# Green Synthesis of Copper Nanoparticles UsingWithania somnifera and Its Antioxidant and Antibacterial Activity

Authors: J. Shanmugapriya; C. A. Reshma; V. Srinidhi; K. Harithpriya; K. M. Ramkumar; Dhamodharan Umpathy; Krishnamoorthy Gunasekaran; R. Subashini
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

This study employed green chemistry to synthesize copper nanoparticles (CuNPs) using fresh root extract ofWithania somnifera. The size, shape, morphology, and stability of resultant CuNPs were investigated by UV-Vis spectroscopy, FTIR, transmission electron microscopy (TEM), and energy-dispersive spectrophotometer. Crude extracts were assessed phytochemically to determine the presence of alkaloids, flavonoids, phenols, saponins, steroids, and tannins. The UV-Vis absorption spectra show maximum absorption at 330 nm, and FTIR showed the presence of biological molecules responsible for reducing Cu+ ions. In TEM and EDS analyses, the average diameter of the synthesized NPs was found to be 6.28±1.13nm. The crystal structure of synthesized nanoparticle was determined using XRD analysis and showed its peak at 36.34°, 43.33°, 50.47°, and 73.57°, which corresponds to the cubic lattice of copper. The synthesized nanoparticles (NPs) were tested for their in vitro antimicrobial activity against bacterial strains E. coli and S. aureus by the agar well diffusion method which showed higher efficiency against S. aureus. The antioxidant activity of the synthesized NPs was evaluated by DPPH assay, and the percentage of inhibition was found to be 62% at the concentration of 400 μg/ml and the IC50 value was 51.53 μg/ml. The present investigation suggests that the biosynthesized CuNPs showed prominent antibacterial and free radical scavenging potential. The result proved that root extract of W. somnifera could be applied for synthesis of nanoparticle CuNPs with high uniform particle size along with potent biological activity.


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## Body

## 1. Introduction

Various physical and chemical means of nanoparticle (NP) synthesis like chemical reduction using metallic salts, microemulsion using surfactants, sonochemistry using ultrasound, microwave using microwave radiation, and electrochemistry using electricity require high-capital input for reagents, radiation, and toxic chemicals which are both environmentally toxic and a tedious protocol [1]. Alternatively, the plant-based green synthesis of NPs has proven a rapid, eco-friendly process with advances over microbe-mediated synthesis in the time taken for culturing and its maintenance. Due to the diverse characteristics of metallic nanoparticles like size, magnetization property, flexibility, and rigidity, metallic nanoparticles are extensively used in various sectors, including drug delivery, biosensors, catalysis, and antibacterial agents [2, 3].On considering electrical conductivity, photocatalytic activity, and a lower electrochemical migration, copper nanoparticles (CuNPs) have gained more interest among several metal NPs and have a wide application, including wastewater treatment plants, DNA analysis, antioxidants, and antibacterial agents [4]. Although CuNPs can be synthesized by physicochemical means, a few previous pieces of literature highlight the effectiveness of green synthesis in attaining better photocatalytic and biological activities. Many plants, not restricted to a single part, have been used to synthesize CuNPs because of their bioactive components. For example, extract from the tea leaf, P. granatus leaf, Aloe vera, henna leaf, and tomato has been reported to be effective in synthesizing CuNPs [5].Ashwagandha (W. somnifera L. Dunal) is widely used in the Indian medicinal system to treat numerous diseases like leprosy, nervous disorders, intestinal infections, and rheumatism. It is evident from the literature that W. somnifera is widely used in the herbal formulation that helps our body to defend against diseases by enhancing cell-mediated immune response due to the presence of biologically active compounds such as saponins, tannins, flavonoids, and phenols [6]. The root part of Ashwagandha has a wide range of benefits, including anti-inflammatory, antiulcer, and antidiabetic properties. Previous studies have demonstrated that the extract has been proven to have free radical scavenging activity that helps the cell protect against oxidative stress and its damage. A previous study showed that Ashwagandha had been used for green synthesis of the Ag, Se, TiO, and ZnO [7]. However, the green synthesis of CuNPs from the root extract of W. somnifera has not been investigated. Therefore, this study extends on understanding of the synthesis of CuNPs by reducing Cu+ present in the solution of Cu(NO3)2 by root extract of W. somnifera. The biologically synthesized CuNPs were characterized using ultraviolet-visible (UV-Vis) spectrophotometry and Fourier transform infrared (FT-IR) spectroscopy, whereas the size of the synthesized CuNPs was determined using transmission electron microscopy (TEM), energy-dispersive spectrometry (EDS) analysis, and X-ray diffraction analysis. The antimicrobial properties of CuNPs against both gram-positive and gram-negative bacteria were determined by Kirby-Bauer agar well diffusion test. Also, the free radical scavenging activity of the synthesized CuNPs from the root extract of W. somnifera was determined by DPPH free radical scavenging assay. Furthermost, this study provides comprehensive data on the synthesis and antimicrobial and antioxidant activities of CuNPs, which helps to foresee its biomedical applications.


