Biosynthesis of silver nanoparticles using Azadirachta indica leaves: characterisation and impact on Staphylococcus aureus growth and glutathione‐S‐transferase activity

Abstract

Silver nanoparticles (AgNPs) are toxic to various microbes, but the mechanism of action is not fully understood. The present report explores Azadirachta indica leaf extract as a reducing agent for the rapid biosynthesis of AgNPs. The effects of AgNPs on the growth, glutathione‐S‐transferase (GST) activity, and total protein concentration in Staphylococcus aureus were investigated, as was its antibacterial activity against seven other bacterial strains. Nanoparticle synthesis was confirmed by the UV‐Vis spectrum and colour change of the solution. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential analysis, and infrared spectroscopy were used to characterise the synthesised nanoparticles. The UV‐Visible spectrograph showed an absorbance peak at 420 nm. DLS analysis showed an average AgNP size of 159 nm and a Polydispersity Index of 0.373. SEM analysis showed spherical particle shapes, while TEM established an average AgNP size of 7.5 nm. The element analysis profile showed small peaks for calcium, potassium, zinc, chlorine, with the presence of oxygen and silver. AgNPs markedly affected the growth curves and GST activity in treated bacteria, and produced moderate antibacterial activity. Thus AgNPs synthesised from A. indica leaves can interrupt the growth curve and total protein concentration in bacterial cells.

1 Introduction

Over the last few years, nanotechnology is gaining increased interest because of its application in many fields, including medicine, electronics, and even space industries [1]. Nanoparticles can be synthesised by various chemical or physical methods using certain noble metals, such as Ag, Au, and Pt with a multitude of biological applications [2, 3, 4], Silver nanoparticles (AgNPs), in particular, have attracted substantial attention as they remain stable for a long time because it develops the least molecular aggregations [5, 6]. The particle size of AgNPs is usually not >100 nm, hence they demonstrate remarkable antibacterial and anticancer activity. Nanoparticles also possess the unique property of a high surface to volume ratio, which increases their utility in the catalytic industry where a high surface area is needed, and thereby makes AgNPs good catalysts [7]. In addition to this, AgNPs are also suitable for other applications, such as drug delivery, water distillation, and in healing processes [8, 9]. One of the mechanisms behind the antibacterial activity of AgNPs may be its ability to inhibit microbial growth and disturb cellular enzyme and protein levels [10, 11]. Normally bacterial growth starts with the lag phase before replication, then exponential growth to reach maximum population density, and finally death phase [12]. The disruption pattern of bacterial growth describes the toxic levels of a particular material [11].

Even though there are many routes for nanoparticle synthesis, green synthesis has recently gained increased importance as it does not need any culture preparation, is eco‐friendly, and inexpensive. Leaf extract can act as a reducing and capping agent during nanoparticle synthesis by rapidly reducing silver ions into AgNPs without any extra effort [13, 14].

AgNPs coated with plant material are not as harmful as AgNO₃ [15]. Several leaves are currently being used for synthesising nanoparticles [16, 17]. However, nanoparticles synthesised from the same plants, but from different regions, usually show different characteristics [18, 19] since the same plants grown under different environmental conditions show significant differences in their primary and secondary metabolites [20]. The change in composition thus acts as a chemical interface between the plant and its environment, which is mediated mainly by the biosynthesis of some secondary metabolites [21]. In the present study, leaf extract of Azadirachta indica from the Riyadh region of Saudi Arabia was used to generate AgNPs. Numerous studies on the same plant have previously been conducted; however, this study reports for the first time on A. indica grown in Saudi Arabia. A. indica, commonly known as Neem tree, is native to Pakistan, India, Bangladesh, and Myanmar, and is now widely grown in almost all African countries. The Neem tree was introduced to the plains of Arafat, Saudi Arabia almost 100 years ago; however, a few mature trees are also found in the regions of Medinah, Taif, and Riyadh [22].

