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Comparative studies of sunlight mediated green synthesis of silver nanoparaticles from Azadirachta indica leaf extract and its antibacterial effect on Xanthomonas oryzae pv. oryzae
⁎Corresponding author. mounilbiotech@gmail.com (Mounil Mankad)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Biogenic synthesis of silver nanoparticle (AgNPs) has attracted worldwide attention as it is cheap and non-toxic. Therefore, in present study sunlight mediated silver nanoparticle was synthesized from Azadirachta indica A. Juss (Neem) leaf extract. Various combinations i.e., reducing agent (5, 10, 15 and 20 ml leaf extract), substrate concentration (1 mM AgNO3) and sunlight durations (5, 10, 15 and 20 min) were assessed for their ability to synthesized stabilized AgNPs. Leaf extract provide both reducing and capping agent, while sunlight served as catalyst for the synthesis process. The green synthesized AgNPs were characterized using change in color due to Surface Plasmon Resonance further analyzed by UV–visible spectroscopy, dynamic light scattering for size, polydispersity index and zeta potential for stability studies and Fourier Transform Infrared Spectroscopy (FT-IR). The smallest size of synthesized AgNPs was 67.94 ± 0.72 nm synthesized using to 20 ml of reducing agent (leaf extract) and 5 min of sunlight exposure. Antibacterial activity of synthesized silver nanoparticle was examined against plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) and showed a good antimicrobial activity compared to 200 mg/l of streptocycline.
Keywords
Sunlight
Azadirachta indica A. Juss (Neem)
Green synthesis
Silver nanoparticles
Dynamic light scattering
FTIR
Xanthomonas oryzae pv. oryzae (Xoo)
1 Introduction
Resistance develop by microbes against synthetic molecules has pose severe issue on the sustainability of these molecules to aid in protection to humans, plants and animals. Every higher living being is in continuous threat of invasion by the microbes. Plants are especially highly vulnerable to various microbial attacks due to constant exposure in environment. Rice is one of the most important cereal crop of world providing more than half a million their daily nutrition intake. Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most severe diseases of rice (Yoshimura and Tahar, 1960). It is also one of the oldest known diseases, first noted by farmers in Kyushu Province, Japan around 1884 (Swings et al., 1990). The disease increases with plant growth, peaking in the flowering stage, while symptoms are noted as early as at the tillering stage (Tagami and Mizukami, 1962). Streptocycline is an aminoglycoside antibiotic which has been extensively utilized in treatment of bacterial diseases of humans and animals (Sundin and Bender, 1993), is also used to prevent bacterial blight of rice (Xu et al., 2013). Development of resistance in Xanthomonas oryzae pv. oryzae against streptocycline was first reported by Shetty and Rangaswami (1971) which has become alarming situation for the crop pathologist to address this issue.
Nanotechnology is one of the emerging areas of research in the field of science. Nanoparticle show novel properties such as morphology of particles, size and distribution (Kaviya and Viswanathan, 2011). Metal nanoparticles have precise surface area due to its distinctive physicochemical characteristics that enhances antimicrobial, electronic, magnetic, catalytic and optical properties (Catauro et al., 2005; Crabtree et al., 2003; Krolikowska et al., 2003; Zhao, 1998). Synthesis of metallic nanoparticles have been carried out by three methods (i) chemical (ii) physical and (iii) biological. Advantages of chemical and physical methods includes precise synthesis, higher recovery and monodispersed particle synthesis. Several biological methods for both intracellular and extracellular nanoparticles synthesis have been reported by means of plants, fungi and bacteria (Mukherjee et al., 2001; Spring, 1995). Plants offers enhanced platform for nanoparticles synthesis because they are free from toxic chemicals and offers natural capping agents. Moreover, the use of plant extracts diminishes the cost of microorganisms isolation and maintenance (Singhal et al., 2011).
Silver is renowned for possessing an inhibitory effect against various bacterial strains and microorganisms usually present in industrial and medical processes (Jiang et al., 2004). In medicines, silver nanoparticles have abundant application including creams and skin ointments that prevent infection of open wounds and burns (Duran et al., 2005) medical implants and tools equipped with silver-impregnated polymers (RO, 1999). Synthesis of nanoparticles using plant extracts is the mainly opted procedure for eco-friendly and green synthesis of nanoparticles has a unique benefit that the plants are usually dispersed, easily accessible, much safer to handle and act as a source of various metabolites (Ankamwar et al., 2005).