## 2. Materials and Methods

### 2.1. Collection of Plant Material

The roots ofW. somnifera (commonly known as Ashwagandha) were collected from the local market, which was then identified and authenticated by the Interdisciplinary Institute of Indian System of Medicine (IIISM), SRMIST, Chennai. The roots were then washed and air-dried for 48 h. Following this, the dried root samples were freeze-dried to obtain a constant mass which was then ground finely and further placed on a desiccator till the extract was collected.


### 2.2. Extraction and Phytochemical Screening

The dried coarse powder ofW. somnifera root was extracted with sterile distilled water by the overnight maceration method. 25 g of dried powder was dissolved in 250 ml of dH2O, closed, and stored at room temperature for 24 h. The contents were then filtered, and the filtrate was kept at 4°C until further use [8].Preliminary phytochemical screening was carried out to determine the presence of secondary bioactive metabolites. Standard procedures, as mentioned elsewhere, for determining alkaloids, flavonoids, saponins, tannins, glycosides, phenols, and steroids were carried out in the aqueous extract [9, 10]. The presence of alkaloids was detected using Wagner’s reagent, and phenols were detected using ferric chloride (FeCl3) solution. The presence of triterpenes was quantified using the Salkowski test. Liebermann-Burchard’s test was performed to check the presence of phytosterols. The saponins were detected by foam test. The tannins were detected using a lead acetate test. Fehling’s test was adopted to detect the presence of glycosides. The lead acetate test was performed to check the presence of flavonoids.


### 2.3. Synthesis of CuNPs

The aqueous extracts ofW. somnifera were mixed with the freshly prepared 0.2 M copper nitrate solution in a ratio of 3 : 1. With a constant stirring, the solution was heated for at least 4 h at 55°C until a color change from blue to sea green was observed [11, 12]. The fully reduced solution was further centrifuged for 15 min at room temperature (5000 rpm), washed, and stored at 4°C until further use [13].


### 2.4. Characterizing the Synthesized CuNPs

To characterize the synthesized CuNPs, UV-Vis spectrophotometer, FT-IR, TEM, and XRD were used as follows.


#### 2.4.1. UV-Vis Spectrophotometer

The UV-Vis spectra of synthesized CuNPs, root extract, and Cu(NO3)2 solution were measured using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) measured in the range of 200-800 nm [14, 15].


#### 2.4.2. FT-IR Analysis

The chemical binding of synthesized nanoparticles was analyzed using FT-IR spectrometer (Shimadzu, Japan). The sample mixture was powdered using heat treatment method and stored in a sterile Eppendorf. Then, the powder was used for the FT-IR analysis from the region of 400-4000 cm1. The sample was irradiated and a specific wavelength was absorbed by a particular compound present in the sample, and a graph was plotted accordingly. The peak values were obtained, which correspond to the particular functional group [16].


#### 2.4.3. Transmission Electron Microscopy

The biosynthesized nanoparticles were centrifuged for 15 minutes at room temperature (5000 rpm), washed, and pelleted out. The pellet was then dried and coated on carbon grids to visualize under TEM 39000x magnification. The energy-dispersive spectrometry patterns of synthesized nanoparticles were analyzed using a high-resolution transmission electron microscope (JEM-2100 Plus, JEOL Japan) [17].


