Environmental and antimicrobial properties of silver nanoparticles synthesized using Azadirachta indica Juss leaves extract
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This study employs a green, eco-friendly and convenient hydrothermal method for the synthesis of stable silver nanoparticles (Ag NPs) using medically beneficial Azadirachta indica A. Juss leaf extract as reducing as well as capping agent. Careful optimization of hydrothermal conditions leads to the formation of spherical Ag NPs of size 9–15 nm. Thus, synthesized NPs are characterized by spectroscopy and microscopy techniques, revealing the morphology and size the NPs. The chemical composition and functionality of the plant extract and NPs is studied using FTIR and EDS analysis. The photocatalytic activity of Ag NPs is demonstrated by decolourization of Rose Bengal under visible light. Ag NPs also show bactericidal activity against E. coli, K. pneumonia, S. epidermidis and S. pneumoniae.
KeywordsHydrothermal Green synthesis Azadirachta indica A. Juss Silver nanoparticles Bactericidal activity
Nanotechnology is attracting tremendous efforts of research because of the unique advantages it posses in various scenarios. The unique properties of metal nanoparticles with AgNPs and drags a lot of attention and used for many applications such as photocatalytic, electrical properties, optical, textile industry, pharmaceutical and bio-medical sciences in specific areas of anti-microbial, anti-viral and anti-fungal activities [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. NPs can be synthesized by different chemical and physical methods. Many of the methods are reasonably expensive and potentially hazardous to the environment [11, 12, 13]. In contrast, the synthesis of NPs using microorganisms like (bacteria, fungi, algae) and plant extracts is non-toxic, and ecofriendly alternative to well-known chemical and physical procedures. The synthesis of NPs using plant extracts is simple procedures, time-reducing and fast compared to microorganisms. In addition, all parts of a plant including leaves, fruits, roots, seeds, and stems can be used for the synthesis of NPs [14, 15, 16, 17]. On the other hand, plant materials contain chemical compounds like amino acids, proteins, polysaccharides, alkaloids, flavonoids and phenolic compounds etc., which can act as a reducing agents and stabilizing agents [18, 19].
According to Nair NC and Henry AN, Neem (Azadirachta indica A. Juss), a meliaceae family tree, is a hardy evergreen tree commonly found in all parts of India . The Azadirachta indica A. Juss plant contained for triterpenoids, steroid, flavonoids, nimbin, salannin, phenolic compounds, and also the plant leaf extracts higher the properties of NPs like antibacterial activity [20, 21, 22, 23, 24, 25]. In case of neem leaves commonly used for much medicinal purpose and wildly handling for tooth brush. Recently, Roy et al.  has been report for this same leaf and its inhibiting to the growth of microbes.
Dyes are a major class of synthetic organic compounds and natural pigments that are widely applied in various industries like paper, paint, plastic, leather, food, cosmetic, textile, printing and pharmaceutical industries [27, 28]. Rose Bengal is an important fluorescein derivative dye widely used in textile industries whose molecular formula C20H4Cl4I4O5. It has severe toxic effects on the human health especially on corneal epithelium. This dye is very dangerous when it comes to contact with skin and causes itchiness, irritation, reddening and blistering. It also affects to eyes like inflammation, eye redness, itching etc. These synthetic dyes mixed with wastewater are directly discharged and pollute to plants, soil, water, and animals. There are several physical and chemical methods like coagulation, filtration, adsorption and reverse osmosis the removal of dye molecules and other water contaminants [29, 30, 31, 32]. But there is a difficult to remove these dyes from water, because of their chemical stability. In recent times, nano-catalysts are widely used for the removal of dye molecules.
The zone of inhibition (ZOI) for different bactericidal concentrations for Ag NPs and the positive control have chosen against E. coli, K. pneumoniae, S. epidermidis and S. pneumonia organisms
Concentration of sample (μg/mL)
Zone of inhibition (mm)
13.3 ± 1.52
13 ± 1.0
12.6 ± 2.08
14.6 ± 0.57
12.3 ± 2.08
13 ± 2.64
14.6 ± 3.05
17.6 ± 2.08
15.6 ± 1.52
12.6 ± 2.08
14.6 ± 1.52
15 ± 2.0
Standard 10 μg
17.6 ± 1.15
16 ± 2.64
11.6 ± 1.52
11.6 ± 1.52
2 Experimental section
Leaf of Azadirachta indica A. Juss were collected from B.S Abdul Rahman university campus and authenticated by Dr. S. Jayaraman, Director of Plant and Anatomy Research Centre, Chennai (Reg. No. PARC/2016/3239). Silver Nitrate (AgNO3) (99.99%) was procured from Sigma Aldrich, Mumbai. RB was obtained from Merck India Ltd. Deionized water has been used as the solvent throughout this experiment. Terephthalic acid (TA) and sodium hydroxide (NaOH) used in the present study were procured from SRL fine Chemicals Ltd. Mumbai, India.
