Indigofera tinctoria leaf extract mediated green synthesis of silver and gold nanoparticles and assessment of their anticancer, antimicrobial, antioxidant and catalytic properties

Abstract

This study reports the synthesis of noble metal nanoparticles, namely silver and gold from their respective salt by leaf extract of a medicinal plant Indigofera tinctoria. This leaf extract plays a dual role as stabilizing and reducing agent for the formation of nanoparticles. The synthesized silver and gold nanoparticles were characterized by UV-vis. spectroscopy, FTIR spectroscopy, XRD, TEM, EDX and AFM analysis. All these techniques confirm the formation of crystalline nanoparticles. The cytotoxic effect of I. tinctoria leaf extract and the nanoparticles were studied on lung cancer cell line A549. It was shown that the cell viability decreases with increasing concentration and nanoparticles has more toxic effect on cancer cell than the pure leaf extract. IC₅₀ value of I. tinctoria leaf extract, AuNP and AgNP respectively, are 71.92 ± 0.76 μg/ml, 59.33 ± 0.57 μg/ml and 56.62 ± 0.86 μg/ml. Antimicrobial activities were tested against both bacterial and fungal strains by agar well diffusion method. The synthesized nanoparticles show high antimicrobial activities towards all tested microbial strains with varying degree. The antioxidant activities of synthesized nanoparticles were analysed by using DPPH method and found that nanoparticles show higher antioxidant activities than the leaf extract. Outstanding catalytic activities of nanoparticles were demonstrated by employing the reduction reactions of o/p-niroanilines by NaBH₄.

Keywords:

• Indigofera tinctoria
• green synthesis
• anticancer
• antimicrobial
• antioxidant
• catalysis

Introduction

Over a last few decades, synthesis and characterization of metal nanoparticles gained numerous attention because of their peculiar properties compared to their bulk counterparts. The major reason for its peculiar properties is their high surface to volume ratio [1]. Among the metal nanoparticles, the noble metal nanoparticles like silver and gold have most promising properties because of their excellent physical and chemical properties. Various methods like chemical, sono chemical, electrochemical, photochemical and micro emulsion, etc. methods are used for their production [2,3]. Many of the methods need complex reaction conditions and several toxic chemicals, which result in many pollution problems, thus these methods are not eco-friendly. Hence, researchers have been trying to develop environmental friendly method for the synthesis of nanoparticles.

In recent times, bio-inspired synthesis emerged as popular method for the synthesis of metal nanoparticles. Biological resources like bacteria [4], fungi [5], yeast [6] and plant extracts [7] were used for this purpose. Use of these resources is expected to reduce metal salt into its nanosized particles. A large number of research papers in the literature have reported the biosynthesis of silver and gold nanoparticles [8]. Among these the plant-based synthesis is very simple, fast, green and does not required any specific conditions as in other methods.

Compared to chemical method, biological method is time-consuming. The microwave-assisted technology overcomes these problems and retains green reaction conditions. This method is very fast, requires less energy and gives better product yield. Uniform heating occurs during microwave irradiation. Several reports are available in the literature describing the plant mediated synthesis of nanoparticles using microwave-assisted method [9].

In this study, we introduce a new medicinal plant namely Indigofera tinctoria for the synthesis of silver and gold nanoparticles employing fast microwave-assisted method. Nanosynthesis using I. tinctoria leaf extract has not been reported in the literature even though it is widely used in traditional medicines. I. tinctoria, a part of Fabaceae family, is enriched with antitoxic, hemostatic and sedative properties. It is used in the treatment of cancer, piles, chronic bronchitis, asthma, healing of ulcers and dropsy. For promoting hair growth, I. tinctoria’s roots, stems and leaves are used [10]. From the leaf extract, indirubin and indigtone are obtained, which are being used in the treatment of hydrophobia [11]. Photochemical analysis shows that aqueous extract of this plants contains alkaloids, amino acids, flavonoids, saponins, steroids, glycosides, carbohydrates, tannins, phenolic group and proteins, which are responsible for the antioxidant, antimicrobial and anticancer activities [12].

