Green biosynthesis, characterisation and antimicrobial activities of silver nanoparticles using fruit extract of Solanum viarum

Abstract

Green synthesis of nanoparticles is considered an efficient method when compared with chemical and physical methods because of its bulk production, eco‐friendliness and low cost norms. The present study reports, for the first time, green synthesis of silver nanoparticles (AgNPs) at room temperature using Solanum viarum fruit extract. The visual appearance of brownish colour with an absorption band at 450 nm, as detected by ultraviolet‐visible spectrophotometer analysis, confirmed the formation of AgNPs. X‐ray diffraction confirmed the AgNPs to be crystalline with a face‐centred lattice. The transmission electron microscopy‐energy dispersive X‐ray spectroscopy image showed the AgNPs are poly‐dispersed and are mostly spherical and oval in shape with particle size ranging from 2 to 40 nm. Furthermore, Fourier transform‐infrared spectra of the synthesised AgNPs confirmed the presence of phytoconstituents as a capping agent. The antimicrobial activity study showed that the AgNPs exhibited high microbial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus susp. aureus, Aspergillus niger, and Candida albicans. The highest antimicrobial activity of AgNPs synthesised by S. viarum fruit extract was observed in P. aeruginosa, S. aureus susp. aureus and C. albicans with zone of inhibition, 26.67 mm.

1 Introduction

Nanotechnology is one of the most promising research areas in the modern science and technology. It deals with the particles having a one‐dimensional size range of 1–100 nm. In the last few decades, research on inorganic nanoparticles has been developing rapidly due to their exceptional electronic, catalytic, optical, magnetic and other physical and chemical properties that are quite different from the bulk one [1] and these are directly related to particle size and shape. To utilise and optimise the chemical or physical properties of nano‐sized metal particles, a large spectrum of research has been focused to control the size and shape, which is crucial in tuning their physical, chemical and optical properties [2, 3]. Several techniques, including chemical and physical means have been developed to prepare metal nanoparticles (MNPs) such as chemical reduction [4, 5, 6], electrochemical reduction [7, 8], photochemical reduction [9, 10], heat evaporation [11, 12], microwave assisted [13, 14], photosynthesis [15] etc.

Although the chemical method of synthesis requires a short period of time for synthesising a large quantity of nanoparticles, this method requires capping agents for size stabilisation of nanoparticles. Chemicals which are being use in synthesis and stabilisation of nanoparticles are generally toxic and produce harmful by‐products. The need for environmental non‐toxic synthetic protocols for nanoparticles synthesis leads to the developing interest in biological approaches which are free from the use of toxic chemicals and generation of by‐products. Thus, there is an increasing demand for ‘green nanotechnology’. Many biological approaches for both extracellular and intracellular nanoparticles synthesis have been reported to date using microorganisms such as bacteria [16, 17], fungi [18, 19] and plants [20]. The literature review revealed that many biosynthesised MNPs using plants such as Aloe vera [21], Mentha pulegium [22] Rosa rugosa [23], Basella alba [24], Diospyros assimilis [25], Atrocarpus altilis [26], Acalypha indica [27], Canarium ovatum [28] etc. show high antimicrobial activities.

Silver nanoparticles (AgNPs) have tremendous applications in the fields of high sensitivity bimolecular detection, diagnostics [29], drug delivery [30], sanitisation [31], water treatment [32], antimicrobials, therapeutics, catalysis and micro‐electronics. A very efficient and green method for the synthesis of AgNPs is very essential. Silver is well known for possessing an inhibitory effect towards many bacterial strains and microorganisms commonly present in medical and industrial processes [33]. AgNPs play a very vast role in medicinal fields as anti‐infection agents with various applications as creams, tropical ointments acting actively in healing wounds, burns [34], medical devices and implants prepared with silver‐impregnated polymers [35]. In textile industry, silver‐embedded fabrics are now used in sporting equipment [36]. Here we have worked out on the use of Solanum viarum as a reducing and stabilising agent for the green synthesis of AgNPs at room temperature. The AgNPs obtained were fully characterised by ultraviolet (UV)‐visible spectroscopy, Fourier transform infrared (FT‐IR), transmission electron microscopy (TEM), energy dispersive X‐ray spectroscopy (EDS) and X‐ray diffraction (XRD). The as‐synthesised AgNPs were studied to observe the antimicrobial activity.

