# Biosynthesis of Biocompatible AgNPs Using Medicinally ImportantCarduus edelbergii Rech.f. Extract for Multifarious Biological Activities

Authors: Shahid Jamil; Ghulam Dastagir; Ahmed Ibrahim Foudah; Mohammad Hamed Alqarni; Hasan Soliman Yusufoglu; Huda Mohammed Alkreathy; Omer Erturk; Rahmat Ali Khan; Muhammad Abdur Rehman Shah
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

In current scenario, plant-mediated synthesis of nanoparticles has received significant interest owing to its cost-effectiveness and eco-friendly methods. This study was planned to find the potential ofCarduus edelbergii Rech.f. extract for the fabrication of silver nanoparticles (AgNPs) and their subsequent antibacterial, antifungal, cytotoxic, and antioxidant activities. The prepared plant extract was analyzed for phytochemicals, elements, and nutrients. The fabricated AgNPs were characterized by spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The synthesized AgNPs and the Carduus edelbergii extract were investigated for their antioxidant, antibacterial, cytotoxic, and antifungal characteristics. The results showed that the plant extract contains a wide range of important nutrients and secondary metabolites. The synthesized AgNPs were crystalline and globular with an average diameter of 36.5 nm and exhibited a localized surface plasmon resonance (LSPR) peak at roughly 420 nm. As compared to Carduus edelbergii extract, the phytofabricated AgNPs had an increased antioxidant and cytotoxic efficacy. The AgNPs showed substantial antibacterial action against Gram-positive (Staphylococcus epidermis and Staphylococcus aureus) and Gram-negative (Klebsiella aerogenes and Escherichia coli) bacteria. The findings revealed that the phytofabricated AgNPs could be an effective antioxidant, antibacterial, and antifungal for treatment of medical conditions.


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## Body

## 1. Introduction

Nanotechnology is among the most explored domains in science and technology in the twenty-first century that has revolutionized every discipline of life. Nanotechnology is concerned with manipulating shape, size, and structure at the nanoscale to bring forth novel characters in materials compared to their bulk counterparts [1]. The nanotechnology is based on nanoparticles (NPs) having unique optical, physical, chemical, electrical, magnetic, thermal, and catalytic properties [2]. Metallic nanoparticles are being used in biomedical, agricultural, and environmental sectors. Metallic nanoparticles with antibacterial properties represent a promising alternative to antibiotics [3].The formulation and synthesis of NPs are being carried out in a top-down and bottom-up manners [3, 4]. The top-down approach or microfabrication method includes physical methods like cutting and grinding to reshape the materials into desirable conformation and pattern [4], which commonly involves lithography, sputtering, etching, and ball mill facilitated grinding [5]. Bottom-up procedure or self-assembly method effectively regulates the size and shape of desirable materials according to the intended use by adjusting the reaction parameters like pH, temperature, or the concentration of precursor material [6].NPs synthesis can be accomplished via physical, chemical, and biological methods [7]. Physical techniques involve the use of variety of methods including laser ablation and evaporation condensation and do not require addition of any external agents which means that the NPs produced would be completely free of organic contaminants that are commonly used in chemical methods [8]. They are extremely costly; require high energy, large space for installing machinery, and increased environmental temperature [9]. The chemical methods of NP synthesis focus on the production of chemical-based NPs with the assistance of inorganic and organic reductants, such as sodium citrate, ascorbic acid, polyols, sodium borohydride, tollen agents, ethylene glycol, and elemental hydrogen [10–12]. Green synthesis or biological synthesis is the most appropriate and sustainable substitute to the physical and chemical methods [7]. Biological production of NPs is fairly simple, environmentally safe, and cheaper in terms of a cost which makes it the method of choice [13]. Green synthesis of NPs can be performed at room temperature and normal pH, which reduces the efforts and time required for the optimization of pH and temperature for NPs synthesis [14]. Various biological molecules like proteins and polypeptides obtained from various biological organisms like plants, bacteria, fungi, yeasts, algae, viruses, and actinomycetes were employed in the biosynthesis of NPs [15, 16].AgNPs are an important tool in nano-science owing to their applications in plethora of fields like nanomedicine, diagnostics, drug delivery, pharmacology, cosmetics, food industry, and optical sensing. AgNPs have been successfully employed as antibacterial, antifungal, and anticancer agents. AgNPs have also been used in the formulation of textile products, biomedical devices, wound dressings, keyboards [17], electrical conductance [18], catalysis, biosensors, bioimaging, delivery of drugs, and photonics [19]. The biocompatibility of AgNPs is their most striking feature which makes it a NP of choice for biomedical applications [20]. AgNPs synthesis has been accomplished using several biological agents like bacteria, fungi, algae, and plants; however, due to stable NPs production and one-step approach, plant-based synthesis approach is the most widely used approach for the biosynthesis of AgNPs. The present study was designed to conduct green synthesis of Carduus edelbergii mediated AgNPs and investigate their chemical and biological charateristics mediated AgNPs.