By using MTT assay, the in vitro anticancer activities of synthesized silver and gold nanoparticles were studied against lung cancer cell line A549. The antimicrobial studies were carried out against both gram positive, gram negative bacterial strains and fungal strains by agar well diffusion method. The antioxidant activities of synthesized silver and gold nanoparticles are carried out by DPPH assay using ascorbic acid as standard. The catalytic activities are analysed by using them in the reduction of o-/p-nitroanilines by NaBH₄.

Materials and methods

Materials

Silver nitrate (AgNO₃), o-nitroaniline, p-nitroaniline, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid and sodium borohydride (NaBH₄) were obtained from Merck Chemicals, India. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄ 3H₂O) was obtained from Sigma Aldrich. All chemicals were used without further purification and all aqueous solutions were prepared in double distilled water.

Plant collection and preparation of the aqueous leaf extract of I. tinctoria

The fresh leaves of I. tinctoria were collected and authenticated. It was washed thoroughly with running tap water and then with double distilled water to remove any dust and other impurities adheres on it. The leaves were chopped into small pieces and boiled with 100 ml double distilled water for 20 min in a round bottom flask fitted with a condenser. The obtained extract was cooled and filtered through Whatman No. 1 filter paper and the filtrate were collected and stored in 250 ml Erlenmeyer flask at 4 °C in refrigerator for further studies.

Synthesis of silver and gold nanoparticles

Silver and gold nanoparticles were synthesized with the aid of microwave irradiation. For the synthesis, 10 ml of I. tinctoria leaf extract were mixed well with 90 ml of 1 mM silver nitrate (AgNO₃)/HAuCl₄ 3H₂O solution in a 250 ml beaker. Then this solution was placed in a domestic microwave oven (Sharp R-219T). Its operating power and frequency were 800 W and 2450 MHz. The formation of nanoparticles was monitored by using UV-vis. spectrophotometer in the range 200–800 nm. The synthesized silver and gold nanoparticles were respectively represented as AgNP- tinctoria and AuNP- tinctoria. The above-mentioned reactions also carried out in the room temperature without the microwave irradiation.

In vitro anticancer study

Cell line and culture conditions

A549 (lung carcinoma) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen). The cell line was cultured in tissue culture flask with DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate, antibiotic solution (penicillin (100 U/ml), streptomycin (100 μg/ml) and amphoteracin B (2.5 μg/ml)). Then, it kept at 37 °C in a humidified 5% CO₂ incubator. The viability of cells were evaluated by direct observation of cells by inverted phase contrast microscope and followed by MTT assay method. Two-day-old confluent monolayer of cells were trypsinized and the cells were suspended in 10% growth medium, 100 μl cell suspension (5 × 10⁴ cells/well) was seeded in 96 well tissue culture plate and incubated (37 °C, 5% CO₂).

MTT assay

After attaining sufficient growth of the cells, the growth medium was removed, freshly prepared samples in 5% DMEM were five times serially diluted by two fold dilution (100 μg, 50 μg, 25 μg, 12.5 μg, 6.25 μg in 100 μl of 5% MEM) and each concentration of 100 μl were added in triplicates to the respective wells and incubated for 24 h (37 °C, 5% CO₂). After incubation entire plates were observed using inverted phase contrast tissue culture microscope (Olympus CKX41 with Optika Pro5 CCD camera) and microscopic observation were recorded as images.

After 24 h of incubation period, the sample content in wells were removed and 30 μl of reconstituted MTT (5 mg/ml in PBS) solution was added to all test and cell control wells, the plate was gently shaken well and incubated for 4 h. MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazole and in living cells it is reduced to purple formazan. Therefore, there is a direct relationship between the formazan produced and the number of viable cells. After the incubation period, the supernatant was removed and 100 μl dimethyl sulphoxide (DMSO) was added into the wells and mixed well to solubilize the formazan crystals. The absorbance values were measured by using microplate reader at a wavelength of 570 nm [13,14].

The percentage of cell viability was calculated using the formula: (OD = optical density)

Cell viability (%) = ([OD of the control − OD of the test]/[OD of the control]) × 100.

