Phyto-Fabrication of Silver Nanoparticles Using Typha azerbaijanensis Aerial Part and Root Extracts

Abstract

Background:

Silver nanoparticles (AgNPs) were phyto-synthesized using Typha azerbaijanensis aerial part and root extracts, and their biological activities were investigated.

Methods:

This study was conducted in the Science and Research Branch, Islamic Azad University, Tehran, Iran in 2019. In this experimental study, silver nanoparticles (AgNPs) were phyto-synthesized and the physicochemical properties of AgNPs were determined using UV-Vis (UV-Vis) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy. Antibacterial and anticancer activity of synthesized AgNPs was determined using microdilution assay, and MTT 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) methods, respectively. The apoptotic effects of AgNPs were investigated using Real-Time PCR and flow cytometry techniques.

Results:

Morphological analysis of the synthesized AgNPs confirmed the spherical shape of AgNPs with an average size of 10.67 to 16.69 nm. The FTIR spectrum confirmed the presence of phytochemicals from T. azerbayenensis extract at the AgNP surface. Antibacterial experiments showed that phyto-fabricated AgNPs had significant antibacterial activity against Gram-negative bacteria. The AgNPs were significantly cytotoxic against breast cancer cell line (MCF-7) through induction of apoptosis.

Conclusion:

The phyto-synthesized AgNPs had biological activities could be useful in pharmaceutical applications.

Introduction

Nanotechnology has promising applications in medicine and drug delivery along with cancer treatment and several other fields (1). Metal nanoparticles, especially silver nanoparticles (AgNP) which have controlled size, shape, and structure have wide applications in medicine (2). In general, there are different physical and chemical methods for synthesizing AgNPs (3–4). However, these methods use different chemical compounds for synthesis that are toxic.

Recently, the phyto-synthesis of AgNP using plant extracts has attracted the attention of researchers. In phyto-synthesis, seeds, gums, fruits, roots, skin, and flowers can be used to synthesize AgNP (5). In addition, the phyto-synthesis of AgNP using plant extracts is cost-effective and environmentally friendly (6). In general, plant extracts contain compounds such as polyphenols, which are secondary compounds that mediate AgNP synthesis (7).

So far, various plant extracts have been used to synthesize AgNP (8–12). In the present study, we used Typha azerbaijanensis extract for AgNP synthesis for the first time. T. azerbaijanensis is a species of the genus Typha that grows in northeastern Iran. The name will not be communicated to the independent Republic of Azerbaijan. The region of Azerbaijan in northeastern Iran may be notified (8–12). These species have different biological properties such as antibacterial, anticancer, antioxidant, anti-diabetic, antiemetic, antihypertensive, hypoglycemic, hypolipidemic effects (13). Phytochemical studies of these species have shown that they are rich in tannins, phenols, flavonoids, carotenoids, and alkaloids, which have been reported to show high antioxidant activities and can be a major factor in reducing silver to AgNP (14).

We performed this study by synthesizing, characterizing, and evaluating the antibacterial, anti-cancer, and antioxidant potentials of phyto-synthesized AgNPs from aqueous silver nitrate (AgNO3) using aerial parts and root extracts of T. azerbaijanensis.

Materials and Methods

Ethical considerations

This study was conducted in the Science and Research Branch, Islamic Azad University, Tehran, Iran in 2019. Ethics approval for this study was obtained from Islamic Azad University, Tehran, Iran permitted this study with number: IR.IAU.REC.1398.029.

Phyto-synthesis of AgNPs

For the synthesis of silver nanoparticles (AgNPs), 4 ml of aqueous and ethanolic extracts of aerial parts and roots of T. azerbaijanensis were added to 100 ml of 0.01 mM silver nitrate (AgNO3) (Merck, Germany). After stirring for 45 minutes at room temperature, the solution became colorless brown, indicating reducing of Ag to AgNP. After 45 minutes, the formed precipitate was washed three times with distilled water by centrifugation at 13000 rpm for 20 minutes and the sediment was kept at 60 °C for 2 h (15).

