Green synthesis and biological activities of gold nanoparticles functionalized with Salix alba

1 Introduction

Metals nanoparticles especially gold and silver have attracted considerable attention in catalysis, optics, sensing, imaging and biomedical devices (Jain et al., 2008). Various physical and chemical processes have been used in the synthesis of nanoparticles; however these processes generate a large amount of hazardous byproducts (Thakkar et al., 2010). Green nanotechnology, that uses biological organisms, plant biomass or extracts is recently considered as an alternative to the conventional chemical and physical methods for the synthesis of nanoparticles in a clean, non-toxic, ecologically sound and environment friendly manner (Wang et al., 2009). The use of plant materials for the synthesis of nanoparticles could be more advantageous, because it does not require elaborate processes as various biomolecules like proteins, phenols and flavonoids in plants play a vital role in the reduction of silver or gold ions (Sathishkumar et al., 2012). In recent years, several plants are successfully used for efficient and rapid extracellular synthesis of stable dispersions of silver and gold nanoparticles (Kumar and Yadav, 2009).

Willow (Salix alba L.), (syn: white willow) belongs to the family Salicaceae and grows primarily on moist soils in cold and temperate regions (Klasnja et al., 2002). Traditionally, it has been used for the treatment of various ailments due to its potent antipyretic, analgesic and anti-inflammatory properties. The Assyrians and ancient Egyptians used the leaves and bark for musculoskeletal pain (Mahdi et al., 2006). The efficacy of this plant is mainly due to its phenolic contents and includes salicylates such as salicin (Fig. 1), which is a precursor of salicylic acid that played an important role in the development of aspirin (acetylsalicylic acid). Other phenolic compounds isolated from S. alba are flavonoids (e.g. quercetin) and tannins (Harbourne et al., 2009). Willow contains certain metabolites which act as tumor inhibitors by causing apoptosis and damages the DNA as well as affect the cell membranes or denature proteins in various human carcinoma cells (El-Shemy et al., 2007). Gold nanoparticles using the bark extract of S. alba have been reported to have high potential as colourimetric sensor for selective recognition and monitoring of cysteine among other amino acids (Bahram and Mohammadzadeh, 2014).

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Figure 1

Chemical structure of salicin.

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The present study reports the green synthesis of gold nanoparticles functionalized with the leaves extract of S. alba (Au-WAs). The gold nanoparticles were characterized with UV–Visible (UV–Vis), Fourier transform infra-red spectroscopy (FTIR), atomic force microscopy (AFM) and scanning electron microscopy (SEM). The effect of sodium chloride (NaCl), pH and reaction temperature on the stability of the biosynthesized gold nanoparticles was studied. Moreover, the gold nanoparticles were screened for various biological activities.

2 Materials and methods

3 Results

3.1

3.1 Characterization of gold nanoparticles

Gold-willow solutions were made in ratios of 8:1, 9:1, 10:1, 15:1 and 25:1. From the recorded UV–Vis spectra of Au-WAs, it was observed that by varying the volume of gold solutions while keeping the volume of the plant extract constant, a change in the intensity of UV–Vis spectra was observed (Fig. 2). The 15 ml of 1 mM gold solution and 1 ml of plant extract (i.e. 15:1) gave a uniform and sharp peak at 540 nm. As shown in the inset of Fig. 2, the change in peaks intensities can be correlated with change in color from purple blue to pink and then finally to ruby red. The appearance of ruby red color indicated the formation of gold nanoparticles in the solution. Further studies were conducted on the 15:1 gold-willow solution.

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Figure 2

UV–Vis spectra of gold nanoparticles synthesized using different volumes of HAuCl₄ solution while keeping the concentration of Salix alba leaves extract constant. The inset photo shows the change in color with varying ratios of gold-extract solutions correspond to their absorption spectra.