Synthesis of silver nanoparticles by plants such as Ziziphora tenuior (Sadeghi and Gholamhoseinpoor, 2015), Solanum tricobatum (Logeswari et al., 2013), Erythrina indica (Sre et al., 2015), Spirogyra varians (Salari et al., 2016), carnivorous plants such as Drosera sp. and D. muscipula (Banasiuk et al., 2020), leaf extract of Acalypha indica with high antibacterial activities (Krishnaraj et al., 2010) and of Sesuvium portulacastrum also reported with nanoparticle size ranging from 5 to 20 nm (Nabikhan et al., 2010) are crammed in literature as a source for the synthesis of nanosilver particles as an substitute to the conventional methods. Leaf extract of Azadirachta indica (Neem) family Meliaceae has been used for bioconversion of silver ions into nanoparticles. Neem is commonly available in India, each and every part of this tree is been utilized as a household remedy against a variety of human ailments from ancient times including treatment against viral, bacterial and fungal infections (Omoja et al., 2011). The method used here is easy, cost effective, simple to perform and sustainable.
The reduction of Ag+ to Ag0 nanoparticles using neem leaf extract was due to the presence of phenolics, flavonoids, terpenoids, alkaloids, lipids, proteins and carbohydrates in the leaf extract (Kesharwani et al., 2009, Mittal et al., 2013). Thus, leaf extract plays dual role (i) reducing agent and (ii) capping agents which overcomes post modification of nanoparticle for stability. Kumar et al., (2015) reported synthesis of silver nanoparticles at room temperature for 120 min from various concentration of neem leaf extract as reducing agent and obtained minimum 146 nm size. Further reduction in time and size for nanoparticle synthesis is required thus, the present study aim to develop a novel method for silver nanoparticle synthesis that are stable with reduced size and consumes less time for synthesis. In this present work, AgNPs were synthesized using neem leaf extract as a source for nanoparticle formation and its antibacterial activity was monitored against Xanthomonas oryzae pv. oryzae.
2 Materials and methods
AR-grade silver nitrate (AgNO3) was purchased from Sigma-Aldrich, USA and fresh, mature neem leaves were collected from campus of Anand Agricultural University, Gujarat, India. Milli-Q water (18.2 MΩ) was used for the synthesis of silver nanoparticles.
2.1 Preparation of plant extract
Neem leaves extract was used for synthesis of silver nanoparticles on the basis of its availability, cost effectiveness and medicinal properties. Fresh leaves were collected from university campus and were cleaned with running tap water to remove dust particles followed by rinsing with double distilled water and air dried under sunlight for 6 h. 5% dried neem leaf extract was prepared by weighing 5 g of dried neem leaves in 100 ml of milli – Q water and boiled for 3 min. The extract was filtered using muslin cloth, cooled at room temperature and centrifuged at 6000 rpm for 10 min. The supernatant was used as extract for green synthesis of silver nanoparticles and stored at 4 °C for further use.
2.2 Effect of various substrate concentration and incubation time on synthesis of silver nanoparticles
1 mM silver nitrate solution was freshly prepared and 5, 10, 15 and 20 ml of neem leaf extract was added to 95, 90, 85 and 80 ml of 1 mM silver nitrate solution, respectively to make up final volume upto 100 ml. The treatments were kept under sunlight for different time period (5, 10, 15 and 20 min). Reduction of Ag+ to Ag0 was confirmed by the change in color of solution from yellowish to reddish brown.
2.3 Characterization of green synthesized silver nanoparticles
The bioreduction of the Ag+ ions was detected using UV–visible spectra of the samples. UV–visible spectroscopy was used for detection silver nanoparticles. The absorbance spectrum of the colloidal sample was obtained in the range of 200–700 nm, using a UV–visible spectrometer Beckman Coulter DU730. The size, PDI and zeta potential measurement were carried out using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., U.K.) All the measures were carried out three times and the data are represented in mean ± S.Em. Potassium bromide (KBr) pellet preparation was carried out using 1 mg of dried AgNPs and 100 mg of KBr and pellet was prepared using KBr press. The pellet was later subjected to FTIR Perkin Elmer Spectrum II spectroscopy for analysis. Scans in the range of 400–4000 cm−1 were collected for each spectrum at a spectral resolution of 4 cm−1.