Antimicrobial study

The antimicrobial activities of synthesized nanoparticles were done by using “agar well” diffusion method [15]. Petri plates containing 20 ml media (nutrient agar for bacteria and potato dextrose agar for fungi) were seeded with tested microorganisms. Wells of 6 mm diameter were created using a sterile cork borer. Then samples (0.1 ml) were added, then fungi were incubated at the room temperature (1 week) and bacteria were incubated at 37 °C (24–48 h). The zone of inhibition were formed around the well, by measuring its diameter the antimicrobial activities were assayed. The distilled water was used as control.

Antioxidant study – DPPH assay

DPPH radical scavenging assay for synthesized AgNP and AuNP were carried out by according to the procedure by Chang et al [16]. Ascorbic acid was used as reference. First, 2.96 ml DPPH (0.1 mM) solution was added to different concentration of samples (12.5 μg/ml – 200 μg/ml) prepared in DMSO. This reaction mixture was incubated at room temperature in dark condition for 20 min. After 20 min, the absorbance of the mixture was measured in a UV-vis. spectrophotometer at 517 nm. DPPH was used as control. The percentage of inhibition can be calculated using the following equation: [(Absorbance_control − Absorbance_sample)/(Absorbance_control)] × 100. The mean and SD were calculated by repeating all the experiments thrice.

Catalytic studies

Reduction of o- and p-nirtoanilines

Reduction reactions of o- and p-nitroaniline were carried out in a quartz cuvette of 1 cm path length. 2.5 ml of 0.06 mM solution of nitroaniline (o-/p-) was taken and mixed well with 0.5 ml of ice-cold 0.06 M NaBH₄ solution. To this mixture, 0.5 ml of 0.02 mg/ml of AgNP- tinctoria/AuNP- tinctoria solution was added and shaken well. By using a UV-vis. spectrophotometer, the time-dependent UV-vis. response of the reduction reaction was analysed in the range 200–800 nm.

Statistical analysis

All the biological analysis was carry out in triplicate and the final values were presented as the mean ± SD. One way analysis of variance (ANOVA) was performed followed by Tukey’s test (using the GraphPad Prism 5.0 software). p < .05 was considered statistically significant.

Characterizations

Several techniques were used to characterize the synthesized silver and gold nanoparticles. For the UV-visible spectral studies Shimadzu UV-2450 spectrophotometer was used in the range of 200–800 nm. The phytochemicals that are involved in metal reduction and capping were identified by FTIR spectroscopy (Perkin Elmer-Spectrum Two spectrometer in the range 500–4000 cm⁻¹). In order to determine the crystalline nature, XRD analysis was carried out on Bruker AXS D8 Advance X-ray Diffractometer with Cu radiation (λ = 1.5406 A°). Transmission Electron Microscopy (TEM) (JEOL JEM-2100) was used to determine the particles size and morphology. EDAX (for determining elemental composition) were also taken on same instrument.

Results and discussions

Synthesis of silver and gold nanoparticles and UV-vis. spectral analysis

UV-vis. analysis plays an important role for the characterization of metal nanoparticles. Due to the interaction of conduction electrons of metal nanoparticles with incident photons, sharp and intense surface plasmon absorption peaks are observed in the UV-vis. spectrum. It is the characteristics to each metal and depends on the size, shape and distribution of nanoparticles [17,18]. The present work deals with the synthesis of silver and gold nanoparticles using the leaf extract of I. tinctoria by a fast microwave-assisted technology. This extract performs as both reducing and stabilizing agent for the nanosynthesis, confirmed by both the visible colour change and well-defined absorption peaks in the UV-vis. absorption spectra of nanoparticles. The presence of secondary metabolites like alkaloids, amino acids, flavonoids, saponins, steroids, glycosides, carbohydrates, tannins and phenolic group in the leaf extract causes the reduction of metal ions and thus corresponding nanoparticles were formed. After 60 s of microwave irradiation, the reaction mixture of silver nitrate solution and leaf extract change its colour from colourless to brownish orange (Figure 1(b)(2)) and, shows SPR band at 427 nm in UV-vis. spectrum (Figure 1(c)). But auric chloride and leaf extract mixture changes its colour to violet (Figure 1(b) (3)) from light yellow solution within 30 s of microwave irradiation and shows SPR peak at 545 nm (Figure 1(d)). The photographs of plant I. tinctoria and its leaf extract are shown in Figure 1(a) and b(1), respectively. If these reactions were carried out in room temperature, both the nanoparticles formed only after several hours. From this, we can explain the relevance of fast microwave-assisted method in synthesis of nanoparticles.