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The AFM (Fig. 3) and SEM (Fig. 4) data revealed the average particle size of 63 nm and 50–80 nm respectively. The formation of gold nanoparticles was also confirmed by FTIR spectroscopy. FTIR spectra of both CEW and Au-WAs were recorded as shown in Fig. 5. FTIR spectrum of CEW has a broad peak at 3210 cm⁻¹ suggesting the presence of OH group which may be responsible for the reduction of Au³⁺ to Au⁰ where as two bands of lower intensities at 2908 cm⁻¹ and 2854 cm⁻¹ indicated the presence of methylene group (CH₂) next to OH or NH₂ functional group. Similarly, bands at 1589 cm⁻¹ and 1456 cm⁻¹ show the C⚌C stretching of an aromatic ring which may indicate the presence of benzene ring, a band at very low intensity at 1660 cm⁻¹ suggests the ortho substitution pattern of benzene ring and is supported by bands at 871 cm⁻¹ and 748 cm⁻¹. Bands at 1361 cm⁻¹ may be due to in plane bending vibration of OH of phenols, while the band at 1273 cm⁻¹ is due to C—O stretching of phenol. These bands are shifted to higher absorption due to conjugation of oxygen with benzene ring. Similarly, spectrum of Au-WAs stabilized with S. alba leaves extract shows a band at 1242 cm⁻¹ which correspond to —C—O—H groups while the bands at 1296 cm⁻¹ and 1635 cm⁻¹ correspond to amides of proteins.

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Figure 3

Atomic force microscopy images of optimized Salix alba gold nanoparticles.

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Figure 4

Scanning electron microscopy (SEM) images of optimized Salix alba gold nanoparticles at different resolutions (A and B).

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Figure 5

FTIR spectra of Salix alba leaves extract and its gold nanoparticles.

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3.2

3.2 Stability of gold nanoparticles

Fig. 6 shows a change in UV–Vis peak intensities with a corresponding color change due to the effect of varying pH on the stability of Au-WAs. The gold nanoparticles were quite stable in acidic medium; however, a gradual decrease in UV–Vis peak intensities showed less stability with an increase in pH of the colloidal solution. Similarly, in acidic pH, the solutions retained their characteristic ruby red color, however a deviation from this color was observed as the pH becomes alkaline. Peak broadening and red shift were noted at pH 12 and 13.

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Figure 6

UV–Vis spectra showing the effect of varying pH on stability of Salix alba gold nanoparticles. The inset photo shows the change in color with different pH correspond to absorption spectra of the gold nanoparticles.

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The effect of salt on the stability of Au-WAs was determined at different volumes of 0.1 M NaCl (Fig. 7). A gradual change of 0.1 M NaCl from 50 to 200 μl has little effect on the peak intensities of Au-WAs except for a decrease in their absorbance. At 250 and 300 μl, Au-WAs exhibited extreme peak broadening, therefore suggesting an aggregation of gold nanoparticles. As shown in the inset of Fig. 7, Au-WAs showed a color change from ruby red with a gradual change in the volume of 0.1 M NaCl solution.

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Figure 7

UV–Vis spectra showing the effect of different volumes of 0.1 M NaCl on stability of the gold nanoparticles. The inset photo shows the change in color of gold nanoparticles solutions with varying salt volume correspond to their absorption spectra.

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Fig. 8 shows the effect of temperature on the stability of Au-WAs. An extreme peak broadening with red shift and a decrease in absorbance were observed after heating Au-WAs for 30 min at 80 °C. This indicates an aggregation of gold nanoparticles after heating the colloidal solution and can be confirmed from the SEM images as shown in Fig. 9.

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Figure 8

UV–Vis spectra showing the effect of temperature on stability of the gold nanoparticles before and after heating at 80 °C for 30 min.

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Figure 9

Scanning electron microscopy (SEM) images showing the effect of temperature on stability of the gold nanoparticles before (A) and after (B) heating at 80 °C for 30 min.

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3.5

3.5 Acetic acid induced writhing test

As shown in Fig. 10, CEW and Au-WAs at the tested doses possessed significant (P < 0.001) antinociceptive activity. A dose dependant effect was observed for both CEW and Au-WAs. The percent analgesic effect of CEW was 66.45% at 50 mg/kg while it was 78.98% at 100 mg/kg. Similarly the percent attenuation of acetic acid induced writhing for Au-WAs was 72.87% and 80.76% at doses of 5 and 10 mg/kg respectively.

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Figure 10

Percent antinociceptive effect of Salix alba extract (CEW) and its gold nanoparticles (Au-WAs). Each bar represents mean ± S.E.M for a group of six animals. ANOVA followed by Dunnett’s post hoc test. ^∗P < 0.05 and ^∗∗P < 0.01 as compared to saline treated control group.

