Biosynthesis, characterization, biological and photo catalytic investigations of Elsholtzia blanda and chitosan mediated copper oxide nanoparticles

2.1 Materials and methods

Copper sulphate pentahydrate (CuSO₄·5H₂O), sodium hydroxide (NaOH) and acetic acid (CH₃COOH) were obtained from Spectrum Reagents and Chemicals Pvt. Ltd. Edayar, Cochin. Chitosan was bought from Sisco Research Laboratories Pvt. Ltd.

2.2 Collection of plant sample

Elsholtzia blanda leaves were collected from Mao area of Manipur, India in April and was taxonomically verified at the Department of Botany, D.G.Vaishnav College, Chennai-106, India.

2.3 Preparation of leaf extract

The leaves were washed thoroughly under running tap water to remove any foreign and dust particles after which they were shade dried. Aqueous extract of the plant was prepared by taking about 2 g of the dried leaves in a 200 mL beaker. Having added 100 mL of double distilled water, the mixture was heated at 80 °C for about 30 mins. After cooling it down, it was filtered with the help of Whatman filter paper no.1 and the filtrate was used as the plant extract for the subsequent synthesis of the desired nanoparticles.

2.4 Synthesis of copper oxide nanoparticles using chitosan and plant extract

Synthesis of E. blanda-chitosan mediated copper oxide nanoparticles (CPCE) was achieved using a modified procedure used by Jayaramudu et al. (Jayaramudu et al., 2019). 1.5 g of chitosan was dissolved in 100 mL 1.5% acetic acid at 60 °C. 1.25 g of CuSO₄·5H₂O was dissolved in 100 mL double distilled water. 60 mL of the CuSO₄·5H₂O solution was taken and 20 mL of aqueous plant extract added to it. The mixture was stirred continuously on a magnetic stirrer for 30 min. 20 mL of 0.6 M NaOH was then added drop wise till pH ∼ 9 was attained. 20 mL of chitosan solution was then slowly added and the stirring continued for 3 h at 70 °C. The change of colour was observed from blue to green and ultimately greenish black. E. blanda mediated copper oxide nanoparticles (PCE) was prepared separately without adding chitosan solution. The prepared solutions containing CuO NPs were cooled, filtered and washed repeatedly with double distilled water. Further, the CuO NPs were dried at 80 °C and then taken for characterisation. Fig. 2 shows the schematic outline of the procedure followed in synthesising the nanoparticles.

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Fig. 2

Schematic representation of the synthesis of CuO nanoparticles.

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2.5 Characterization

The characterisation of CuO NPs is so important to confirm the biological feasibility of the synthesis of the nanoparticles and the UV–Vis spectra was examined by UV spectrophotometer (Jasco V-670 Serial No. B072061154) in the wavelength range between 200 nm and 800 nm with 1 nm data interval. The functional groups and the Cu-O bond was indicated by FTIR (Nicolet iS50, Thermo Scientific, USA) using KBr pellet in the range of 4000 – 400 cm⁻¹ with a resolution of 4 cm⁻¹. X-ray diffraction were recorded by X-ray diffractometer (D8 advance, Bruker, Germany) which has 2.2KW Cu-anode ceramic tube as its source in 2θ range from 10⁰ to 90⁰. The surface morphology and elemental composition of the samples were observed by FESEM and EDAX respectively. The shape and grain size of the synthesised nanoparticles were analysed by HRTEM which was scanned at 200 kV voltage and equipped with SAED pattern.

