Citrus limetta Risso peel mediated green synthesis of gold nanoparticles and its antioxidant and catalytic activity

2.1 Preparation of fruit peel extract

Sweet lime (Citrus limetta Risso) was purchased from the local market of Tenkasi district of Tamil Nadu, India. Peel from fruits was taken off, washed thoroughly in distilled water, and dried in shade. Dried fruit peels were boiled in distilled water for 30 min. After cooling macerated in distilled water and filtered through a clean muslin cloth. Then, chilled acetone was added and the resulting supernatant was collected by centrifugation at 10,000 rpm for 10 min. The collected supernatant (extract) was stored at 4 °C for further experiments.

2.2 Green preparation of gold nanoparticles

Gold nanoparticles were synthesized by boiling 1 mM chloroauric acid (HAuCl₄) (Sigma-Aldrich, St. Louis, USA) in double distilled water and fruit peel extract for 10 min. The reaction mixture was monitored until the light-yellow solution turned into ruby red (UV–vis spectroscopy). At the end reaction time, the sample was dried at 100 °C for 2 h, then ground into fine powder for characterization and study of different activities.

2.3 Green gold nanoparticles characterization

The colloidal suspension of green synthesized gold nanoparticles was characterized by UV–vis spectroscopy (PerkinElmer). Crystallinity of gold nanoparticles was analyzed by XRD (Diffractometer system XPERT-PRO). Functional groups present on the surface of green synthesized gold nanoparticles were examined by FTIR (Bruker TENSOR 27). The morphology of the prepared sample was characterized by FESEM (Tescan VEGA) and AFM (Nanosurf Easyscan 2).

2.4 Various assays to examine the antioxidant activity of green gold nanoparticles

DPPH radical scavenging activity of gold nanoparticles was assayed using the protocol of Shimada et al. (1992). Protocol of Halliwell and Gutteridge (1981) was applied to examine the hydroxyl radical scavenging potential of green gold nanoparticles. Nitric oxide radical inhibition assay was done using the method of Ebrahimzadeh et al. (2010) with slight modifications. Hydrogen peroxide (H₂O₂) scavenging activity of gold nanoparticles was determined by following the method of Nabavi et al. (2008). Superoxide anion scavenging activity of gold nanoparticles was assayed as described by Nishikimi et al. (1992). Lipid peroxidation inhibition ability of gold nanoparticles was evaluated by the ammonium thiocyanate method (Mitsuda et al 1966). ABTS radical scavenging assay was performed according to the method of Siddhuraju and Becker (2003).

2.5 Assay of reducing power

Reducing power of gold nanoparticles was determined as reported earlier (Yen and Chen, 1995). Different concentration of gold nanoparticles (20–100 μg/ ml) was added into 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1 %w/v). The reaction mixture was incubated at 50 °C for 20 min. Then, 2.5 ml trichloroacetic acid (10 % w/v) was added to terminate the reaction. Reaction mixture was then centrifuged at 3000 rpm for 10 min and collected the supernatant. After that 2.5 ml of supernatant was mixed with 2.5 ml distilled water, and 0.5 ml ferrous chloride (0.1 %, w/v). The absorbance of the reaction mixture was measured at 700 nm. Higher absorbance indicates higher reducing power of the gold nanoparticles. Ascorbic acid was used as a positive control.

2.6 Catalytic reduction of 4-nitrophenol

Briefly, 0.2 ml of 4-nitrophenol (1 mM) was mixed with 3.6 ml of freshly prepared sodium borohydride (16.5 mM) and 0.2 ml of gold nanoparticles suspension in a 10-mm quartz cuvette. The UV–vis Spectroscopy was used to record the progress of reaction at different time intervals (0–80 min) and absorbance was recorded between 200 and 600 nm to calculate the reduction of 4-nitrophenol to 4-aminophenol.

3.1 UV–vis spectroscopy

Formation of gold nanoparticles by the fruit peel extract was observed with change of color of solution from light yellow to ruby red. The excitation of surface plasmon vibrations in gold nanoparticles is the reason for this characteristic color change. The size and shape of the metal nanoparticles are responsible for the optical properties of the metal nanoparticles. According to the Mie theory, the small gold nanoparticles exhibit only one surface plasmon resonance (SPR) absorption band, whereas anisotropic particles show two or three SPR bands (Pechyen et al., 2021). Fig. 1 shows UV–vis spectra of the gold nanoparticle solution and the SPR band appears at 530 nm. Colloidal suspension gold nanoparticles were stable without agglomeration for several weeks (See Fig. 2).