Figure 1. (a) Photograph of plant Indigofera tinctoria, (b) (1) Photograph of Indigofera tinctoria leaf extract, (2) silver nanoparticles (AgNP- tinctoria), and (3) gold nanoparticles (AuNP- tinctoria), (c) UV-vis. spectrum of AgNP- tinctoria, and (d) UV-vis. spectrum of AuNP- tinctoria.

FTIR spectral analysis

FTIR spectroscopy is very important in probing the chemical composition of chemical compounds. Here it is used for characterizing plant extract and nanoparticles synthesized from it. For the analysis, samples of synthesized AgNP- tinctoria and AuNP- tinctoria were centrifuged at 10,000 rpm for 15 min and the obtained pellets were then washed thoroughly with double distilled water thrice to get rid off the unreacted plant extract residues. The isolated AgNPs and AuNPs were then freeze dried and used for FTIR analysis in the range 500–4000 cm⁻¹. Figure 2(a) shows the FTIR spectra of I. tinctoria leaf extract (1), AgNP- tinctoria, (2) and AuNP- tinctoria (3). The three spectra are more or less similar. The peak at 3206 cm⁻¹ is due to O-H vibrations. The one at 2909 cm⁻¹ is C-H stretching, peaks at 1587 cm⁻¹ and 1393 cm⁻¹ correspond to C = C stretching and C-O stretching respectively. C-N stretching of amines is at 1017 cm⁻¹. Peaks at 739, 708 and 523 cm⁻¹ are due to = C-H stretching (aromatic). It strongly support our hypothesis that various phytochemical constituents like alkaloids, amino acids, flavonoids, saponins, steroids, glycosides, carbohydrates, tannins and phenolic compounds and they take part in the formation and stabilization of nanoparticles. There is a change in intensity and small shift was observed in the spectrum of nanoparticles. This is due to the co-ordination of phytochemicals with metal surface [19].

Figure 2. (a) FTIR spectrum of (1) Indigofera tinctoria leaf extract, (2) AgNP- tinctoria, and (3) AuNP- tinctoria (c) XRD pattern of AgNP- tinctoria, and (d) XRD pattern of AuNP- tinctoria

XRD analysis

In order to study the crystalline nature of biosynthesized silver and gold nanoparticles, the XRD analysis were performed (Figure 2(b,c)). XRD patterns of nanoparticles showed four distinct Bragg reflections at 38.31°, 44.44°, 64.19° and 77.60° in AgNP- tinctoria and at 38°, 44.11°, 64.38° and 77.26° in AuNP- tinctoria. This was due to the reflections from the lattice planes of (111), (200), (220) and (311) of crystalline silver and gold nanoparticles. From this, it is confirmed that both the synthesized nanoparticles have face-centred cubic structure. It is also evident from spectra that the three planes (200), (220) and (311) are small compared to (111) plane. These indicate that the synthesized nanoparticles are highly oriented in (111) plane.

TEM analysis

The TEM analysis was carried out to visualize the size, shape and morphology of the nanoparticles. TEM images of synthesized AgNP- tinctoria and AuNP- tinctoria are given in Figure 3. From the TEM images of the AgNP- tinctoria (Figure 3(a–c)), it is clear that obtained nanoparticles are highly stabilized and are spherical in shape with smooth surface. The particles size lies between 9 nm to 26 nm with average particle size 16.46 nm (Figure 3(f)). The nanoparticles are highly stabilized and no agglomeration was found.