#### 2.4.4. X-Ray Diffraction

The XRD pattern of synthesized copper nanoparticles was obtained using an X-ray diffractometer (D8 Advance, BRUKER, USA). The XRD was set up at the step size of 0.05°, time of 1 s from angle 10° to 110° (2Ө) [18]


### 2.5. Antibacterial Screening

The bacterial strainsE.coli (gram-negative) and S. aureus (gram-positive) were inoculated in nutrient broth in an orbital shaker maintained at a constant temperature of 37°C for 24 h and used to test the antibacterial efficacy of the root extract [19]. Agar well diffusion was performed to find the antibacterial susceptibility. The Mueller Hinton Agar plate was poured and inoculated with test strains. After solidification, four wells were punctured (6 mm) using cork borer, and the wells were filled with 25 μl, 50 μl, and 100 μl of synthesized nanoparticle solution of concentration 0.5 μg/ml. For the experiment, sterile water was used as the negative control and a standard antibiotic, ciprofloxacin (5 μg/ml), disk was taken as a positive control. The zone of inhibition was measured in the experimental plates after incubation. All the tests were done in triplicates, and the mean value of the zone of inhibition was determined [20, 21].


### 2.6. Antioxidant Assay

To determine the antioxidant potential of the root extract, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was performed [22]. Absorbance was recorded after incubating the samples in the dark for about half an hour at 517 nm. The free radical scavenging percentage of the extract was determined by the below given formula [23].
(1)%inhibition=absorbanceofthecontrol−absorbanceofthesampleabsorbanceofthecontrol×100.


## 2.1. Collection of Plant Material

The roots ofW. somnifera (commonly known as Ashwagandha) were collected from the local market, which was then identified and authenticated by the Interdisciplinary Institute of Indian System of Medicine (IIISM), SRMIST, Chennai. The roots were then washed and air-dried for 48 h. Following this, the dried root samples were freeze-dried to obtain a constant mass which was then ground finely and further placed on a desiccator till the extract was collected.


## 2.2. Extraction and Phytochemical Screening

The dried coarse powder ofW. somnifera root was extracted with sterile distilled water by the overnight maceration method. 25 g of dried powder was dissolved in 250 ml of dH2O, closed, and stored at room temperature for 24 h. The contents were then filtered, and the filtrate was kept at 4°C until further use [8].Preliminary phytochemical screening was carried out to determine the presence of secondary bioactive metabolites. Standard procedures, as mentioned elsewhere, for determining alkaloids, flavonoids, saponins, tannins, glycosides, phenols, and steroids were carried out in the aqueous extract [9, 10]. The presence of alkaloids was detected using Wagner’s reagent, and phenols were detected using ferric chloride (FeCl3) solution. The presence of triterpenes was quantified using the Salkowski test. Liebermann-Burchard’s test was performed to check the presence of phytosterols. The saponins were detected by foam test. The tannins were detected using a lead acetate test. Fehling’s test was adopted to detect the presence of glycosides. The lead acetate test was performed to check the presence of flavonoids.


## 2.3. Synthesis of CuNPs

The aqueous extracts ofW. somnifera were mixed with the freshly prepared 0.2 M copper nitrate solution in a ratio of 3 : 1. With a constant stirring, the solution was heated for at least 4 h at 55°C until a color change from blue to sea green was observed [11, 12]. The fully reduced solution was further centrifuged for 15 min at room temperature (5000 rpm), washed, and stored at 4°C until further use [13].


## 2.4. Characterizing the Synthesized CuNPs

To characterize the synthesized CuNPs, UV-Vis spectrophotometer, FT-IR, TEM, and XRD were used as follows.


### 2.4.1. UV-Vis Spectrophotometer

The UV-Vis spectra of synthesized CuNPs, root extract, and Cu(NO3)2 solution were measured using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) measured in the range of 200-800 nm [14, 15].


### 2.4.2. FT-IR Analysis

The chemical binding of synthesized nanoparticles was analyzed using FT-IR spectrometer (Shimadzu, Japan). The sample mixture was powdered using heat treatment method and stored in a sterile Eppendorf. Then, the powder was used for the FT-IR analysis from the region of 400-4000 cm1. The sample was irradiated and a specific wavelength was absorbed by a particular compound present in the sample, and a graph was plotted accordingly. The peak values were obtained, which correspond to the particular functional group [16].


### 2.4.3. Transmission Electron Microscopy

The biosynthesized nanoparticles were centrifuged for 15 minutes at room temperature (5000 rpm), washed, and pelleted out. The pellet was then dried and coated on carbon grids to visualize under TEM 39000x magnification. The energy-dispersive spectrometry patterns of synthesized nanoparticles were analyzed using a high-resolution transmission electron microscope (JEM-2100 Plus, JEOL Japan) [17].


