Abstract
Nanoparticles are important advanced materials with numerous uses in a variety of fields. Novel antibacterial nanocomposites with synergistic capabilities can be created by combining metal nanoparticles with biopolymers of various functionalities. This research evaluates an antimicrobial and antioxidant-rich chitosan-based silver nanocomposite synthesized by using citrus lemon extract as a reducing and capping agent. UV-vis spectrophotometer, scanning electron microscope, elemental dispersive analysis, X-ray diffraction assay, atomic force microscope, Fourier transform infrared spectroscopy, UV-near infrared spectroscopy, and transmission electron microscopy were used to characterize the chitosan-based silver nanocomposite (CS-Ag nanocomposite). The nanocomposite synthesized is used to demonstrate antioxidant and antimicrobial activity against fungal pathogens.
1. Introduction
Nanoparticles and nanocomposites are employed in a variety of applications across a wide range of industries. Silver nanoparticles have a wide range of applications as antimicrobial agents, including wastewater treatment, food preservation, clothe and textile preservation, detergents, and medical implants [1]. Plant extracts can be used to make silver nanoparticles that are both environmentally friendly and cost-effective [2]. Plant extracts such as Boerhavia diffusa leaves [3], Piper nigrum stem and leaf [4], Avicennia marina mangrove plant [5], Cissus quadrangularis [6], Parthenium hysterophorus [7], Manilkara zapota seeds [6], and Chrysanthemum flower extracts [8] and fruit extracts of pomegranate [9], Dillenia indica [10], Solanum xanthocarpum [11], and Crataegus douglasii [12] were employed, and it was used for antibacterial, antifungal, and antioxidant properties. Chitosan is a harmless, biodegradable polymer found in natural resources such as crab and prawn shells, as well as other marine and terrestrial invertebrates. Because of its excellent antibacterial and biocompatibility qualities, it has a wide range of biological uses [13]. Once silver nanoparticles are mixed with other polymers, they can generate nanocomposites with a variety of benefits. The antimicrobial activity of silver with agar against Listeria monocytogenes and Escherichia coli [14], the antimicrobial activity of polysulfone with silver to inhibit biofilm growth on downstream membrane surfaces [15], and the bacterial cellulose with silver magnetic nanocomposites are used for high antimicrobial activity against the model microbes Escherichia coli and Bacillus subtilis [16], and agar-based silver nanocomposite films are prepared using red alga Gracilaria dura to control Bacillus pumilis bacterial growth [17]. Chitosan-based silver nanocomposites, which include chitosan with silver nanoparticles, are being developed for a variety of commercial uses. The Bacillus subtilis-based silver nanocomposites were tested against disease-causing pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans [18], and the chitosan-based silver and fluoride nanocomposites were tested against disease-causing pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans using microdilution technique [19]. In this research, we employed citrus lemon juice to synthesize chitosan-based silver nanocomposites (CS-Ag nanocomposite). UV-vis spectrophotometer, scanning electron microscope, elemental dispersive analysis, X-ray diffraction assay, atomic force microscope, Fourier transform infrared spectroscopy, and UV-near infrared spectroscopy were used to characterize the nanocomposites. Finally, antioxidant and antibacterial activity against fungal infections is performed to the nanocomposites which have been synthesized.
2. Materials and Methods
2.1. Preparation of Fruit Extract
Lemon fruit was purchased from Vellore market and washed thoroughly under tap water. The lemons were cut into four pieces, and the fresh juice was extracted, filtered using Whatman No.1 filter paper.
2.2. Synthesis of CS-Ag Nanocomposite
To synthesize CS-Ag nanocomposite, 1 mM of silver nitrate was added to 95 ml of distilled water. 5 ml of filtered fresh lemon juice extract was added to 95 ml of silver nitrate solution. The reaction mixture was placed on a magnetic stirrer at 700 rpm for 3 h to detect color changes. Figure 1 depicts a visual observation of the synthesized silver nanoparticle solution. After 3 h, 0.25 g of chitosan was added to the solution and stirred for 3 h in a magnetic stirrer at 700 rpm. The CS-Ag nanocomposite solution was then centrifuged for 10 minutes at 8000 rpm. The pellet was collected after centrifugation, and the supernatant was discarded.
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2.3. Characterization of CS-Ag Nanocomposite
During the reduction of Ag+ to Ag NPs, UV-vis absorption analysis was carried. It was often scanned in the 300-500 nm wavelength range. XRD measurements at 40 kV and 30 mA, equipped with a Cu anode and a ceramic X-ray tube, were used to assess the crystalline nature and structure of AgNPs incorporated with chitosan. The functional groups present in the CS-Ag nanocomposite were analyzed using FT-IR in the range of 4000-500 cm-1. The microstructure of lemon extract mediated CS-Ag nanocomposite was analyzed using a scanning electron microscope (SEM) and atomic force microscope (AFM) to determine the size shape and morphology of the CS-Ag nanocomposite. Energy-dispersive X-ray analysis spectrum was also recorded.