Figure 3. (a, b and c) TEM images of AgNP- tinctoria, at different magnification, (d) HR-TEM image of AgNP- tinctoria, (e) SAED pattern of AgNP- tinctoria, and (f) particle size distribution histogram of AgNP- tinctoria, (g) EDX Spectrum of AgNP- tinctoria, (h, i and j) TEM images of AuNP- tinctoria, at different magnification (k) SAED pattern of AuNP- tinctoria, (l) HR-TEM image of AuNP- tinctoria, and (m) particle size distribution histogram of AuNP- tinctoria, and (n) EDX Spectrum of AuNP- tinctoria.

In the case of gold nanoparticles (Figure 3(h–j)), the particle size is also in nano-range and but different morphologies like spherical, triangular and hexagonal are found. The particle size is between 6 nm and 29 nm, with average particle size 19.73 nm (Figure 3(m)). The crystalline nature of the silver and gold nanoparticles are also confirmed from the HRTEM images (Figure 3(d,l)), and show the clear lattice fingers. The selected area diffraction patters (SAED) of silver and gold nanoparticles are shown in Figure 3(e,k). In this pattern bright circular rings are observed. This is due to the reflection from the lattice planes (111), (200), (220) and (311) of crystalline nanoparticles. The crystalline nature is also evident from the XRD studies.

EDX analysis

The EDX spectra of silver and gold nanoparticles are given in Figure 3(g,n). In two respective spectrums the strong signals of silver (2–4 keV) and gold (2–2.5 keV, 8–8.5 keV and 9.5–10 keV) at characteristic energy are observed.

Anticancer studies

The cytotoxic effect of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria were examined on lung cancer cell line (A549) by MTT assay. Figure 4(a,b,c) respectively represents the morphological changes induced by I. tinctoria leaf extract, AgNP- tinctoria, and AuNP- tinctoria on lung cancer cell line (A549). There is shrinking of cells at varying degree were observed in three cases, it gives the indication about the cytotoxicity of the samples. The previous studies on I. tinctoria proves that the flavanoidal fraction in their extract causes anticancer effect on lung cancer cell line A549 [20]. Figure 4(d) shows the cytotoxicity effect of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria. The samples hinder the expansion of cancer cells in a dose dependent manner, that is, as the concentration of samples increases, the percentage of cell viability decreases. IC₅₀ value of I. tinctoria leaf extract, AgNP- tinctoria, and AuNP- tinctoria respectively are 71.92 ± 0.76 μg/ml, 56.62 ± 0.86 μg/ml and 59.33 ± 0.57 μg/ml. From this, it is clear that the concentration required to inhibit 50% lung cancer cell line (A549) is low for AgNP- tinctoria and AuNP- tinctoria nanoparticles than the I. tinctoria leaf extract. This may be due to the reactive oxygen species induced by nanoparticles, it causes damages to the cellular component and leads to the cell death [21]. Being more spherical in shape, activities of AgNP- tinctoria are higher than AuNP- tinctoria.

Figure 4. (a) Morphological changes induced on treated A549 cancer cell by (a) Indigofera tinctoria, (b) AgNP- tinctoria, and (c) AuNP- tinctoria, and (d) Cytotoxicity studies for Indigofera tinctoria leaf extract, AgNP- tinctoria, and AuNP- tinctoria towards A549 cancer cells.

We have compared anticancer activities of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria by using ANOVA test. The test results for various concentrations are provided in Table 1. The ANOVA test is significant for all five different concentrations analysed signifying that the mean values of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria are statistically different. Furthermore, we have conducted post-hoc test (Tukey’s test). Tukey’s test results show that I. tinctoria leaf extract has the highest mean value followed by AuNP- tinctoria and AgNP- tinctoria is having the lowest mean value.

Table 1. The results of ANOVA and Tukey’s test (for anticancer studies).