## 2. Materials and Methods

### 2.1. Plant Collection and Extract Preparation

Aerial parts of young and healthyCarduus edelbergii plants were gathered from the district Bajur, Khyber Pakhtunkhwa, Pakistan. The plants were identified by Prof. Dr. Ghlum Dastagir, Chairman Department of Botany and voucher specimen number Shahid Jamil Bot 180 (PUP) was preserved in the departmental herbarium for future reference. For the removal of contaminants like dust, the plant materials were rinsed with tap water. The aerial parts were shade dried for 25 days and pulverized. Powdered plant material (150 g) was weighed, put into flask, poured 450 mL methanol (70 %), and placed for 72hours with frequent agitation. The extract was filtered with Whatman filter paper and kept for future use.


### 2.2. Phytochemical Screening

Phytochemical screening of the crude methanol extract of aerial parts ofCarduus edelbergii was studied according to standard protocols [21, 22].


### 2.3. Elemental Determination

Both the macro and microelements present in the aerial parts ofCarduus edelbergii were determined by Atomic Absorption Spectrophotometry.


### 2.4. Proximate Analysis

TheCarduus edelbergii powder were analyzed using AOAC techniques [23, 24] for the measurement of dry matter, moisture, crude fat, protein, and fibers as well as total ash. Plant materials were weighed, and moisture and ash contents were estimated based on the variation in mass. After the samples were digested and devoid of fat, the fiber content was estimated by weighing the dry residue after it was ignited. The crude fat content was obtained from the samples by extracting them with petroleum ether in a Soxhlet extractor. All the proximate values were presented in percentages.Carbohydrate contents were analyzed by the standard protocol [25]. The sample extract (45 mL) was diluted to 450 mL with dH2O. 1 mL of the diluted filtrate, water, and glucose was put into experimental, control, and standard test tubes, respectively. Freshly prepared Anthrone reagent (0.1%, 5 mL) was poured to each test tube and mixed thoroughly by gently shaking, kept in water bath (30 °C) for 12 min and then cooled to room temperature. The absorbance was measured at 630 nm spectrophotometrically. The stable green color for about 2 h indicates the presence of glucose.Total percent carbohydrates and total gross energy values were computed by using the formula:(1)%Carbohydrates=100−%protein+%fibres+%fats+%moisturecontents+%ashcontents,(2)TotalgrossenergyvalueKcal/100g=4.2×%carbohydrates+8.37×%fats+2.62×%proteins.


### 2.5. AgNPs Biosynthesis

To synthesize AgNPs, AgNO3 was employed as a precursor salt. AgNPs were synthesized by combination of 5 mL extract with 95 mL AgNO3 solution and placed at room temperature for 24 hours. They were isolated by centrifugation for 12 minutes using a Labnet 24D Spectrofuge in 1 mL Eppendorf tubes at 12000 rpm. The supernatant was removed and collected the pallet. To clean any unattached phytochemicals or unreacted precursor salt from the resultant AgNPs, they were rigorously rinsed 4 times with distilled water.