2.4. Antibacterial Activity
The agar well diffusion method was used to test antibacterial activity. The Mueller-Hinton agar was sterilized and prepared. The Muller-Hinton agar was poured and solidified in aseptic condition on clean and sterilized Petri plates. Swabs of pathogenic organisms such as Streptococcus sp, Rhizobium radiobacter, Klebsiella pneumoniae, and Escherichia coli were cultured in liquid broth. After 5 minutes, wells were punched in the agar plate with a 5 mm gel puncher. The results were seen after different concentrations of nanoparticles of 25 μl, 50 μl, and 75 μl were loaded into each well and incubated for 24 hours. The antibacterial activity was determined by measuring the diameter of the inhibitory zone generated around the well and recording the mean results.
2.5. Antifungal Activity
The agar well diffusion method was used to test antifungal activity. Rose Bengal Agar was sterilized and prepared. The Rose Bengal Agar was poured and solidified in sterile conditions on clean and sterilized plates. Cultures of pathogenic organisms like Aspergillus niger, Fusarium oxysporum, and Aspergillus fumigatus were inoculated and swabbed. After 5 minutes, wells were punched in the agar plate with a 5 mm gel puncher. Different concentrations of nanoparticles of 25 μl, 50 μl, and 75 μl were loaded into each well and incubated for 24 hours before the data were analyzed. The antifungal activity was determined by measuring the diameter of the inhibitory zone that formed around the well and recording the mean results.
2.6. Antioxidant Activity of Nanocomposites
Based on previous findings [8], the free radical scavenging activity of the lemon juice mediated CS-Ag nanocomposite was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. 0.78 mg of DPPH was added to 10 ml of methanol to make DPPH. Five different concentrations of extracts (20 μl, 40 μl, 60 μl, 80 μl, and 100 μl) were added to 2 ml of this DPPH solution. The antioxidant ascorbic acid was utilised as a standard. The samples were incubated for 30 min in dark. The absorbance was measured using a spectrophotometer at 517 nm. The blank was prepared without the addition of extract. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the following equation:
where control is the absorbance of the control reaction and test is the absorbance in the presence of the sample of the extracts.
3. Result and Discussion
3.1. Color Change and Visual Observation
Figure 1(a) shows the silver nitrate solution, which changed color from pale yellow to brown after adding lemon juice, indicating the synthesis of silver nanoparticles. Fruit extracts are a great source for making silver nanoparticles because they quickly change the color of silver nitrate solution into silver nanoparticles [9]. AgNPs were expected to form in the CS-Ag nanocomposite as a result of AgNO3 reduction. Depending on the concentration of AgNPs, the chitosan films loaded with AgNPs turned from pale brown to dark brown. Figure 1(c) depicts the color change of silver nanoparticles in chitosan solution to turbid solution.
3.2. UV-Visible Spectroscopy
The UV-vis absorption spectra of Ag nanoparticles and AgNP-based chitosan nanocomposites are shown in Figure 2. Figure 2 displays a peak at 420 nm, due to the plasma vibrations of silver nanoparticles and another peak at 470 nm showing some possible interaction between chitosan and silver nanoparticles. The absorption peaks of the UV-vis spectra shifted to a longer wavelength when the particle diameters were increased [20]. Based on the size of the AgNPs, the SPR band at 420-460 nm represents silver nanoparticles, and the peak increases when chitosan is added to the solution [21, 22]. The nanocomposite was again analyzed for its surface plasmon resonance at 48 h and 7th day. The peaks are near to the value of after the addition of chitosan values confirms the stability of nanocomposites.
3.3. X-Ray Diffraction Analysis
Figure 3 shows the XRD patterns of the CS-Ag nanocomposite spheres. The (111), (121), and (200), crystallographic planes of the face-centered cubic silver crystals might be attributed to three distinctive peaks of silver nanoparticles corresponding to , (32.18°), and (46.16°). The position and relative intensity of all the diffraction peaks of the samples were compatible with the crystalline pattern of silver, indicating that the produced nanoparticles were silver nanoparticles. Furthermore, the chitosan utilised in the synthesis procedure did not induce silver oxides to develop. Since the chitosan polymer and silver nanoparticles were present, the shape of each peak was broadened.
3.4. FTIR Spectroscopy
FTIR spectra of CS-Ag nanocomposites are shown in Figure 4. The band between 3273.20 cm-1 was related to the stretching vibration of =C-H from the alkyne group. The band between 1728.22 cm-1 was related to stretching vibration of C=O from ester groups. The band between 1543.05 cm-1 was related to the bending vibration of N-H from the amine secondary group. The band between 1367.53 cm-1 was related to bending vibration of N=O from the nitro group. The band between 1215.15 cm-1 and 1020.34 cm-1 was related to the stretching vibration of C-O from the ester group. The silver nanoparticles were bonded by protein, which served as a stabilizing agent, either through the free amine group or cysteine residues. These proteins could reduce AgNO3 ions to form silver nanoparticles [4, 23].