2.6 Biological activities of CuO nanoparticles

2.7 Antioxidant assay

The in vitro antioxidant activity of the synthesised CuO NPs using chitosan and plant extract (CPCE) and using plant extract alone (PCE) was tested with the help of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay following the method of Lakhsmanan et al. (Lakshmanan et al., 2016). Methanolic solution of DPPH exhibits a strong purple colour with strong absorption at 517 nm and when reduced the colour changes to yellow. An aliquot of 1 mL of 0.004% DPPH solution in methanol was mixed with 3 mL of sample solutions at varied concentrations (5, 10, 15, 20, 25 μg/mL). After a vigorous shake, the mixture was kept in dark at room temperature for 30 min. The absorbance, to measure the decolourization of DPPH was taken at 517 nm using UV–VIS spectrophotometer (LMSP-UV1000B). The percentage inhibition of DPPH radicals by the samples was determined by comparing the absorbance values of the control and the experimental samples. $$\mathbf{D}\mathbf{P}\mathbf{P}\mathbf{H}\mathbf{s}\mathbf{c}\mathbf{a}\mathbf{v}\mathbf{e}\mathbf{n}\mathbf{g}\mathbf{i}\mathbf{n}\mathbf{g}\mathbf{a}\mathbf{c}\mathbf{t}\mathbf{i}\mathbf{v}\mathbf{i}\mathbf{t}\mathbf{y}(\%)=\frac{\mathit{A}\left(\mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l}\right)-\mathit{A}(\mathit{s}\mathit{a}\mathit{m}\mathit{p}\mathit{l}\mathit{e})}{\mathit{A}(\mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l})}\mathbf{x}100$$ Where A (control) is absorbance value of control and A (sample), absorbance value of sample.

2.8 Photocatalytic activity

The photocatalytic degradation efficiency of the biosynthesized CuO NPs was assessed by degradation of congo red dye according to the method of Thyagarajan et al. (Thyagarajan et al., 2017). A stock solution of 1 mg/mL of the dye was prepared from which working solutions of 100 μg/mL were further prepared. The dye decolourization was studied by two parameters (i) keeping time constant while varying the concentration and (ii) keeping the concentration constant while varying the time. In the former case, different concentrations (10, 20, 30, 40, 50 μg) of synthesized CuO NPs were added to equal volumes of the working solutions at room temperature for 10 h while in the latter the working solution with a constant concentration (50 μg/mL) of the nanoparticles was exposed to light for different time intervals (0, 2, 4, 6, 8, 10 h). The decolourization was monitored by measuring absorbance at 497 nm which is the absorbance value of congo red using double beam UV–Visible spectrophotometer (ELICO-SL218). In both, three trials were carried from which the average value was drawn.

3.1 Ultraviolet–Visible spectroscopy (UV–Vis) study

The E. blanda and chitosan mediated formation of CuO NPs was monitored by UV–Vis spectrophotometry. The change in colour to blackish green and the absorbance value in agreement with the literature (Sarkar et al., 2020) pointed out to the completion of reduction process and the formation of the desired nanoparticles. The characteristic blackish green colour is attributed to the excitation of the surface plasmon resonance of copper oxide. The UV–Vis spectra of CPCE and PCE in Fig. 3 exhibited absorbance peaks at 286 nm and 278 nm respectively which clearly indicates the formation of copper oxide nanoparticles. Sankar et al. reported that the strong absorbance between 250 and 300 nm suggested the formation of copper oxide nanoparticles (Sankar et al., 2014). Bibi et al. reported a similar UV–Vis absorbance peak for CuO NPs (Bibi et al., 2021). Due to a slight increase in average particle size, it is observed that the surface plasmon resonance of CPCE shifts to longer wavelength and becomes broader which is in good agreement with literature (Link and El-Sayed, 1999). The increase in plasmon bandwidth with increase in particle size is due to extrinsic size effect. Moreover excitation of different multiple modes, which peak at different energies contribute too to the increased line width. On the other hand PCE displayed a blue shift due to its smaller size and more spherical structures. An increase in intensity of the peak with respect to PCE is observed which may be due to an increase in the number of nanoparticles. Goh et al. reported that the intensity of absorbance decreases with increase in size due to decrease in nanoparticle concentration and vice versa (Goh et al., 2014).

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Fig. 3

DR-UV–Vis spectra of (a) CPCE (b) PCE and inset Tauc plot of (c) CPCE (d) PCE.

