Biogenic synthesis of silver nanoparticles and their antioxidant and antibacterial activity
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Nanomedicine utilizes biocompatible nanomaterials for diagnostic and therapeutic purposes. The present study reports the use of Helicteres isora root extract for the synthesis of silver nanoparticles (AgNPs). The synthesized AgNPs were initially noticed through visual color change from yellow to reddish brown and further confirmed by surface plasmonic resonance (SPR) band at 450 nm using UV–visible spectroscopy. Morphology and size of AgNPs were determined by transmission electron microscopy (TEM) analysis. X-ray diffraction (XRD) study revealed crystalline nature of AgNPs. The prolonged stability of AgNPs was due to capping of oxidized polyphenols and carboxyl protein which was established by Fourier transform infrared spectroscopy (FTIR) study. In addition, the synthesized AgNPs were tested for antioxidant and antibacterial activities. It showed good antioxidant activity as compared to butylated hydroxytoluene (BHT) and ascorbic acid as standard antioxidant. It could be concluded that H. isora root extract can be used efficiently in the production of potential antioxidant and antibacterial AgNPs for commercial application.
KeywordsGreen synthesis Silver nanoparticles Helicteres isora, antioxidant Antibacterial TEM & FTIR
Recent years researchers are interested on developing efficient method for the large-scale synthesis of nanoparticles (NPs). Nanomedicine is a rapidly developing and promising field that makes best use of inert metals like silver, gold and platinum to synthesize metallic nanoparticles with high therapeutic potential for various biomedical applications. Among all metal nanoparticles, silver nanoparticles (AgNPs) have much attention due to the surface plasmon resonance (SPR) (strong absorption in the visible region), which can be easily observed by UV–visible spectrophotometer (Krishnaraj et al. 2010). Silver with its potent antimicrobial activity has been used in the synthesis of silver nanoparticles which finds extensive use in the preparation of food processing, topical ointments and medical implants (Weiss et al. 2006; Wong 2012). Though the synthesis of silver nanoparticles has been carried out by various methods such those based on reduction in solution, chemical and photochemical reactions (Henglein 1998), decomposition of silver compounds by thermal method (Viet Quang and Hoai Chau 2013) and microwave-assisted process (Jiang et al. 2006), they involve the use of noxious chemicals. The green synthesis methods using plant extracts have been shown to be more advantageous owing to their simple methodology and eco-friendly nature (Muthukrishnan et al. 2015; Ramalingam et al. 2014; Kanipandian et al. 2014; Singh et al. 2013). Green synthesis of silver nanoparticles using various medicinal plants including, Acacia leucophloea (Murugan et al. 2014), Aegle marmelos (Nithya Deva Krupa and Raghavan 2014), Alstonia scholaris (Shetty et al. 2014), Solanum trilobatum, Syzygium cumini, Centella asiatica and Citrus sinensis (Logeswari et al. 2013), Crataegus douglasii (Ghaffari-Moghaddam et al. 2014) has been reported. Such green synthesized silver nanoparticles from Dillenia indica (Singh et al. 2013), Morinda pubescens (Inbathamizh et al. 2013), and Ceropegia thwaitesii (Muthukrishnan et al. 2015) have also been shown to exhibit in vitro antioxidant and antibacterial activities. With these evidences, this study was designed to synthesize AgNPs using aqueous Helicteres isora root extract and assess their antioxidant and antibacterial activity.
H. isora fruits are used as vermifuge, astringent, stomachic, vulnerary and useful in bowel gripes (Chopra et al. 1956). H. isora plant extracts possess anticancer properties (Mathew and Unnithan 1992). Usually, the root juice and bark were used against emphysema and diabetes. It is also used as expectorant, astringent, to condense gripping and a cure for snakebite (Kirtikar and Basu 1993; Singh et al. 1984). In traditional medicine, the root juice and bark are claimed to be useful in snake bite, diabetes, asthma, blood disorder, cough, colic, diarrhea, dysentery, stomach affections, intestinal infections, emphysema, and also as a urinary astringent (Shriram et al. 2008). The extract from the root and bark possess insulin-sensitizing, hypolipidemic activity and has the potential for use in the treatment of type-2 diabetes (Kumar et al. 2007). Moreover, the root extracts exhibited significant antihyperglycemic activity and the effect was comparable with that of glibenclamide (Venkadesh et al. 2004). Here, we report on the green synthesis of silver nanoparticles (AgNPs) from H. isora root extract, their physical characterization and their antioxidant and antibacterial activities.