2.4 Antibacterial activity of green synthesized silver nanoparticles
The antibacterial activity of the synthesized silver nanoparticles was assessed against bacterial plant pathogen, Xoo by agar well diffusion method (Nanda and Saravanan, 2009; Mankad et al., 2016). Briefly, inocula (100 µl) of overnight single colony bacterial suspension were spreaded onto the agar plates and were allowed to get solidified. A sterile cork borer four wells of 6 mm diameter were made on a nutrient agar plate and 100 µl of each different dilutions of silver nanoparticles solution (i.e., commercial silver nanoparticle (20 µg/ml), green synthesized AgNPs (10 and 20 µg/ml)) along with standard streptocycline (200 mg/l) as a control, followed by incubation of plates at 37 °C for 24 h. The green synthesized silver nanoarticles were dried overnight and desired dilutions of AgNPs were prepared in autoclaved Milli Q water. After incubation, the plates were observed for the zone of inhibition which was measured in terms of diameter (mm). Three test plates for each treatment were used and the results were expressed in mean ± S.Em.
3 Result and discussion
3.1 Characterization of silver nanoparticles
3.1.1 Effect of various substrate concentration and incubation time
Green synthesis of silver nanoparticles was carried out by mixing aqueous 1 mM AgNO3 and different volume of neem leaf extract i.e., 5 ml, 10 ml, 15 ml and 20 ml respectively under sunlight exposure for 5, 10, 15 and 20 min. With increasing sunlight exposure period, the color changed from yellow to reddish brown. The intensity of color increases in proportion to time due to reduction of Ag+ (Das et al., 2017). The change in color is due to the excitation of Surface Plasmon Resonance (SPR) in solution (Fig. 1a–d) (Mulvaney, 1996).
3.2 UV–visible spectrophotometry
Green synthesized silver nanoparticles were characterized by UV–visible spectrometry. The peak ranges between 420 and 440 nm (λ max) of different concentration of neem leaf extract (5, 10, 15 and 20 ml) with increasing exposure time (5, 10, 15 and 20 min) which corresponds to the absorbance of silver nanoparticles (Fig. 1a–d). From the studies carried out by Kumar et al., (2015) for silver nanoparticles synthesized from neem leaf extract, the maximum peak found at 435 nm which resembles to data obtained in current study. The resonance between the frequency absorption peak at 300 nm with a varying intensity and band width resulting from the varying size and size distribution of the particles. As far as different shapes in the UV–visible spectra above 600 nm are concerned it represents different extents of aggregation (Desai et al., 2012). The shift might be because of blue shift and depends on the particle shape and size (Kelly et al., 2003; Lee and Sayed, 2006). According to Njagi et al., (2011), the excitation of surface plasmon results in vibration of bands corresponding to the absorption by colloidal silver nanoparticles in the region (400–450 nm).
3.3 Dynamic light scattering (DLS)
The particle Z-average size (nm) and polydispersity index (PDI) of synthesized silver nanoparticles was determined using DLS measurements which are shown in Figs. 2 and 3, respectively. DLS is a quantitative analytical technique which measures the velocity of dispersed particle by measuring fluctuations of light scattering intensity due to Brownian movement of particles. Results show that size of nanoparticles is influenced by extract concentration and incubation time. Distribution of size ranges from 67.94 nm to 133.2 nm. The lowest size was recorded in 20 ml plant extract exposed for 5 min under sunlight. The nanoparticle synthesized at the end of 5 min exposure was found to be at par among all the plant extract concentration. Further, as the exposure time increases, the size of nanoparticle also increases. This clearly suggest that the exposure time has profound effect on particle size. In addition, the plant extract concentration used also plays important role in the conversion of Ag+ to Ag0. The highest deviation among the exposure and the plant extract was observed for 20 ml plant extract and followed in decrement concentration. Maximum size increase of 82.19% was recorded for 20 ml; followed by 57.17%, 40.62% and 19.78% for 15 ml, 10 ml and 5 ml, respectively. This deviation in particle size clearly suggest that at higher plant extract concentration, there is more presence of reducing agents present in the extract compared to reactant which tends to increase the particle size upon longer exposure duration. The present findings contradicts the earlier report by Habibi et al., (2017) that at higher concentration, size of nanoparticle tends to decrease. Further, the polydispersity index (PDI) obtained for different treatments ranges from 0.156 to 0.272 which clearly depicts the effectiveness of treatments for synthesis of stabilized nanoparticles.