		ANOVA results
		Concentration 6.25 μg/ml (F = 165.6***)	Concentration 12 μg/ml (F = 172.1***)	Concentration 25 μg/ml (F = 43.49***)	Concentration 50 μg/ml (F = 38.07***)	Concentration 100 μg/ml (F = 167.1***)
Post-hoc Tukey’s test results
		Mean difference (I–J)
(I) type	(J) type	Concentration6.25 μg/ml	Concentration12 μg/ml	Concentration25 μg/ml	Concentration50 μg/ml	Concentration100 μg/ml
Indigofera tinctoria leaf extract	AgNP- tinctoria	15.10***	16.01***	7.753***	10.07***	10.39***
Indigofera tinctoria leaf extract	AuNP- tinctoria	9.573***	11.15***	3.830***	3.993*	6.997**
AgNP- tinctoria	AuNP- tinctoria	−5.527**	−4.853**	−3.923**	−6.073**	−3.393***

***, **and *indicate significance at 1% level, 5% level and 10% level, respectively.

Antimicrobial studies

Several papers are available in the literature describing the antimicrobial activities of silver [22] and gold nanoparticles [23]. The antimicrobial activities of synthesized nanoparticles are carried out by agar well diffusion method. For this, two gram positive, two gram negative and two fungal strains were used. They are following, Bacillus pumilis and Staphylococcus aureus (gram positive bacteria), Pseudomonas sp and Escherichia coli (gram negative bacteria), Aspergillus fumigatus, and Aspergillus niger (fungi). The antimicrobial activities of I. tinctoria plant extract, AgNP- tinctoria and AuNP- tinctoria are shown in Figure 5(a,b). There are several mechanisms proposed for the antimicrobial properties of nanoparticles [24]. The smaller size and high surface to volume ratio of nanoparticles enables them to interact closely to the microbial membrane [25]. It diffuses into the medium and prevents the bacterial growth in the region of the well. This is because of the high affinity of nanoparticles towards the sulphur- and phosphorus-containing biomolecules in the cell membrane [26]. This results in the membrane rapture and killing of microorganisms. The bacterial efficacy is calculated by measuring the diameter of inhibition zone formed around the well. From the figure, it is clear that that the zone of inhibition of gold nanoparticles are smaller than that of silver nanaparticles this is due to the immobilization of gold nanoparticles due to aggregation on the agar plate [14]. The gram positive bacteria are less affected by silver and gold nanoparticles than gram negative bacteria. This is because of the presence of large number of peptidoglycon layers on the walls of gram positive bacteria than gram negative bacteria. This peptidoglycon layers somewhat prevent the nanoparticles to reach cytoplasmic membrane than gram negative bacteria [27]. The leaf extract of I. tinctoria is also shows some antimicrobial effect. This is due to the presence of biologically active compounds like saponins, flavanoids and alkaloids [28,29]. Therefore, we can conclude that the synthesized AgNP- tinctoria and AuNP- tinctoria nanoparticles can effectively be used to control microorganism and to prevent harmful infections.

Figure 5. (a) Illustration of the antimicrobial activity of AgNP- tinctoria and AuNP- tinctoria against various human pathogenic microorganisms using agar well diffusion method. [(A) Control, (B) Indigofera tinctoria leaf extract, (C) AuNP- tinctoria, and (D) AgNP- tinctoria] (b) Bar diagram showing zone of inhibitions (mm) of Indigofera tinctoria leaf extract, AgNP- tinctoria, and AuNP- tinctoria and (c) Antioxidant activities of AgNP- tinctoria, AuNP- tinctoria, and Indigofera tinctoria extract compared to ascorbic acid.

Antioxidant studies

The antioxidant activities of I. tinctoria plant extract and synthesized silver and gold nanoparticles are evaluated by a simple and less expensive DPPH assay using ascorbic acid as standard. The DPPH is a stable free radical with red colour and shows absorption at 517 nm. It reacts with antioxidative compounds which donate hydrogen and get reduced. During this reaction, colour changes to yellow consequently absorbance at 517 nm get decreased. Most of the phenolic compounds in the plant extract show high levels of antioxidant activity due to the presence of OH group [30]. As shown in Figure 5(c), the potent inhibitory actions of synthesized silver and gold nanoparticles are higher than the plant extract ranging from12.5 μg/ml to 200 μg/ml. The percentage of inhibition was increased with increasing concentration of the substrate [31]. The increased antioxidant activity of nanoparticles compared to leaf extract can be attributed to the adsorption of bioactive compounds of leaf extract over spherically shaped nanoparticles. The AgNPs are more spherical in shape compared to AuNPs and having a larger surface area thus its activity is higher than AuNPs. The IC₅₀ values of I. tinctoria extract, AgNP- tinctoria, and AuNP- tinctoria are 177.52 ± 0.43 μg/ml, 10.04 ± 0.51 μg/ml and 68.05 ± 0.71 μg/ml, respectively. IC₅₀ values denotes the concentration that requisite for 50% inhibition. We have compared antioxidant activities of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria by using ANOVA test. The test results for various concentrations are provided in Table 2. The ANOVA test is significant for all five different concentrations analysed signifying that the mean values of I. tinctoria leaf extract, AgNP- tinctoria and AuNP- tinctoria are statistically different. Furthermore, we have conducted post-hoc test (Tukey’s test). Tukey’s test results show that AgNP- tinctoria has the highest mean value followed by AuNP- tinctoria and I. tinctoria leaf extract is having the lowest mean value.

