Antibacterial and dye degradation potential of zero‐valent silver nanoparticles synthesised using the leaf extract of Spondias dulcis

Abstract

The present study reports a simple and low cost synthesis of zero‐valent silver nanoparticles (ZVSNPs) from silver nitrate using the leaf extract of Spondias dulcis. The ZVSNPs showed a unique peak at 420 nm in UV–vis spectrum. The SEM image portrayed cuboidal shaped particles. The EDX spectrum designated the elemental silver peak at 3 keV. In XRD, a sharp peak at 32.47° denoted the existence of (1 0 1) lattice plane and the average crystallite size was calculated as 48.61 nm. The lattice parameter was determined as 0.39 nm. The FTIR spectra of the leaf extract and ZVSNPs showed shifts in the specific functional group bands which ascertained the involvement of phytoconstituents in the formation and capping of nanoparticles. The average hydrodynamic size was measured as 59.66 nm by DLS method. A low PDI, 0.187 witnessed the monodispersity. A negative zeta potential value of −15.7 mV indicated the negative surface charges of the nanoparticles. The bactericidal action of ZVSNPs was demonstrated against two pathogens S.typhimurium and E.coli during which a dosage dependent zone of inhibition results was observed. Additionally, the catalytic potential of ZVSNPs was examined for the degradation of methylene blue dye in which an accelerated degradation of the dye was observed.

1 Introduction

A myriad of applications of zero‐valent silver nanoparticles (ZVSNPs) in various fields of science and technology have made many scientists to indulge in the research of this surging area. A few of the appealing applications include antibacterial [1], antioxidant [2, 3], anticancer [4, 5], anticoagulant [6], larvicidal [7], antileishmanial [8, 9, 10], bioimaging [11], biosensor [12, 13] and pollutant removal [14, 15].

Therefore, it is essential to develop an eco‐friendly, low‐cost and rapid method of synthesis of nanoparticles. In the recent past, the plant‐mediated synthesis (PMS) of nanoparticles is gaining attention because of the above‐mentioned features. In fact, the annual publication of articles about the PMS of nanoparticles increased exponentially since 2000 [16]. Recently, our research group also have utilised the leaf extract of a variety of plants [17, 18, 19, 20] and the pod extract of Peltophorum pterocarpum [21] for the synthesis of nanoparticles.

In contrast to the conventional synthesis of nanoparticles, the PMS method does not necessitate the harmful chemicals, which may lead to environmental pollution and severe reaction conditions. These points are of the utmost priorities for a green synthesis method. Furthermore, another green synthesis method, known as ‘microbe‐mediated synthesis method’ has the disadvantages such as time consuming, costly media and maintenance of aspectic conditions during the growth. Consequently, the PMS method is the recommended method for the synthesis of nanoparticles.

As far as we know, there is no published article on the synthesis of ZVSNPs using the aqueous extract of Spondias dulcis leaves. S. dulcis is a tropical tree, which is commonly found in the southern states of India. It is commonly known as ambarella. The leaves and fruits of this plant are highly rich in antioxidants such as flavonoids and polyphenols. The antioxidant, antimicrobial, cytotoxic and thrombolytic activity of this plant has already been reported [22]. We speculate that the polyphenolic compounds inherent in the leaves of this plant could play a role in the formation and stabilisation of ZVSNPs.

Hence, for the first time, we present a simple and rapid process for the production of ZVSNPs from the silver nitrate using the leaf extract of S. dulcis without the addition of any separate reducing or capping agent. The synthesised ZVSNPs were characterised by various techniques. The bactericidal efficacy of the ZVSNPs was checked against common human pathogens. In addition, the catalytic ability of the ZVSNPs to degrade a pollutant dye – methylene blue (MB) was also demonstrated.

2 Materials and methods

2.1 Preparation of Spondias dulcis leaf extract (SDLE)

Fallen leaves of Spondias dulcis (Fig. 1) were collected from the campus of MIT, Manipal. The leaves were rinsed thoroughly with distilled water to remove all the dust. 10 g of dried leaves were heated with 100 ml of distilled water for 20 min at 90°C in a water bath. The resultant mixture was cooled to ambient conditions and filtered to remove the particulate matter. The filtrate obtained was named as SDLE and was stored at 4°C for further use.

Fig. 1.

Leaves of S. dulcis tree used in this study

2.2 Synthesis of ZVSNPs

10 ml of SDLE was mixed with 90 ml of 1 mol/m³ AgNO₃ solution in a glass vessel. The pH of the reaction mixture was adjusted to 5 and the beaker was incubated at 90°C for 20 min in a water bath. The change in the colour to reddish brown indicated the formation of ZVSNPs.