2 Experimental

2.1 Materials and method

The S. viarum leaves used in the experiment were fresh and collected from NIT Silchar campus, Assam. Silver nitrate was procured from Fischer Scientific (India). 25 g of leaves sample was taken, washed three times with distilled water, dried and cut into fine pieces. The fine cut leaf pieces were boiled in 500 ml flask along with 100 ml sterilised distilled water for 30 min. Then plant extract was filtered through Whatman filter paper no. 1. The plant extract thus collected was stored at 4°C and used further for synthesis of AgNPs.

2.2 Preparation of 1 mM silver nitrate solution

For the preparation of 1 mM silver nitrate (AgNO₃), 0.02 g of AgNO₃ was added to 100 ml of double distilled water. The solution was mixed thoroughly and stored in an amber coloured bottle in order to prevent auto oxidation of silver.

2.3 Synthesis of AgNPs using leaf extracts

5 ml of S. viarum leaf extract was added to 45 ml of 1 mM aqueous AgNO₃ solution in a 250 ml Erlenmeyer flask at room temperature. After 10 min the light yellowish reaction mixture changed to reddish brown which visually identify the synthesis of AgNPs.

2.4 Screening of antibacterial property of synthesised AgNPs

2.4.1 Test microorganisms

The present study of in vitro antimicrobial screening incorporates the following bacterial strains; Bacillus subtilis [microbial type culture collection (MTCC) 1427], Escherichia coli (MTCC 1195), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus susp. aureus (MTCC 1430) and Streptococcus pneumonia (MTCC 2672), Klebsiella pneumoniae [American Type Culture Collection (ATCC) 700603] and two fungal strains; Candida albicans (MTCC 4748) and Aspergillus niger (lab isolates). The MTCC and ATCC numbers were obtained from Institute of Microbial Technology (IMTECH), Chandigarh, Punjab and Haryana, whereas the lab isolated fungus was collected from Downtown Hospital, Guwahati, Assam, India. The stains were sub‐cultured, based on requirement, by Muller–Hinton broth and maintain at 4°C.

2.4.2 Antimicrobial assay

Antimicrobial activity was determined by applying the agar well diffusion method [37], with slight modifications. Freshly prepared molten Muller–Hinton agar (MHA) media for bacterial culture and potato dextrose agar (PDA) media for fungal culture were poured in the 9 cm petri plates to uniform depth of 5 mm and allowed to cool at room temperature. After solidification, wells were made in MHA media by using a 6 mm sterilised cork borer. 1–2 drops of media poured in the bottom with the help of sterile micropipette. 1–2 × 10⁷ cfu/ml of inoculums was spread by sterile swab on the solid agar plates. 50 µl of samples of three different concentrations were then filled in three wells. Four antibiotics for bacteria, i.e. ciprofloxacin, ceflexin, azithromycin and metronidazole; two antibiotics for fungi i.e. nystatin and ketoconazole were taken as positive control; whereas, dimethyl sulphoxide was used as negative control. The plates were then incubated for 24 h at 37°C for bacterial culture and 48 h at 25°C for fungal culture. At the end of incubation, zones of inhibition (ZOIs) formed around the well were measured by a transparent ruler in millimetre (mm).

2.4.3 Minimum inhibitory concentration (MIC)

MIC is defined as the lowest concentration of material that inhibits the growth of an organism [38] and the values were determined by broth dilution methods [39] with few modifications. Serial dilutions of each test sample performed in 96 well micro‐plates. 50 µl of each concentration was pipetted to individual wells after that 50 µl of standardised inoculums (1–2 × 10⁷ cfu/ml) were added. The plates were incubated at 37°C for 24 h for bacterial culture and 48 h at 25°C for fungal culture. After incubation, growth of inhibition was observed in the well plates where the minimum concentration that did not have any turbidity was taken as MIC.

