Novel green synthesis of silver nanoparticles using clammy cherry (Cordia obliqua Willd) fruit extract and investigation on its catalytic and antimicrobial properties
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In this study, highly monodispersed, exceptionally stable, spherical silver nanoparticles (AgNPs) were successfully synthesized by the microwave assisted rapid and cost-effective green method. Aqueous extract of clammy cherry (Cordia obliqua Willd) fruit was used as the green reductant, and capping agent for the synthesis of AgNPs and the effect of different synthesis parameters on the optical properties of the synthesized AgNPs was also studied. The characterization of synthesized AgNPs by Fourier transform infrared spectroscopy, X-ray diffraction studies, UV–visible spectroscopy, scanning electron microscopy and transmission electron microscopy (TEM) revealed the formation of small AgNPs with narrow size distribution. TEM studies corroborated that the AgNPs are highly crystalline and spherical with an average diameter of 7.13 nm. The cyclic voltammetry profile of AgNPs modified electrode in NaOH depicted prominent redox peaks evidencing an impressive electrochemical response. The AgNPs manifested high catalytic activity towards reduction of methyl orange and rhodamine blue with apparent rate constant 0.3038 min−1 and 0.1542 min−1 respectively. Additionally, the prepared AgNPs exhibited strong antibacterial efficacy against the tested microbes.
KeywordsSilver nanoparticles Microwave Green Reduction Catalysis Surface plasmon resonance Methyl orange Rhodamine blue
Nanotechnology is a fast developing research field making its impact on all spheres of human life. Currently, the synthesis of noble metal nanoparticles has drawn considerable attention by nanochemists because of their exceptional physio-chemical characteristics and their wide biomedical, sensing and catalytic applications [1, 2, 3, 4, 5]. Silver nanoparticles (AgNPs) are extensively studied compared to nanoparticles of Au, Pt, and Pd because of its ease of synthesis and lower cost. AgNPs shows excellent optical, electronic, antimicrobial and catalytic properties [6, 7, 8]. Versatile organic transformations for the production of industrially and therapeutically important organic molecules have been successfully carried out with high efficiency and selectivity using nanosilver based catalytic systems [9, 10, 11]. The catalytic activity of metal nanoparticles has extensively investigated in the field of water pollution remediation. Many research groups have demonstrated the effective utilization of noble metal NPs or metal NP incorporated composites systems as a suitable redox catalyst for the degradation of various organic pollutants like dyes, drugs and aromatic nitro compounds [12, 13, 14, 15, 16, 17, 18, 19]. Additionally unique electrochemical features and high conductivity of AgNPs identifies AgNPs as a good candidate for selective and sensitive monitoring of biomolecules and pharmaceuticals [20, 21, 22]. Earlier reports have shown that size, shape, and surface features are critical in deciding performance of their so far mentioned catalytic and sensing applications [21, 22, 23, 24, 25]. It is reported that the cytotoxicity of AgNPs against diseases like HIV and cancer are also size dependent [2, 26, 27]. In this context, the method of synthesis, especially the nature of stabilizing agents, reducing agent and other synthesis parameters have a critical role in deciding the properties of the AgNPs.
Due to the potential applications in diverse fields, there is a constant urge for the development of the fast synthesis of AgNPs with controllable size and uniqueness in good yield. To date, a number of chemical reduction procedures have been reported for the synthesis of AgNPs [7, 28]. Even if this synthetic route allows the fast synthesis of AgNPs in bulk scale, most of the reagents used as reducing and/stabilizing agents are toxic and not biocompatible. Hence the AgNPs obtained by such chemical routes cannot be used for the biomedical application, such as drug carrier and or in vivo imaging studies.
