Phyto‐mediated synthesis, biological and catalytic activity studies of gold nanoparticles

Abstract

In the present study, a phyto‐mediated synthesis of gold nanoparticles (AuNPs) using an isoflavone, Dalspinosin (5,7‐dihydroxy‐6,3′,4′‐trimethoxy isoflavone) isolated from the alcoholic extract of roots of Dalbergia coromandeliana is reported. It is observed that Dalspinosin itself acts both as a reducing and a capping agent in the synthesis of the nanoparticles (NPs). An ultraviolet–visible (UV–Vis) spectral study showed a surface plasmon resonance band at 526 nm confirming the formation of AuNPs. The NPs formed were characterised by UV–Vis spectroscopy, Fourier transform‐infrared spectroscopy, X‐ray diffraction (XRD), high‐resolution transmission electron microscopy (HR‐TEM) with energy‐dispersive x‐ray spectroscopy (EDX) and dynamic light scattering. HR‐TEM analysis showed the synthesised AuNPs were spherical in shape with a size of 7.5 nm. The AuNPs were found to be stable for seven months when tested by in vitro methods showed good antioxidant and anti‐inflammatory activities. They also showed moderate anti‐microbial activities when tested against Gram positive (Staphylococcus aureus and Streptococcus sp), Gram negative bacterial strains (Klebsiella pneumonia and Klebsiella terrigena) and fungal strain (Candida glabrata). The biosynthesised AuNPs showed significant catalytic activity in the reduction of methylene blue with NaBH₄ to leucomethylene blue.

1 Introduction

Life style modification and pollution in the environment create an imbalance between pro‐oxidants and antioxidants inside the cells, prompting to the surplus production of free radicals in the body [1]. Many different analytical studies have been attempted in the study of free radicals and reactive oxygen species (ROS) such as superoxide anion (O₂ ⁻), hydroxyl radical (OH^•), and hydrogen peroxide (H₂ O₂) which are harmful by‐products of cellular energy production. Free radicals are atoms, molecules or ions with unpaired electrons that are highly unstable and active towards chemical reactions with other molecules. Over production of free radicals during metabolism and other activities beyond the antioxidant capacity of a biological system leads to oxidative stress [2]. Excess free radicals attacking the immune system in the body play key roles in many degenerative diseases such as neurodegenerative disorders, cancer, aging, inflammatory and cardiovascular diseases, renal disorders and eye diseases [3, 4]. Free radicals are normally deactivated before attacking the cells by antioxidant enzymes present in the body [5]. Researchers have already reported that phytochemicals acting as reducing agents restrain the production of ROS thereby making phyto‐mediated nanoparticles (NPs) less toxic. Isoflavones such as Genistein and Daidzein isolated from soy exhibit significant antioxidant activities [6, 7]. The development of new resistant strains of microorganisms to current antibiotics has become a serious problem in the environment. Hence there is a strong incentive to develop new bactericides and this has received considerable attention among the researchers. Most of the polyphenolics isolated from the plants exhibit a potent activity against Gram‐positive bacteria [8]. NPs have a distinct property of exhibiting a larger surface area to volume ratio and gold NPs (AuNPs) are gaining great interest in environmental remediation nowadays [9]. Researchers are concentrating on the green synthesis of NPs of nobel metals viz. silver, gold, palladium and platinum to explore their applications in different fields like catalysis, optical sensors, biomedical applications and also in the treatment of some cancers [10, 11, 12]. The nano‐scale size of AuNPs offers small size, high surface area, and high surface reactivity. Combination of green chemistry approach with nanotechnology plays a vital role in nanoscience research as they are helpful in minimising the toxic methods in nano material synthesis [13]. Many conventional methods are available for the synthesis of AuNPs wherein gold cations (Au¹⁺ or Au³⁺) are reduced to zerovalent gold (Au⁰) with suitable reducing agents. Use of materials like plant extracts, algae, bacteria, fungi, viruses and yeasts for the synthesis of NPs is considered as an environmentally benign method as it does not include toxic chemicals [12, 14]. Medicinal plants have been found useful in the synthesis of AuNPs [15] and plant extracts containing bioactive alkaloids, polyphenols, phenolic acids, proteins, sugars, and terpenoids are believed to play an important role not only in reducing the metallic ions but also in stabilising them [16]. Green synthesised AuNPs are being used in the degradation of compounds in many chemical reactions. The catalytic potential of the AuNPs highly depends upon variation in their size [17, 18]. AuNPs show unique catalytic activities at mild conditions facilitating them to be considered as catalysts due to their higher stability and less poisoning when compared with other NPs [19]. Frequent exposure to hazardous pollutants from various industries to air and water makes the environment and the living organisms’ toxic [20]. Photocatalytic degradation is one of the best methods for detoxification of dyes and pollutants. In the present work, we have aimed to develop a green process for the preparation of AuNPs by using Dalspinosin, an isoflavone isolated from the roots of Dalbergia coromandeliana and our study revealed that Dalspinosin can very well act as a green reducing as well as a stabilising agent in the synthesis of AuNPs. The AuNPs synthesised found to be stable were characterised by spectroscopic techniques such as ultraviolet–visible (UV–Vis) spectroscopy, high‐resolution transmission electron microscopy (HR‐TEM) with energy dispersive spectrum (EDX) and selected area diffraction (SAED) pattern, X‐ray diffraction (XRD), Fourier transform‐infrared (FT‐IR) spectroscopy and dynamic light scattering (DLS). The in vitro antioxidant and anti‐inflammatory studies of the synthesised AuNPs revealed their good free radical scavenging and anti‐inflammatory activities. The AuNPs showed antifungal activity also. They showed effective catalytic activity in the degradation of organic pollutants like methylene blue (MB) also. The metal NPs were shown to be effective against both aerobic and anaerobic bacteria by precipitating bacterial cellular proteins and by blocking the microbial respiratory chain system.