Characterization of AgNPs

UV-Vis analysis of phyto-fabricated AgNPs by spectrophotometer at wavelengths of 200–700 nm was performed to investigate the maximum absorption. Scanning electron microscopy (SEM) and transmission electron microscope (TEM) was used to determine of shape and size of synthesized AgNPs. FTIR spectroscopy was used to identify functional groups involved in AgNP synthesis. FTIR spectra between 4000 cm − 1 and 400 cm − 1 were recorded at a resolution of 4 cm −1 in transmission

Antibacterial activity

Antibacterial activity of phyto-fabricated AgNPs was performed using the micro-broth dilution method to determine minimum inhibitory concentration (MIC) value. Briefly, various concentrations of AgNP including 100, 50, 25, 12.5, 25.2, 3.125, and 1.56 μg/ml were poured into 96-well plates. Subsequently, bacterial suspensions including Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 23857, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 were added to each well at a concentration of 0.5 McFarland. MIC is defined as the lowest inhibitory microbial growth concentration (17).

MTT assay

Cytotoxic effects of AgNPs against breast cancer cell line (MCF-7) was evaluated using MTT (3- (4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazole bromide) colorimetric method. Initially, 10⁴ cells were seeded in 96-well plates, followed by 96-well plates in a CO₂ incubator at 37 °C for 24 h. Concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.39 μg/ml from AgNPs were prepared and added to the wells and the plates were incubated for 24 h, then MTT (Sigma Aldrich, Germany) was added to the well and the reaction mixture was kept at 37 °C for 4 h in a 5% CO₂ atmosphere. The MTT dye was then removed and all wells were dissolved using isopropanol. Finally, the absorbance rate of the samples was measured using an ELISA reader at 570 nm (Oraganon Teknika, Netherlands) in which the cell toxicity was calculated using:

$$\%\hspace{0.17em}\text{Cell}\hspace{0.17em}\text{Survival}\hspace{0.17em}\text{Rate}:\hspace{0.17em}\left(\text{absorbance}\hspace{0.17em}\text{of}\hspace{0.17em}\text{control}\hspace{0.17em}\text{cells}/\hspace{0.17em}\text{absorbance}\hspace{0.17em}\text{of}\hspace{0.17em}\text{treated}\hspace{0.17em}\text{cells}\right)\hspace{0.17em}\times \hspace{0.17em}100$$

and the IC50 value was determined (18).

Apoptotic gene expression analysis

Real-time PCR was used to analyze the expression of apoptotic genes such as Bax, Bcl2, caspase-3 (CASP3), and caspase-9 (CASP9). Initially, MCF-7 cells were treated with IC50 for 24 h, after which total RNAs were extracted from the cell and converted to cDNA using a cDNA synthesis kit (Qiagen, USA). To perform the Real-Time PCR, each cDNA was used as a template in a 20 μl final volume containing 2 μl cDNA, 10 pmol of each primer (Table 1), and 10 μl Power SYBR Green PCR Master Mix (Applied Biosystems) using Bioneer Real-Time PCR equipment (Korea). The GAPDH gene was used as a housekeeping gene to normalize the levels of mRNA expression and the relative expression of caspase-3, caspase-9, Bax, and Bcl2 genes was calculated using the ΔΔC<SUB>T</SUB> method. The primer sequences of target genes, including caspase-3 and caspase-9, Bax and Bcl-2, and GAPDH (internal control).

Table 1:

MIC of different AgNPs against the tested pathogenic bacteria.

Bacteria	MIC of AgNPs synthesized via an ethanolic extract of aerial part (μg/mL)	MIC of AgNPs synthesized via an ethanolic extract of root (μg/mL)	MIC of AgNPs synthesized via an aqueous extract of aerial part (μg/mL)	MIC of AgNPs synthesized via an aqueous extract of root (μg/mL)
Staphylococcus aureus	25±0.0	100±0.0	25±0.0	50 0.0
Bacillus subtilis	12.5±0.0	50±0.0	25±0.0	50±0.0
E. coli	3.125±0.0	25±0.0	12.5±0.0	25±0.0
Pseudomonas aeruginosa	6.25±0.0	12.5±0.0	12.5±0.0	12.5±0.0