Materials and methods
Roots of H. isora were collected from Western Ghats of Tamil Nadu, washed with sterile distilled water and dried, then make it powder using mortar and pestle. 1 g of root powder was mixed with 100 ml of water and kept on orbital shaker at 120 rpm for 12 h. After that, the extracts were filtered with Whatman No. 1 filter paper and stored at 4 °C in refrigerator until further use.
Synthesis of silver nanoparticles (AgNPs)
AgNPs were synthesized following the procedure of Geethalakshmi and Sarada (2010) with slight modification. AgNPs were synthesized by mixing aqueous AgNO3 solution (1 mM) and root extracts in the ratio of 1:1 and incubating the mixture at room temperature for 6 h. Following incubation, the AgNPs formed were collected by centrifugation at 18,000 rpm for 20 min. The collected pellet was washed three times with double distilled water, transferred to a Petri plate and dried at room temperature.
Characterization of AgNPs
The bioreduction of Ag+ ion in solution was monitored using UV–visible spectrophotometer (UV-160v, Shimadzu, Japan). The size distribution of synthesized AgNPs in solution was analyzed by DLS particle size analyzer [ZETA Seizers Nanoseries (Malvern Instruments Nano ZS)]. The studies on size, morphology and composition of silver nanoparticles were performed by transmission electron microscopy (JEOL JEM2100 TEM) and energy dispersive X-ray spectrum (EDX). The purified AgNPs were examined for the presence of biomolecules using FTIR spectrum (Thermo Scientific Nicolet 380 FT-IR Spectrometer) and crystalline nature of AgNPs was determined by X-ray diffraction (XRD) analysis.
Antibacterial activity of synthesized AgNPs was determined using disc diffusion method. The overnight inoculated bacterial cultures were spread over the freshly prepared Mueller-Hinton agar plates. The 6-mm sterile discs (Himedia) were kept on at Center of plate and different concentration of AgNPs (12.5, 25, 50 and 100 µg/mL) was poured on disc. The streptomycin disc (reference disc) was also kept on the plate incubated at 37 °C for 24 h. The antimicrobial property of AgNPs was determined by measuring the zone of inhibition around the discs in diameter (millimeter) after incubation.
In vitro antioxidant assays
DPPH free radical scavenging assay
Hydrogen peroxide scavenging assay
The H2O2 scavenging activity was assayed by the modified method (Pick and Mizel 1981). In brief, different concentrations (10, 20, 30, 40, 50, 75 and 100 µg/mL) of AgNPs and ascorbic acid (control) were mixed with 50 µL of 5 mM H2O2 solution (SD Fine Chem, Mumbai) and incubated at room temperature for 20 min. The absorbance was measured at 610 nm. The percentage of H2O2 scavenging was calculated using Eq. (1).
Nitric oxide radical scavenging assay
Nitric oxide radicals generated from sodium nitroprusside in aqueous at physiological pH interacts with oxygen to produce nitrite ions, which were measured by using the Griess reaction reagent was evaluated by modified method of Sousa et al. (2008). In brief, nitric oxide radicals, which were generated from 100 µl of 20 mM sodium nitroprusside, were incubated with 100 µl (10, 20, 30, 40, 50, 75 and 100 µg/mL) of AgNPs for 60 min, at room temperature. BHT and NO• scavenger were used as a positive control. Nitric oxide radical scavenging assay was calculated by Eq. (1).
Reducing power assay
The reducing power was determined by Oyaizu’s method (1986) with slight modification. In brief, different concentrations (10, 20, 30, 40, 50, 75 and 100 µg/mL) of AgNPs solution were mixed with 2.5 mL of phosphate buffer (200 mM, pH 6.6) and 2.5 mL of 1 % potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and then cooled rapidly. Subsequently, 2.5 mL of 10 % TCA was added with the above-mentioned solution and centrifuged at 3000 rpm for 8 min. The collected supernatant was mixed with equal amount of Millipore Milli-Q water. Finally, 1 mL of 0.1 % ferric chloride was added with the upper layer and the absorbance was measured spectrophotometrically at 700 nm. The obtained results were compared with BHT which was used as a positive control. The percentage of reducing power was calculated by Eq. (1).
Results and discussion
It is generally recognized that UV–Visible spectroscopy could be used to examine size and shape of controlled NPs in aqueous suspensions. This analysis showed the sharp absorbance at around 450 nm (Fig. 1a), which was specific for AgNPs. The UV–Vis absorption band in the current visible light region (420–450 nm) is an evidence of the presence of surface plasmon resonance (SPR) of AgNPs (Ramalingam et al. 2014; Muthukrishnan et al. 2015; Kanipandian et al. 2014). A single SPR band resembles to the spherical nanoparticles, whereas two or more SPR bands correspond to the anisotropic molecules (Krishnaraj et al. 2010). In the present study, two SPR band exhibited by the reaction mixture reveals the cubic shape (with Oh symmetry) of the AgNPs (Sands 1993). The intensity of the SPR peak increased with reaction time indicating the increasing concentration of AgNPs. The reduction was ascribed to the steroids, terpenoids, alkaloids, carbohydrate and phenolic compounds present in the extract (Suthar et al. 2009).