Recently, the biosynthesis of metal nanoparticles (MNPs) have been proposed as a cost-effective and eco-friendly substitute to chemical methods. Many studies have been demonstrated the biosynthesis of AgNPs mediated by the microorganism or by phytochemicals or enzymatic synthesis [11, 18, 19, 29, 30, 31, 32, 33]. Among the biological methods, photosynthesis which involves reduction by phytochemicals derived from plants seems to be the superior choice. Phytochemical mediated synthesis is considered as a green alternative as it utilizes nontoxic, green reducing and stabilizing agent, thus making the method simple, economical environment-friendly and biocompatible. However, synthesis of AgNPs by the phytochemical reduction methods are rather slow compared to conventional chemical reduction and it was considered as the major limitation that faced during the earlier periods of green synthesis. Currently, by coupling plant-mediated synthesis with microwave (MW) assisted synthetic techniques, the biosynthesis can be conveniently carried out rapidly with good yield [4, 34, 35]. MW assisted chemical transformations a simple yet versatile eco-friendly process to achieve the fast synthesis of metal NPs.
Here, we investigated the role of fruit extract of Cordia obliqua Willd as the green reductant and stabilizing agent during the formation of AgNPs Cordia obliqua Willd commonly known as clammy cherry is a plant that belongs to Boraginaceae family which has widely distributed warmer regions of India and Ceylon [36, 37]. Traditionally the plant is of great medicinal value. The phytochemical screening studies on clammy cherry extract reported earlier revealed the presence of numerous poly-functional molecules like carbohydrates, proteins, amino acids, flavonoids, phenolic compounds, alkaloids, glycosides and sugar in different part of the plant [36, 37].
We have demonstrated the ability of this clammy cherry derived phytochemicals as reducing and capping agent during the formation of AgNPs. In the present work, we could synthesize spherical AgNPs with narrow size distribution under microwave irradiation of few minutes. The use of water for extraction and reaction medium is a further add-on to the green chemistry policy. The effect of the MW power, irradiation time, and concentration of silver nitrate solution on AgNPs characteristics was also investigated. Impressive electrochemical response and high catalytic activity towards the reduction of organic dyes are highly promising for its futuristic applications in diverse fields including catalysis and sensing. This study has established a rapid, cost-effective and eco-friendly procedure for the synthesis of highly stable AgNPs having hopeful application potentials.
2.1 Materials and methods
The chemicals, silver nitrate (AgNO3), sodium borohydride (NaBH4), graphite powder, paraffin liquid and sodium hydroxide used in the study were purchased from Merck Chemicals Ltd, Mumbai, India. The organic dyes methyl orange (MO) and rhodamine blue (RhB) have purchased from Nice Chemicals, India. Microwave oven [wave (LG) Model 1S2021CW at 2450 MHz] was used for the AgNPs synthesis.
2.2 Preparation of clammy cherry extract
Ripened clammy cherry fruits were collected from the Maharajas college campus, Kerala, India. 10 g of fruit was washed thoroughly with deionized water several times, then peel was removed, and the pulp along with seed was refluxed with 100 mL water under microwave heating for 2 min at 350 W. The aqueous extract is cooled, filtered with Whatmann 40 filter paper and filtrate is used for synthesis.
2.3 Synthesis of silver nanoparticles (AgNPs)
Silver NPs was prepared by heating a solution of AgNO3, and clammy cherry extract in a domestic MW oven. In a typical procedure, 10 mL of the clammy cherry extract (10 g/100 mL) was mixed well with 20 mL of 1 mM AgNO3 solution and irradiated in MW oven at 2.45 GHz under 350 W for about 8 min. Wherein, the color of the solution changes to light yellowish to reddish brown indicating the formation of the AgNPs. The effect of different parameters such as MW power, the period of exposure, the concentration of AgNO3 and composition of clammy cherry extract were also investigated. The entire process was monitored by recording the UV–Vis spectrum using the reaction mixture collected at regular intervals.