Table 2. The results of ANOVA and Tukey’s test (for antioxidant studies).

		ANOVA results
		Concentration 12.5 μg/ml (F = 977.7***)	Concentration 25 μg/ml (F = 2654***)	Concentration50 μg/ml (F = 1932***)	Concentration100 μg/ml (F = 3562***)	Concentration 200 μg/ml (F = 4812***)
Post-hoc Tukey’s test results
		Mean difference (I–J)
(I) type	(J) type	Concentration12.5 μg/ml	Concentration25 μg/ml	Concentration50 μg/ml	Concentration100 μg/ml	Concentration200 μg/ml
Indigofera tinctoria leaf extract	AgNP- tinctoria	−20.67***	−28.98***	−31.22***	−36.13***	−39.10***
Indigofera tinctoria leaf extract	AuNP- tinctoria	−10.23***	−9.137***	−6.047***	−13.47***	−19.56***
AgNP- tinctoria	AuNP- tinctoria	10.45***	19.84***	25.17***	22.66***	19.54***

***, ** and *indicate significance at 1% level, 5% level and 10% level, respectively.

Catalytic studies

Reduction of o- and p- nitroaniline

Aromatic nitroaniline compounds are mainly used as intermediate in the synthesis of pesticides, antioxidants, antiseptic agents, azo dyes, corrosion inhibitors and fuel additives [32,33]. They are easily soluble in water and alcohol. These are dangerous to human beings and aquatic organisms [34]. Therefore, the reduction of nitroaniline is environmentally very important. Here we report the reduction of o- and p-nitroanilines using the green synthesized AgNP- tinctoria and AuNP- tinctoria nanoparticles.

The catalytic activities of AgNP- tinctoria, AuNP- tinctoria on the reduction of o- and p-nitroaniline by sodium borohyride (NaBH₄) was studied by using UV-vis. spectroscopy. Characteristic absorption peak of p-nitroaniline is found at 380 nm and for o-nitroaniline two peaks are found at 412 and 283 nm in UV-vis. spectrum [35].

Uncatalyzed reactions were carried out to analyse the capability of NaBH₄ with o- and p- nitroanilines. It shows only a small percentage of degradation as shown in Figure 6(a,b). This is due to the presence of kinetic barrier, but the reaction is thermodynamically favourable. When the synthesized AgNP- tinctoria, AuNP- tinctoria nanoparticles are added to the nitroaniline solution it overcomes this kinetic barrier by facilitating the electron transfer from donor BH₄^? ions to accepter nitroanilines [36].

Figure 6. (a) and (b) Reduction reactions of o-nitroaniline and, p-nitroaniline using NaBH4 without nanocatalysts; (c) and (d) reduction reactions of o-nitro aniline using AgNP-tinctoria and AuNP-tinctoria nanocatalysts; (e) and (f) reduction of p-nitroaniline using AgNP-tinctoria, and AuNP-tinctoria nanocatalysts.

