Biogenic synthesis of silver nanoparticles by leaf extract of Cassia angustifolia

The leaves of C. angustifolia used in the present study were procured from Vellalanvilai in Tiruchendur Taluk (Tamil Nadu) India. The dirt and other foreign materials were removed by thorough washing with tap water and finally with double-distilled water. To prepare the leaf extract 5 g of thoroughly washed and finely cut leaves were boiled in 100 ml of sterile distilled water for 5 min in a 250 ml Erlenmeyer flask and the solution was finally decanted. Fresh leaf extract was always used to do the experiments. All the experiments were carried out with this extract unless otherwise mentioned.

About 5 ml of the leaf extract was added to 100 ml of 1 mM aqueous silver nitrate solution (1:20 ratio) at room temperature. The formation of AgNPs was indicated by the development of its characteristic yellowish-brown colour. In the variation of pH, first the pH of the extract was adjusted before adding silver nitrate solution. In sunlight induced reactions, the extract was first exposed to sunlight for 10 min and silver nitrate solution was added to start the reaction.

UV–Vis spectral analysis was performed on a JASCO, V-530 spectrophotometer. TEM analysis was done on a PHILIPS, CM 200 instrument operated at operating voltages 200 kV, resolution 2.4 Å. FTIR spectrum was recorded with Thermo Electron Corporation, USA, Nexus 670 spectrometer centaurms 10 × . Particle size distribution was determined by photon correlation spectroscopy, which analyzes the fluctuations in light scattering due to Brownian motion of the particles, using a Zetasizer 1000 HS (Malvern Instruments, UK). Light scattering was monitored at 25 °C at a 90° angle. Particle size distribution studies were performed at a fixed refractive index of the respective formulation and also the stability of the prepared particles was assessed by measuring Zeta potential.

It is now well known that silver nanoparticles exhibit yellowish-brown colour in aqueous solution. This colour arises due to combined vibration of free electrons of silver nanoparticles in resonance with light wave, which give rise to a surface plasmon resonance (SPR) absorption band in the visible region of electromagnetic radiation [47]. The development of silver nanoparticles at different leaf extract quantities, silver nitrate concentrations, temperature, pH and contact time was monitored by UV–Vis spectroscopy by measuring the absorption of SPR band. Figure 2(a) shows the UV–Vis spectra recorded from the aqueous silver nitrate-C. angustifolia leaf broth reaction medium as a function of time.

Zoom In Zoom Out Reset image size

Colloidal silver nanoparticles of diameters (10–40 nm) give sharp peaks in the visible region. The reduction of the silver ions occurred within 10 min of mixing at room temperature, as indicated by the appearance of yellowish-brown colour. The silver SPR band is centred at around 420 nm and almost linearly increases in intensity with time of contact (figure 2(b) inset). The silver nanoparticles were stable in solution for 6 months of synthesis with a very small red shift in due course of time. The surface plasmon band at 420 nm indicates the presence of spherical nanoparticles [11], which was further confirmed by TEM analysis.

3.1.1. Effect of the quantity of leaves.

3.1.2. Effect of silver ion concentration.

3.1.3. Effect of volume of extract.

3.1.4. Effect of temperature.

3.1.5. Effect of pH.

To see the effect of pH on formation and the stability of AgNPs, the reactions were carried out at different pH ranging from 2 to 12 by adjusting with 0.1 M H₂SO₄ and 0.1 M NaOH in acid and basic medium, respectively. Synthesized NPs were stable in this range of pH. At pH 2 no colour was developed, at pH 4 yellowish brown colour was observed, with further increase of pH the colour became reddish brown and the intensity of the colour was also increased (figure 3(a)). AgNPs show high absorbance and narrow peaks at higher pH (pH 10 and 11), which may be due to the formation of monodispersed and smaller sized AgNPs.

Zoom In Zoom Out Reset image size

TEM of AgNPs synthesized using C. angustifolia at pH 6 are given in figure 4. TEM picture clearly shows that the silver nanoparticles are spherical in shape. Capping of silver nanoparticles by bio-component from the plant extract is visual from the pictures. The bio-component encapsulates AgNPs and forms a colony of approximately 10–15 silver nanoparticles and some of them consist of 4–6 silver nanoparticles. Most of the particles in the TEM pictures are not in contact with each other, but they are bound to the bio-organic material which is responsible for their stability.

Zoom In Zoom Out Reset image size

The bio-organic component of the C. angustifolia leaf broth forms a crystalline shell around the silver nanoparticles and makes them stable for 6 months. The selected area diffraction pattern (SAED) recorded from one of the nanoparticles confirms the crystalline nature of AgNPs. The average size of the AgNPs at pH 6 was found to be 42.4 nm.