3.4 Zeta potential (mV)
Zeta potential was carried out to study the stability of silver nanoparticles as it is very important for various applications. Criteria of stability of Nps are measured when the values of zeta potential ranged from higher than +30 mV to −30 mV (Zhang et al., 2008). This behavior of particles clearly suggests the presence of strong electric charges on the particle surfaces to hinder agglomeration. The values that fall in the negative side, showed the effectiveness of the capping materials in stabilizing the nanoparticles by providing intensive negative charges that keeps all the particles away from each other (Haider and Mehdi, 2014). The zeta potential for green synthesized silver nanoparticles range between −19.6 and −22.8 mV. The strong negative values for the AgNPs clearly suggest stability of nanoparticles at room temperature and therefore could be utilized effectively for its downstream applications like antimicrobial (Table 1).
| Treatments | Zeta potential (mV) (mean ± S.Em) | |
|---|---|---|
| Plant extract (ml) | Incubation time (minutes) | |
| 5 | 5 | −22.05 ± 0.64 |
| 10 | −22.00 ± 0.42 | |
| 15 | −20.25 ± 0.64 | |
| 20 | −22.45 ± 0.35 | |
| 10 | 5 | −21.90 ± 0.42 |
| 10 | −22.80 ± 0.14 | |
| 15 | −21.25 ± 0.92 | |
| 20 | −21.50 ± 0.28 | |
| 15 | 5 | −21.35 ± 0.21 |
| 10 | −21.25 ± 1.06 | |
| 15 | −19.60 ± 0.14 | |
| 20 | −20.25 ± 0.78 | |
| 20 | 5 | −22.35 ± 0.49 |
| 10 | −21.05 ± 0.35 | |
| 15 | −20.05 ± 0.21 | |
| 20 | −19.80 ± 0.56 | |
3.5 FTIR analysis
FTIR is highly informative technique for revealing the biomolecules present in plant extract which had played important role in formation and stabilization of nanoparticle (Senthilkumar and Sivakumar, 2014). The FTIR spectrum of green synthesized silver nanoparticles by the neem leaves extract, Fig. 4 shows strong spectra at 3415, 1578 and 1384. A broad peak at 3415 cm−1, corresponds to stretching vibrations of hydroxyl (—OH) group while a peak at 1384 cm−1 is attributed due to O—H bending vibrations of polyols present in leaf extract like flavanoids. Flavanoids and terpenoids absorbed on metal nanoparticles surfaces may be due interaction of carbonyl groups or π-electrons in absence of optimum ligating agents (Gericke and Pinches, 2006). Further, possible reduction of metal ions from bulk to nano scale by terpenoids occurs through oxidation of aldehyde groups to carboxylic acids (Ali et al., 2015). The spectral bands (1450–1600 cm−1) show presence of proteins which are accountable for the reduction of metal ions or affinity for metal nanoparticles. From the study of the FTIR spectrum, carboxyl group were found adsorbed on the particles surface, hence; this confirms the presence of biomolecules like terpenoids, flavonoids which acts as a capping agent for the synthesized nanoparticles. Capping of nanoparticles by protein stabilizes silver nanoparticles and prevents agglomeration in the medium (Lalitha et al., 2013). Over the years, stability of nanoparticles is one of the most important factors limiting usage of these nanoparticles. These issue is generally addressed through binding of stabilizing agents like citric acid (Hindi et al., 2009) and polyvinylpyrrolidone (Van der Zande et al., 2012). However, natural coating by biomolecules present in leaf extract is one of the effective alternative to overcome these post-synthesis modification as reported by Qu et al. (2014) and Ali et al. (2015) using Agrimoniae herba and Eucalyptus globulus leaves extracts, respectively.