Thermoscientific evolution 160 UV–VIS Spectrometer is used for recording the UV–Vis spectrum of the periodically collected reaction mixtures. The surface morphology of AgNPs was analyzed by scanning electron microscope (SEM) using VEGA3 TESCAN. Transmission electron microscopic images were done using a JEOL JEM-2100 microscope. X-ray diffraction (XRD) studies were carried out using powdered AgNPs which are collected by ultracentrifugation followed by drying in a vacuum oven for 24 h. Fourier transform infrared spectroscopy (FTIR) spectra of the vacuum dried clammy cherry extract and AgNPs were recorded in the range 4000–450 cm−1 using Perkin Elmer FTIR spectrometer.
2.5 Antibacterial assay
Biological studies were conducted for determining the bactericidal activity of synthesized AgNPs against four human pathogenic bacteria namely Escherichia coli, Bacillus circulans, Pseudomonas aeruginosa, and Staphylococcus aureus. Mueller–Hinton agar (MHA) well diffusion method was employed to investigate the antibacterial activity of clammy cherry stabilized silver NPs. 25 µL spore suspension (10–100 spore/mL) of S. aureus, E. coli, B. circulans, P. aeruginosa were added to a sterile Muller Hinton medium fast before solidification, then poured into sterile Petri dishes (9 cm in diameter) and spread using a cotton swab. Sterilized disc (6 mm) is taken, and 10 µL of nanoparticles solution was dropped into it and was kept in the Petri dish. Sealed the Petri dish with cling film and incubated for 16 h. Inhibition zones were detected around the disc and measured.
Minimum inhibitory concentration (MIC) is the lowest concentration of a chemical which prevents visible growth of a bacterium. MIC was determined by resazurin based microtiter dilution assay (RMDA) method. In a standard procedure, RMDA was done using, 96 well microtiter plates (HiMedia) under sterile conditions. The first row of the plate is filled with 100 μL of AgNPs (1 mg/mL) dissolved in sterilized water. All the wells of microtiter plates are packed with 50 μL of Luria broth. By transferring 50 μL AgNPs solution from the first row to the next wells in the next row of the same column, a two-fold serial dilution is achieved and so that each well has 50 μL of test material in serially descending concentrations. 2 μL of resazurin indicator solution was added into each well to impart purple color. Finally, 10 μL bacterial suspension was added to each well to achieve a concentration of 5 × 106 CFU/mL. Then each plate was covered with cling film to avoid the dehydration of bacterial culture. There was a set of 3 controls for each microtiter plate, a first column with the positive control (Ampicillin), a second column with all reagents but without AgNPs solution and the third column with all solutions along with 10 μL of Luria broth instead of the bacterial solution. The entire plates are incubated at 37 °C for 1 day. Any color change observed from purple was taken as positive. The lowest concentration of the sample at which no color change occurred was recorded as the MIC value. All the experiments are performed in triplicates and average was taken as the MIC of synthesized AgNPs.
2.6 Catalytic activity studies
The catalytic activity of the clammy cherry stabilized AgNPs was evaluated by monitoring the reduction of organic pollutants like methyl orange (MO) and rhodamine blue (RhB) under pseudo-first order condition in presence of relatively high concentration of sodium borohydride. The reaction is monitored by recording the absorption spectra in each min in the range of 200–800 nm at room temperature using Thermo scientific evolution 160 UV–Vis Spectrophotometer.
2.7 Electrochemical studies
The electrochemical response of the AgNPs was studied by cyclic voltammetric (CV). The CV was recorded with AgNPs modified carbon Paste Electrode (CPE) was carried out in Metrohm Auto lab Potentiostat/Galvanostat (Model No. AUT87141) furnished with NOVA 2.1 software. A three-electrode electrochemical setup containing modified carbon paste electrodes as working electrode, Pt wire as a counter electrode and Ag/AgCl reference electrode is used for recording CV. Carbon paste prepared by thorough mixing graphite powder and paraffin oil (weight ratio of 70:30) was packed in a clean glass tube. Silver wire was inserted into carbon paste for electrical contact. The CPE surface was modified by drop casting 10 µL of the AgNPs to get AgNPs/CPE.