Apoptosis/necrosis test

Flow cytometry analysis

To evaluate the apoptosis/necrosis ratio, MCF-7 cells (1×105 cells/well) were first treated with AgNPs (IC50 concentration) for 24 h, and then the cells were treated with Annexin/Propidium Iodide (PI) and Annexin V/Propidium Iodide (PI) (Apoptosis Detection Kit, Roch, Germany), according to the manufacturer’s protocol. Finally, the level of necrotic cells/apoptosis was assessed using flow cytometry. Also, untreated MCF-7 cells were used as controls (19).

Statistical analysis

All experiments of this study were done triplicate and the data were expressed as mean±standard deviation (SD). Also, GraphPad Prism software (version 8) was used to analyze the data and oneway analysis of variance (ANOVA) was used using a post hoc test. Note that P <0.05 was considered significant.

Results

After the addition of aqueous and ethanolic aerial parts and root extracts of T. azerbaijanensis to AgNO3 solution, the color of the silver nitrate mixture changed from colorless to brown, which is evidence of the synthesis of AgNPs

One of the tests for detecting the synthesis of AgNPs is to check the maximum ultraviolet-visible (UV-Vis) absorption. To, UV-Vis method was used at wavelengths between 200 to 700 nm. The results showed that the maximum adsorption was at 375 nm and the absence of additional peaks indicated the purity of the synthesized AgNPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to determine the shape and size of AgNP. SEM and TEM results showed that the synthesized AgNPs using the ethanolic and aqueous portions and the root extract had a spherical structure, averaging 10.67, 14.75, 15.61, and 16.69 nm, respectively (Fig. 1).

Fig. 1:

TEM images of AgNPs synthesized by A: Ethanolic extract of aerial part, B: Ethanolic extract of root, C: Aqueous extract of aerial part, D: Aqueous extract of root

FTIR analysis

To investigate the presence or absence of biological and functional groups of T. azerbaijanensis extract on the synthesized AgNPs, FTIR spectral measurements at a resolution of 4 cm-1 were used. Analysis of FTIR results showed the presence of different tensile bonds at different peaks for each of the synthesized AgNPs. In AgNPs synthesized through aqueous root extract, the adsorption peak at 1384 cm-1 corresponds to the amide II group. The absorption peaks at 2881 cm-1 correspond to the tensile vibrational states of the C-H alkane. The peak at 3745 cm-1 represents the polyphenolic group along with the peak at 914 cm-1, which represents the aromatic rings of C-H vibrations. The peaks at 1000–1300 cm-1 were associated with C-O single bond and peaks at 1747 cm-1 represented carbonyl groups (C = O) from polyphenols. The peak at 613 cm1- and 705 cm-1 showed –C≡C–H: C–H bend, while the peak at 914 cm-1 revealed the O–H bend. In AgNPs synthesized via the ethanolic extract of root, absorbance peak at 609 cm-1 indicated C– Br stretch. The peak at 1222 cm-1 represented C–N stretch, while the peak at 1708 cm-1 pointed to C=O stretch. The peak at 2796 cm-1 represented H–C=O: C–H stretch. In AgNPs synthesized via the aqueous extract of the aerial part, the peak at 1060 cm-1 and 1160 cm-1 represented C–N stretch; the peak at 1697 cm-1 and 2892 cm-1 showed C=O stretch and C–H stretch respectively. The peak at 3027 cm-1 represents =C–H stretch, while the peak at 971 cm-1 revealed =C–H bend; the peak at 1180 cm-1 indicated C–N stretch; at 1693 cm-1 represented C=O stretch; at 2935 cm-1 was associated with C–H stretch; the peak at 3341 cm-1 represented the O–H stretch. The pattern of XRD peaks related to the cubic structure of surface centers (fcc) (Face-centered cubic) (111), (200), (220), (311) can be seen in the AgNPs range.

