min and 85 °C).
EDS analysis representing the compositional analysis of SPE-AgNPs (40 mM, 60 min and 85 °C).
3.5 Particle size distribution and zeta potential measurement
The average diameter of total particles and stability of green synthesized SPE-AgNPs were determined by dynamic light scattering (DLS) and zeta potential measurements, as shown in Fig. 7. The negative charges of the products play a key role in the agglomeration formation and thus increase the stability of the products (Rajkumar et al., 2021). The average particle size of the colloidal nanoparticles produced with the aqueous extract of S. pachycarpa extract was reported as 107–150 nm. Furthermore, the result of zeta potential reports high stability (due to electrostatic repulsion) of nanoparticles equal to −12.38 mV. The results obtained from the DLS analysis confirm that SPE-AgNPs has an adequate surface charge for electrostatic stability to prevent aggregation.
(a) DLS and (b) zeta potential measurement of SPE-AgNPs (40 mM, 60 min and 85 °C).
3.6 Analysis of antibacterial and antifungal activities
In this study, the MIC assay was used to evaluate the antibacterial and antifungal activities of synthesized silver nanoparticles using S. pachycarpa extract against Gram-positive bacteria (three strains), Gram-negative bacteria (three strains), and C. albicans fungi. The results for the synthesized nanoparticles, the antibiotic ceftriaxone, and the antifungal amphotericin B are given in Table. 2. According to the results, silver nanoparticles had an antibacterial effect on all bacterial strains, so that the lowest inhibitory concentration was observed for E. coli (MIC value of 0.39 µg/ml) and the highest for S. epidermidis and S. aureus (MIC values of 6.25 µg/ml). In addition, the broad-spectrum antibiotic ceftriaxone had the most significant effect on K. pneumoniae (MIC value of 1.95 µg/ml) and the most minor effect on E. faecalis (MIC = 15.62 µg/ ml). Comparison of the results for MIC and MBC in strains showed that the effect of silver nanoparticles on Gram-negative bacteria is more significant than on Gram-positive ones (Fig. 8). The effect of nanoparticles varies in bacterial strains; such variations can be due to the structure, and chemical composition of the cell wall and the mechanisms affecting the activity of various microorganisms studied (Ravichandran et al., 2019). However, the mechanism of antibacterial effect of silver nanoparticles is still unknown. Morones et al. showed that silver nanoparticles penetrate bacterial and fungal cells, interact with sulfur and phosphorus-containing compounds such as DNA and ultimately destroy microbes (Morones et al., 2005). The results of the present study showed the antifungal activity of biogenic silver nanoparticles against fungi studied (Table. 3). This nanoparticle even appeared to be superior to amphotericin B, as it had the lowest MIC (0.78 µg/ml) compared to amphotericin B (4 µg/ml). The comparison of the MIC and MBC in bacterial strains also indicated that silver nanoparticles synthesized with S. pachycarpa extract had a more substantial antimicrobial effect than the extract alone. The resulting silver nanoparticles not only exhibited promising antimicrobial effects, but also outperformed the broad-spectrum antibiotic ceftriaxone, albeit at lower concentrations. Bacterial resistance has become a severe problem due to improper and widespread use of antibiotics for prevention or therapeutic purposes regardless of actual medical symptoms (García et al., 2020). As a result, nanoparticles seem to be promising alternatives to antibiotics due to their unique properties in combating bacteria (Rajeshkumar et al., 2019).
Microorganism
SPE-AgNPs
Extract
Ceftriaxone
MIC (µg/ml)
MBC (µg/ml)
MIC (mg/ml)
MBC (mg/ml)
MIC (µg/ml)
MBC (µg/ml)
P. aeruginosa ATCC7853
0.78
12.5
50
> 100
3.9
31.25
K. pneumoniae ATCC9997
0.78
1.56
> 100
> 100
1.95
15.62
E. coliATCC25922
0.39
0.39
50
> 100
3.9
15.62
S. epidermidisATCC12228
6.25
200
50
> 100
7.81
62.5
E. faecalis ATCC15753
1.56
200
> 100
> 100
15.62
125
S. aureus ATCC16538
6.25
200
50
> 100
7.81
31.25
Photograph of MBC experiments of various bacteria.