2.4.4 Minimum bactericidal concentrations (MBC)

For MBC, 10 µl of broth medium were taken from each well of the MIC micro‐plate. These were spread on sterile MHA plates for incubation at 37°C. The incubation period was 24 h. After the completion of incubation period, the lowest concentration that has no observable bacterial growth on the agar plates was selected for MBC.

2.4.5 Minimum fungicidal concentrations (MFC)

For MFC, 10 µl of broth medium were taken from each well of the MIC micro‐plate. These were spread on sterile PDA plates for incubation at 25°C for 48 h. After the completion of the incubation period, the lowest concentration that has no observable fungal growth on the agar plates was selected for MFC [36, 37].

2.4.6 Statistical analysis

All experiments were carried out in triplicate (for MIC, MBC and MFC, duplicate), determinations ± standard error, constructed from independent measurements. Results were analysed by IBM SPSS statistics v21 software.

3 Results and discussion

3.1 Visual identification

The preliminary confirmation of the ability of the plant extract in nanoparticle synthesis is the identification of colour change. On incubation with AgNO₃ solution, a visual colour change from light yellowish to reddish brown was observed indicating the formation of AgNPs (Fig. 1). Initially, the reaction mixture showed no colour change and turned into yellowish brown colour after 15 min of incubation. The colour change in the solution was due to the reduction of Ag⁺ to Ag⁰ by the bioactive components present in the aqueous leaves extract of S. viarum.

Fig. 1.

Visual identification of AgNPs synthesised by S. viarum. Leaf extract as recorded at different functional times

(a) Initial, (b) 2 h, (c) 4 h, (d) 8 h. The formation of reddish brown colour revealed the formation of AgNPs in the reaction mixture

3.2 UV‐Visible spectra analysis

The formation of AgNPs in aqueous solution was confirmed by the positioning of the surface plasmon resonance (SPR) band in UV‐visible spectroscopic analysis. The MNPs have free electrons, which give the SPR absorption band, due to the combined vibration of electrons of MNPs in resonance with light wave. A single strong characteristic absorption peak at 450 nm was observed for synthesised AgNPs using S. viarum. The reduction of the metal ions occurred fairly rapidly; more than 90% reduction of Ag⁺ ions was complete within 4 h after addition of the metal ions to the plant extract. The metal particles were observed to be stable in solution even 4 weeks after their synthesis. By stability, we mean that there was no observable variation in the optical properties of the nanoparticle solutions with time (Fig. 2).

Fig. 2.

UV‐vis absorption spectra of biosynthesised AgNPs from S. viarum leaves extract at different time intervals

3.3 TEM and EDS analysis

The morphology and size of the resultant nanoparticles were elucidated with the help of TEM analysis. The TEM images at different magnifications and selected area electron diffraction (SAED) patterns are depicted in Fig. 3. The TEM image showed that the synthesised AgNPs are polydispersed and are mostly spherical and oval in shape with particle size ranging from 2 to 40 nm. The SAED pattern confirms the polycrystalline nature of nanoparticles. Four rings arise due to the reflection from (111), (200), (220), (311) lattice planes of fcc silver which was further confirmed by XRD analysis.

Fig. 3.

AgNPs synthesised from Solanum viarum leaves extracts'

(a),(b) TEM images at 20 nm and 10 nm scale, (c) HRTEM image at 2 nm scale and (d) SAED pattern

EDS spectra of AgNPs are shown in Fig. 4. It revealed a strong signal in the silver region and confirmed the formation of AgNPs. Typically, the optical absorption peak of the metallic AgNPs, which is obtained roughly at 3 keV, is because of the surface plasmon resonance. The EDS profile showed a strong signal for silver along with weak oxygen, carbon and some other peaks which may have originated from the biomolecules that are bound to the surface of AgNPs, indicating the reduction of silver ions to elemental silver. The other peak corresponding to Cu in the energy dispersive X‐ray is an artefact of the Cu‐grid on which the sample was coated.

Fig. 4.