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4 Discussion

The synthesis and characterization of nanoparticles and their applications represent a rapidly growing concept and an emerging trend in science and technology (Gupta and Gupta, 2005). The use of plant materials for the synthesis of nanoparticles could be more advantageous, because it does not require elaborate processes (Song and Kim, 2009). In this study, gold nanoparticles were efficiently synthesized using the leaves extract of S. alba. The reaction between gold ions and S. alba extract was monitored by color change and UV–Vis absorption spectra. When a leaf extract solution of S. alba was added to 1 mM HAuCl₄·3H₂O solution, the color of solution changed from golden yellow to crimson red and then finally to ruby red at an optimized ratio of 15:1 (15 ml of 1 mM gold solution and 1 ml of S. alba leaves extract). The appearance of ruddiness color is indicative of formation of colloidal gold nanoparticles in the medium. Gold nanoparticles exhibit a ruby red color in aqueous media due to excitation of surface plasmon vibrations in the metal nanoparticles which give rise to surface plasmon resonance band centered at about 536 nm (Jensen et al., 2000). The plasmon resonance can be pictured as a ‘‘wave’’ of electrons sloshing over the surface of a metal nanoparticle. As a result, an enhanced electromagnetic field at and near the metal nanoparticle surface is set up. The position of the plasmon band (extinction spectrum) is best measured on a conventional UV–Visible spectrophotometer, and appears as a band with extremely high extinction coefficients (Murphy et al., 2008). In this study, it was observed from the UV–Vis spectra that the sharpness of absorption peak was dependent on the volume ratio of extract, thus being sharper with a higher ratio. No peak at a wavelength of 540 nm appeared for the S. alba extract however, in comparison, the gold nanoparticles functionalized with S. alba extract gave uniform and sharp peaks with varying intensities. It was noticed that the 15:1 ratio of gold solution and leaves extract in the reaction mixture is effective for the generation of gold nanoparticles as the UV–Vis peak becomes sharper and has maximum absorbance with SEM measured particle size ranging between 50 and 80 nm, which is in good agreement with the AFM analysis particle size measurement i.e. 64 nm. The UV–Vis spectra of Au-NPs at other ratios i.e. 8:1, 9:1, 10:1, 20:1 and 25:1 are either having lower intensities or exhibit broad peaks with red shifts at a wavelength corresponding to the plasmon resonance of gold nanoparticles (540 nm). This might be due to formation of large anisotropic particles or aggregation of nanoparticles in the medium. The surface plasmon resonance absorbance is extremely sensitive to the nature, size and shapes of the particles formed and their inter-particle distances (Sun and Xia, 2002). The size and concentration of gold nanoparticles can be determined directly from the UV–Vis spectra (Haiss et al., 2007; Murphy et al., 2008). Furthermore, the frequency of the plasmon band will differ from that of the simple isolated sphere if the nanoparticle is nonspherical in shape, and if it is close to other particles. Moreover, aggregation of noble metal nanoparticles red-shifts and broadens the plasmon bands (Murphy et al., 2008).

FTIR spectroscopic studies were carried out to investigate the possible capping and bio-reducing functional groups present in the extract. The FTIR spectrum of S. alba leaves extract shows absorption bands at 3210 cm⁻¹ (—OH or —NH₂), 2908 cm⁻¹ and 2854 cm⁻¹ (—CH₂), 1589 cm⁻¹ and 1456 cm⁻¹ (C⚌C stretching of an aromatic ring), 1361 cm⁻¹ (bending vibration of OH of phenols) and 1273 cm⁻¹ (C—O stretching). Likewise the FTIR spectrum of gold nanoparticles shows absorption bands at 1242 cm⁻¹ (—C—O—H), 1296 cm⁻¹ and 1635 cm⁻¹ that correspond to amide bands of proteins. This indicates that the gold nanoparticles synthesized using S. alba extract are surrounded by some proteins and metabolites having functional groups of amines, alcohols, ketones, aldehydes, and carboxylic acids. S. alba contains flavonoids like quercetin and tannis however, the major phenolic contents present is salicin. All these compounds contribute to the metal ion reduction processes and also stabilized the gold nanoparticles. The presence of salicin may be responsible for the formation of gold nanoparticles as this compound have hydroxyl group and glucosidal linkages which helps in the reduction of Au³⁺ to Au⁰ (Villanueva-Ibáñez et al., 2015). Flavanones could be adsorbed on the surface of metal nanoparticles, possibly by interaction through carbonyl groups or π-electrons in the absence of other strong ligating agents in sufficient concentration (Shankar et al., 2004). Moreover, proteins could possibly form a coat covering the gold nanoparticles, thus preventing agglomeration of the particles and provide stability in the medium (Nel et al., 2009).