### 2.6. Characterization of NPs

#### 2.6.1. Spectrophotometric Analysis

The biosynthesis of AgNPs was confirmed spectrophotometrically by using Halo DB-20 UV-Vis Double Beam Spectrophotometer. The testing mixtures were examined in the wavelength (λ) ranging between 350 and 800 nm. The incidence of λmax between 400 and 480 shows the existence of AgNPs in the reaction mixture.


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

The AgNPs generated usingCarduus edelbergii extract were investigated using an X-ray Diffractometer in the range of 10°-80°, with 0.03°/s scanning step size, and 0.55 seconds step time (Model-D8 Advance, Germany). It was found that the Cu and Kα radiation (tube current: 30 mA; generator voltage: 40 kV; wavelength: 1.5406 Å) provided the best diffraction results. The powdered AgNPs were analyzed using around 1 mg of powdered AgNPs. The size of AgNPs was computed using the Debye-Scherrer equation as follows.
(3)D=kλβCosθ,where θ= Bragg’s angle, k = shape factor (0.94), β = full width at half maximum in radians, and λ = X-ray wavelength (1.5421 Å).


#### 2.6.3. Fourier Transformed Infrared Spectroscopy (FTIR)

An infrared spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) with KBr was operated to explore the functional groups bound to the prepared nanoparticles within spectral range of 500 to 3500 cm-1.


#### 2.6.4. Scanning Electron Microscopy (SEM)

The surface morphology of the produced NPs was examined with the help of SEM (Jeol JSM-6510LV). The ionic conductivity of the NPs was improved by sputtering them with platinum after they had been fastened to support.


### 2.7. Antioxidant Potential ofCarduus Edelbergii

The antioxidant activity ofCarduus edelbergii was assessed using the following assays.


#### 2.7.1. DPPH Free Radical Scavenging Assay

In order to determine the antioxidant capacity of NPs, the commonly used antioxidant assay known as the DPPH (2,2-diphenyl-1-picrylhydrazyl) test was carried out in accordance with the recommended [26] protocol. Unique working solutions of ascorbic acid and NPs were made (100, 150, 300, 500, and 1000 μg/mL). The DPPH solution (3 mg in 100 mL) was produced, kept at 25 °C in a container completely covered with aluminum foil for 30 minutes, and then its absorbance at 517 nm was measured and calibrated to <1. The absorbance was spectrophotometrically determined after the sample/ascorbic acid solutions were poured to the DPPH solution. The capability of ascorbic acid/AgNPs to scavenge free radicals was calculated by using the formula below:
(4)Inhibition%=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


#### 2.7.2. ABTS Free Radical Scavenging Assay

The ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) activity was carried out according to Re et al.’s [27] protocols. The stock solution was prepared by combination of 2.4 mM K2S2O8 and 7 mM ABTS solutions. The resultant mixture was diluted with methanol to attain the desired absorbance 0.900 (±0.02). The prepared solution was placed over night at 37 °C in total darkness and then measured the absorbance at 745 nm. After combining the ascorbic/NPs solution (300 μL; 100, 150, 300, 500, and 1000 μg/mL) with the ABTS working solution and incubation for 6 minutes, the absorbance was measured. Ascorbic acid was used as a reference in the control. The following equation was used to compute the % scavenging of ABTS free radicals:
(5)Percentscavenging=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


### 2.8. Antibacterial Activity

Five distinct strains of bacteria, includingEscherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Staphylococcus epidermis, and Acetobacter spp., were evaluated against the produced AgNPs and plant extract. The agar-well diffusion test was used to conduct an in vitro investigation of their antibacterial potential. Using a cotton swab, bacteria were inoculated onto agar plates in a quick manner. To the appropriate wells, 20 μL of each sample, i.e., standard antibiotic amoxicillin (10 mg/mL), plant extract (10 mg/mL), and Carduus edelbergii–mediated AgNPs (10 mg/mL), and DMSO were introduced. Prior to usage, all equipments to be utilized in the experiment were adequately sterilized. Plates were stored in incubator for 24 hours at room temperature. The zones of inhibition were measured in millimeters after a 24-hour incubation period.