2.2 Preparation of leaves extract
Fresh and healthy leaf of Azadirachta indica A. Juss were collected locally and rinsed thoroughly first with tap water followed by distilled water to remove all the dust and unwanted visible particles, cut into small pieces and shade-dried. About 5 g of leaves were weighed and transferred into 250 mL standard flask containing 100 mL distilled water and kept under magnetic stirring for 24 h at room temperature. The extracts were then filtered thrice through Whatman filter paper to remove particulate matter and thus obtained clear solutions were refrigerated in 100 mL Erlenmeyer flasks for further experiments.
2.3 Hydrothermal preparation of the Ag NPs
In a typical synthesis, 2 mL of aqueous extract of Azadirachta indica A. Juss leaf was added to 18 mL of 1 mM aqueous AgNO3 solution. The solution mixture was transferred into a 100 mL Teflon-lined autoclave and heated at 150 °C for 3 h and then gradually cooled to room temperature. A block solid product at the bottom of the vessel indicated the formation of Ag NPs. The collected product was washed with distilled water several times and centrifuged to remove remnant starting materials in the reaction mixture, and finally stored in a desiccator for further use.
2.4 Antimicrobial activity
The antibacterial activity of the Ag NPs was determined by the diffusion method against Gram-negative E. coli, K. pneumonia, Gram-positive S. epidermidis and S. pneumonia bacteria on Mueller–Hinton Agar, according to the Clinical and Laboratory Standards Institute (CLSI). The media plates (MHA) were streaked with bacteria 2–3 times by rotating the plate at 60° angles for each streak to ensure the homogeneous distribution of the inoculums. After inoculation, discs (6 mm Hi-Media) loaded with 75 μL/mL, of the test sample was placed on the bacteria-seeded well plates using micropipettes. The plates were then incubated at 37 °C for 24 h. The inhibition zone around the well was measured and recorded. Amoxicillin (Hi-Media) was used as the positive controls against Gram-negative E. coli, K. pneumoniae Gram-positive S. epidermidis and S. pneumonia bacteria to compare the efficacy of the test samples.
2.5 Photocatalytic activity study
Photocatalytic was investigated by following the procedure mentioned in Gnanamoorthy et al.  and Ramar et al. . The Photocatalytic performance of Ag NPs was testing by a Rose Bengal organic dye with using visible light irradiation, in a procedure: 20 mg of Ag NPs was dispersed after 1 mL of dye with remaining 99 mL of water also added. Finally, this suspension was stirred in 5 min in dark medium after lights on and further samples were collected in different time intervals with monitored by using UV–Vis spectrophotometer.
2.6 Hydroxyl (·OH) radical detection
Terephthalic acid (TA) was used in order to find out the generation of the main active species is hydroxyl (OH·) radicals during the photoreaction process. To determine the production of OH· radicals, 0.5 mg of the photocatalyst was added to a 10 mL mixture of TA solution (0.415 mg) dissolved in NaOH solution (0.4 mg) and magnetic stirred for 30 min. After the production of OH· radicals under UV–visible light (30 min) irradiation, the reacted solution was centrifuged and then monitored by Fluoromax-4 Spectrophotometer. TA readily reacts with OH• hydroxyl radicals to generate highly fluorescent 2-hydroxyterephthalic acid (TAOH) which emits fluorescence around 461 nm on excitation at 350 nm.
Synthesis of NPs and their photocatalytic activity were monitored with the help of a UV–visible, single beam spectrophotometer (Jasco V-770). Powder X-ray diffraction (PXRD) was carried out using Bruker D8 diffractometer [λ (Cu-Kα) = 1.54 Å]. Fourier-Transform Infrared Spectroscopy (FTIR) results were obtained from Jasco 6300 spectrometer (ATR mode) in the range of 400–4000 cm−1. Field emission scanning electron microscopy (FESEM) images were recorded using an Ultra 55 Carl Zeiss instrument with an operating voltage of 10 kV. Samples for the FESEM analysis were mounted on a stub using a conductive carbon tape. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) images were recorded using an FEI Technai G220 STEM instrument operated at an acceleration voltage of 200 kV. The hydrodynamic size distributions of NPs were analysed using dynamic light scattering (DLS) instrument (Zetasizer Nano-S90). The photoluminescence (PL) emission and excitation spectra were recorded on a Horiba Jobin–Yvon model FL3-22 Fluoromax-4 Spectrophotometer.
