Green‐route mediated synthesis of silver nanoparticles (AgNPs) from Syzygium cumini (L.) Skeels polyphenolic‐rich leaf extracts and investigation of their antimicrobial activity

Abstract

Medicinal plants are widely utilised by the African population since they have no harmful side effects and low cost compared with different treatments. The field of nanotechnology is the most active part of research in modern material's science. Though there are several chemicals as well as physical methods, however, green synthesis of nanomaterials is the most emerging method of synthesis. Conventionally, chemical reduction is the most often applied approach for the preparation of metallic nanoparticle's however, in most of the synthesis protocols it cannot avoid the utilisation of toxic chemicals. Hence, the authors report an environmentally friendly, cost effective and green approach for synthesis of 1 mM AgNO₃ solution using the polyphenolic‐rich leaf extracts of Syzygium cumini (S. cumini) (L.) Skeels as a reducing and capping agent. The synthesised AgNPs are characterised by UV‐Vis spectroscopy and Fourier transform infrared (FTIR) spectroscopy. FTIR analysis revealed that the AgNPs were stable due to eugenols, terpenes, and other different aromatic compounds present in the extract. The green biosynthesised S. cumini AgNPs significantly inhibited the growth of human pathogenic both gram‐positive Staphylococcus aureus (1.40 mm) and gram‐negative bacteria Escherichia coli (2.75 mm) and Salmonella typhimurium (1.45 mm) showing promising antimicrobial activity.

1 Introduction

Metal nanoparticles are centred in researches regarding their distinctive electrical, chemical, electronic, optical and mechanical properties that are appreciably different from those of large materials. Biosynthesis of silver nanoparticles (AgNPs) has attracted attention in science due to their several properties and uses together with antimicrobial and antibacterial activities, magnetic and optical polarisation, electrical conductivity, catalysis, DNA sequencing, and surface‐enhanced Raman scattering [1]. Particles displaying a characteristic size of 1–100 nm are defined as nanoparticles and they show completely new particles based on their size, distribution and morphology [2]. A strong surface plasmon resonance absorption is revealed in the UV‐Visible region by the metallic nanoparticle [3]. Metallic nanoparticles are exploited broadly because of their excellent antimicrobial properties, anti‐inflammatory activities, anti‐angiogenic, and anti‐cancer agents, by enhancing the action of the active compounds from plant extracts and up their efficiency and activity [4, 5, 6]. AgNPs are applied new technologies advances in the fields of electronics material science and medicine [7]. Nanoparticles are synthesised either by size reduction from a suitable starting material typically involving physical and chemical types of production and/or the nanoparticles are designed from smaller entities by linking atoms and molecules, because it includes the utilisation of chemical and biological protocol of production [8]. Biological methods of synthesis of nanoparticles are benign, environment friendly and it is a cost effective method resulting in toxic chemicals being eliminated. They include microbial and the plant extracts synthesis but microorganism's synthesis process is more expensive than the production of plant extracts [9]. Plant mediated nanoparticle synthesis using whole plant extract or by living plant was reported in the literature [10].

Syzygium cumini (S. cumini) (L.) Skeels belongs to family Myrtaceae usually called Jamun, Jambu and Black Plum in Hindi, Sanskrit and English, respectively. It is an evergreen tree indigenous to India and conjointly, may be found distributed through the Asian landmass, South America, Eastern Africa and Madagascar [11]. Diverse parts of the plants are used for numerous economic importance. The ripe fruits that are juicy are used to make jams, jellies, juice, vinegar, puddings and also vital in making wine in large quantities in the Philippines. The woods of Black Plum are water and termite resistant and really strong and useful in the installation of motors in wells, and a good source of fuel and charcoal. Essential oil's from the leaf are used as a fragrance in soaps and is mixed to make perfumes [12]. The seeds of S. cumini have been shown to possess an anti‐diabetic activity by increasing body weight and decreasing the fasting blood sugar level in alloxan‐induced diabetic rats after days of treatments with ethanol extracts of the seeds [13]. The ethanol extract of the leaves of S. cumini was proved to exhibit a potential growth inhibitory activity against multi‐drug resistant Vibrios and thence, perhaps useful to combat cholera while the fruit extract exhibited reducing power and DPPH radical scavenging activity [14, 15]. Most reports are on the characterisation and application of the synthesised AgNPs in catalytic activity, electronic properties; however, the antimicrobial effects of AgNPs using polyphenolic extracts are investigated rarely. Hence, this study deals with the synthesis of AgNPs by bioreduction of Ag⁺ through the green method using polyphenol‐rich S. cumini (L.) Skeels leaf extract and enhancing the antibacterial activities against some human pathogenic bacterial strains.

2 Materials and methods

2.1 Chemicals

AgNPs were bought from Sigma (Sigma‐Aldrich) representative in Nigeria. All chemicals used for the experiment were of analytical grade.

2.2 Collection of plant material

S. cumini (L.) Skeels leaves were collected and obtained from Bisi market, Ado‐Ekiti, identified and authenticated at the Department of Plant Science, Ekiti State University, Nigeria. All plant names in this study correspond to the good practices in publishing studies on herbal materials, as described in [16]. The leaves were washed, air‐dried and pulverised with an electric blender into a fine powder and stored in an airtight container until further use.