2.3 Characterisation of ZVSNPs

The formation of ZVSNPs was confirmed by taking the absorption spectra of the colloidal solution at regular time intervals using an ultraviolet–visible (UV–Vis) spectrophotometer (Shimadzu). The colloidal solution was centrifuged at 10,000 rpm for 10 min and the denser fluid settled at the bottom of the centrifuge tube was collected. One drop of this solution was put on a glass slide of the dimension of 1.2 cm × 1.2 cm. This slide was then dried in a hot air oven at 80°C for 15 min. The cured sample was used for scanning electron microscopy (SEM)–energy dispersive X‐ray (EDX; EVO MA18) to examine the surface morphology and the elemental composition of the nanoparticles. The same sample was used to determine the crystalline nature by using an X‐ray diffractometer (Rigaku Miniflex 600). Dynamic light scattering (DLS) analysis was performed with a Malvern Zetasizer nanosizer to measure the particle size distribution and zeta potential of the colloidal nanoparticles. A Fourier transform infrared (FTIR; Shimadzu‐8400S) spectrum of dried ZVSNPs was recorded to examine the involvement of specific functional groups of SDLE in the production and stabilisation nanoparticles.

2.4 Antibacterial activity

With the objective of checking the antibacterial efficacy of the nanoparticles, two bacterial pathogens, namely, Escherichia coli MTCC 40 and Salmonella typhimurium MTCC 98 were tested by the agar‐well‐diffusion assay. Both the pathogens were maintained in agar‐slants and periodically sub‐cultured in the lab. The inoculums were prepared from the single pure colony and the overnight culture of each pathogen was uniformly spread on separate sterile nutrient agar plates. Three circular wells were made on each plate using a sterile cork‐borer of 5 mm diameter. A known quantity of ZVSNPs was loaded into each well and the plates were incubated at 37°C for overnight in a temperature‐controlled incubator. The zone of inhibition (ZOI) around each well was visually monitored from each plate after 24 h incubation.

2.5 Catalytic activity of ZVSNPs

The MB dye reduction experiments were performed to evaluate the catalytic activity of the synthesised ZVSNPs. In this procedure, 3 ml of a known concentration of the MB dye solution was taken in two clean cuvettes. In the first cuvette, only 200 µl of 100 mM NaBH₄ was added. In the second cuvette, in addition to NaBH₄, 200 µl of freshly synthesised ZVSNPs was also added. The reaction was conducted at room temperature. After the addition of the reactants, both cuvettes were mixed vigorously with the help of a micropipette and the absorbance spectra were noted at regular time intervals. The change in the absorbance at a maximum wavelength of 663 nm was also recorded.

3 Results and discussions

3.1 Visual monitoring and UV–Vis spectroscopy

It is a known concept that the ZVSNPs appear colloidal brown in an aqueous medium because of surface plasmon resonance (SPR) vibrations. Hence, the change in the colour of the silver nitrate from colourless to yellow and further to reddish brown after the addition of SDLE with heating indicated the formation of ZVSNPs. During our experiment, a yellow hue started to appear after a period of 5 min.

On analysing the solution through UV–Vis spectroscopy, the spectrum exhibited a peak at 420 nm (Fig. 2) which is consistent with the published results for the fabrication of ZVSNPs using Ficus panda leaf extract [23]. This peak was not present in the spectrum of the SDLE as shown in Fig. 2. The phenomenon of SPR of ZVSNPs causes the maximum absorption to occur in the range of 400–480 nm [8]. This SPR peak serves as a fingerprint for the characterisation of a substance since every particle transmits light of different wavelengths in a specific manner.

Fig. 2.

UV–Vis spectra of the SDLE and ZVSNPs synthesised using SDLE (inset plot shows the spectrum of the ZVSNPs for various time intervals)

Additionally, the stability of the nanoparticles was assessed by continuous monitoring of the spectrum over a period of one week (Fig. 2 inset). On observing the spectrum at different time intervals, it was noted that the trend of the spectrum remained more or less unchanged (λ _max between 420 and 425 nm). The constant spectrum implied the stability of nanoparticles to be quite high. This makes the nanoparticles suitable for storage and use over a long period‐of‐time at ambient temperature.

3.2 SEM and EDX

The SEM image of ZVSNPs (Fig. 3) shows the morphology and size of nanoparticles. At a magnification of 21.71kX, small aggregates and individual nanoparticles were observed with cuboidal shape. The formation of small aggregates may be due to the procedures involved in the sample preparation. Similar kind of small aggregate has been reported in [24, 25] for the green synthesis of ZVSNPs.

Fig. 3.

SEM image of the ZVSNPs synthesised using SDLE

In Fig. 3, the size of an individual nanoparticle was shown as 68.82 nm, which ascertained the desired range (1–100 nm) of nanoparticles. The variation in size, shape of the nanoparticles and aggregation can be attributed to the availability and non‐uniform distribution of stabilising and capping agents present in the SDLE.