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Fig. 1

UV–vis spectrum of green synthesized gold nanoparticles.

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Fig. 2

FTIR spectra of green synthesized gold nanoparticles.

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3.2 X-ray diffraction analysis

Crystallite size and structure of green prepared gold nanoparticles were determined by XRD spectra. Sweet lime peel extract mediated synthesized gold nanoparticles showed four strong diffraction peaks at the 2θ values of 38.52°, 44.03°, 64.53°, and 78.65°, corresponding to plane (1 1 1), (2 0 0), (2 2 0), and (3 1 1), respectively. These planes indicate the gold nanoparticles have face cantered cubic (fcc) crystalline structure. The spectrum of synthesized gold nanoparticles was compared with standard spectrum published by Joint Committee on Powder Diffraction Standards (JCPDS file: 04–0783). The comparison of this spectrum with the standard confirmed that green prepared gold nanoparticles have crystalline nature. The Debye-Scherrer’s equation (Patterson, 1939) was used to calculate the crystallite size of green synthesized gold nanoparticles. $$D=\frac{K\lambda }{\beta \cos \theta }$$ where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-ray wavelength (1.5418 Å), β is the width of the XRD peak at half height, θ is the diffraction angle, and K is the Scherrer coefficient (0.89). The average crystallite size of the as synthesized gold nanoparticles was found to be around 64 nm (size range: 50–80 nm). The present XRD report is well matched with the standard gold and previous report (Shukla et al., 2010).

3.3 Fourier transform infrared spectroscopy

FTIR spectra of green synthesized gold nanoparticles is presented in Fig. 3. Functional groups responsible for the synthesis of gold nanoparticles are denoted by the corresponding peaks arising due to the infrared radiations (Table 1). FTIR data of green gold nanoparticles are well matched with previous report (Donga et al., 2020; Yang et al., 2019).

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Fig. 3

XRD pattern of green synthesized gold nanoparticles.

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3.4 Field emission scanning electron microscopy and atomic force microscopy

Field emission scanning electron microscope (FESM) was used to identify the surface morphology of green synthesized gold nanoparticles. Fig. 4A–D show the FESEM micrographs of gold nanoparticles at different scale bar. These micrographs suggested that green prepared gold nanoparticles were uniformly distributed and most of the particles were irregular in shape with the size range of 50–80 nm (average size 64 nm). Atomic force microscopy (AFM) images of green gold nanoparticles are shown in Fig. 5. The particles are viewed at 2 and 4 µm distances and also in 3D pattern. AFM images further supported the FESEM results.

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Fig. 4

FESEM images of green synthesized gold nanoparticles. (A) 5 µm scale. (B) 500 nm scale. (C) 1 µm scale. (D) 300 nm scale.

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Fig. 5

AFM images of green synthesized gold nanoparticles.

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3.5 Antioxidant activity

Antioxidant activity (free radicals scavenging activity) of green prepared gold nanoparticles was examined by a series of assays. DPPH is a stable synthetic free radical that is being applied for the assessment of antioxidant activity of various materials (Medhe et al., 2014). This is a fast, easy and reliable method. This method is based on the principal that DPPH can easily receive an electron from antioxidant molecules to become a stable diamagnetic molecule (Soares et al., 1997). In this study, DPPH radical scavenging activity of gold nanoparticles was analyzed at different concentration (20–100 μg/ml) to identify the percentage inhibitory concentration. Results showed that gold nanoparticles exhibited significant DPPH scavenging activity in a dose-dependent manner (Fig. 6A). The 50 % DPPH radical scavenging activity was observed at 51.84 μg/ml concentration of gold nanoparticles.

