Degradation of Toxic Dye Using Phytomediated Copper Nanoparticles and Its Free-Radical Scavenging Potential and Antimicrobial Activity against Environmental Pathogens

Abstract

The present investigation deals with the green synthesis of copper nanoparticles in an ecofriendly manner using leaf extract of Andrographis paniculata. Green-synthesized copper nanoparticles were studied for their antibacterial, antioxidant, and catalytic activity. The leaves were powdered and extracted with water and added to copper sulphate solution. The reduction of copper ions to nanoparticles was preliminarily identified by the color change of the reaction mixture. The synthesized nanoparticle was characterized by using a UV-Vis Spectrophotometer at a different wavelength with different time intervals. Functional groups available on the surface of the nanoparticle were identified by Fourier transform infrared spectroscopy (FTIR). Surface roughness was characterized by atomic force microscopy (AFM). X-ray diffraction (XRD) analysis showed six distinct intense peaks indicating the crystalline nature of synthesized copper nanoparticles (CuNPs). A scanning electron microscope (SEM) demonstrated polydispersed nanoparticles formed in the reaction process. The antibacterial activity of the nanoparticles was evaluated by an agar well diffusion assay against pathogenic bacteria. The antioxidant activity showed the excellent reduction of DPPH free radicals by nanoparticles. These results confirmed that copper nanoparticles serve as an alternative therapeutic agent over conventional drugs. Moreover, copper nanoparticles were also used to study the effect on the dye degradation process of methyl red and eosin dyes. Copper nanoparticles effectively remove the dyes with high efficiency up to 92% and 95% of methyl red and eosin dye, respectively.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Nanotechnology creates nanomaterials with desired properties and small size falling into the nanometer scale [1]. The small-sized nanoparticles have a high surface area compared to bulk materials. Development of bioinspired nanoparticles is an immensely growing field in modern technology [2]. Various methods are available for fabrication of bioinspired nanoparticles such as physical, chemical, and biological methods. Moreover, the biological method includes using bacteria [3], fungi [4], virus and algae [5], and yeast and plants [6]. Microbe-mediated synthesis required frequent microbial cell culture maintenance and adequate handling experiences. But, among the methods, plant-derived nanoparticles are highly acceptable and preferable due to their environment friendliness, no usage of toxic chemicals, eliminating culture maintenance process, and no need of physical and chemical parameters [7]. Phytomediated synthesis of nanoparticles is based on the concept of phytoremediation of metals. Selection of plant for the nanoparticles synthesis is based on the heavy-metal resistance capability [8].

Among the noble metals, copper is abundantly available in the earth crust and exists in low stable metallic form. Copper is a crucial trace element in the biological system. Copper is extensively used because of high costs other noble metals compared to copper. Currently, copper nanoparticles have been attaining great attention due to their catalytic, optical, and electrical properties [9]. Therefore, copper is mostly preferred for the synthesis process because of low cost and high conductivity. Copper nanoparticles are widely used as antimicrobial, antioxidant, anticancer, anti-inflammatory, and antihepatotoxic agents. Currently, copper nanoparticles are used as an alternative feed additive in animal feed, especially poultry diet [10].

Andrographis paniculata (king of bitters) belongs to the family Acanthaceae, widely grown in Asian countries. In ancient medicinal system, this herb was used in therapeutic formulations to treat liver disorders. This plant has andrographolide, a bitter taste-giving major compound, and also has diterpene lactone [11] which is responsible for its pharmacological activities [12]. A. paniculata has various beneficial activities such as antioxidant [13], anticancer [14], hepatoprotective [15], antimicrobial [16], antidiabetic [17], antifertility [18], and antimalarial activity [19].

Methyl red is an azo dye with the molecular formula C₁₅H₁₅N₃O₂. Typically, methyl red dye is used in various industries such as textile, pharmaceutical, cosmetics, and tannery. The effluent releases from the industries are very toxic to the human beings.

The molecular formula of eosin is C₂₀H₆Br₄Na₂O₅. Eosin is also commonly named as acid red 87, eosin yellowish, and so on. It is used for diverse purpose in the industries such as pharmaceutical, cosmetics, and textile. It is also used for tissue stain as well as counter stain. The existence of the hazardous dye in the terrestrial and aquatic environment leads to harmful health effects to the human beings such as cancer, tumor, and skin diseases.

In previous decades, physical and chemical methods were predominantly used for the degradation of dye. However, they have many draw backs. In order to avoid those issues, nowadays, biological methods are widely used for the degradation of hazardous dyes. Nowadays, nanoparticles are greatly receiving attention in degrading toxic dyes due to their large-surface-area properties [20]. In this study, copper nanoparticle was prepared by the simple green method and characterized. Synthesized nanoparticle was applied in various targeted applications such as antimicrobial, antioxidant, and catalytic activity.

2. Materials and Methods

2.1. Preparation of Plant Extract

The leaves of A. paniculata were collected from Vellore district. The freshly collected leaves were washed thoroughly with distilled water thrice and shade-dried for a few days at room temperature, and then, a fine powder was obtained using a blender. In this study, 5 g of plant powder was accurately weighed and added into a 250 mL conical flask containing 100 ml of double distilled water. The mixed suspension were boiled in a microwave oven for 5 minutes followed by double filtration accomplished with Whatman No.1 filter paper, and the consequent suspension was stored at 4°C for the further experiments.

2.2. Green Synthesis of Copper Nanoparticles

For the biosynthesis of CuNPs, 25 ml of aqueous leaf extract was assorted with 75 ml of distilled water containing 10 millimolar of copper sulphate. The prepared solutions were incubated in a shaker at room temperature for 24 h. After the incubation time, the change in the color of the solution owing to the formation of copper nanoparticles was observed by using a UV-Visible spectrophotometer.