3.6 Antibacterial activity of green synthesized silver nanoparticles
AgNPs has shown good antibacterial activity against wide bacterial species which has drawn attention of several researchers to evaluate and assess these nanoparticles for control of various diseases including crop diseases. Because of extremely smaller size, these particles could be effectively utilized in control of microbes without developing resistant microbes (Zhang et al., 2016). The zone of inhibition (mm) was found higher for most of the green synthesized AgNPs as compared to antibiotic streptocycline, as shown in Table 2. From the results it can be established that the maximum zone of inhibition was 30.50 ± 0.41 mm for synthesized 20 µg/ml AgNp from 5 ml to 10 min and 10 ml-15 min. In addition, good antimicrobial activity was also reported at 10 µg/ml for two treatments 5 ml–10 min (27.00 ± 2.45 mm) and 10 ml- 15 min (21.50 ± 0.41 mm). Further, the mean antimicrobial effect of green synthesized AgNPs for various treatments are also depicted in Table 2. The zone of inhibition was found to be more for the treatments with 5 ml of extract and which tends to decreases as the concentration of plant extract utilized for synthesis increases (Fig. 5). This clearly depicts that green synthesis of silver nanoparticles using lower concentration of plant extract has clear advantages for both size as well as its antimicrobial efficacy. Compared to currently utilized streptomycin concentration (600 µg/ml) for control of bacterium (Xu et al., 2010), the green synthesized AgNPs possess excellent antimicrobial activity even at lower concentration. Maximum and minimum zone of inhibition for X. campestris was found to be 24.8 ± 0.1 mm and 12.2 ± 0.1 mm, respectively using red algae mediated green synthesized AgNPs (Vadlapudi and Amanchy, 2017).
| Treatment Code | Treatments | Commercial AgNPs (20 µg/ml) | Streptocycline (200 mg/l) | Green synthesized AgNPs | General Mean of treatment | |||
|---|---|---|---|---|---|---|---|---|
| Plant extract (ml) | Incubation time (minutes) | 20 µg/ml | 10 µg/ml | 20 µg/ml | 10 µg/ml | |||
| S1 | 5 | 5 | 00.00 ± 0.00 | 21.00 + 0.41 | 28.00 ± 0.82 | 24.00 ± 0.00 | 29.38 ± 0.92 | 23.75 ± 0.81 |
| S2 | 10 | 30.50 ± 0.41 | 27.00 ± 2.45 | |||||
| S3 | 15 | 29.50 ± 0.41 | 21.50 ± 0.41 | |||||
| S4 | 20 | 29.50 ± 2.04 | 22.50 ± 0.41 | |||||
| S5 | 10 | 5 | 25.00 ± 0.00 | 20.50 ± 0.41 | 27.75 ± 0.61 | 20.5 ± 0.20 | ||
| S6 | 10 | 28.00 ± 1.63 | 20.00 ± 0.00 | |||||
| S7 | 15 | 30.50 ± 0.41 | 21.50 ± 0.41 | |||||
| S8 | 20 | 27.50 ± 0.41 | 20.00 ± 0.00 | |||||
| S9 | 15 | 5 | 25.00 ± 0.00 | 20.00 ± 0.00 | 24.12 ± 0.30 | 18.75 ± 0.41 | ||
| S10 | 10 | 22.50 ± 0.41 | 15.50 ± 0.41 | |||||
| S11 | 15 | 24.50 ± 0.41 | 19.00 ± 0.82 | |||||
| S12 | 20 | 24.50 ± 0.41 | 20.50 ± 0.41 | |||||
| S13 | 20 | 5 | 21.50 ± 1.22 | 19.50 ± 0.41 | 22.00 ± 0.61 | 18.62 ± 0.30 | ||
| S14 | 10 | 21.50 ± 1.22 | 20.00 ± 0.00 | |||||
| S15 | 15 | 25.00 ± 0.00 | 20.50 ± 0.41 | |||||
| S16 | 20 | 20.00 ± 0.00 | 14.50 ± 0.41 | |||||
4 Conclusion
A simple, rapid and environmental friendly method for synthesis of silver nanoparticles was developed using sunlight and leaf extract of neem. Initial silver nanoparticle synthesis was confirmed by using change in color from light yellow to reddish brown. Furthermore, UV–visible spectroscopic analysis was also conducted which resembles the reported peak in between 420 and 440 nm. The size of green synthesized silver nanoparticles was monitored using Zeta sizer and 5 min of sunlight exposure was found to be optimum for the least particle synthesis irrespective of plant extract concentration. Further, the present study for the first time successfully demonstrates the effect of plant extract and clearly showed that the plant extract at higher concentration is not effective in reducing the particle size. Zeta potential was performed to check the stability study of green synthesized silver nanoparticles, and it was observed that for all the treatments a strong negative zeta values was recorded which confirms the effectiveness of plant extract and the green synthesized method developed in the present study. Presence of functional group that are responsible for synthesis and stability of silver nanoparticle (capping agent) from neem were also identified. Antibacterial activity of synthesized silver nanoparticle was optimal at 5 ml plant extract and 10 min sunlight exposure treatment.
Competing interest
The authors declare that they have no competing interest.
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