A. indica is not only used in Ayurveda and homeopathic medicine but has also gained a foothold in modern medicine [23]. Hundreds of compounds, which are used to treat human diseases, including inflammation, infections, skin diseases, and dental disorders, have been isolated from different parts of this tree; especially from the leaves [24]. A. indica leaves are rich in quercetin, which can act as a capping and reducing agent, and play a vital role in stabilising nanoparticles [25]. This paper presents the characteristics and antibacterial activity of biologically prepared AgNPs from an aqueous extract of A. indica leaves. The effects of AgNPs on the growth pattern and level of glutathione‐S‐transferase (GST) in Staphylococcus aureus were also investigated. S. aureus was selected as the study bacterium since it is one of the main pathogens associated with skin infections, soft tissue, wound infections etc.

2 Materials and methods

2.1 Leave extract

Fresh green leaves of A. indica were selected and washed with distilled water. Twenty grams of healthy green leaves were finely chopped and boiled with 0.2 L of double‐distilled water for half an hour. After cooling to room temperature, the broth was filtered and stored at 4°C.

2.2 Biosynthesis of AgNPs

AgNPs were prepared by mixing leaf extract with 5 mM AgNO₃ to catalyse the reduction of Ag⁺ ions. The reduction process was indicated by change in colour from yellowish‐green to brownish‐black over time (Fig. 1 (I)). This was recorded by scanning through a UV‐visible spectrophotometer at different time intervals for a total of 3 h (Fig. 1 (II)).

Fig. 1.

I‐colour change; II‐UV–Vis spectra showing absorbance at different time intervals with AgNPs synthesised from A.indica leaves extract

2.3 Characterisation of biosynthesised AgNPs

The techniques described below were used to characterise the synthesised AgNPs:

2.3.1 UV‐Vis spectra analysis

The optical properties and colour change of the synthesised nanoparticles were analysed by Biochrom UV‐Vis spectroscopy (LibraS22, England). Following addition of AgNO₃ to the aqueous extract of A. indica, 1 mL of the mixture was collected at different time intervals to monitor the bio‐reduction of Ag⁺. Absorbance from 200 to 700 nm at 10 min, 30 min, 60 min, 120 min, and 3 h were analysed to determine the absorbance peak.

2.3.2 Average size of biosynthesised AgNPs

• Zeta potential analysis: Zetasizer Nano Series ZS (ZEN3600) was used to measure the hydrodynamic size and zeta potential of AgNPs. The scattering angle was fixed at 173 degrees and the red laser set at a wavelength of 633 nm for all measurements.

• Scanning electron microscope (SEM) and transmission electron microscopy (TEM): SEM analysis was by Oxford Instruments and transmission electron microscope analysis by JEOL JEM‐1400 Japan. These measurements were used to determine the average size of the synthesised particles.

• Infrared (IR) spectroscopy: A Thermo Scientific Nicolet 6700 FT‐IR spectrometer was used for exploring the IR spectroscopy of synthesised AgNPs. IR spectroscopy was used to determine the nature of bio‐reducing functional groups.

2.4 Effect of synthesised AgNPs on bacterial growth kinetics

S. aureus cells were cultured at 37°C in Luria Bertani (LB) media overnight. Freshly grown cells (1.5% inoculum) were transferred to 30 mL LB, along with various concentrations AgNPs (0, 2.5, 5.0, and 10 µg/mL) and incubated at 37°C with continuous agitation. Growth trends were monitored at 30 min intervals by measuring OD at 600 nm on a Biochrom UV‐Vis Spectrophotometer (LibraS22, England).

2.5 Effect of synthesised AgNPs on GST and total protein in exposed bacteria

S. aureus cells were treated with 2.5 µg/mL synthesised AgNPs for 24 and 48 h, and then immediately harvested and washed. Cells were lysed by sonication in an ice‐water bath for 3 s, followed by cooling for 3 s. Cell debris was removed by centrifugation at 15,000 g for 10 min. The supernatant was used for protein concentration determination [26] and the GST activity assay [27].