EDX diffraction is an analytical technique used for elemental and chemical characterisation and quantification of a sample. Fig. 4 shows EDX spectra recorded for ZVSNPs, which designated the elemental silver peak at 3 keV. This corroborated the presence of elemental silver in the sample. The silicon and calcium peaks could be the result of the glass used as a base for the preparation of the test sample. This is in accordance with the EDX images shown in [25, 26] for ZVSNPs. The chlorine peak might have originated from the SDLE that was bound to the surface of the ZVSNPs [27]. As can be seen from Fig. 4, the relative amounts of Si, Ca and Cl are much lesser than silver.

Fig. 4.

EDX spectrum of the ZVSNPs synthesised using SDLE

3.3 X‐ray diffraction (XRD) analysis

XRD was employed to determine the crystalline structure and size of the individual nanoparticles. A face‐centred‐cubic crystalline structure was observed for the ZVSNPs synthesised with respect to the Bragg peaks. A single, sharp peak at 32.47° (Fig. 5) denoted the existence of (1 0 1) lattice plane (JCPDS no. 04–0783). This finding is in good agreement with the published literature [28, 29, 30]. The absence of other peaks confirmed that only zero‐valent nanosilver particles were formed in the study. The average crystallite size was calculated as 48.61 nm by using the Scherrer equation. A mean crystallite size of 48.6 nm has been estimated by Balavigneswaran et al. [31] for the ZVSNPs synthesised using the leaf extract of Anacardium occidentale. Moreover, the lattice parameter was calculated as 0.39 nm, which was approximately equal to the standard value of 0.40729 nm for metallic silver [21].

Fig. 5.

XRD pattern of the ZVSNPs synthesised using SDLE

3.4 FTIR

FTIR spectroscopic analysis was done to examine the functional groups of biomolecules that uniquely made contact with synthesised nanoparticles. FTIR spectra of both leaf extract and ZVSNPs are shown in Fig. 6. It can be visualised from the figure that both have a shift in the band location. The shifts in bands (cm^–1) 3440–3452, 3269–3278, 2900–2923, 1602–1623, 1789–1780, 1406–1427 and 1045–1076 belong to O–H stretching of alcohols, N–H stretching of amides, C–H stretching of hydrocarbon part of biomolecules, N–H bending of amide II, non‐conjugated C=O stretching vibrations, C–C stretching of aliphatic groups and C–O stretching of carboxylic acids present in the SDLE, respectively [32]. Similarly, the shifts in bands (cm^–1) 1199–1224 and 786–781 connote to C–O stretching of esters and C–H bending vibrations of phenyl rings, respectively [17]. The presence and shifts in these bands endorse the involvement of alcoholic groups, amides, amino, esters, phenyl and carboxylic groups of SDLE in the synthesis and stabilisation of ZVSNPs. This phenomenon is in accordance with the finding of [33], who described that the presence of proteins in the plant extract bind to ZVSNPs through free amine groups of proteins and thus render the stability to them.

Fig. 6.

FTIR spectrum of the leaf extract and the ZVSNPs synthesised using SDLE

Moreover, as mentioned in the introduction section, the SDLE contains many flavonoids and polyphenol compounds. According to the literature [34, 35], the flavonoids have a potential to reduce the Ag⁺ to ZVSNPs and other phytochemicals may act as capping agents. The FTIR analysis substantiated the presence of these compounds in the ZVSNPs and therefore involved in the synthesis and capping of nanoparticles.

3.5 DLS studies

The DLS is one of the renowned methods to determine the hydrodynamic size and the histogram of the size of the colloidal nanoparticles. The particle size distribution of the colloidal nanoparticles is shown in Fig. 7. A various range of particles from 21 to 106 nm can be visualised and the average hydrodynamic size of the nanoparticles was measured as 59.66 nm. An average size of 62.09 nm was reported by Patil et al. [36] for the synthesis of ZVSNPs using the Jatropha gossypifolia latex. Similarly, for the biosynthesis of nanoparticles using bacterial culture supernatants, an average size of 50 and 52.5 nm was documented by the authors of [37, 38], respectively.

Fig. 7.

Particle size distribution of the ZVSNPs synthesised using SDLE

Du et al. [39] reveal that a polydispersity index (PDI) value <0.5 is desirable for the nanoparticle solutions to be monodispersed. In the current study, a PDI value of 0.187 was obtained which witnessed the monodispersity. This is backed‐up by the single‐intense peak obtained (Fig. 7) in the investigation.

The extent of the stableness of the synthesised ZVSNP solutions was assessed from the zeta potential value (Fig. 8) which was −15.7 mV in the present study. The negative value indicated the negative surface charges of the nanoparticles. A zeta potential value of −15.8 mV was reported for the seed extract mediated colloidal ZVSNPs [40].

Fig. 8.