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The Tauc’s plot obtained by extrapolating the (αhν)^1/n versus photon energy, hν plot to α = 0 depicted in the inset of Fig. 3 (c) and (d) shows the band gap energy to be 3.8 eV for both CPCE and PCE . The band gap energy was calculated using Tauc’s equation: $${(\alpha h\nu )}^{1/n}=K(h\nu -E_g)$$ where α is the optical absorption coefficient, K a constant called the band tailing parameter, h the Planck’s constant, ν the frequency of incident photons, E_g the optical band gap, and n the type of transition in the semiconductor which can have different values (2, 3, 1/2 and 1/3) (Mir, 2014).

3.2 Plausible mechanism for the formation of CuO NPs

The main advantage of using a plant which contain a variety of phytochemicals is to facilitate the oxidation of metal-to-metal oxide nanoparticles in vitro. The possible mechanism involved in the formation of CuO NPs with the help of plant extract is explained as: plant leaf extract which possess different phytochemicals such as alkaloids, flavonoids, terpenoids, polyphenols, aldehydes , ketones , sugars, amides, etc. are responsible for their dual role of stabilizing and reducing agents in synthesising nanoparticles. These phytochemicals serve as a natural source to reduce the metal salts by chelation to zero-valent states. The–OH group present in polyphenols plays a vital role in the synthesis of nanoparticles as they can form coordinated complex with metal ions. When heating was carried out, the bond breakage between the metal and –OH group resulted in the formation of metal oxide nanoparticles by removing water thus enabling the formation of metal oxide nanoparticles (Jaafar et al., 2015; Nasrollahzadeh and Mohammad Sajadi, 2015; Singh et al., 2018). Chitosan has played the dual role of being a controller of nucleation as well as a stabilizer.

3.3 Fourier Transform Infrared spectroscopy (FT-IR)

FT-IR analysis was used to establish the presence of the probable biomolecules in the aqueous extract of E. blanda which facilitated the synthesis of CuO NPs, especially to identify the functional groups and that of Cu-O bond present in the nanoparticles. Fig. 4 shows the IR spectra of the plant extract, CPCE and PCE. The broad band ranging from 3270 to 3364 cm⁻¹ observed in all the three spectra is attributed to the O-H stretching frequency due to physisorption of water (Arun et al., 2015). The absorbance values from 1020 to 1099 cm⁻¹ is indicative of C-O stretching vibrations, confirming the presence of aliphatic amines and its derivatives (Arun et al., 2015). The absorption peak at 1557 cm⁻¹ present in CPCE corresponds to N-H bending of the amide group while the band at 1404 and 1409 cm⁻¹ are related to C = C vibrations (Murthy et al., 2021). On the other hand, the peaks at 417 , 476 , 481, 598 and 601 cm⁻¹ point to the stretching vibration of Cu-O bond which supports the monoclinic phase of CuO NPs which is also in agreement with Siddique et al. (Uddin et al., 2020). The FTIR spectrum of the aqueous plant extract depicted in Fig. 4 (c) indicates the presence of certain functional groups. The bands at 2969 , 1557 , 1397 and 1023 cm⁻¹ can be due to saturated hydrocarbon, amide group characteristic of proteins (Rehana et al., 2017), CH₃ from proteins and lipids (Mostaço-Guidolin et al., 2010) and –OH in phenolic group respectively.

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Fig. 4

Infrared spectroscopy (IR) of (a) CPCE (b) PCE and (c) Plant extract.

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3.4 X-Ray diffraction (XRD)