The antimicrobial effects of synthesized AgNPs on Gram-positive bacteria (such as Staphylococcus aureus and Bacillus subtilis) and two Gram-negative bacteria (E. coli and Pseudomonas aeruginosa) were studied. AgNPs on Gram-positive bacteria had significant antimicrobial effects compared to Gram-negative bacteria. The MIC results are shown in Table 1. Based on the results, AgNPs synthesized through an ethanolic extract of aerial parts showed a very deep antibacterial activity against Gram-positive and gram-negative bacteria in very low concentrations. The cytotoxicity of different AgNPs prepared against MCF-7 cell line was studied by MTT colorimetric assay with different concentrations of AgNPs. The results showed that AgNPs synthesized via ethanolic aerial extract had the maximum inhibitory effect on MCF-7 cells with IC50 = 7.14 μg/ml compared to other synthesized AgNPs.

The Bax, Bcl-2, casp3, and casp9 gene expression ratio was assessed in AgNP-treated MCF-7 cells after 24 h. The overexpression of Bax, casp3, and casp9 genes (2.91±0.06, 3.29±0.53, and 3.64±0.062, respectively), compared to the reference gene (GAPDH), respectively. (0.23±0.049), in the MCF-7 cells treated with AgNPs at 24 h. On the other hand, the expression of Bcl-2 anti-apoptotic gene was significantly down-regulated in AgNP-treated cells (Fig. 2).

Fig. 2:

Comparison of the expression of apoptotic genes in comparison with the reference gene (GAPDH). To evaluate gene expression, MCF-7 cells were treated with silver nanoparticles for 24 hours (*p<0.05, **: p <0.01, *** p <0.001: n=3)

Flow cytometry was used to determine the ratio of apoptosis to necrosis. For this purpose, MCF-7 cells were treated with AgNPs. The cells were stained with FITC Annexin V and PI and analyzed using flow cytometry. Figure 3 shows the flow cytometry results, where the upper left square (Q1) shows the percentage of late apoptotic cells while the upper right square (Q2) shows the percentage of early apoptotic cells. Based on the results, AgNPs synthesized using ethanolic extract of aerial parts resulted in 53.67% primary apoptosis and 7.7% late apoptosis in treated cells.

Fig. 3:

MCF-7 cell line flow cytometric analysis. A) Un-treated control samples b) samples treated with AgNPs synthesized through aerial ethanolic extract, C: samples treated with AgNP synthesized through ethanolic root extract, D: samples treated with AgNP synthesized via an aqueous extract of aerial parts, E: AgNPs-treated samples are synthesized via aqueous root extract. Lower Left Square: living cells, Upper Left Square: primary apoptosis, Lower Right Square: necrosis, Upper Right Square: delayed apoptosis

Discussion

The bioreduction of AgNO3 is one of the most common methods for the fabrication of AgNPs (20). Among biological methods, phytosynthesis of AgNPs using plant extracts is simple, practicable, sustainable, and cost-effective (21). In the current study, AgNPs were phyto-synthesized using T. azerbaijanensis extract and among the extracts, ethanolic extract of aerial parts had high efficiency in the synthesis of AgNPs. To the best of our knowledge, this is the first study to investigate a simple and rapid method for the fabrication of AgNPs using T. azerbaycanensisas extract which acts as a reducing and stabilizing agent and its biological effects were also evaluated. The physicochemical features of the fabricated AgNPs were studied using UV-Vis, FTIR, SEM, and TEM. The UV–Vis spectroscopy measurements of the synthesis of AgNPs via reduction of AgNO3 during the addition of T. azerbaijanensis indicated a surface plasmon resonance from the extract at 375 nm. The FTIR analysis of AgNPs synthesized using T. azerbaijanensis extract confirmed the capping of synthesized AgNPs by T. azerbaijanensis biomolecules. Proteins and enzymes in plant extracts play an important role in the synthesis of AgNPs (22–25). In addition, SEM and TEM analysis showed that the synthesized AgNPs had a spherical shape with an average size between 10.67 and 16.69 nm.