Zeta potential distribution of the ZVSNPs synthesised using SDLE

3.6 Antibacterial activity

It is shown by many researchers [41, 42, 43] that the silver ions are toxic to the microbial population and hence used as antimicrobial agents. In the current investigation, the antimicrobial efficacy of the synthesised ZVSNPs was tested against E. coli MTCC 40 and S. typhimurium MTCC 98 using well‐diffusion method (Fig. 9). Three different concentrations of ZVSNPs were used for both pathogens. After making the bacterial lawn and loading of ZVSNPs, the plates were incubated for 24 h at a specified temperature and then, the ZOI in each well was measured. An increase in the ZOI with respect to the concentration of ZVSNPs was noticed in all the wells. For E. coli (Fig. 9 A), the ZOI (mm) values were 14, 17 and 19 for three different concentrations of ZVSNPs (v/v) viz., 60, 70 and 80%. Similarly, another set of ZOI (mm) of 11, 12 and 18 was observed for S. typhimurium (Fig. 9 B) for the concentrations of 50, 60 and 100% ZVSNPs (v/v). From these results, it can be inferred that the antibacterial activity depends on the dosage of the nanoparticles used. A similar kind of result has been shown for both Gram‐ positive and Gram‐negative bacteria [44].

Fig. 9.

Antibacterial activity of the ZVSNPs synthesised using SDLE

(A) E. coli, (B) Salmonella spp

The bactericidal action of ZVSNPs and the mechanism have been well‐documented [41]. According to this report, the accumulation of ZVSNPs in the bacterial cell membrane increases the permeability of the cell contents and thus leading the cell death. Moreover, the deactivation of the respiratory enzymes inside the cell due to the formation of stable bonds between the thiol groups and ZVSNPs may lead to the reactive oxygen species production [43]. In addition, the smaller size and the larger specific‐surface area of the ZVSNPs allows better interactions with the microorganisms [42]. Therefore, the above study clearly suggests that the SDLE‐mediated ZVSNPs can be effectively used against bacterial pathogens as antibacterial agents.

3.7 Catalytic activity of ZVSNPs

The degradation of MB dye in the presence of NaBH₄ is shown in Fig. 10. The main absorption peak at 663 nm and the shoulder peak at 614 are characteristic features of the pure MB dye solution [45]. The absorbance and the position of both the peaks started decreasing slowly after the addition of the reducing agent, NaBH₄, as portrayed in Fig. 10. The absorption spectra at regular time intervals were monitored for 20 min. It can be figured out from the spectra that the complete degradation of MB dye would take a longer time.

Fig. 10.

Sequential UV–Vis spectra of degradation of MB dye in the presence of NaBH₄

Nevertheless, there was an accelerated decrement in the absorbance and spectra of MB dye solution in the presence of NaBH₄ and ZVSNPs. It is evident from Fig. 11 that the complete degradation of MB dye was completed within 11 min. The initial blue colour of the MB dye (0th min) was completely lost at the end of the degradation process (@11th min). This witnessed the effective role of ZVSNPs in the catalytic degradation of MB dye. The kinetic data were fitted to a first‐order degradation model as explained by Vidhu and Philip [46], which showed a linear relationship between ln [A/A₀] and time (Fig. 11 inset). The first‐order degradation rate constant was calculated as 0.31 min^–1.

Fig. 11.

Sequential UV–Vis spectra of degradation of MB dye in the presence of NaBH₄ and ZVSNPs synthesised using SDLE (inset plot shows the first‐order linear plot of ln [A/A₀] versus time)

A valid point to be emphasised here is the non‐changing peak of ZVSNPs (@ 420 nm) during the degradation process, which corroborated the ZVSNPs role as a catalyst. The accelerated degradation is due to the high‐surface area and more active sites present on the surface of ZVSNPs [23, 39]. The ZVSNPs assist in the electron relay from BH₄ ^– to MB dye to form colourless leucomethylene blue [46, 47, 48]. It is evident from the present study that the ZVSNPs have excellent catalytic potential and may find their applications in the development of new nanocatalysts.

4 Conclusions

Green synthesis of ZVSNPs using a plant waste, S. dulcis leaves has been developed. The phytoconstituents inherent in the leaf extract reduced the silver nitrate to form ZVSNPs in a shorter time and hence may be used as an alternate to the existing conventional methods. The synthesised ZVSNPs have been characterised by using various methods, which revealed the shape, structure, size, stability and the involvement of specific functional groups of the nanoparticles. The synthesised nanoparticles were found to have a potent dosage‐dependent antibacterial effect against S. typhimurium and E. coli. In addition, the higher specific‐surface area of the nanoparticles accelerated the degradation of MB dye in the presence of NaBH₄. The findings of the current investigation disclose that the plant‐mediated synthesised nanoparticles can be used in the development of new antibiotics and new nanocatalysts.