2.6 Antimicrobial activity

The antimicrobial activity of synthesised AgNPs was determined by disc diffusion method against eight different bacterial strains, namely Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella typhi, S. aureus, Staphylococcus epidermis, and Streptococcus pneumonia.

3 Results and discussion

UV‐Vis spectroscopy is one of the best and fastest techniques used for exploring AgNPs [28]. Reduction of silver ions by the plant extract was monitored by colour change, from yellowish‐green to brownish‐black, as shown in Fig. 1 (I). The UV–Vis absorbance spectrum showed a sharp peak at 420 nm (Fig. 1 (II)), indicating AgNPs were able to shift the maximum peak of absorption to the visible light region [29]. The change in colour, after adding the AgNO₃ solution, was due to the surface plasmon resonance phenomenon. This occurs due to the combined vibration of free electrons in the metal nanoparticles resonating with the light waves [30]. Furthermore, the maximum spectrum band observed at 420 nm was similar to AgNPs synthesised from the same plant in India, which showed an absorption band in the 420 to 450 nm range [19].

Interactions between particles and biomolecules are linked to their zeta potential, which plays an important role in aggregation and colloidal stability. Suspended particles with large negative or positive zeta potentials, small size, and low‐density repel each other, and thereby create a relatively stable system that resists aggregation [31]. Dynamic light scattering (DLS) was used to determine particle size distribution of AgNPs synthesised with A. indica (Fig. 2). The Z‐average mean of the synthesised AgNPs was 159 nm, and the Polydispersity Index was 0.373. Overall, the size of the nanoparticle was acceptable, since Polydispersity Index values >0.7 indicate a broad size distribution [19]. Nanoparticles are energetically very unstable because of their small size, thus they undergo agglomeration/aggregation and acquire charge potentials on their surface to gain stability. The charge, or Zeta, potential of the AgNPs synthesised in the current study was 0.249 mV (Fig. 3).

Fig. 2.

DLS result for the AgNPs synthesised from A.indica leaves extract

Fig. 3.

Zeta Analysis result for the AgNPs synthesised from A.indica leaves extract

The IR spectrum of green nanoparticles was examined to identify the biomolecules responsible for the capping and efficient stabilisation of the synthesised metal nanoparticles (Fig. 4). The IR spectrum of the AgNPs showed a band at 3281.32 cm⁻¹, which corresponds to stretching of the hydroxyl groups as a functional group of carbohydrates, phenols, flavonoids, and alcohols; the peak at 1635.51 cm⁻¹ shows the stretching of the C = O (carboxylic or amide), C = C, and C − H (Alkenyl) groups. The IR spectrum for the same plant extract from India showed bands at 3454, 2083, 1636, and 1113 cm⁻¹ [32], which differed slightly from our observations, suggesting climate can change the plant's phytochemical composition [21, 33]. From an analysis of the IR studies, we confirmed that the hydroxyl, amino, carbonyl, and amide moieties present within A. indica have a strong ability to bind metal and thereby provide capping and prevent agglomeration of the AgNPs. Leaf extract of A. indica thus performs the dual role of reducing and capping agent.

Fig. 4.

IR spectra of (I) aqueous extract; (II) synthesised AgNPs from A.indica leaf

TEM images of AgNPs synthesised from aqueous A. indica leaf extract were spherical in shape, as seen in Fig. 5 (I). TEM is widely used for particle sizing as it shows the 2D structure of the sample. Ultrathin sample imaging is possible by absorption of a beam of electrons through the sample. The average size of AgNPs synthesised in this study were in the range of 5–10 nm (Fig. 5 (I)). SEM is typically used for determining the surface morphology and 3D structure of a material. As per the SEM micrograph (Fig. 5 (II)), synthesised AgNPs were spherical and aggregated. However, the markedly smaller size recorded by TEM confirms the cluster of AgNPs shown by SEM micrograph [34].

Fig. 5.