FTIR spectral analysis
Mechanism of reduction of AgNO3 to AgNPs by the phytoconstituents
Measurement of H2O2 scavenging assay
In living systems, uninhibited accumulation of H2O2 leads to the development of oxygen free radicals like peroxide and hydroxyl radicals which causes huge damage to cell membranes. The hydrogen peroxide scavenging activity of AgNPs was quantified spectrophotometrically using ascorbic acid as a standard and is shown in Fig. 7b. The concentrations at 100 µg/mL inhibition were found to be 93.31 and 85.35 % for the AgNPs and ascorbic acid, respectively. Interestingly, H2O2 free radical was consistently higher than those obtained for DPPH scavenging activity. Surprisingly, the AgNPs exhibited comparatively better reducing power than ascorbic acid due to the structure and characterization of the AgNPs. In the presence of hydrogen peroxide, the dispersed AgNPs can induce reactive oxygen species like hydroxyl radicals. Hydrogen peroxide inside a cell at a low dose can accelerate the dissolution of AgNPs and produce much stronger oxidative stress (He et al. 2012). AgNPs can produce greater accounts of hydrogen peroxide and induce greater inflammasome formation because they can cause stronger leakage of cathepsins from impaired lysosomes and efflux of K+ ions may contribute to the production of superoxide and hydrogen peroxide in the membranes of mitochondria (Yang et al. 2012). Our results are in good accordance with an earlier report on the H2O2 scavenging effect of leaf extract of Abutilon indicum (Mata et al. 2015).
Nitric oxide scavenging activity
Nitric oxide (NO·) is an important bioregulatory molecule in the nervous, immune and cardiovascular systems (Rees et al. 1989). The biosynthesized AgNPs showed a concentration-dependent activity in NO· scavenging activity and the best activity 80.46 % scavenging was observed at a higher concentration of 100 μg/mL (Fig. 7c). The above-observed NO activity was lesser than that of the standard BHT (81.35 %). It may be the interaction between AgNPs and nitric oxide (NO·) under anhydrous, anaerobic conditions at room temperature and the NO· radical which is very less stable with high electronegativity can easily accept electron from silver nanoparticles. (Rodriguez-Gattorno et al. 2002).
The reducing power
Figure 7d shows the dose-dependent response for the reducing powers of the biosynthesized AgNPs of root extracts. Reducing power was increased consistently with increasing the concentration of AgNPs. Surprisingly, the AgNPs exhibited comparatively better reducing power than standard (BHT) due to the presence of phytoconstituents in the extracts. However, these phytoconstituents like steroidal saponins also have electron-donating antioxidant capacity (Lin et al. 1996). This result was correlated with biosynthesized AgNPs of Iresine herbstii (Dipankar and Murugan 2012).
AgNPs have emerged as a typical antimicrobial nanomaterial applied in industry, daily life, and medicine. Due to the strong activity of AgNPs and release of Ag ions, the biological properties and safety thereof have attracted tremendous attentions from scientists in recent era. A simple, stable and eco-friendly method of biosynthesizing AgNPs was successfully developed using H. isora root extract. H. isora root contains more triterpenes that play major roles as reducing as well as capping agents for use in synthesis of AgNPs. The extract acts as both reducing and stabilizing agent which was confirmed by FTIR studies. TEM and XRD reports revealed that synthesized AgNPs were crystalline in nature with an average particle size of 30–40 nm. This biosynthesized AgNPs were found to be multifunctional with good antioxidant activities. This biosynthesized method facilitates best alternative for both chemical and other physical methods. Hence, this method can be employed in large-scale production and can be used in many medicinal and technological applications.
The work was financially supported by University Grant Commission-Rajiv Gandhi National Fellowship (UGC-RGNF) (No: F1-17.1/2013-14/RGNF-2013-14-SC-TAM-44942. (SA-III)) University Grant Commission New Delhi, India to the first author. We thank sophisticated analytical instrument facility (SAIF), North-Eastern Hill University (NEHU), Shillong for accessing TEM facility. The authors wish to thank the following individuals who provided valuable advice in the final stage of the revision process: V. Ramalingam, Research Scholar (Department of Marine science, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India), N. Kanipandian & KS Rajkumar (Research Scholars, Department of Animal Science, Bharathidasan University, Tiruchirappalli, 620 024, India).
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