2.3 Extraction of polyphenol

Polyphenol‐rich extract from S. cumini (L.) Skeels leaves was obtained by dissolving 4 g of the plant powder in 40 ml of an aqueous acetonic solution (70%, v/v). It was further incubated at 4°C for 90 min and centrifuged at 3500 rpm at 4°C for 20 min. The polyphenol‐rich supernatants were collected and stored for further analysis [17].

2.4 Synthesis of AgNPs from polyphenol extract of S. cumini (L.) Skeels (PES)

The desired volume of 1 mM aqueous solution of silver nitrate (AgNO₃) was prepared and then 1.0, 2.0, 3.0, 4.0 and 5.0 ml of PES were added separately to 10 ml aqueous silver nitrate solution kept separately at room temperature. Also, a portion of 10 ml of PES and no silver nitrate solution was also prepared. The solutions were kept in a dark chamber until the colour of the solutions changes from yellow to dark yellow. Then after, 15 min, the solution turns yellow to yellow‐red or dark brown indicating AgNPs formation. The UV spectrophotometer was used in monitoring the bio‐reduction of silver ions by periodic sampling [18, 19].

2.5 Characterisation of AgNPs

The formation of AgNPs ensuing from an indication of a colour change from pale yellow to dark brown upon incubation owing to surface plasma resonance vibration was observed. The UV‐Visible spectroscopy and Fourier transform infrared (FTIR) spectroscopy were carried out according to [20].

2.5.1 UV‐Vis spectroscopy analysis

The bio‐reduction of Ag⁺ ions in solutions was monitored by measuring the UV‐VIS spectrum of the reaction medium. The UV‐VIS spectral analysis of the sample was done by using a 6715 Jenway spectrophotometer at room temperature operated at a resolution of 1 nm between 200 and 800 nm ranges.

2.5.2 FTIR spectroscopy analysis

In FTIR spectroscopy measurements, the bio‐transformed products present in the cell‐free filtrate were freeze‐dried and diluted with potassium bromide in the ratio of 1:100. The FTIR spectrum of samples was recorded on a FTIR instrument with a diffuse reflectance mode (DRS‐8000) attachment. All measurements were carried out in the range of 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹.

2.6 Antimicrobial activities

The disc diffusion method was used to assess antimicrobial activities of synthesised AgNPs against Staphylococcus aureus, Escherichia coli, Candida albicans and Salmonella typhimurium. The microbes were sub‐cultured and incubated at 37°C for 24 h. Fresh cultures were used and spread on Mackonkey agar plates to cultivate bacteria. Sterile paper discs of 5 mm diameter were saturated with double distilled water (as control), plant extract and synthesised AgNP solution were placed in each agar plate and incubated again at 37°C for 24 h. After 24 h, the antimicrobial activities were then studied according to the inhibition zones around the discs saturated with plant extract and synthesised AgNPs.

3 Results

Fig. 1 shows the photographs of an aqueous solution of 1 mM AgNO₃ before and after polyphenolic‐rich S. cumini (L.) Skeels leaf extract. The first beaker (AgNO₃ without plant extract) does not show any colour change. The second tube (1 mM concentration AgNO₃) shows the change in colour of the reaction medium as an effect of the presence of AgNPs.

Fig. 1.

Photographs showing colour changes

(a) After adding AgNO₃ before reaction, (b) After reaction time

Fig. 2 shows the UV‐Vis spectrum of colloidal solutions of AgNPs synthesised from polyphenolic‐rich S. cumini (L.) Skeels leaf extract that have the characteristic absorbance peaks at 340 nm at 1 mM AgNO₃.

Fig. 2.

UV‐Vis absorption spectra of AgNPs synthesised from S. cumini at 1 mM silver nitrate (AgNO₃)

The FTIR spectrum of biosynthesised AgNPs is shown in Fig. 3 which manifest absorption peaks located in the region about 500 and 3500 cm⁻¹ in order to identify the functional groups of the extract involved in the reduction of the synthesised AgNPs. The absorption peak located at 500 cm⁻¹ –C–I– stretch (alkyl halide), 1000 cm⁻¹ –C–F– stretch (alkyl halide), 1324 cm⁻¹ –C–F– stretch (alkyl halide), 1526 cm⁻¹ –N–H– bending (amide), 1625 cm⁻¹ –C–C– stretch (alkene), 3000 cm⁻¹ –C–H– stretch (aromatic), 3500 cm⁻¹ –O–H– stretch, free (alcohol) functional groups. Biological components are known to interact with metal salts through these functional groups and mediate their reduction to nanoparticles.

Fig. 3.

FTIR spectrum of the synthesised AgNPs

Figs 7, 6, 5, 4 show that biosynthesised AgNPs exhibited excellent antimicrobial activity against S. aureus, E. coli, C. albicans, and S. typhimurium. The anti‐bactericidal activity is estimated by the zone of inhibition. The diameter of the zone of inhibition around the antibiotic disc with AgNPs against the test strains is shown in Table 1. AgNPs synthesised showed inhibition zone against all the studied bacteria. The maximum zone of inhibition was found to be 2.75 mm in E. coli and minimum of 1.40 mm in S. aureus, Table 1.