### 2.9. Antifungal Activity

The slant approach was used for antifungal activity. A conical flask’s was sealed with a cotton stopper after pouring 50 mL of dH2O to 3.5 g SDA to prepare SDA media. By autoclaving the medium, test tubes, and wire loop for 15 minutes at 121 °C and 15 psi, all of the instruments were thoroughly sterilized. The slants within the laminar flow hood were established by pouring 60 mL of SDA medium into test tubes. To assess the zones of inhibition, 60 μL from each prepared sample, i.e., Carduus edelbergii–mediated AgNPs, plant extract, standard antibiotic (Terbinafine; Saffron Pharmaceuticals Pvt. Ltd.), and DMSO, was put into the appropriate wells.


### 2.10. Cytotoxic Assay

Methanol extract and AgNPs were tested for their cytotoxic effects utilizing brine shrimp lethality bioassays. In a dark chamber, 1 mg shrimp eggs were cultured in 4% seawater (w/v). A central porous wall allowed shrimp to enter the lit chamber after hatching. Methanol was used to generate working solutions of all samples with varying concentrations (100-1000μg/mL). A total of three trials were conducted for each of the mentioned concentration. For the removal of methanol, the test tubes were held at 25 °C for 24 hours. 10 shrimp were put in each test tube and incubated for 24 hours at 25 °C. After that, the shrimps in each test tube were counted, and Abbot’s equation was applied to compute the percentage of death. Then, the experimental and control groups were compared.
(6)%Mortality=Experimental−control/control×100.


### 2.11. Statistical Analysis

GraphPad Prism software was used for statistical analysis.


## 2.1. Plant Collection and Extract Preparation

Aerial parts of young and healthyCarduus edelbergii plants were gathered from the district Bajur, Khyber Pakhtunkhwa, Pakistan. The plants were identified by Prof. Dr. Ghlum Dastagir, Chairman Department of Botany and voucher specimen number Shahid Jamil Bot 180 (PUP) was preserved in the departmental herbarium for future reference. For the removal of contaminants like dust, the plant materials were rinsed with tap water. The aerial parts were shade dried for 25 days and pulverized. Powdered plant material (150 g) was weighed, put into flask, poured 450 mL methanol (70 %), and placed for 72hours with frequent agitation. The extract was filtered with Whatman filter paper and kept for future use.


## 2.2. Phytochemical Screening

Phytochemical screening of the crude methanol extract of aerial parts ofCarduus edelbergii was studied according to standard protocols [21, 22].


## 2.3. Elemental Determination

Both the macro and microelements present in the aerial parts ofCarduus edelbergii were determined by Atomic Absorption Spectrophotometry.


## 2.4. Proximate Analysis

TheCarduus edelbergii powder were analyzed using AOAC techniques [23, 24] for the measurement of dry matter, moisture, crude fat, protein, and fibers as well as total ash. Plant materials were weighed, and moisture and ash contents were estimated based on the variation in mass. After the samples were digested and devoid of fat, the fiber content was estimated by weighing the dry residue after it was ignited. The crude fat content was obtained from the samples by extracting them with petroleum ether in a Soxhlet extractor. All the proximate values were presented in percentages.Carbohydrate contents were analyzed by the standard protocol [25]. The sample extract (45 mL) was diluted to 450 mL with dH2O. 1 mL of the diluted filtrate, water, and glucose was put into experimental, control, and standard test tubes, respectively. Freshly prepared Anthrone reagent (0.1%, 5 mL) was poured to each test tube and mixed thoroughly by gently shaking, kept in water bath (30 °C) for 12 min and then cooled to room temperature. The absorbance was measured at 630 nm spectrophotometrically. The stable green color for about 2 h indicates the presence of glucose.Total percent carbohydrates and total gross energy values were computed by using the formula:(1)%Carbohydrates=100−%protein+%fibres+%fats+%moisturecontents+%ashcontents,(2)TotalgrossenergyvalueKcal/100g=4.2×%carbohydrates+8.37×%fats+2.62×%proteins.