2 Experimental methods

2.1 Materials

Hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄. 3H₂ O) purchased from Sigma Aldrich was used as such. MB, sodium borohydride, DPPH (2,2‐diphenyl‐1‐picrylhydrazyl hydrate), phosphate buffer, diclofenac and ascorbic acid were purchased from Merck, Mumbai, India. Millipore water was used to prepare all the solutions in the present investigation. The various chemicals utilised as a part of this present study were of analytical grade and used directly without any purification.

2.2 Plant collection, isolation and characterisation of Dalspinosin

D. coromandeliana (Family: Leguminosae), a shrub, identified by Dr R. Ramasubbu, Assistant Professor, Department of Biology, Gandhigram Rural Institute, Gandhigram was collected from Palamedu, Madurai District, Tamil Nadu and a specimen is kept in the Department of Chemistry, Gandhigram Rural Institute‐Deemed University for further reference. In the present study, the alcoholic extract of roots of D. coromandeliana was subjected to column chromatography over a silica gel column built in petroleum ether and elution of the column with ethyl acetate:petroleum ether (3:7, v/v) gave a cream coloured solid, identified as Dalspinosin (5,7‐dihydroxy‐6,3′,4′‐trimethoxy isoflavone) (DLS, Fig. 1) by UV–Vis, IR, ¹ H and ¹³ C nuclear magnetic resonance and liquid chromatography‐mass spectrometry studies [21].

Fig. 1.

Structure of Dalspinosin (DLS)

2.3 Synthesis of AuNPs

All the glass wares were thoroughly washed with a freshly prepared aqua regia (HCl:HNO₃, 3:1) and rinsed with Millipore water. Dalspinosin‐mediated AuNPs (DLS‐AuNPs) were prepared by mixing of HAuCl₄ ·3H₂ O (2.5 × 10⁻³ M) with Dalspinosin (2.7 × 10⁻⁴ M) in ethanol and 10 ml of Millipore water in a 100 ml conical flask with constant stirring. After 10 min the colourless solution turned red indicating the formation of AuNPs. Stirring was continued for another 15 min and it was stored in a refrigerator at 4°C for further studies.