3.5. Scanning Electron Microscopy
SEM analysis was used to determine the size and shape of nanocomposites. Figure 5(a) clearly shows the SEM micrograph of the silver nanocomposite. The composite particle has an average diameter of about 200 nm and a uniform particle size. The morphology of the chitosan spheres is shown in Figure 5(b), which have relatively smooth structures when compared to the CS-Ag nanocomposite spheres. The background of chitosan and silver nanoparticle binding is clearly shown in Figures 5(b) and 5(c). Youssef et al. previously reported to synthesize chitosan-based silver and gold nanocomposites [18] in which SEM images clearly show the binding of chitosan and silver nanoparticles.
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3.6. EDX Spectrum Analysis
The EDX analysis was used to determine the elements in chitosan-based silver nanocomposites, and the results are shown in Figure 6. The EDX analysis confirmed that the nanocomposites contained approximately 15% silver and chitosan, 35% carbon, 44% oxygen, and 4% nitrogen. The metallic element, Ag, was present in all of the chitosan-based silver nanocomposite films, and the amount of elemental silver in the CS-Ag nanocomposite films increased as the content of AgNP increased.
3.7. TEM Analysis
The morphology of nanoparticles and nanocomposites prepared using green synthesis methods is greatly aided by TEM analysis [24, 25]. The various shapes and sizes produced by silver nanoparticles with chitosan are clarified by TEM analysis. Silver nanoparticles with sizes ranging from 15 to 25 nm have been discovered, and the size of the nanocomposite is 150 to 200 nm. The chitosan nanocomposites were clearly observed to be embedded with silver nanoparticles, confirming the nanocomposite formation. Figures 7(a) and 7(b) show different spherical-shaped nanoparticles and the background light color, which was discovered to be chitosan.
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3.8. AFM Analysis
AFM was used to perform topographical imaging on the surface roughness of the film sample. In Figure 8, an AFM image of silver nanoparticles with chitosan-formed nanocomposites clearly matches the SEM and TEM images.
3.9. Antibacterial Activity
Figure 9 depicts the antibacterial activity of CS-Ag nanocomposite against bacterial pathogens. At a concentration of 75 μl, the maximum zone of inhibition was obtained in the gram-positive bacteria Streptococcus, with a zone diameter of . At a concentration of 25 μl, the gram-negative bacteria Klebsiella pneumoniae had the smallest zone of inhibition, with a zone diameter of . The difference in inhibition is due to the arrangement of cell walls in gram-positive and gram-negative organisms. Streptococcus pneumoniae, which causes pneumonia, wound infection, and skin infection, can be killed by CS-Ag nanocomposites. Silver nanoparticles are an excellent antibacterial agent, and when combined with chitosan, they may be even more effective against other bacterial pathogens [21, 22, 26].
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3.10. Antifungal Activity
Figure 10 depicts the antifungal activity of chitosan/silver nanocomposites against fungus. It had the highest fungal activity against Aspergillus niger and the smallest inhibition zone against Aspergillus fumigatus and Fusarium oxysporum. The fungus’ cell wall is very strong, and it may prevent fungal growth from being killed. Silver nanoparticles have been shown to suppress fungal infections on their own [27].
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3.11. Antioxidant Activity by DPPH Assay
The silver nanoparticles synthesized using plant extracts are having very good antioxidant property [8, 28]. DPPH has been used extensively as a stable free to evaluate reducing substances, and it is a useful reagent for investigating free radical scavenging activity of the component. The free radical scavenging activity by DPPH in graphical representation is shown in Figure 11. It demonstrates the highest antioxidant activity at higher concentration such as 100 μl which implies a dose-dependent manner (Bedlovicova et al.,2020).
4. Conclusion
Lemon juice extract was shown to form a silver nanoparticle-based chitosan nanocomposite. Characterization of the material using XRD, UV-vis spectroscopy, and FT-IR techniques confirmed the production of the nanocomposite. AFM, SEM, and TEM were used to analyze the morphology of nanocomposite. The nanocomposites were found to be spherical using SEM. The synthesis of the nanocomposite was confirmed by the formation of chitosan in the presence of silver nanoparticles in TEM. Agar well diffusion was used to test the antibacterial properties of nanocomposites against Streptococcus, Rhizobium radiobacter, Klebsiella pneumoniae, and Escherichia coli. The findings demonstrated that silver nanoparticles with chitosan layer exhibited outstanding antibacterial and antioxidant properties, suggesting that they could be used as advanced biomedicine in the future.
Data Availability
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that there is no conflict of interest.
Authors’ Contributions
SR designed, carried out research, and wrote the manuscript.
Acknowledgments
The study is funded by DST-SERB (file no. YSS/2014/000931), New Delhi, India.