Fungal strain
SPE-AgNPs
Extract
Amphotericin B
MIC (µg/ml)
MFC (µg/ml)
MIC (mg/ml)
MFC (mg/ml)
MIC (µg/ml)
MFC (µg/ml)
C. albicans
0.78
0.78
> 100
25
4
8
3.7 Antioxidant activity
In recent years, a variety of methods have been reported to investigate antioxidant activity, one of the most significant of which is DPPH. The DPPH method was used to evaluate the antioxidant properties of the synthesized silver nanoparticles. The DPPH radical is purple when dissolved in an organic solvent, while turns yellow when exposed to a reducing agent or a hydrogen donor. Such a change can be measured at the wavelength of 517 nm. Chemical and biological compounds with regenerative properties can neutralize such radicals, and the decrease in color intensity at the wavelength of 517 nm indicates stronger neutralizing effect. In this research, the percentage of inhibition of DPPH by S. pachycarpa extract and SPE-AgNPs were studied in three concentrations. Our results showed that silver nanoparticles at a concentration of 0.16 µg/ml had an antioxidant effect 15.5% and that higher concentrations led to a more substantial antioxidant property (Fig. 9). Comparing the antioxidant effect in the two groups of silver nanoparticles and S. pachycarpa extract showed that synthesized silver nanoparticles using extract had a better antioxidant effect at much lower concentrations than the pure extract. However, the current research is similar to findings that have emerged from Jalilian et al. (Jalilian et al., 2020).
DPPH inhibition percentage at different concentrations of SPE-AgNPs.
3.8 Antitumor effects on K562 cells
To assess the toxic effects of SPE-AgNPs on the K562 cell line, MTT colorimetry was used. For this purpose, 20,000 cells were cultured in each micro-well and treated with different concentrations of the medication for 24 h. The results confirmed the antitumor effect on reducing the cell growth compared to the control group. The results also showed that such an effect had a significant association with the concentration, so that within 24 h of treatment at a concentration of 50 µg/ml, the highest decrease in cell growth was observed (97%). The IC50 value of the medication was determined (IC50 of 19.5 µg/ml Calcusyn software was used). Our cytostatic results demonstrated SPE-AgNPs decreased viability of K562 cells in a dose-dependent manner (Fig. 10). Similarly, a 2014 study by Mousavi on the apoptotic features and cytotoxicity of S. pachycarpa showed significant toxicity against the HeLa, HL-60, MCF-7, A549, and PC3 cell lines (Mousavi et al., 2014).
Cytotoxic effect of SPE-AgNPs (sample no. 11) against K562 cancer cell line.
3.9 Photocatalytic properties
The synthesized nano-scale AgNPs using S. pachycarpa extract as capping, stabilizing, and reducing agents were used as UV and sun-light responsive photocatalytic to degrade the methylene blue and eriochrome black T pollutants as common organic pollutant released from industrial wastewater. The photocatalytic performance of all the samples at various time intervals of irradiations was studied. As expected, no degradation has occurred without the usage of SPE-AgNPs as a nanocatalyst. The photocatalytic results showed that the photo-degradation of EBT and MB contaminants was 96.23% and 90.06% under UV irradiation and 84.57% and 79.18% under sunlight irradiation after 80 min (Fig. 11). High uniformity, small size, and good surface area can be reasons for superior action of biogenic silver nanoparticles (SPE-AgNPs) in dyes degradation under sun-light and UV irradiations (Zinatloo-Ajabshir et al., 2019). Upon closer examination of the results, we conclude that the photodegradation of methylene blue as a cationic pollutant is higher than eriochrome black T as an anionic pollutants. This increase could be due oxygen group and positive charge in the molecular structure of cationic contaminant. The possible mechanism of degradation of cationic and anionic pollutants using synthesized silver nanoparticles can be displayed as follows (Scheme.1):
Photocatalytic degradation of EBT and MB organic dyes by optimal SPE-AgNPs (40 mM, 60 min and 85 °C).
Mechanism of photocatalytic degradation of organic dyes by SPE-AgNPs.
SPE-AgNPs (huv) + H2O → Ag0 + H + OH (6)
4 Conclusion
Spherical and oval-like silver nanoparticles with an average size of 30–40 nm were synthesized using S. pachycarpa extract. XRD pattern showed that the SPE-AgNPs were in the face-centered cubic (fcc) structure. FTIR spectra confirmed the capping behavior of the S. pachycarpa extract. The synthesized SPE-AgNPs exhibited high antibacterial, antioxidant, anticancer and catalytic activities. SPE-AgNPs demonstrated stronger antibacterial and antifungal activities than ceftriaxone and amphotericin B, respectively. High anticancer activity of SPE-AgNPs was obtained on K562 cancer cells with the IC50 value of 19.5 µg/ml. Furthermore, the results showed that the SPE-AgNPs as a nanocatalyst had excellent photocatalytic activity for degradation of EBT and MB under sun-light and UV irradiations. This study suggests that SPE-AgNPs can be effective nanoparticles for environmental and biological applications.
Acknowledgment
In this investigation, the entire procedures were conducted according to the Helsinki Declaration and ethical standards of the institutional research committee. The ethics code was taken from Birjand University of Medical Sciences (IR.BUMS.REC.1399.126).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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