### 2.4.4. X-Ray Diffraction

The XRD pattern of synthesized copper nanoparticles was obtained using an X-ray diffractometer (D8 Advance, BRUKER, USA). The XRD was set up at the step size of 0.05°, time of 1 s from angle 10° to 110° (2Ө) [18]


## 2.4.1. UV-Vis Spectrophotometer

The UV-Vis spectra of synthesized CuNPs, root extract, and Cu(NO3)2 solution were measured using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) measured in the range of 200-800 nm [14, 15].


## 2.4.2. FT-IR Analysis

The chemical binding of synthesized nanoparticles was analyzed using FT-IR spectrometer (Shimadzu, Japan). The sample mixture was powdered using heat treatment method and stored in a sterile Eppendorf. Then, the powder was used for the FT-IR analysis from the region of 400-4000 cm1. The sample was irradiated and a specific wavelength was absorbed by a particular compound present in the sample, and a graph was plotted accordingly. The peak values were obtained, which correspond to the particular functional group [16].


## 2.4.3. Transmission Electron Microscopy

The biosynthesized nanoparticles were centrifuged for 15 minutes at room temperature (5000 rpm), washed, and pelleted out. The pellet was then dried and coated on carbon grids to visualize under TEM 39000x magnification. The energy-dispersive spectrometry patterns of synthesized nanoparticles were analyzed using a high-resolution transmission electron microscope (JEM-2100 Plus, JEOL Japan) [17].


## 2.4.4. X-Ray Diffraction

The XRD pattern of synthesized copper nanoparticles was obtained using an X-ray diffractometer (D8 Advance, BRUKER, USA). The XRD was set up at the step size of 0.05°, time of 1 s from angle 10° to 110° (2Ө) [18]


## 2.5. Antibacterial Screening

The bacterial strainsE.coli (gram-negative) and S. aureus (gram-positive) were inoculated in nutrient broth in an orbital shaker maintained at a constant temperature of 37°C for 24 h and used to test the antibacterial efficacy of the root extract [19]. Agar well diffusion was performed to find the antibacterial susceptibility. The Mueller Hinton Agar plate was poured and inoculated with test strains. After solidification, four wells were punctured (6 mm) using cork borer, and the wells were filled with 25 μl, 50 μl, and 100 μl of synthesized nanoparticle solution of concentration 0.5 μg/ml. For the experiment, sterile water was used as the negative control and a standard antibiotic, ciprofloxacin (5 μg/ml), disk was taken as a positive control. The zone of inhibition was measured in the experimental plates after incubation. All the tests were done in triplicates, and the mean value of the zone of inhibition was determined [20, 21].


## 2.6. Antioxidant Assay

To determine the antioxidant potential of the root extract, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was performed [22]. Absorbance was recorded after incubating the samples in the dark for about half an hour at 517 nm. The free radical scavenging percentage of the extract was determined by the below given formula [23].
(1)%inhibition=absorbanceofthecontrol−absorbanceofthesampleabsorbanceofthecontrol×100.


## 3. Results

Table1 depicts the preliminary phytochemical tests of the root extract of W. somnifera. The presence of alkaloids, saponins, tannins, glycosides, phenols, and steroids was observed. At the same time, a trace or a negligible amount of phytochemicals was estimated in the root extracts of W. somnifera.Table 1
Phytochemical screening of aqueous root extract ofW. somnifera.
S. no.Preliminary phytochemical testResult1Alkaloids++2Flavonoids−3Saponins++4Tannins++5Glycosides++6Phenols+7Triterpenes++8Phytosterols+++++: high; ++: medium; +: poor; −: not found.


### 3.1. UV-Vis Spectrophotometer

The synthesized nanoparticle was confirmed as a copper nanoparticle using a UV-Vis spectrophotometer. The copper nanoparticle showed an absorbance peak at 330 nm, whereas the aqueous root extract showed a peak at 225 nm and the copper nitrate solution showed an absorbance peak at 235 nm, as shown in Figure1.Figure 1
UV-Vis spectrophotometer analysis of synthesized CuNPs.