FTIR spectrum was used to identify the possible biomolecules responsible for the reduction of the Ag⁺ ions and capping of the bio-reduced AgNPs. The silver nanoparticles were centrifuged at 10 000 rpm for 30 min to isolate the silver nanoparticles from plant materials or other compounds present in the solution, and the residue was collected, washed three times with double-distilled water and dried at room temperature in a Petri dish. The solid residue was scraped and mixed with KBr, ground and pressed into a clear disc which was mounted and examined directly. Figure 5 represents the FTIR spectrum of AgNPs synthesized using C. angustifolia leaf broth.

Zoom In Zoom Out Reset image size

The peak at 1037 cm⁻¹ can be assigned to –C–O [48], the peak at 1608 cm⁻¹ may be assigned to chelated carbonyl group or –OH from carboxylic group in the sennosides [49]. The band at 1510 cm⁻¹ may be due to –${\rm C}=\!\!\!={\rm C}$– from aromatic ring. The bands at 2875 and 2930 cm⁻¹ may be assigned to –C–H stretching. The band at 3413 cm⁻¹ can be assigned to –OH group.

Particle size distribution studies were performed at a fixed refractive index of the respective formulation, and also the stability of the prepared particles was determined by measuring their zeta potential. All samples were analyzed three times (triplicates). The average particle size of AgNPs formed at pH 6 was 42.4 nm and the zeta potential was –34.5 mV, whereas the average particle size in pH 11 was 21.4 nm and the zeta potential was –36.4 mV.

On adding aqueous leaf extract of C. angustifolia, silver nanoparticles were formed within 10 min as indicated by its signatory yellowish brown colour. On monitoring the formation of AgNPs with time by UV–Vis spectrophotometer, initially the rate of formation was rapid and tended towards attaining a constant absorbance, but the absorbance did not become constant and it was increasing almost linearly with time. The linear increase in absorbance with time appears to be the effect of sunlight. Since the reaction was carried out in daytime in the laboratory, the diffused sunlight might have some impact on the reaction. In order to prove this, the reaction was also studied in the presence of sunlight and it was noticed that the reaction was very fast in the sunlight (figure 6(a)).

Zoom In Zoom Out Reset image size

The active constituents of senna are anthraquinone derivatives such as sennoside A, B, C, D, aloe-emodin, rhein, emodin, chrysophanol, kaempferol and phytosterols [50]. Sennosides are glycosides of 1, 8-dihydroxyanthracene derivatives, among all sennosides A and B (figure 7) are active and present up to 8.5% in the leaves and seeds of different cassia species [51].

Zoom In Zoom Out Reset image size

In alkaline pH, the formation of AgNPs was instantaneous and an intense SPR band was observed. The increase of pH presumably facilitated the ionization of –OH and –COOH groups of biomolecules (probably sennosides) present in the extract. The thus formed –O^– could reduce Ag⁺ ions into AgNPs, which in turn got oxidized to –${\rm C}=\!\!\!={\rm O}$. The AgNPs thus formed are stabilized by –COO^– groups present in the molecules, which is evidenced by the better stability of particles formed under alkaline pH. The average particle size of nanoparticles formed in pH 11 was less (21.6 nm) than those formed under pH 6 (42.4 nm) which may be due to the instant reduction, and AgNPs thus formed do not get time to form clusters. Andreescu et al [52] reported a similar pH effect and instantaneous reduction at elevated pH. The AgNPs formed under alkaline pH are monodispersed with sperical shape (figure 8).

Zoom In Zoom Out Reset image size

This argument is further supported by UV–Vis spectra of the extract before and after reduction (figure 6(b)). The UV–Vis spectrum of extract has two peaks in the UV region, one at 266 nm and the other at 347 nm, whereas the UV–Vis spectrum after the formation of AgNPs has peaks at 263 nm and 417 nm. The intensity of peak at 266 nm is reduced and there is a minor shift in λ_max. π–π* and n–π* transitions generally occur around 266 nm and this indicates the presence of –OH and/ or –C = O groups in the biocomponent of the extract. In the UV region, substituted anthraquinones show intense quinonoid bands in a range from 260 to 290 nm and hydroxyl anthraquinones show an absorption band between 220 and 240 nm [53]. Most natural anthraquinones have complex structures with several substituents, which modify their absorption spectra. Ionization of a hydroxyl group results in a bathochromic shift [54]. Further, the decrease in intensity of these peaks indicates the involvement of this group in the reduction of Ag⁺ to Ag^o.

The AgNPs prepared using C. angustifolia were assessed for antimicrobial activities in two types of bacteria via Escherichia coli and Staphylococcus aureus. Among the two bacterial species the AgNPs inhibit the growth of S. aureus (22 mm) more effectively than E. coli (16 mm). The action of silver nanoparticles is broadly similar to that of silver ion. The slightly enhanced activity of AgNPs was due to effective contact of bacteria over a large surface area of the particles. Such a large contact surface is expected to enhance the extent of bacterial elimination.