I‐ TEM micrograph; II‐ SEM micrograph of the synthesised AgNPs from A. indica leaf

Energy dispersive X‐ray spectroscopy was used to validate the presence of specific elements present in the sample. Our results show small peaks for calcium, potassium, zinc, and chlorine with the specific element of oxygen (Fig. 6). The strong signal in the silver region confirms the presence of AgNPs. The presence of these elements in the plant extract may thus act as a capping substance around the synthesised nanoparticles. Table 1 shows the quantitative analysis of synthesised AgNPs for A. indica.

Fig. 6.

EDX elemental analysis of synthesised AgNPs from A.indica leaf

Table 1.

Quantitative analysis of synthesised AgNPs for A. indica

Element	Weight%
oxygen	52.01
chlorine	7.08
potassium	7.04
calcium,	7.51
zinc,	1.63
silver	24.73

AgNPs are currently widely used in disinfectants, detergents, and soaps as an antimicrobial agent. These particles are able to attack the bacteria through the surface of the cell membrane to impede its respiratory function, resulting in bacterial cell instability [35]. Furthermore, the negative charge on the cell surface of some bacteria is easily extricated by Ag⁺ ions, resulting in cell death [36]. In the present study, we exposed S. aureus to the synthesised AgNPs for two days and monitored its growth curve and enzyme activity, as shown in Figs. 7 and 8, respectively. As shown in Fig. 7, AgNPs inhibited the growth of S. aureus. The observed growth trends of S. aureus exposed to AgNPs indicate reactive oxygen species (ROS) generation, which can damage the cells and decrease bacterial growth [37]. Exposure to AgNPs for one day decreased total protein concentration, but markedly increased the GST activity. Results recorded on the second day showed the same trend in total protein concentration, but with a concomitant decrease in GST activity, as compared to the control (Fig. 8). One of the major modes of detoxification in bacterial cells is through the consumption of GSH via a GST‐catalysed reaction. In our study, AgNPs are likely to induce GST activity as a potent protection mechanism. The decrease in total protein concentration demonstrated the presence of AgNP‐induced oxidative stress, since cellular protein concentrations are usually imbalanced under such conditions due to proteolysis [38]. Eight bacterial strains were tested against the AgNPs prepared from aqueous A. indica leave extract, as shown in Fig. 9 and Table 2. The most sensitive gram‐positive strains were found to be S. aureus and B. subtilis, with inhibition zones of 13 and 12 mm, respectively. Among the gram‐negative bacteria, E. coli and P. aeruginosa were found to be the most sensitive strains with inhibition zones of 12 mm each. Overall, gram‐positive strains were found to be more sensitive to AgNPs compared to gram‐negative strains. This may be due to the presence of different membrane structures [39].

Fig. 7.

Growth curves of an S.aureus grown in presence of AgNPs synthesised from A.indica leaves extract

Fig. 8.

Total protein and GST activity of AgNPs treated cells of S.aureus as compared to control

Fig. 9.

Antibacterial activity of AgNPs synthesised from A.indica leaves extract by disc diffusion assay

Table 2.

Zone of Inhibition (mm) of AgNPs synthesised from A. indica aqueous extract against different gram +ive and gram −ive bacterial strains

	Bacteria	Inhibition zone, mm
gram +ive	S.aureus.	13 ± 0.30
	B. subtilis	12 ± 0.20
	S.epidermidis	11 ± 0.30
	S.pneumonia	11.5 ± 0.20
gram −ive	E.coli.	12 ± 0.30
	S. typhi	11.5 ± 0.30
	P. aeruginosa	12 ± 0.10
	K.pneumoniae	10.5. ± 0.20

4 Conclusion

To the best of our knowledge, we herein report, for the first time, the biosynthesis and characterisation of AgNPs using A. indica leaves native to the Riyadh region of Saudi Arabia. AgNPs formations were confirmed by SEM, TEM, and FTIR. This process of synthesis did not require any surfactants, capping or stabilising agent, indicating that all these factors were provided by the leaf extract. Increased GST activity and decreased total protein concentration in AgNP‐treated cells indicated the presence of cellular oxidative stress.