2.4 Instrumentation

UV–Vis absorption spectra were recorded by using a Perkin Elmer Lambda 35 spectrophotometer. The reduction of Au ions in solution was observed in UV–Vis spectra. The FT‐IR spectra of both DLS and DLS‐AuNPs were observed for identification of different functional groups using a JASCO FTIR 400 in the range of 4000–400 cm⁻¹. The crystalline structure of AuNPs was confirmed by XRD analysis. The shape and size of DLS‐AuNPs were identified from HR‐TEM carried out using a JEOL JEM3010 operating at 200 kV. HR‐TEM samples are prepared by dropping the AuNPs on a copper grid. To find out the size distribution of AuNPs, the DLS measurements were carried out using Malvern (United Kingdom).

2.5 Free radical scavenging ability by DPPH method

DLS and biosynthesised DLS‐AuNPs were explored for their antioxidant activity by DPPH (1,1‐diphenyl‐2‐picryl‐hydrazil) radical scavenging according to the method of McCune and Johns [22]. The activity was measured at different concentrations (0.5–2.5 µg/ml) of DLS and DLS‐AuNPs individually mixed with 1.0 ml of DPPH in ethanol (0.3 mM) and 1 ml of ethanol and kept for 30 min at room temperature. The absorption was measured at 517 nm using a UV–Vis spectrophotometer in each case. A similar procedure was carried out simultaneously with a standard ascorbic acid. DPPH radical scavenging activity (RSA) was calculated by using the following equation:

$$\text{\%}\mathrm{RSA}=\frac{A_{\mathrm{Control}}-A_{\mathrm{Sample}}}{A_{\mathrm{Control}}}\times 100,$$

where A _control is the absorbance of the blank and A _Sample is the absorbance of the test sample.

2.6 Hydrogen peroxide (H₂ O₂) radical scavenging assay

The ability of hydrogen peroxide radical scavenging for DLS and DLS‐AuNPs was assessed by following the method described by Ruch et al. [23]. In this assay, freshly prepared hydrogen peroxide (40 mM) in phosphate buffer solution (50 mM, pH 7.4) was prepared and different concentrations (0.5–2.5 µg/ ml) of DLS and DLS‐AuNPs were added into 0.5 ml of H₂ O₂ solution individually. A blank was also performed with phosphate buffer without H₂ O₂. After 30 min the absorbance spectra were determined for all the samples at 230 nm. The percentage of H₂ O₂ scavenging activity was calculated by using the following equation:

$$\text{\%}\mathrm{of}{\mathrm{H}}_2{\mathrm{O}}_2\mathrm{scavenging}\mathrm{activity}=\frac{A_{\mathrm{Control}}-A_{\mathrm{Sample}}}{A_{\mathrm{Control}}}\times 100,$$

where A _control is the absorbance of the blank and A _Sample is the absorbance of the test samples.

2.7 Human red blood cell membrane stabilisation method (HRBC method)

The HRBC stabilisation method [24] was used for evaluation of the anti‐inflammatory activity of DLS and DLS‐AuNPs. Blood collected from healthy human volunteers was mixed with equal volume of Alsever's solution (0.8% sodium citrate, 2% dextrose, 0.5% citric acid and 0.42% sodium chloride) and centrifuged at 3000 rpm for 30 min and the packed cells were separated and washed with isosaline solution and a 10% suspension was made with isosaline. This HRBC suspension was used for the estimation of in vitro anti‐inflammatory activity. Different concentrations of DLS, DLS‐AuNPs, and control were mixed individually with 2 ml of phosphate buffer, 4 ml of hyposaline and 1 ml of HRBC suspension. The samples were incubated at 37°C for 45 min and centrifuged at 3000 rpm for 10 min. The supernatant liquids were decanted and the haemoglobin content was estimated for each test solution at 560 nm by using a UV–Vis spectrophotometer. Diclofenac was used as the standard and a control was performed omitting the test drugs. The % of inhibition was calculated using the following equation

$$\text{\%}\mathrm{of}\mathrm{Inhibition}=\left(A_{\mathrm{Blank}}-A_{\mathrm{Sample}}/A_{\mathrm{Blank}}\right)\times 100,$$

where A _Blank is the absorbance of the blank and A _Sample is the absorbance of the test sample.