Synthesis of stable nanoparticles is an important aspect of nanotechnology as the stability of nanoparticles plays an important role in modulating their biological properties (Gupta and Gupta, 2005). A reduction in stability can leads to total or partial loss of their nanoscale properties which may alters their cellular uptake and modifies their bioavailability and toxicity (Zhu et al., 2012). The most relevant processes that govern the stability and mobility of nanoparticles in the biological environment are agglomeration, aggregation, dispersion, sedimentation, and dissolution. These processes are dependent on the particle physicochemical properties that in turn are influenced by environmental parameters such as pH, temperature, ionic strength and presence of ligands or natural organic matter (Stankus et al., 2010; Levard et al., 2012). In this study, UV–Vis spectroscopy was used to efficiently evaluate the stability of gold nanoparticles. It was observed that the UV–Vis absorption spectra were significantly dependent on the size of gold nanoparticles with an increase in diameter, produced a dramatic red shift in their absorption spectra which might be due to shortening of distance or aggregation of gold nanoparticles. The gold nanoparticles were quite stable in pH range of 2–11 as there is almost no change in their UV–Vis spectra. This indicates their strong stability against either certain acidic or alkaline environments. The effects of NaCl concentration on the stability of gold nanoparticles were studied by using 0.1 M NaCl in quantities of 50–300 μl. It was observed from the UV–Vis absorption spectra that the S. alba mediated gold nanoparticles were relatively stable when the volume of salt increased from 50 μl to 200 μl. Their stability against varying volumes of salt can also be confirmed from the corresponding color of the gold nanoparticles solutions. Temperature is one of the important environmental factors that influence the stability, activity and chemical characteristics of materials (Vorkapic and Matsoukas, 1998). The effect of temperature on the stability of gold nanoparticles was investigated by heating the nanoparticles at 80 °C for 30 min. An increase in temperature facilitated aggregation of gold nanoparticles was observed as evidenced from the SEM images however, the gold nanoparticles still showed the typical plasmon resonance peak but with a red shift. Colloidal stability of nanoparticles is desired in most of the biological applications and in several other analytical applications (Dykman and Khlebtsov, 2012). Hence, understanding the stability under a wide range of environmental conditions, such as high salt concentration, extremes of pH, high temperature and buffer solutions is essential, which likely exhibits the fate of nanoparticles. The enhanced stability toward varying pH, ionic strength and temperature will enable us to explore different formulations of S. alba gold nanoparticles for potentially effective and safe therapy.

Considerable efforts are being made to develop and implement eco-friendly technologies for production of herbal based consumer products for providing better healthcare solutions (Chauhan et al., 2012). Gold and silver are frequently used for the synthesis of stable dispersions of nanoparticles using the extracts of herbs or other medicinal plants (Sadowski, 2010). In this study, the biosynthesized gold nanoparticles although devoid of antibacterial activity, showed excellent antifungal activity against A. solani, A. niger and A. flavus, while in comparison the S. alba extract was found to have moderate antibacterial activity but was inactive against these fungal strains. Gold nanoparticles possess well developed surface chemistry, chemical stability and a large surface to volume ratio due to which more number of drug molecules gets adsorbed on their surfaces via electrostatic attraction between the amine groups of drugs and nanoparticles. The gold nanoparticles surrounded by a number of drug moieties now act as a single group against the microbial organisms thereby increasing the microbial activity (Burygin et al., 2009).