The crystallinity of the synthesised nanoparticles were investigated by X-ray diffraction (XRD). The XRD pattern in Fig. 5 (a) and (b) shows the crystalline nature of the nanoparticles. CPCE shows diffraction peaks at 2θ = 35.7⁰, 38.9⁰ and 48.9⁰ which corresponds to ( −1 1 1), (2 0 0) and ( −2 0 2) respectively. PCE exhibited 2θ at 32.6⁰, 35.7⁰, 38.7⁰, 48.9⁰, 58.5, 61.7⁰, 66.3 and 68.3⁰ which were assigned the planes (1 1 0), ( −1 1 1), (1 1 1), ( −2 0 2), (2 0 2), ( −1 1 3), ( −3 1 1) and (2 2 0) respectively. The corresponding peaks agreed with the Joint Committee on Powder Diffraction Standards (JCPDS card no. 00–005-0661). It was observed that the use of both chitosan and plant extract together had an exploitable effect on the crystallinity of the nanoparticles. For, CPCE that used both chitosan and leaf extract exhibited less sharp peaks, hence indicating suppressed crystallinity. Revathi et al. too reported a similar decrease in crystallinity of the substance at the usage of both chitosan and plant extract (Revathi and Thambidurai, 2019). Yudyanto et al. in their work found that the addition of chitosan resulted in decrease of crystallinity and increase of pore size (Effendi and Gustiono, (n.d.)). When chitosan was mixed with copper sulphate solution and the aqueous leaf extract, it was probable that Cu²⁺ ions get attached to chitosan macromolecules due to electrostatic interaction. Chitosan which possess electron-rich oxygen atoms in their polar hydroxyl groups interact with electropositive metal cations.

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Fig. 5

High Angle X-Ray Diffraction Pattern of a) CPCE, and b) PCE.

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The crystallite size of the nanoparticles were calculated by employing Debye Scherrer equation: $$d=K\lambda /\beta cos\theta$$ where d = crystallite size in nm, K = 0.9 (Scherrer constant), λ = 0.15406 nm (wavelength of the X-ray sources), θ = the Bragg diffraction angle, and β = full width at half maximum (FWHM) of the respective diffraction peak. Using Scherrer equation, the crystallite size of CPCE was calculated as 16.20 nm and that of PCE as 13.87 nm. The experimental results were in consonance with previously reported CuO NPs (Sarkar et al., 2020; Manyasree et al., 2017; Shi et al., 2017).

3.5 Field Emission Scanning Electron microscope (FE-SEM) with EDAX

The surface morphology and size of the synthesised CuO nanoparticles were established by Field Emission–Scanning Electron Microscopy. FE-SEM images of CPCE in Fig. 8 (a) showed short rod-like structures while some were spherical. PCE on the other hand showed spherical morphology (Fig. 8 (b)). CuO NPs with rod-like morphology were reported by George et al. and Mari et al. (Mari et al., 2020; George et al., 2020). Spherical morphology of CuO NPs too were found in literature (Karuppannan et al., 2021; Chowdhury et al., 2020). Particle size of CPCE was ranging from 30.76 nm to 69.08 nm while that of PCE was found to be 15.97 nm to 49.61 nm. The particle size distribution of the CuO NPs is portrayed in Fig. 6 (a & b). The average diameter of CPCE and PCE were found to be 47.71 nm and 36.07 nm respectively. The EDAX analysis revealed that the prepared CuO NPs were highly pure containing only Cu, O and C moieties. The weight compositions for copper and oxygen were 76.32% and 12.71% respectively for CPCE as shown in Fig. 7 (a). Besides these the presence of 10.97% carbon is observed which may be due to the presence of chitosan. Fig. 7 (b) represents the EDAX spectral analysis of PCE where 74.62% and 25.38% were the weight compositions of copper and oxygen respectively.

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Fig. 6

Histogram showing the particle size distribution of (a) CPCE and (b) PCE.

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Fig. 7

EDAX spectral analysis of (a) CPCE and (b) PCE.

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Fig. 8

(a) (c) (e) SEM, TEM and SAED images of CPCE and (b) (d) (f) SEM, TEM and SAED images of PCE respectively.