3 Results and discussion
3.1 Powder X-ray diffraction (PXRD)
3.2 Fourier transform infrared spectroscopy (FTIR)
3.3 UV–visible diffuse reflectance spectroscopy
3.4 Field emission scanning electron microscopy–energy dispersive spectroscopy
EDS analysis also confirms the presence of a strong peak of elemental composition silver (69.34 wt%), oxygen (10.18 wt%), carbon (14.37 wt%) and a weak nitrogen (6.11 wt%) peak as shown in Fig. 4d. The oxygen, carbon and nitrogen peaks might be due to the presence of biomolecules binding to the surface of hydrothermally synthesized Ag NPs .
3.5 Transmission electron microscopy (TEM) and dynamic light scattering (DLS)
3.6 Photocatalytic performance
3.7 Mechanism on enhancement of photo decolourization activity
To more explain these photocatalytic observations, we propose a charge transfer mechanism as illustrated in Fig. 9. When Ag nanoparticles absorb visible irradiation, the surface electrons of the 4d band can be excited to the 5sp states due to SPR effect [43, 44, 45, 46]; these SPR effect by Ag NPs efficiently generated electrons and holes. The electrons are readily accepted by the oxygen (O2) molecules to form superoxide radicals (O 2 · ) which attack and promote the decolourization of RB dye molecules. Also, the holes generated in the 4d orbital and react with adsorbed H2O to produce hydroxyl radicals (OH·) which attack the RB dye molecules. In addition to the decolourization of the dyes by the radicals, the holes generated in the 4d orbital of the Ag NPs capture electrons from the adsorbed dye molecule leading to further decolourization of the RB dye. These activated radical species (OH·, O 2 · ) are responsible for RB decolourization and the generation of transitional products CO2, H2O. Thus, the Ag NPs are known for absorption of whole of the visible spectrum due to SPR effect and the interband transition of 4d electrons to 5sp band.
3.8 Antimicrobial activities
The zone of inhibition values are obtained for the hydrothermally synthesized Ag NPs against two different micro organisms like Gram-negative E. coli, K. pneumoniae Gram-positive S. epidermidis and S. pneumonia bacteria. The antibacterial activities of Ag NPs are more efficient against Gram-positive S. epidermidis and S. pneumonia bacterial than the Gram-negative E. coli, K. pneumoniae bacteria. Different sensitivities of Gram-positive and Gram-negative bacteria against the Ag NPs is because say for example, E. coli, K. pneumonia has a more negatively charged and more rigid surface than S. epidermidis and S. pneumonia bacteria.
The specific mechanism for the antibacterial activity of Ag NPs is still not well established. The interaction of NPs with bacteria often produces reactive oxygen species (ROS), mostly hydroxyl radicals and singlet oxygen, which cause damages to proteins and nucleic acids in bacteria by inducing oxidative stress. It has been proposed that Ag NPs can slowly kill E. coli, K. pneumoniae and S. epidermidis and S. pneumonia leading to the death of the bacteria and these obtained results are higher antibacterial activity when compare to the previous reports [49, 50, 51, 52].
Ag NPs were successfully synthesized by the hydrothermal green synthesis method with the use of aqueous leaf extract of Azadirachta indica A. Juss under the reaction condition. The formation of Ag NPs was confirmed using UV–Visible diffuse reflectance spectroscopy (DRS) absorption peaks at 442 nm and small band gap (2.1 eV). The PXRD result confirmed that the Ag NPs possessed an fcc crystal structure. In addition, this also revealed that Ag was present in the nanoparticles without any contamination peaks. The TEM images showed that the Ag NPs were in spherical shape and the average diameters of the particles size is 9–15 nm. The antimicrobial activity of silver nanoparticle was excellent as indicated by zone of inhibition against E. coli, K. pneumonia, S. epidermidis and S.pneumonia.To conclude, Ag NPs showed excellent photocatalytic activity of RB in the presence of visible light irradiation.
Special thanks are due to B.S. Abdur Rahman University, Chennai for providing laboratory facilities to carry out this study. Authors are thankful to the Jamal Mohamed College and KIRND Institute of Research and development-Trichy for providing necessary facilities for the present study.
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