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Fig. 6

A series of assays demonstrating the antioxidant activity of green synthesized gold nanoparticles. (A) DPPH radical scavenging activity. (B) Hydroxyl radical scavenging activity. (C) Nitric oxide radical scavenging activity. (D) Hydrogen peroxide scavenging activity. (E) Superoxide anion scavenging activity. (F) Lipid peroxidation inhibition activity. (G) ABTS scavenging activity. (H) Reducing power. Data are presented as the % of mean ± SD of triplicate experiments (n = 3).

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As we can see in Fig. 6B, hydroxyl radical scavenging activity of gold nanoparticles was observed in a concentration-dependent manner. The IC₅₀ value of gold nanoparticles and standard (vitamin E) were 45.15 μg/ml and 35.5 μg/ml, respectively.

Nitric oxide (NO) is a reactive free radical generated from sodium nitroprusside in aqueous solution and reacts with oxygen to form nitrite. It is well known that NO radical plays an important role in various inflammatory processes such as carcinomas, juvenile diabetes, multiple sclerosis, arthritis, and ulcerative colitis (Hazra et al., 2008). In this study, NO radical scavenging activity of green synthesized gold nanoparticles increases with increasing the concentration of nanoparticles (Fig. 6C). The IC₅₀ value of gold nanoparticles for NO scavenging activity was 59.39 μg/ml.

The scavenging of hydrogen peroxide (H₂O₂) by gold nanoparticles is shown in Fig. 6D. Gold nanoparticles scavenge the H₂O₂ radical dose-dependently. The 50 % H₂O₂ scavenging activity was observed at 56.58 μg/ml concentration of gold nanoparticles. Gold nanoparticles contain phenolic groups that donate electron to H₂O₂ and thus neutralizing H₂O₂ into water (Mathew and Abraham, 2006). Superoxide anion is a highly toxic radical that generate oxidative damage to biological molecules (Ahamed et al., 2021b). Superoxide anion scavenging activity of green gold nanoparticles is presented in Fig. 6E. Decrease of absorbance at 560 nm with the increment of gold nanoparticles concentrations indicates dose-dependent superoxide anion scavenging efficacy. The IC₅₀ value of gold nanoparticles in superoxide anion scavenging activity was 74.25 μg/ml.

Fig. 6F shows lipid peroxidation inhibition activity of gold nanoparticles. Results showed that gold nanoparticles effectively inhibit the lipid peroxidation reaction. Lipid peroxidation inhibition activity was increases with increasing the concentration of gold nanoparticles (20–100 μg/ml). The IC₅₀ value of gold nanoparticles was 39.64 μg/ml.

2,2′‐Azinobis (3‐ethylbenzothiazolin‐6‐sulphonic acid) (ABTS) radicals scavenging activity of gold nanoparticles was increases in a dose–response manner from 28.21 % to 79.77 % from the concentration of 20 μg/ml to 100 μg/ml (Fig. 6G). The IC₅₀ value for the gold nanoparticles was found to be 54.39 μg/ml. This result shows that gold nanoparticles have the ability to scavenge the free ABTS radicals generated by the reaction ABTS molecule and potassium persulfate (Venkatesh et al., 2014). It prevents ABTS radicals through chain‐breaking reaction. The reducing power of gold nanoparticles was analyzed due to its electron donating ability and therefore, may serve as a strong antioxidant agent. Fig. 6H shows the reducing potential of gold nanoparticles along with standard butylated hydroxyl toluene. The reducing power of gold nanoparticles increases with increasing the amount of prepared sample. These results suggested the antioxidant activity of gold nanoparticles synthesized from fruit lime peel extract.

3.6 Catalytic activity

Transition metal nanoparticles prepared via green route have shown potential in catalytic reduction of organic pollutants because of their unique physicochemical properties (Gao et al., 2014). The catalytic conversion of 4-nitrophenol to 4-aminophenol by gold nanoparticles is presented in Fig. 7. The characteristic absorption peak (400 nm) of 4-nitrophenol decreases over the time and when the final product 4-aminophenol is produced, obtained the peak shifts to 300 nm. The shifting of absorption peak from 400 nm to 300 nm was due to the catalytic reduction of 4-nitrophenol to 4-aminophenol by gold nanoparticles. The catalytic activity of green-prepared gold nanoparticles is also supported by earlier investigations (Dash et al., 2014).

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Fig. 7

Catalytic activity of green synthesized gold nanoparticles.

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