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3.6 Transmission Electron Microscopy (TEM) with SAED pattern

The shape, morphology and size of the CuO nanoparticles was established with the help of TEM as shown in Fig. 8. (c) for CPCE. The nanoparticles exhibited rod-like with some spherical morphology with sizes varying from 30 nm to 70 nm. TEM images for PCE are portrayed in Fig. 8. (d) which showed spherical morphology in agreement with SEM images, possessing sizes varying from 16 nm to 50 nm. The polycrystalline nature of the nanoparticles was clearly indicated by SAED which showed bright circular spots as seen in Fig. 8. (e) and (f)). The inter planar d-spacing values was found to be 0.21 nm, 0.27 nm and 0.29 nm for CPCE while for PCE the inter planar d-spacing was calculated as 0.21 nm, 0.26 nm and 0.27 nm. This is in accordance with earlier literatures that reported about the polycrystalline nature of CuO NPs (V. k., 2021; Mohamed et al., 2021). The bigger average size of CPCE is credited to the use of chitosan. Chitosan, due to its viscosity contribute to the increase in pore size and hence the particle size too increased. Zhang et al. reported that increase in concentration of chitosan increased the viscosity of the sample which in turn increased the particle size (Zhang and Kawakami, 2010).

3.7 Antibacterial activity

Antibacterial activity is well established for nanoparticles which prevents bacteria from growing by destroying the cell wall or by inhibiting protein or nucleic acid synthesis (Lee et al., 2015). Antibacterial property of CPCE and PCE, the two biosynthesized CuO NPs was evaluated against two-gram positive (Enterococcus faecalis and Staphylococcus aureus) and two-gram negative (Escherichia coli and Salmonella typhi) bacteria using agar well diffusion method. Ciproflaxin was used as standard. The nanoparticles exhibited different zone of inhibitions at different concentrations of the sample. The zone of inhibition surrounding the wells was used to measure the antibacterial efficiency. The diameter of the zone of inhibition in mm is depicted in Table 1 and the images of the tested bacteria with their zone of inhibition at three different concentrations is presented in Fig. 9. The control without any nanoparticles showed no zone of inhibition. The highest zone of inhibition was 18 mm exhibited by CPCE against Enterococcus faecalis at the concentration of 100 μg/mL. CPCE showed good antibacterial effect against gram negative Salmonella typhi too whereas it showed mild inhibition against Staphylococcus aureus and Eschrichia coli at the concentration of 100 μg/mL only. PCE comparatively demonstrated better antibacterial activity against all four bacteria.

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Fig. 9

(a-h). Antibacterial activity of biosynthesized CPCE and PCE against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Salmonella typhi respectively at different concentrations.

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A possible mechanism is that Cu²⁺ released from CuO binds to thiol groups found in enzymes and proteins on the cellular surface. This leads to interference with cell division and disruption of cell wall, ultimately leading to bacterial cell death. There are many research reports that give evidences of the effective antimicrobial activity of chitosan coated copper oxide nanoparticles (Effendi and Gustiono, (n.d.); Manyasree et al., 2017; Shi et al., 2017).

3.8 Antioxidant activity

Bioactive components like glycosides, reducing sugar, carbohydrates, phenolic acids, steroids, terpenoids, flavonoids, acid/base/neutral compounds, α-amino acids, tannins, alkaloids and saponins are reported to be actively present in E. blanda (Nwe et al., 2020). These components are known for their therapeutic effects like antioxidant, antidiabetic, anti-inflammatory and cytotoxic activities. Free radicals are extremely reactive atoms, molecules or ions which have one or more unpaired electrons, produced during oxidation reactions. Antioxidants aid in scavenging these free radicals from the bodily cells and in preventing the damage caused by their chain reactions. Some recent studies show that decline in antioxidants in the bodies result slowly in the onset of diseases and sicknesses. Free radicals can be hazardous to the body and cause sicknesses such as cancer, stroke, heart diseases, aging and aging related ailments. The highly reactive ‘Reactive Oxygen Species’ (ROS) are produced during mitochondrial oxidative metabolism (Salehi et al., 2018). An excess of ROS leads to oxidative stress that result in cellular damage or abnormal cell growth. Antioxidants therefore help in preventing or reducing the ROS-induced oxidative damage either by direct reaction with free radicals or by constraining the activities of the free radical generating enzymes (Gulcin, 2020). In the present study, the biosynthesised CuO NPs display significant scavenging activity against DPPH. DPPH is a stable free radical molecule, commonly used to quantify the free radical scavenging capacity of compounds. Its stability is based on the presence of a spare electron that is delocalized over the molecule thereby preventing formation of dimers (Thakar et al., 2021). Taking ascorbic acid as the standard, CPCE showed (Table 2) higher scavenging activity than the standard at 20 and 25 μg/mL concentrations with scavenging potential of 73% and 89% respectively (Fig. 10). A comparative study of CPCE and PCE pointed towards CPCE possessing higher efficiency in their antioxidant activity.