2.8 Antimicrobial activity study by agar well diffusion method

The agar well‐diffusion method was followed to determine the antibacterial and antifungal activities. The antimicrobial activity of both DLS and DLS‐AuNPs was assessed by the agar well diffusion method [25]. In this method, nutrient agar and potato dextrose agar plates were spread with the help of an L‐rod inside the laminar air flow chamber with an 8 h old broth culture of bacteria and fungi. Wells with 6 mm diameter and about 2 cm apart were made in a medium of Petri plate each using a sterile cork borer. Stock solutions of the test drugs DLS/DLS‐AuNPs synthesised were prepared at a concentration of 100 µg/ml. The test drugs were added individually to the wells with the help of a sterile syringe and allowed to diffuse at room temperature (30°C) for 2 h. Ampicillin (100 mg/ml) served as a positive control. Control experiments comprising inoculation without test drugs were conducted and the plates were incubated at 37°C for 18–24 h for microorganisms. The diameter of the inhibition zone (mm) was measured by using an antibiotic scale (Himedia) and the activity index was calculated for triplicates of each experiment.

2.9 Catalytic activity of DLS‐AuNPs

The catalytic activity of synthesised DLS‐AuNPs was studied in the reduction of MB to leucomethylene blue (LMB). MB appears in the UV–Vis spectrum around 664 nm with a small shoulder at 614 nm in aqueous medium [26]. The catalytic activity of synthesised DLS‐AuNPs was established by mixing freshly prepared MB solution (2 mM) (0.2 ml) with a NaBH₄ solution (0.01 M) (0.5 ml) and 200 µL of AuNP catalyst at room temperature. The progress of rapid degradation of MB was studied by observing UV–Vis spectra taken at regular intervals.

3 Results and discussion

3.1 Characterisation of DLS‐AuNPs

Various plant species have been reported previously in the synthesis of AuNPs and the phyto‐constituents of the plants play an important role in the formation of NPs [27]. In the present investigation, AuNPs synthesised using aqueous HAuCl₄ in the presence of DLS showed a UV–Vis absorption maximum at 268 and 291 nm (Fig. 2 a), characteristic of an isoflavone [28]. Addition of DLS to AuCl₄ ⁻ solution resulted in a visual colour change from yellow to ruby red within 20 min indicating the formation of AuNPs. The change in colour arises due to the coherent oscillation of the electron around the surface of NPs resulting in surface plasmon resonance (SPR) [29, 30, 31]. The concentration of HAuCl₄ varied from 0.5 to 3 mM with a constant DLS concentration (2.7 × 10⁻⁴ M). The formation of DLS‐AuNPs was confirmed by the ruby red colour and the progress of the reaction was monitored by an UV–Vis spectrum (Fig. 2 b). Similarly, the concentration of DLS was varied from 0.7 × 10⁻⁴ to 3.7 × 10⁻⁴ M with a constant concentration of HAuCl₄ (2.5 × 10⁻³ M) which is shown in Fig. 2 c. From the above results, the optimised concentration of HAuCl₄ and DLS was 2.5 × 10⁻³ and 2.7 × 10⁻⁴ M, respectively. This result in lower λ _max at 526 nm (Fig. 3 a), which confirms the smaller size and high stability of the NPs formed. Moreover, the UV–Vis spectrum showed an SPR band for AuNPs at 526 nm and the reduction of AuCl₄ ⁻ ions reached saturation within 2 h of the reaction. The SPR band of AuNPs depends on the NP size, shape and medium of the reaction. Further continuous observation of the UV spectral behaviour of DLS‐AuNPs showed no appreciable changes for seven months and the ruby red colour of the solution remained the same showing the high stability of DLS‐AuNPs synthesised (Fig. 3 b). DLS not only reduced the gold salt (Au³⁺) to metallic gold (Au⁰) but also acted as a stabilising agent to prevent the aggregation of AuNPs.

Fig. 2.

UV–Vis absorption spectra of

(a) DLS, (b) Different concentration of HAuCl₄, (c) Different concentration of DLS

Fig. 3.