### 3.2. Transmission Electron Microscope

TEM analysis of biosynthesized copper nanoparticles from the root extract ofW. somnifera displayed size below 20 nm and appears to be spherical, as shown in Figure 2(a). The particle size distribution was determined by taking the diameter of the CuNPs from different parts. The size of the synthesized copper nanoparticle from the aqueous root extract of W. Somnifera particles was in the range between 5 and 9 nm, as shown in Figure 2(b), with an average size 6.28±1.13nm. The energy-dispersive spectroscopy pattern of synthesized copper nanoparticles was recorded and confirmed the presence of copper element in the biosynthesized CuNPs as shown in Figure 3.Figure 2
(a) TEM image of CuNPs. (b) Average particle size of CuNPs.
(a)(b)Figure 3
EDS spectra of Cu element.


### 3.3. FT-IR Analysis

To identify the key factors for the copper ion (Cu+) reduction into copper nanoparticles (CuNPs) in the aqueous root extract of W. somnifera, FTIR spectroscopy analyses were carried out. FT-IR analyses show the shifts in the absorbance peak of copper nanoparticles with different points ranging from 561 to 3390 cm-1 as shown in Figure 4. It is evident that there is presence of functional groups such as alkaloids, phenols, halo compounds, and primary and secondary amines in the extract. The IR spectrum of the biosynthesized CuNPs showed the peak value at 561, 600, 679, 1360, 1644, and 3390 cm-1. The peak value 561 cm-1 relates to the Cu-O bond in the CuO nanostructure group. A peak at 600 cm-1 and 679 cm-1 relates to the C-I stretch and C-Cl bond in halo compound group. The peak at 1350 cm-1 was assigned to be C-H bend in the methane group, 1644 cm-1 relates to the C-C stretch in the aromatic group and peak at 3390 cm-1 O-H stretch in the primary and secondary amide group.Figure 4
FT-IR spectrum of synthesized CuNPs.


### 3.4. X-Ray Diffraction (XRD)

The crystalline structure of the synthesized nanoparticle was done using the XRD technique that confirms the presence of CuNP by the peak at2Ө values of 36.34°, 43.33°, 50.47°, and 73.57° corresponding to 111, 111, 200, and 220 (Figure 5) planes that confirm the cubic lattice of copper (according to JCPDS, copper file no. 04-0836). The minor peaks were observed at 2Ө value of 42.282° and 61.4° indicating the source material Cu2O.Figure 5
X-ray diffraction of synthesized CuNPs.


### 3.5. Antibacterial Screening

Antibacterial activity of CuNPs was shown against both the test organisms. From the mean zone of inhibition, it was evident that the inhibition activity of CuNPs at concentrations of 25, 50, and 100μg/ml against S. aureus was higher when compared to the zone of inhibition for E. coli along with that of positive control. As the concentration of the copper nanoparticle increases, the ability of the CuNP inhibitory activity against the test organism also increases, as shown in Figure 6.Figure 6
Zone of inhibition of synthesized CuNPs againstE.coli and S. aureus. Values are presented as mean±SD (n=3). Statistical analysis was performed by Student’s t-test. ∗∗P<0.01; ∗∗∗P<0.001.


### 3.6. Antioxidant Assay

It was observed that among five different concentrations of CuNPs tested, the maximum of 62% of activity inhibition was observed at 400μg/ml and a minimum of 44.8% of inhibition at the lowest concentration, 25 μg/ml as shown in Figure 7. The IC50 for the synthesized CuNPs was 51.53 μg/ml and is effective in scavenging 50% of the DPPH.Figure 7
Percentage of inhibition DPPH free radical by synthesized CuNPs. Values are presented asmean±SD (n=3). Statistical analysis was performed by Student’s t-test. ∗∗P<0.01; ∗∗∗P<0.001.


## 3.1. UV-Vis Spectrophotometer

The synthesized nanoparticle was confirmed as a copper nanoparticle using a UV-Vis spectrophotometer. The copper nanoparticle showed an absorbance peak at 330 nm, whereas the aqueous root extract showed a peak at 225 nm and the copper nitrate solution showed an absorbance peak at 235 nm, as shown in Figure1.Figure 1
UV-Vis spectrophotometer analysis of synthesized CuNPs.


## 3.2. Transmission Electron Microscope

TEM analysis of biosynthesized copper nanoparticles from the root extract ofW. somnifera displayed size below 20 nm and appears to be spherical, as shown in Figure 2(a). The particle size distribution was determined by taking the diameter of the CuNPs from different parts. The size of the synthesized copper nanoparticle from the aqueous root extract of W. Somnifera particles was in the range between 5 and 9 nm, as shown in Figure 2(b), with an average size 6.28±1.13nm. The energy-dispersive spectroscopy pattern of synthesized copper nanoparticles was recorded and confirmed the presence of copper element in the biosynthesized CuNPs as shown in Figure 3.Figure 2
(a) TEM image of CuNPs. (b) Average particle size of CuNPs.
(a)(b)Figure 3
EDS spectra of Cu element.