3 Result and discussion
3.1 Synthesis of AgNPs and UV–Vis spectral studies
The stability of prepared AgNPs is a valuable parameter to be analyzed and achieved. Excellent stability and durability of AgNPs are highly appreciated for their sensing and catalytic applications. It is observed that the Clammy cherry reduced AgNPs are stable for more than 6 months without coagulation under refrigeration. The UV–Vis spectra of the AgNPs recorded after 6 months presented in Fig. 3 (blue line) still shows SPR with absorption maxima at 420 nm confirming the existence of highly dispersed AgNPs.
3.2 FTIR studies
3.3 XRD studies
3.4 SEM analysis
3.5 TEM analysis
3.6 Cyclic voltammetry studies
3.7 Antibacterial studies
MIC values of prepared AgNPs obtained from RMDA method
Minimum inhibitory concentration (MIC) (µg/mL)
Standard drug ampicillin (µg/mL)
Antibacterial activity of silver nanoparticle and silver ions are well known, but still, an exact mechanism of action is not precise. Different research groups have suggested various modes of bacteriostatic action. It is proposed that the bacterial cell death is due to the structural and morphological changes induced by AgNPs. When AgNPs comes in contact with the bacteria, they adhere to the cell wall and cell membrane. Once bound, some of the silver may penetrate through the cell wall and interacts with DNA and RNA and blocks the bacterial cell’s replication [2, 4, 29, 41]. Although the proposed mechanism varies for every type of cell, as there is great variation in their cell wall composition, AgNPs with an average size of 10 nm or less shows better interactions that significantly increase their bactericidal activity. It has been reported that the size of the particle significant role in antimicrobial activity [26, 29, 42]. The smaller particles can penetrate the cell wall and interact with the cell metabolites quickly. Here the excellent bactericidal activity of as-synthesized AgNPs is attributed to the smaller size and high surface to volume ratio.
3.8 Catalytic studies
Comparison of catalytic activity of the synthesized AgNPs towards the reduction of MO and RhB with reported catalytic systems
Rate constant (min−1)
Methyl orange (MO)
ZnO/carbon black-cellulose acetate
Rhodamine blue (RhB)
It is evident from the comparison that the clammy cherry stabilized AgNPs has competing catalytic efficacy towards the reduction reaction. The good catalytic activity is being attributed to the better stabilization, and small size of the AgNPs synthesized by Clammy cherry mediated reduction. Therefore, clammy cherry protected stable AgNPs based catalytic systems can be conveniently used for the effective removal of dye pollutants in the water bodies.
Here, the green synthesis of AgNPs by reduction of silver ions using clammy cherry extract under microwave irradiation is reported for the first time. The synthesis of AgNPs was comparatively faster and prepared AgNPs colloid was exceptionally stable for a considerably longer period of time. The formation AgNPs of the average size of 7.13 nm and narrow size distribution established that the polyfunctional molecules present in the clammy cherry extract are effective for reduction and proper stabilization of AgNPs. The clammy cherry reduced AgNPs exhibited excellent antibacterial activity, high catalytic efficacy, and better electrochemical response. It is concluded that the present study is highly promising as it could demonstrate a simple and fast synthesis method for the size and shape controlled AgNPs, having unique characteristics for biological, catalytic and sensing applications.
The authors would like to thank the financial assistance to Femina K.S. Granted by University Grants Commission (under Faculty Development Program: Grant No. FIP/12th Plan/KLMG 009 TF 12 dated 20/04/2017), Government of India. The authors would like to thank SAIF STIC and Biotechnology Department of CUSAT, and Kerala, India for characterization facilities. The authors thank Dr. Bindu Sarmila and Dr. Neena George, Maharajas College, Ernakulam, Kerala, India for their valuable suggestions during the manuscript writing.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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