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Fig. 10

DPPH scavenging activity of standard, CPCE and PCE.

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This could be attributed to the collective effect of both chitosan and plant extract, since the properties of metal oxides can be improved by combining with chitosan and the product can be employed for different applications. CuO NPs biosynthesized using chitosan and neem seed showed excellent antioxidant activity according to Revathi et al (Revathi and Thambidurai, 2019). Thakar et al. reported the effective antioxidant activity of Cissus vitiginea mediated CuO NPs (Thakar et al., 2021).

3.9 Photo catalytic activity

Cationic and anionic dyes play a very important role in various manufacturing industries. Yet they contribute significantly to environmental pollution, hence the need to check their effluents. Metals and their oxide nanoparticles are reported to be good photo catalysts. The photo catalytic efficiency of the fabricated CuO NPs was assessed by congo red dye decomposition. The test was carried out based on two parameters, concentration and contact time. Dye degradation was identified by decolourization using a UV–Vis spectrophotometer. A clear picture of the efficiency of CuO NPs in dye degradation at different concentrations and different time is shown in Fig. 11. In the Fig. the y-axis gives us information regarding the concentration of the dye and the percentage of degradation of the same. The red plot is the concentration of the dye (congo red) in μg/mL while the black plot is the percentage of degradation. As the concentration of the nanoparticle is increased in case 1, the concentration of the dye decreased while the percentage of degradation increased. In case 2 where the x-axis is time, with increase in time, the dye concentration decreased and percentage of degradation increased. For CPCE, at the highest concentration (50 μg/mL) the percentage of dye degradation was 94% and at the longest time of light irradiation (10 h) it was 95% while for PCE the percentage of dye degradation at 50 μg/mL was 74% and 74% at 10 h. The equation used for calculating the dye percentage degradation was: $$\eta =\left(\frac{{{\mathbf{A}}_{\mathbf{o}}-\mathbf{A}}_{\mathbf{t}}}{{\mathbf{A}}_{\mathbf{o}}}\right)\times 100$$ Where A_o and A_t are absorbance of control and absorbance of test respectively. A comparative study of CPCE and PCE indicated that CPCE had greater degradation efficiency, attributed to the synergistic effect of both chitosan and plant extract along with the nanoparticles. Table 3 Similar findings of effective photocatalytic degradation of congo red were reported.

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Fig. 11

Photocatalytic efficiency of CPCE and PCE respectively at varying concentration and at varying time.

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The mechanism involved in the photocatalytic degradation is graphically described in Fig. 12. When the green synthesized CuO NPs are irradiated with light, electrons from the valence band jumps to the conduction band, creating holes in the valence band (h⁺) and increasing the negativity in the conduction band (e^-). This paves the way for photocatalytic active sites on the surface of the nanoparticles. The valence band holes react with chemisorbed water molecules to create HO^●─ .The excited electrons too react with dissolved molecular oxygen (O₂) leading to the formation of superoxide radical anion (O₂ ^●─) which in turn produces HO₂ ^●─ radicals. These reactive oxygen species (ROS) attack the dye molecules, leading to their degradation (Badr et al., 2008; Manoharan, 2020).

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Fig. 12

Schematic representation showing the possible mechanism of degradation of dye by CuO NPs.

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3.10 Reusability of the materials

The nanoparticles prepared were found to be stable over a long period of time as various biological and catalytic activities were performed at various time intervals. Further, the material is subjected to test its catalytic activity on selective organic reactions which may throw some light on its reusability and the results are awaited. Hence it is presumed that the material is recoverable and reusable.