Fig. 7.

Zone of inhibition against S. typhimurium

Fig. 6.

Zone of inhibition against C. albicans

Fig. 5.

Zone of inhibition against E. coli

Fig. 4.

Zone of inhibition against S. aureus

Table 1.

Antimicrobial activity of AgNPs synthesised using polyphenol‐rich extract from S. cumini (L.) Skeels leaves

Zone of inhibition
Name of bacterial species	AgNPs, mm
Staphylococcus aureus	1.40 ± 0.07
Escherichia coli	2.75 ± 0.35
Candida albicans	2.25 ± 0.07
Salmonella typhimurium	1.45 ± 0.14

4 Discussion

The reduction of silver ions to silver particles when polyphenolic‐rich S. cumini (L.) Skeels leaf extract was exposed to Ag⁺ ions (AgNO₃, 1 mM), the colour of the reaction mixture changed to yellowish brown and then to dark brown, which was in agreement with the earlier studies, and was thought as the formation of AgNPs [21, 22]. The appearance of dark brown was attributable to the excitation of surface plasmon resonance in the nanoparticles. The current study emphasises the employment of medicinal plants for the synthesis of AgNPs with a potent antimicrobial impact. Nanoparticles were mainly characterised by UV‐Vis spectroscopy, which was shown to be an appropriate method for the analysis of nanoparticles.

Reduction of Ag⁺ ions in the aqueous solution of the silver complex during the reaction with the components present in the plant leaf extracts detected by the UV‐Vis spectroscopy exposed that AgNPs in the solution may be correlated with the UV‐Vis spectra. At the time the leaf extracts were mixed with the aqueous solution of the silver ion complex, it was transformed into dark yellowish‐brown colour as a result of excitation of surface plasmon vibrations, which revealed the formation of AgNPs [23]. The UV‐Vis spectrograph of the colloid of AgNPs was recorded as a function of time by employing a quartz cuvette with silver nitrate as the reference. In the UV‐Vis spectrum, the broadening of the peak revealed that the particles are poly dispersed. The reduction of silver ions and the formation of stable nanoparticles occurred quickly within the reaction time making it one among the quickest bio reducing protocol to produce AgNPs [24]. A surface plasmon band in the AgNPs solution remains close to 340 nm throughout the reaction period suggesting that the particles are scattered in the aqueous solution, with no proof for aggregation.

The FTIR bands of 1342 and 500 cm⁻¹ were attributable to the strong stretching vibrations of C–H alkyl halides. FTIR band intensities in numerous regions of the spectrum for the synthesised AgNPs were analysed and the spectra showed individual peaks in the range 500, 1000, 1324, 1526, 1625, 3000, and 3500 cm⁻¹. The spectrum of the FTIR peak at 3500 was referred as the strong stretching vibration O–H functional group [25]. The vibrational mode at 1625 cm⁻¹ corresponds to C=C variables present in the plant [26, 27] have conjointly recognised several organic extracts in the samples suggesting that these groups may function as a reducing or a capping agent.

The antibacterial properties of AgNPs were obvious on both gram‐negative and gram‐positive bacteria [28, 29]. Biosynthesised AgNPs’ antimicrobial activity was evaluated against human pathogenic microorganisms. AgNPs show a significant inhibition activity against both gram‐positive (S. aureus and C. albicans) and gram negative (E. coli and S. typhimurium) bacteria. Primarily, the maximum zone of inhibition was noted against E. coli, a gram negative bacterium (Fig. 5). Consequently, minimum zone inhibition was measured against S. aureus, a gram positive bacterium (Fig. 4). The difference in inhibition activity of AgNPs against gram positive and gram negative bacteria is attributable to the composition of the cell wall. The mechanisms of antibacterial activity of AgNPs involve binding the membrane of microorganisms through electrostatic interactions, disruption of the cell wall and affecting the intracellular processes such as nuclei acids and protein synthesis [30, 31]. In this study, the gram‐positive bacteria showed the lower zone of inhibition, compared with gram‐negative bacteria. This may be due to the cell wall of gram‐positive bacteria composed of a rigid thick numerous layer of peptidoglycan complex because it stopped AgNPs from entering the cell wall [32, 33, 34].

5 Conclusion

AgNPs were successfully produced using polyphenolic‐rich S. cumini (L.) Skeels leaf extract. Characterisation by UV visible and FTIR techniques confirmed the reduction of silver ions to AgNPs. The very good results of antibacterial activity reveal the biomedical application of AgNPs for diseases related to both gram‐positive and gram‐negative bacterial strains. In the present study, we found that leaves can be a very good source of biosynthesis of AgNPs. The synthetic methods based on naturally occurring biomaterials give an alternative means for getting the nanoparticles. The use of plants in the biosynthesis of nanoparticles is novel leading to a truly ‘green chemistry’ route. The antimicrobial activity of biologically synthesised AgNPs was evaluated against S. aureus exhibiting effective bactericidal activity.