EDS spectrum of AgNPs

3.4 XRD and FT‐IR spectral analysis

The size, structure and crystallinity of the synthesised AgNPs were determined by XRD analysis. The biosynthesised AgNPs from the S. viarum leaf extract was studied for checking the respective characteristic peaks in the XRD image (Fig. 5). The four distinct diffraction peaks of the 2θ values of 38.32°, 44.54°, 64.70° and 77.67° can be assigned to the planes of (1 1 1), (2 0 0), (2 2 0), and (3 1 1), respectively, which indicates that the AgNPs are fcc and crystalline in nature. The broadening of Bragg's peaks indicates the formation of nanoparticles. It might be thought that the few unassigned peaks were due to the presence of phytochemical compounds in the aqueous extract of the plant leaves.

Fig. 5.

XRD profile of AgNPs

FT‐IR measurements were carried out to identify the presence of active biomolecules in the synthesised AgNPs which act as an effective capping agent and efficient stabilisation was measured by FT‐IR analysis. In the case of S. viarum plant extract and its synthesised AgNPs (Fig. 6), the observed peaks at 3427 and 3441 cm⁻¹ correspond to –OH stretching of phenolic compounds. The peak at 2928 cm⁻¹ may be attributed to the C–H stretching vibrations in alkanes. 1631 cm⁻¹ arises from the C=O stretching mode in amine I group which is commonly found in the protein and the medium broad band at 1384 cm⁻¹ is the C–N stretching mode of the aromatic amine group. The C–O–C and C–OH vibrations of the protein in the leaf appeared as a very strong IR band at 1026 cm⁻¹.

Fig. 6.

Fourier transform‐infrared spectrum of

(a) S. viarum leaf extract and, (b) AgNPs

The FT‐IR spectra of aqueous leaf extract and synthesised AgNPs showed change in peak intensities and peak shift indicated the participation of carboxyl, hydroxyl, nitro, amino and amide groups in the formation of AgNPs and their stabilisation. The reduction and stabilisation of AgNPs may proceed through coordination between N of the amide group and silver ions. The FT‐IR studies confirmed that the carboxyl group of amino acids and the amide group of proteins have strong affinity to bind the metal indicating that proteins could possibly form a layer or coating on the surface of AgNPs to prevent agglomeration [40]

3.5 Screening of antibacterial property of synthesised AgNPs

3.5.1 Antimicrobial assay

To compare the activity of synthesised AgNPs, a broad‐spectrum standard antimicrobial susceptibility test was performed on six bacteria and two fungi. Furthermore, four antibiotics, ciprofloxacin, ceflexin, azithromycin and metronidazole, were used for the susceptibility test, likewise, two antibiotics, nystatin and ketoconazole, were used for fungi (Fig. 7). In Fig. 8, B. subtilis did not exhibit any activity except for ciprofloxacin, the ZOI measured was 27.67 ± 0.33 mm. 29.33 ± 1.45 mm ZOI was noted for E. coli at 0.1 M ciprofloxacin. Moreover, maximum ZOI (32.67 ± 1.33 mm) was noted for P. aeruginosa at 0.1 M ciprofloxacin. S. aureus susp. aureus did not show any activity against metronidazole; the highest ZOI observed against ciprofloxacin with 28.33 ± 0.66 mm. The lowest ZOI was witnessed against S. pneumonia at 0.1 M metronidazole with 12.66 ± 0.33 mm. The highest activity with ZOI of 15.33 ± 0.33 and 14.71 ± 0.33 mm was shown by A. niger and C. albicans, respectively, against nystatin at 0.1 M. Ketoconazole at 0.1 M exhibited ZOI of 11.58 ± 0.82 and 10.23 ± 0.82 mm for A. niger and C. albicans, respectively.

Fig. 7.

Antibiotic susceptibility test against two pathogenic fungi, A. niger and C. albicans

Fig. 8.