UV–Vis absorption spectra of

(a) DLS‐AuNPs, (b) DLS‐AuNPs freshly prepared and after seven months

The IR band intensities for DLS and also DLS‐AuNPs in different regions of the spectra were analysed (Fig. 4). The IR spectrum of the DLS showed a strong band at 3387 cm⁻¹ (stretching vibration for hydroxyl groups), a band at 2944 cm⁻¹ (alkenes) and a band at 1654 cm⁻¹ for the carbonyl group [32]. The band at 1473 cm⁻¹ represented the aromatic –CH stretching vibration. The FT‐IR spectra of DLS‐AuNPs depicted typical bands at 3424, 2925, 2841 and 1631 cm⁻¹. The characteristic bands of the DLS for OH and C=O at 3387 cm⁻¹ and 1654 cm⁻¹ are shifted to lower frequency regions (3424 and 1631 cm⁻¹) and became broader after the formation of DLS‐AuNPs [12]. The FT‐IR spectra of DLS‐AuNPs showed the disappearance of some bands (bands at 1274, 1170, 859 and 607 cm⁻¹), decrease in intensity of the band at 1631 cm⁻¹ with an appearance of the new band at 1719 cm⁻¹ corresponding to the carbonyl group as in aldehydic or ketonic component, respectively [33, 34]. The bands at 859 and 607 cm⁻¹ are related to the characteristics of C=CH₂ and C–H bending vibrations [35]. The bands observed at 1274 and 1170 cm⁻¹ are associated with the stretching vibrations for C–O groups from polyols. The total disappearance of bands at 1257 cm⁻¹ and 1170 cm⁻¹ after the formation of DLS–AuNPs can be attributed to the reduction of Au³⁺ ions coupled with oxidation of phenolic components of DLS [36, 37]. The carbonyl groups of the polyphenolic isoflavone DLS has a stronger ability to bind with Au ions, suggesting that polyphenolic compounds could form a coat over the metal NPs to prevent their aggregation [38, 39]. The linkage between carbonyl groups and hydroxyl groups with gold ion was proposed by using the FT‐IR spectrum. Phenolic compounds are known to have high electron donating property resulting in the formation of H radicals that subsequently reduce gold ions (Au³⁺) to nanosize (Au⁰) particles [40].

Fig. 4.

FT‐IR spectra of

(a) DLS, (b) DLS‐AuNPs

XRD analysis used to determine the crystalline structure of the AuNPs synthesised showed five clear Bragg reflections (Fig. 5) appearing at 38.28, 44.4, 64.7, 77.6 and 81.8° that could be indexed to (111), (200), (220), (311) and (222) set of lattice planes observed for face centred cubic gold, respectively (JCPDS file 04–0784) [41]. This result was also in agreement with the observation of the crystalline nature of the particles, shown by selected area electron diffraction (SAED) studies.

Fig. 5.

XRD pattern of DLS‐AuNPs

The average dimension of nanocrystalline size of the prepared AuNPs has been calculated using the Debye–Scherrer equation and is given as

$$D=\frac{K\lambda }{\beta \cos \theta },$$

where K is the Scherrer constant which depends on the how the width is determined, the shape of the crystal, and the size distribution (K  = 0.94), λ is the wavelength of the source (1.54060 Å in the case of CuK _α 1), θ is angle of emergence and β is the full width at half maximum intensity of the peak (in Rad). The nanocrystallite size of the prepared sample is found to be 11 nm using XRD which is in agreement with the particle size obtained from HR‐TEM [42].

HR‐TEM was used to determine the shape and size of the AuNPs [43] and the HR‐TEM images of the synthesised AuNPs (Figs. 6 a and b) showed that the shape of the AuNPs was almost spherical [44]. The histograms of AuNPs (Fig. 6 c) showed the average particle size to be 7.5 nm. The crystalline nature of the AuNPs was further evidenced by the SAED patterns (Fig. 6 d) showing bright circular spots corresponding to (111), (200), (220), (311) and (222) planes. Strong signals observed in EDX analysis (Fig. 6 e) in the gold region confirmed the presence of elemental gold in the AuNPs’ solution. By using a DLS particle size analyser, the average size of synthesised DLS‐AuNPs was found to be 11 nm (Fig 7) which is nearer to that of the HR‐TEM analysis. The stability of the DLS‐AuNPs was also tested in this study by HR‐TEM and the size and shape of the DLS‐AuNPs were examined after six months (Fig. 8). The results indicated that the prepared DLS‐AuNPs were highly stable in an aqueous medium and did not show any signs of aggregation after six months. Moreover, the size of the NPs was found to be 7.6 nm. These results were in good agreement with the results obtained from UV–Vis spectroscopy.