Hence, in the case of o-nitroaniline peak at 412 nm decreased and is reduced to 1,2 benzediamine. This reaction was monitored using UV-vis. spectrometer at different time intervals t. With increasing reaction time, the peak at 283 nm shifted to 289 nm, which point out the steady reaction of o-nitroaniline to benzene diamine (Figure 6(c,d)). It was also evident from the decolouration of the initial yellow colour of o-nitroaniline. In the case of AgNP- tinctoria catalyzed reaction, the reduction completed within 10 min and but for AuNP- tinctoria catalyst it took 18 min for complete reduction. Here the concentration of NaBH₄ used was very high compared to o-nitroaniline and remains constant during the course of a reaction, so pseudo first-order kinetics was assumes in this case. Therefore, the rate equation can be written as k = 1/t ln [A_o]/[A], k is pseudo first-order rate constant, [A_o] is initial concentration of nirtoanilines and [A] is the concentration at time t. Therefore, the plot of ln [A] versus time “t” is straight line. The rates constant were directly calculated from the slope of these plots. Therefore, plot of ln [A] versus time graph is a straight line with correlation coefficient (R²) 0.9839 for AgNP- tinctoria and 0.9840 for AuNP- tinctoria catalyzed reactions (the plot is shown in the inset of corresponding reactions). The rate constants (k) of the reactions are 0.0975 min⁻¹ for AgNP- tinctoria and 0.0673 min⁻¹ for AuNP- tinctoria-catalyzed reaction.

The reduction of p-nitroaniline by AgNP- tinctoria and AuNP- tinctoria catalyst are also studied using UV-vis. spectroscopy. As soon as nanocatalyst added to the reaction mixture, the intensity of the peak at 380 nm decreased and it was accompanied by the appearance of a new peak of 4-phenylenediammine at 240 nm [37]. The reduction reaction occurs by the transfer of electrons from donor BH₄⁻ ion to accepter nitroaniline after absorbing both of the reactant on the catalytic surfaces. The hydride ion generated from the donor hydride ion after electron relay process reduce nitroaniline [38]. These catalytic activities are due to the available large surface area on the catalytic surface and removing the kinetic barrier for the reduction reaction. From Figure 6(e,f), it is clear that AgNP- tinctoria catalyzed reaction completed within 10 min and whereas the AuNP- tinctoria catalyzed reaction required 18 min for complete reduction. An isobestic point at 309 nm also gives clear indication of conversion of p-nitroaniline to phenelenediamine. Here also pseudo first order was assumed and rate constants (0.1823 min⁻¹ and 0.0824 min⁻¹) and correlation coefficient (R²) (0.9979 and 0.9707) are calculated from the linear plot of ln [A] versus time graph (shown in inset) respectively for AgNP- tinctoria and AuNP- tinctoria catalyzed reactions.

From the above catalytic reactions, it is clear that the rate of AgNP- tinctoria catalyzed reaction is very fast and rate is higher than the AuNP- tinctoria catalyzed reactions. This may be due to the small size and spherical nature of silver nanoparticles resulting in the availability large numbers of binding sites for the chemisorptions of reactants and then enhancing the reaction rate [39]. The gold nanoparticles were anisotropic and have different morphology and it affects the rate of reaction. The spherical nature of AgNP- tinctoria improves the adsorption of reactants on its surface and thus enhances the reaction rate.

Conclusions

In this study, we have synthesized silver and gold nanoparticles by a simple, fast, green, and cost-effective method using a popular medicinal plant I. tinctoria. The nanoparticles are characterized by using several techniques like UV-vis., FTIR, XRD, TEM, EDAX and AFM. Gold and silver nanoparticles show good anticancer properties against lung cancer cell line A549 than the plant extract. Therefore, I. tinctoria functionalized silver and gold nanoparticles can be effectively used as a powerful weapon against cancer. Nanoparticles also exhibit remarkable antimicrobial and antioxidant properties. Due to these properties, synthesized nanoparticles have remarkable applications in biomedical field. The nanoparticles also show excellent catalytic activities towards the reduction nitroanilines.

Acknowledgements

Remya Vijayan is thankful to University Grants Commission (UGC), Government of India, for financial assistance (JRF).

Disclosure statement

The authors hereby declare that there is no conflict of interest regarding this paper.

Additional information

Funding

Remya Vijayan is thankful to University Grants Commission (UGC), Government of India, for financial assistance (JRF).