Antibiotic susceptibility test on five pathogenic bacteria: B. subtilis (Bs), E. coli (Ec), P. aeruginosa (Pa), S. aureus susp. aureus (Sa), S. pneumoniae (St pn), and K. pneumonia (Kl pn)

3.5.2 Susceptibility of the microbes against AgNPs

Out of eight microbes, the synthesised AgNPs showed high activity except for S. pneumonia and K. pneumonia. Interestingly, the highest activity was observed in P. aeruginosa, S. aureus susp. aureus and C. albicans at 1 mM with ZOI of 26.67 mm. Furthermore, at 0.25 mM no ZOI was observed in B. subtilis, E. coli, S. pneumonia and K. pneumonia. At 0.5 mM, the highest ZOI was noted in C. albicans of 16.33 ± 0.88 mm followed by 16.00 ± 0.58 mm in P. aeruginosa, 15.67 ± 0.33 mm in A. niger, 14.33 ± 0.33 mm in B. subtilis, 14.00 ± 1.00 mm in S. aureus susp. aureus and 13.00 ± 0.58 mm in E. coli as shown in Figs. 9 and 10. Additionally, the antimicrobial effect of AgNPs may be due to the microcidal destruction of cell membranes, blockage of enzyme pathways, alterations to cell walls, and nucleic materials pathway [41].

Fig. 9.

Effect of AgNP on growth of seven pathogenic microbes (Bs, B. subtilis; Ec, E. coli; Pa, P. aeruginosa; Sa, S. aureus susp. aureus; St pn, S. pneumonia; Kl pn, K. pneumonia: As, A. niger; Ca, C. albicans)

Fig. 10.

ZOI caused by S. viarum AgNPs against five pathogenic bacteria

(a) B. subtilis, (b) E. coli, (c) P. aeruginosa, (d) S. aureus susp. aureus, (e) S. pneumonia, (f) A. niger, (g) C. albicans, (h) K. pneumonia. Key: a, 0.25 mM; b, 0.50 mM; c, 1 mM

MIC, MBC and MFC of AgNP : in Table 1, the lowest MIC and MFC value of 0.10 ± 0.00 and 0.13 ± 0.01 mm, respectively, was observed for C. albicans. The highest MIC and MBC value of 0.41 ± 0.00 and 0.44 ± 0.01 mM, respectively, was noticed for the Gram‐negative bacteria. However, the highest MFC value of 0.13 ± 0.01 mM for A. niger was observed. Moreover, the MIC and MBC values were not witnessed at the highest concentration (i.e. 1 mM) for S. pneumoniae and K. pneumonia.

Table 1.

MIC, MBC and MFC of AgNPs

Microbes		MIC, mM	MBC/MFC, mM
bacteria	Bacillus subtilis	0.37 ± 0.01	0.41 ± 0.04
	Escherichia coli	0.41 ± 0.00	0.44 ± 0.01
	Pseudomonas aeruginosa	0.15 ± 0.02	0.16 ± 0.01
	Staphylococcus aureus susp. aureus	0.13 ± 0.01	0.14 ± 0.01
	Streptococcus pneumoniae	>1	>1
	Klebsiella pneumonia	>1	>1
Fungi	Aspergillus niger	0.12 ± 0.01	0.13 ± 0.01
	Candida albicans	0.10 ± 0.00	0.13 ± 0.01

The values are the mean of two independent replications ± standard error.

4 Conclusion

For the development of a clean and green future, nanotechnology is extensively being used. For the first time, an environmentally benign, novel and rapid method of synthesis of AgNPs using aqueous fruit extract of S. viarum was developed. The water soluble phytochemicals in the extract helped in reduction and stabilisation of the AgNPs. The AgNPs formed were spherical in shape, with a size ranging in between 2–40 nm. FT‐IR analysis confirmed the bioreduction of Ag⁺ ions to AgNPs by various functional groups. The characterisation studies of the synthesised AgNPs using XRD, scanning electron microscopy, TEM and EDS revealed valuable information about the synthesised AgNPs such as their size, shape and crystalline nature. The synthesised nanoparticles exhibit a pronounced antibacterial activity against tested microorganisms. Maximum ZOI was identified at 1 mM AgNPs when compared with 0.5 and 0.25 mM.