## 3.3. FT-IR Analysis

To identify the key factors for the copper ion (Cu+) reduction into copper nanoparticles (CuNPs) in the aqueous root extract of W. somnifera, FTIR spectroscopy analyses were carried out. FT-IR analyses show the shifts in the absorbance peak of copper nanoparticles with different points ranging from 561 to 3390 cm-1 as shown in Figure 4. It is evident that there is presence of functional groups such as alkaloids, phenols, halo compounds, and primary and secondary amines in the extract. The IR spectrum of the biosynthesized CuNPs showed the peak value at 561, 600, 679, 1360, 1644, and 3390 cm-1. The peak value 561 cm-1 relates to the Cu-O bond in the CuO nanostructure group. A peak at 600 cm-1 and 679 cm-1 relates to the C-I stretch and C-Cl bond in halo compound group. The peak at 1350 cm-1 was assigned to be C-H bend in the methane group, 1644 cm-1 relates to the C-C stretch in the aromatic group and peak at 3390 cm-1 O-H stretch in the primary and secondary amide group.Figure 4
FT-IR spectrum of synthesized CuNPs.


## 3.4. X-Ray Diffraction (XRD)

The crystalline structure of the synthesized nanoparticle was done using the XRD technique that confirms the presence of CuNP by the peak at2Ө values of 36.34°, 43.33°, 50.47°, and 73.57° corresponding to 111, 111, 200, and 220 (Figure 5) planes that confirm the cubic lattice of copper (according to JCPDS, copper file no. 04-0836). The minor peaks were observed at 2Ө value of 42.282° and 61.4° indicating the source material Cu2O.Figure 5
X-ray diffraction of synthesized CuNPs.


## 3.5. Antibacterial Screening

Antibacterial activity of CuNPs was shown against both the test organisms. From the mean zone of inhibition, it was evident that the inhibition activity of CuNPs at concentrations of 25, 50, and 100μg/ml against S. aureus was higher when compared to the zone of inhibition for E. coli along with that of positive control. As the concentration of the copper nanoparticle increases, the ability of the CuNP inhibitory activity against the test organism also increases, as shown in Figure 6.Figure 6
Zone of inhibition of synthesized CuNPs againstE.coli and S. aureus. Values are presented as mean±SD (n=3). Statistical analysis was performed by Student’s t-test. ∗∗P<0.01; ∗∗∗P<0.001.


## 3.6. Antioxidant Assay

It was observed that among five different concentrations of CuNPs tested, the maximum of 62% of activity inhibition was observed at 400μg/ml and a minimum of 44.8% of inhibition at the lowest concentration, 25 μg/ml as shown in Figure 7. The IC50 for the synthesized CuNPs was 51.53 μg/ml and is effective in scavenging 50% of the DPPH.Figure 7
Percentage of inhibition DPPH free radical by synthesized CuNPs. Values are presented asmean±SD (n=3). Statistical analysis was performed by Student’s t-test. ∗∗P<0.01; ∗∗∗P<0.001.