2.3. Characterization of Green-Synthesized Copper Nanoparticles

Synthesis of copper nanoparticles was carried out by forming an SPR band measured in a double-beam UV-Vis Spectrophotometer. Crystalline structure and morphology of nanoparticles were observed by X-ray diffraction and scanning electron microscope analysis, respectively. The surface roughness and topography character were evaluated by atomic force microscopy analysis. Functional groups present on the surface of the nanoparticle were characterized by Fourier transform infrared spectroscopy.

2.4. Various Applications of Copper Nanoparticles

2.4.1. Antibacterial Activity

Antibacterial activity of synthesized copper nanoparticles was determined by the agar well diffusion method. In this assay, about five microorganisms were used, Bacillus sp., E. coli, Klebsiella sp., S. aureus, and Proteus sp. Fresh overnight cultures of the abovementioned organisms were separately and evenly spread using a sterile cotton swab on a Muller Hinton Agar plate. About 6 mm diameter-sized four wells were made on the plate using gel puncture. Each well was filled with 50 μL of leaf extract, 50 μL (50 μg/ml) of copper sulphate, 50 μL (50 μg) of copper nanoparticles, and 15 μL (15 μg) of standard (ampicillin). Plates were incubated at 28–30°C for 24 hours, and the zone formation around the well was measured.

2.4.2. Antioxidant Activity (DPPH Scavenging Assay)

DPPH radical scavenging assay was performed using green-synthesized copper nanoparticles. DPPH solution was freshly prepared by mixing 0.004 g of DPPH in 95% methanol. 2.9 ml of DPPH solution was taken in four test tubes, and 0.1 ml of different concentrations (250–1000 μg) of nanoparticles were added in each test tube. The reaction mixture was shaken vigorously and incubated for 30 min, and the resulting solution was measured at 517 nm in a UV-Vis spectrophotometer. Control was prepared containing the same concentration of standard ascorbic acid, and 95% methanol was used as blank. The scavenging property was calculated by using the following formula:$$\begin{array}{}\text{(1)}& \%\text{inhibition}=\frac{{\text{A}}_{\text{cont}}-{\text{A}}_{\text{test}}}{{\text{A}}_{\text{cont}}}\times 100,\end{array}$$where A_cont is the absorbance of the control reaction and A_test is the absorbance in the presence of samples.

2.4.3. Catalytic Reduction of Dyes

In this investigation, 10 mg of methyl red and eosin dye were prepared in 1 liter of double-distilled water in two conical flasks. From this stock solution, 100 mL of methyl red and eosin dye solutions were withdrawn and added into two conical flasks. 10 mg of green-synthesized copper nanoparticles was added into each conical flask, and control was maintained without addition of nanoparticles. The reaction solution was magnetically stirred and periodically measured by taking aliquots of 3 mL suspension and filtered. Filtered samples were monitored using a UV-Vis spectrophotometer at the wavelength from 300 nm to 800 nm.

3. Results and Discussion

3.1. Visual Observation and UV-Vis Spectroscopy Studies for Copper Nanoparticles

Preliminarily, synthesis of copper nanoparticles was observed by color change in the reaction mixture. Initially, the copper sulphate solution was in light blue color. Consequently, the color of the copper sulphate solution changes from light blue color to green on addition of leaf extract of A. paniculata, and eventually, dark greenish brown color was formed owing to the formation of CuNPs, as shown in Figure 1. The occurrence of the color changes in the aqueous solution is a result of the surface plasmon resonance phenomenon. The leaf extract of A. paniculata acts as a reducing agent as well as stabilizing agent, which reduced the copper sulphate into copper sulphide. The synthesis of copper nanoparticles was observed using UV-Vis spectra. The spectral readings for the concerned sample mixture were taken from 0 to 24 hours. Predominantly, the surface plasmon resonance of CuNPs exhibits the highest absorption peak at 530 nm, as displayed in Figure 2. Similarly, in previous studies, it has been reported that the surface resonance plasmon of CuNPs is in the range of 500–600 nm. On the other hand, it has been revealed in the range of 550–650 nm [21, 22].

[figure omitted; refer to PDF]

(3) Catalytic Degradation of Eosin Dye. The catalytic activity of the biosynthesized copper nanoparticles was investigated against the water containing eosin dye. The kinetic reaction was observed using the UV-Visible spectrophotometer technique in the wavelength of 300–800 nm. Figure 11 shows that the maximum peak value was obtained in the range of 500–515 nm. As per the previous study, it also reported that the absorption spectrum of eosin was recorded at 517 nm [27]. Gradually, the peak disappears when the reaction time increases. Therefore, it indicates that eosin dye has been degraded in the existence of copper nanoparticles, as depicted in the figure.

[figure omitted; refer to PDF]

4. Conclusions

Phytomediated synthesis of copper nanoparticles is a simple and ecofriendly method. In this study, plant leaf powder was used to prepare aqueous extract which was mixed with copper sulphate solution. The extract was reduced to copper sulphate and into copper nanoparticles. Synthesized copper nanoparticles were characterized by positioning of SPR band confirmed using UV-Vis Spectra. The crystalline structure and morphological characters were identified using XRD and SEM analysis. AFM image shows that topography characters of green-synthesized copper nanoparticles. The functional groups involved in the reduction of copper sulphate to copper nanoparticles were characterized by FTIR spectrum analysis. Hence, the synthesized nanoparticle was studied for medical applications such as antibacterial and antioxidant activity against pathogenic microorganisms and DPPH free radical. Other than that, the synthesized nanoparticle was utilized in environmental applications such as the toxic dye degradation process.

Authors’ Contributions

SR designed the study, and SR and MV carried out research, and SR, MV, and KA wrote and corrected the manuscript.

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