Fig. 6.

HR‐TEM images of gold nanoparticles at

(a) 50 magnification, (b) 5 magnification, (c) Particle size distribution histogram of AuNP, (d) SAED and (e) EDX images of AuNPs

Fig. 7.

DLS particle size analyser shows distribution of DLS‐AuNPs

Fig. 8.

HR‐TEM images of AuNPs after six months with different magnifications

(a) 50 nm, (b) 10 nm and (c) Histogram

3.2 Antioxidant assays

The DPPH radical assay has been used extensively as a model system to examine the radical scavenging activities of compounds. Several researchers have reported the antioxidant activity of isoflavones [45, 46, 47]. DPPH radicals were scavenged by DLS‐AuNPs through the donation of hydrogen from the antioxidant‐coated isoflavone molecules and reduced the formation of DPPH molecules. After the reduction of DPPH radicals, the colour of the solution was changed from purple to yellow, which was quantified by its decrease in absorbance at 517 nm. The DPPH scavenging assay showed effective inhibition activity for both DLS and DLS‐AuNPs at the different concentrations tested when compared with standard ascorbic acid (Fig. 9). It was observed that at a concentration of 2.5 µg/ml, DLS‐AuNPs showed the highest radical‐scavenging activity (88.3%) when compared with DLS (80.4%) and standard ascorbic acid (98.0%) at the same concentration. The DPPH activity of the NPs was found to increase in a dose‐dependent manner.

Fig. 9.

DPPH scavenging assay of DLS, DLS‐AuNPs and ascorbic acid (std)

The antioxidant ability of DLS‐AuNPs was determined by the H₂ O₂ scavenging method (Fig. 10) was found to be the highest (91.6%) at 2.5 µg/ml concentration of AuNPs when compared with ascorbic acid (95.4%). The results indicated that DLS‐AuNPs exhibited good scavenging effect on the H₂ O₂ radical and increase in the concentration of DLS‐AuNPs increased the percentage of inhibition showing that the antioxidant activity of DLS‐AuNPs behaved in a dose‐dependent manner.

Fig. 10.

H₂ O₂ scavenging assay of DLS, DLS‐AuNPs and ascorbic acid (std)

The antioxidant activity mechanism may possibly be due to the presence of poly phenolic groups in DLS and DLS‐AuNPs that could contribute electrons to hydrogen peroxide radicals neutralising them in the water.

3.3 Anti‐inflammatory activity of DLS‐AuNPs

The results of the in vitro anti‐inflammatory activity study indicated that both the test drugs DLS and DLS‐AuNPs showed significant anti‐inflammatory activity at various tested concentrations of 5, 10, 15, 20 and 25 µg/ml. While DLS‐AuNPs showed moderate anti‐inflammatory activity, DLS showed lesser activity than the standard drug (Fig. 11).

Fig. 11.

In vitro anti‐inflammatory assay of DLS, DLS‐AuNPs and diclofenac (std) by HRBC method

The neutrophil lysosomal constituents include protease and bactericidal enzyme, which upon extracellular biosynthesis create additional tissue inflammation and damage. From the results, it is clear that DLS‐AuNPs showed a maximum inhibition efficiency of 90.81% at 25 µg/ml while the standard drug showed a maximum inhibition of 94.15% at the same concentration. Although the precise mechanism of membrane stabilisation is not known exactly, it is possible that the test drug produced an effective surface area/volume ratio of the cells, which could be brought about by an expansion of the membrane or the shrinkage of the cells and an interaction with membrane proteins.

3.4 Antibacterial assay of DLS and DLS‐AuNPs

The results of an antimicrobial assay performed against five selected pathogens Staphylococcus aureus, Streptococcus sp., Klebsiella pneumonia, Klebsiella terrigena and Candida glabrata by the Agar well diffusion method given in Table 1 and presented in Fig. 12 showed that the zone of inhibition of DLS‐AuNPs is higher than that of DLS and maximum zone of inhibition of 17 mm was observed against K. terrigena and C. glabrata at 100 µg/ml concentrations of DLS‐AuNPs, while the standard ampicillin showed 38 mm. The exact mechanism of the bactericidal effect of AuNPs is not known clearly, but DLS‐AuNPs may attach to the surface of the cell membrane of the pathogen and disturb its power function such as permeability and respiration [25].