## 4. Discussion

Biosynthesis of nanoparticles from the plant extract has gained the limelight in nanotechnology, which possesses significant biological activity such as antioxidant, antimicrobial, and antipyretic activities. Among various medicinally beneficial plats,W. somnifera has an array of applications in traditional medicine because of its rich antimicrobial, antioxidant, and enhancement of cell-mediated immune response. The current study emphasizes the synthesis of CuNPs from the root extract of W. somnifera with potent biological activity such as free radical scavenging and antibacterial efficacy. Green copper nanoparticle synthesis from the reduction of copper ion, further exposing to the plant biological extract, the color change could be analyzed. Due to the plasmon resonance phenomena, the synthesized copper nanoparticle exhibits sea-green color in the aqueous solution. Then, the synthesized CuNPs were primarily characterized by UV-Vis spectrometry where the sharp peak indicated the existence of CuNPs.The reduction of copper ions (Cu+) in the presence of plant extract has occurred within 4 hours of reaction, claimed to be the fastest biosynthesis method of the NPs. The plasmon surface resonance band of CuNPs was indicated at the peak absorbance at 330 nm. A similar peak value was seen in the green synthesis of CuNPs using Ageratum houstonianum Mill. [13]. The synthesized CuNPs predominantly appear to be spherical in shape. The TEM analysis image showed the relative spherical NPs typically the size between 5 and 9 nm. Similar phenomena were reported in Mali et al. [17]. EDS was performed for the samples to check the existence of Cu element in the synthesized NPs.To identify the key factors for the Cu+ reduction into CuNPs in the aqueous root extract of W. somnifera, FT-IR spectroscopy analysis was carried out. The shifts in the absorbance peak of CuNPs with different points ranging from 561 to 3390 cm-1 were observed in FT-IR analysis. This predicts the possible functional groups involved in reducing the Cu+ to CuNPs. FT-IR spectrum detects several absorption peaks, specifically for C-C stretch, indicating alkenes, and a strong C-I stretch and C-Cl bond attributes to halo compounds. The broad primary and secondary amines groups (O-H) show a strong stretching vibration at 3390 cm-1. Spherically shaped CuNPs were observed in the biosynthesized sample, with an average size of 6.28±1.13nm. The EDS microanalysis was recorded from the synthesized CuNPs. From the EDS analysis, it is confirmed that the existence of Cu element with the peak absorption range observed in the synthesized CuNPs using W. somnifera aqueous root extract has the weight percentage of copper as 20.98%. EDS peaks from C, O, N, Cl, Na, and S may be due to the emission of X-ray from proteins and enzymes of aqueous root extract. From the XRD peaks of synthesized CuNPs confirm the presence of copper nanoparticle with the 2Ө value of 36.34°, 43.33°, 50.47°, and 73.57° [24]. The peaks were then confirmed using the JCPDS database. It is evident that the peaks obtained from the XRD analysis correspond to the cubic lattice of copper [25, 26].From the preliminary phytochemical tests, the existence of alkaloids and flavonoids was confirmed, which could be correlated with the effect of the extract on bacterial strains. In our study, agar well diffusion method of antibacterial screening of CuNPs againstE.coli and S. aureus was performed. The results from our current study showed a concentration-dependent effectiveness which coincided with some of the earlier literary findings where the antimicrobial activity was shown to have a direct relation with the increasing concentration of biosynthesized CuNPs [27]. Synthesized nanoparticle has high antibacterial efficacy against S. aureus than E.coli. This may be due to small size and high surface area to volume ratio that allows the copper nanoparticle to interact with the membrane of the bacteria [25]. In accordance with DPPH free radical scavenging assay, it has been revealed that the synthesized CuNPs have greater antioxidant potential. The antioxidant potential of synthesized CuNPs has increased in a concentration-dependent manner with 62% of scavenging activity at maximum concentration and 44% at minimum concentration. The observed IC50 value showed the synthesized CuNPs exhibited the highest antioxidant potential (51.53 μg/ml). According to the previous study [28], the compound which exhibits IC50 value ranging from 50 to 100 ppm reflects the intermediate antioxidant ability, whereas a compound with 10 to 50 ppm of IC50 is considered to possess strong antioxidant potential.


## 5. Conclusion

In conclusion, this study presented the eco-friendly, cost-effective green synthesis of copper nanoparticles usingW. somnifera aqueous root extract as a reducing agent in the presence of copper nitrate. Though the preliminary confirmation of the formation of CuNPs was the change in solution color, UV-Vis spectroscopy confirmed its formation with an absorption peak of 330 nm. FTIR results showed the presence of CuO nanostructure at the peak value of 561 cm-1. FTIR also confirmed the presence of functional groups responsible for the reducing Cu+ ions. TEM image showed that the average size of copper nanoparticles was 6.28±1.13nm. Synthesized nanoparticles were further confirmed by XRD analysis at 2Ө values of 36.34°, 43.33°, 50.47°, and 73.57° corresponding to the planar copper cubic lattice. Antibacterial assay performed showed that copper nanoparticles have good antibacterial activity against S. aureus. Synthesized copper nanoparticles exhibited good antioxidant activity, which was found by the DPPH assay. Hence, using the study, potent antimicrobial and antioxidant principles from biosynthesized CuNPs can be formulated in the future for the clinical development of therapeutic drugs against infectious diseases.



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*Source: 7967294-2022-07-25.xml*