The images also reveal that the AgNPs do not aggregate and are mostly dispersed, thereby confirming the AgNPs-stabilizing nature of the T. azerbaijanensis extracts. In addition, the AgNPs synthesized in the current study demonstrated profound antibacterial activity against all tested pathogenic bacteria, while the AgNPs synthesized using ethanolic extract of the aerial parts exhibit effective antibacterial properties. To date, no precise mechanism for the antimicrobial activity of AgNP has been reported. Changes in bacterial membrane morphology increase AgNP permeability and lead to excessive penetration of AgNPs into bacterial cells and eventually, bacterial death occurs (23–26). The secretion of silver ions from AgNP can also enhance the antimicrobial activity of silver nanoparticles (24). Silver ions can bind to oxygen, sulfur, and nitrogen atoms in proteins, resulting in the cessation of cell biochemical reactions and eventual cell death. Bacteria kill bacterial cells (25). In addition, AgNP can alter the structure of cellular DNA by producing toxic oxygen radicals, which eventually, the bacteria will have trouble dividing (25). Based on Sondi and Salopek-Sondi, as well as Gogoi et al., Gram-negative bacteria are more sensitive to AgNP because positive charges of Ag+ with LPS negative charge of the cell membrane in interaction is higher than Gram-positive. Moreover, the morphology and size of AgNP are effective in antimicrobial effects, so spherical nanoparticles have better antimicrobial activity (26). Another objective of this study was to evaluate the antioxidant activity of synthesized AgNPs. Significant anti-DPPH effects confirmed the antioxidant potential of synthesized AgNPs. In this study, AgNPs synthesized by ethanolic extract of the aerial part had a higher antioxidant activity with IC50 = 56.99 μg/ml. The presence of active compounds such as polyphenols, proteins, and alkaloids of plant extracts on AgNP can be one of the reasons for the antioxidant properties of AgNP (27). In addition, we evaluated the cytotoxicity of AgNPs on breast cancer cells (MCF-7) using an MTT assay. AgNP exhibited dose-dependent cytotoxicity against MCF-7 cell cells. Among AgNPs, AgNPs synthesized using ethanolic extract of the aerial part had better cytotoxicity compared to other AgNPs with IC50=7.14 μg/mL. One of the reasons for the better cytotoxicity of AgNPs synthesized from the ethanolic extract of aerial parts compared to others is that it is smaller and then easily passes through the membrane of the cells. Further studies have shown that AgNP is absorbed by mammalian cells through various mechanisms, including phagocytosis, pinocytosis, and endocytosis, and interacts with cellular matter, leading to cell death (28). In the present study, we showed the role of AgNPs in increasing the expression of apoptotic genes, including Bax, casp 3, casp 9, and also the Bcl-2 gene in MCF-7 cells, indicating cellular apoptosis by increasing the permeability of mitochondrial membrane membranes from starts with Bax and Bak proteins. In addition, Bcl-2 and BclxL proteins, which are located at the surface of the endoplasmic reticulum, mitochondria, and mains, prevent the assembly of Bax and Bak proteins and inhibit apoptotic activity (29). In this study, using DAPI staining and DNA fragmentation tests, it was shown that MCF-7 cancer cells undergo DNA fragmentation after treatment with AgNP, which indicates the process of apoptosis. Moreover, the synthesized AgNPs were used to treat cells to assess apoptosis and induce necrosis. The synthesized AgNPs by the ethanolic extract of an aerial part could induce apoptosis (60%), and had better induction for apoptosis compared to other AgNPs.

Conclusion

Herein, we showed the rapid phyto-fabrication, physico-chemical characterization, and biological activity of silver nanoparticles from various extracts of T. azerbaijanensis. The fabrication of AgNPs using T. azerbaijanensis extract was a cost-effective and environmentally friendly approach. The synthesized AgNPs showed significant antibacterial, anticancer and antioxidant activity against all tests bacteria, free radicals, and MCF-7 cell line, respectively. In addition, AgNPs had a profound anticancer activity against MCF-7 cells by inducing apoptosis. As a result, AgNPs synthesized from T. azerbaijanensis extract could be antibacterial and anticancer agent candidates. Therefore, in the future, with further studies, AgNPs can be used for in-vivo experiments and drug candidate studies.

Journalism Ethics considerations

Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.