S. alba is famous for analgesic effect due to the presence of salicin which have potent analgesic and anti-inflammatory properties. In this study significant antinociceptive effect was produced by the leaf extract of S. alba at doses of 50 mg/kg (66.45%) and 100 mg/kg (78.98%). Similarly, significant antinociceptive effect was also produced by the gold nanoparticles, but at much lower doses compared to that of leaf extract i.e. 72.87% and 80.76% at 5 and 10 mg/kg respectively. Moreover, the S. alba leaves extract (50 and 100 mg/kg) as well as the gold nanoparticles (5 and 10 mg/kg) produced significant muscle relaxant effect in both chimney and traction tests. These results suggest that the gold nanoparticles possess antinociceptive and muscle relaxant properties comparable to the leaves extract but at much lower doses. No sedative effect was produced by both S. alba leaves extract and its gold nanoparticles. A natural product having analgesic as well as muscle relaxant properties is beneficial as in majority of cases pain is usually accompanied with muscle stretching for which muscle relaxants are mostly prescribed along with analgesics. Pain and inflammation can result in unrelenting symptoms, misery, stress and sometimes disablement for the sufferer. Inflammation is the response to an injurious stimulus that can evoked by a wide variety of noxious agents. The classic inflammatory response includes warmth, pain, redness and swelling. Many mechanisms and mediators are involved in the promotion of the inflammatory process. Prostaglandins are important mediators of inflammation, fever and pain. They are synthesized by the constitutive enzyme, cyclo-oxygenase-1 (COX-1), and its isoform enzyme COX-2, which is induced in peripheral tissues by cytokines, growth factors and other inflammatory stimuli. Although in some situations prostaglandins contribute to pain by directly activating nociceptors, they are generally considered to be sensitizing agents. Prostaglandins increase the levels of cyclic AMP and may enhance nociceptor sensitization by reducing the activation threshold for TTX-R sodium channels via a protein kinase A pathway. They sensitize primary afferent neurons to bradykinin and other mediators and are likely to be involved at multiple sites along the nociceptive pathway (Kidd and Urban, 2001). The beneficial effect of willow plant to ease pain and reduce inflammation has been attributed to the presence of salicin among other active components including polyphenols and flavonoids. The presence of salicylates in willow provided foundation for the discovery of aspirin, the acetylated form of salicylic acid which subsequently leads to the synthesis of other effective analgesic and anti-inflammatory drugs i.e. non-steroid anti-inflammatory drugs (NSAIDs). Aspirin and NSAIDs inhibit the activity of COXs which inhibit the formation of prostaglandins and therefore relieve inflammation, swelling, pain and fever (Vane and Botting, 2003).

Keeping in view the well-known ethno-medicinal use of S. alba, the use of its gold nanoparticles is far better than prescribing two synthetic drugs for musculoskeletal pain as each drug has its own side effects. Furthermore, gold is usually prescribed for various joints pain conditions (Panyala et al., 2009). This added advantage along with its own potent analgesic and muscle relaxant properties compared to the leaves extract and diclofenac sodium, greatly promotes the use of S. alba gold nanoparticles as replacement herbal therapy for the clinical management of different pain conditions. Nanotechnological strategies change a substance’s properties and behavior in a biological environment and can potentiate the actions of plant extracts, promote sustained release of active ingredients, reduce the required dose, decrease side effects and improve activity (Bonifácio and da Silva, 2014). Drug delivery system fetched a novel drug delivery system, a novel approach to overcome the drawbacks of the traditional drug delivery systems (Ansari and Farha Islam, 2012). Green nanotechnology greatly promotes the development of biological medicine and bioavailability enhancement of herbal drugs (Bhadoriya et al., 2011).

5 Conclusions

We have developed a green method to synthesize gold nanoparticles using the leaves extract of S. alba that act as both reducing and stabilizing agent by avoiding the use of hazardous and toxic solvents. The nanoparticles are mostly in the size of 50–80 nm. FTIR analysis indicates the possible involvement of amines, amides and aromatic groups in the reduction process and may act as capping agents. Phytochemicals capping the nanoparticles make them colloidally stable in different media like salt and in various pH solutions but were relatively less stable at elevated temperature as revealed from their UV–Vis absorption spectra. The good antifungal activity along with significant attenuation of pain as well muscle relaxant effect of gold nanoparticles can be potentially applied in various products such as topical preparations. The process for the synthesis of nanoparticles in large scale using S. alba leaves extract may have several advantages such as cost-effectiveness and compatibility for biomedical and pharmaceutical applications.