Table 1.

Antibacterial assay of DLS and DLS‐AuNPs by the agar well diffusion method

S. no.	Test drug	Concentration, μg/ml	Zone of inhibition, mm
			S. aureus	Streptococcus sp.	K. pneumonia	K. terrigena	C. glabrata
1	DLS	100	5	4	6	3	6
2	DLS‐AuNPs	100	12	13	16	17	17
3	ampicillin (std)	10	22	24	35	35	38

Fig. 12.

Antibacterial assay of

(a) Ampicillin (std), (b) DLS‐AuNPs, (c) DLS, (d) Control against S. aureus (I), Streptococcus sp, (II), K. pneumonia (III), K. terrigena (IV) and C. glabrata (V)

3.5 Catalytic activity of DLS‐AuNPs on reduction of MB

The catalytic efficiency of DLS‐AuNPs in the reduction of MB to LMB was studied in the presence of NaBH₄. MB exhibited an absorption peak at 664 nm [48, 49] corresponding to n –π * transition and a shoulder at 614 nm in an aqueous medium (Fig. 13). The catalytic reaction was monitored spectrophotometrically by the constant decrease in absorbance at 664 nm with regular time. The MB reduction as a function of time has been analysed in the presence of sodium borohydride and absorbance is shown in Fig. 13 a. Interestingly, a strong decrease of the UV–Vis absorbance intensity of MB (Fig. 13 b) observed within 15 min indicated the DLS‐NPs improved the reduction rate of MB to LMB suggesting that DLS‐AuNPs acted as a catalyst. The UV–Vis absorption spectra recorded at different time intervals over a period of 60 min for the reduction of MB by NaBH₄ alone the reduction of MB was very slow [50]; however, with DLS‐AuNPs acting as a catalyst, the intensity decreased abruptly suggesting a faster reaction rate for the reduction of MB to LMB. In this case, AuNPs acted as an electron relay, aiding in the transfer of electrons from the BH₄ ⁻ ions to the dye thereby causing a reduction in the intensity of the absorption.

Fig. 13.

UV–Vis absorption spectra for degradation of MB dye in the presence of

(a) NaBH₄, (b) NaBH₄ and DLS‐AuNPs and (c) kinetics spectra of MB

The reaction kinetics of reduction process is calculated using the following equation:

$$\ln (A_t/A_0)=-K_{a}t,$$

where A_t and A ₀ are the absorptions of dye at time t and initial absorption, respectively, K_a is the rate constant. Fig. 13 c shows the plot of ln (A_t /A ₀) versus time for catalytic degradation of MB by AuNPs, which follows pseudo first‐order reaction kinetics [51, 52] and rate constant obtained is 2.76 × 10⁻³ S⁻¹.

4 Conclusion

Isoflavones are antioxidant and anti‐inflammatory compounds with wide potential applications in the treatment of various diseases. The present phyto‐mediated synthesis of DLS‐AuNPs using DLS is a green, eco‐friendly, cost‐effective method taking place at ambient conditions. The findings of the present study showed the formation and stabilisation of the DLS‐AuNPs possessing potent therapeutic efficacy in terms of antioxidant and anti‐inflammatory activities and moderate antimicrobial activity against the selected Gram positive, Gram negative and fungi pathogens. The study also established the catalytic ability of DLS‐AuNPs in the reduction of MB to LMB by NaBH₄. The rate of degradation of the dye was found to be very slow in the absence of AuNPs and was enhanced considerably with the addition of AuNPs indicating the catalytic potential of DLS‐AuNPs. The kinetic studies revealed that the degradation of MB follows the pseudo first‐order reaction and rate constant found to be 2.76 × 10⁻³ S⁻¹. To our knowledge, this is the first report on the study of the synthesis and biological applications and catalytic activity of AuNPs synthesised using an isoflavone. This method of synthesis of AuNPs may pave way for newer applications in the field of medicine etc.