## 2.5. AgNPs Biosynthesis

To synthesize AgNPs, AgNO3 was employed as a precursor salt. AgNPs were synthesized by combination of 5 mL extract with 95 mL AgNO3 solution and placed at room temperature for 24 hours. They were isolated by centrifugation for 12 minutes using a Labnet 24D Spectrofuge in 1 mL Eppendorf tubes at 12000 rpm. The supernatant was removed and collected the pallet. To clean any unattached phytochemicals or unreacted precursor salt from the resultant AgNPs, they were rigorously rinsed 4 times with distilled water.


## 2.6. Characterization of NPs

### 2.6.1. Spectrophotometric Analysis

The biosynthesis of AgNPs was confirmed spectrophotometrically by using Halo DB-20 UV-Vis Double Beam Spectrophotometer. The testing mixtures were examined in the wavelength (λ) ranging between 350 and 800 nm. The incidence of λmax between 400 and 480 shows the existence of AgNPs in the reaction mixture.


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

The AgNPs generated usingCarduus edelbergii extract were investigated using an X-ray Diffractometer in the range of 10°-80°, with 0.03°/s scanning step size, and 0.55 seconds step time (Model-D8 Advance, Germany). It was found that the Cu and Kα radiation (tube current: 30 mA; generator voltage: 40 kV; wavelength: 1.5406 Å) provided the best diffraction results. The powdered AgNPs were analyzed using around 1 mg of powdered AgNPs. The size of AgNPs was computed using the Debye-Scherrer equation as follows.
(3)D=kλβCosθ,where θ= Bragg’s angle, k = shape factor (0.94), β = full width at half maximum in radians, and λ = X-ray wavelength (1.5421 Å).


### 2.6.3. Fourier Transformed Infrared Spectroscopy (FTIR)

An infrared spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) with KBr was operated to explore the functional groups bound to the prepared nanoparticles within spectral range of 500 to 3500 cm-1.


### 2.6.4. Scanning Electron Microscopy (SEM)

The surface morphology of the produced NPs was examined with the help of SEM (Jeol JSM-6510LV). The ionic conductivity of the NPs was improved by sputtering them with platinum after they had been fastened to support.


## 2.6.1. Spectrophotometric Analysis

The biosynthesis of AgNPs was confirmed spectrophotometrically by using Halo DB-20 UV-Vis Double Beam Spectrophotometer. The testing mixtures were examined in the wavelength (λ) ranging between 350 and 800 nm. The incidence of λmax between 400 and 480 shows the existence of AgNPs in the reaction mixture.


## 2.6.2. X-Ray Diffraction (XRD)

The AgNPs generated usingCarduus edelbergii extract were investigated using an X-ray Diffractometer in the range of 10°-80°, with 0.03°/s scanning step size, and 0.55 seconds step time (Model-D8 Advance, Germany). It was found that the Cu and Kα radiation (tube current: 30 mA; generator voltage: 40 kV; wavelength: 1.5406 Å) provided the best diffraction results. The powdered AgNPs were analyzed using around 1 mg of powdered AgNPs. The size of AgNPs was computed using the Debye-Scherrer equation as follows.
(3)D=kλβCosθ,where θ= Bragg’s angle, k = shape factor (0.94), β = full width at half maximum in radians, and λ = X-ray wavelength (1.5421 Å).


## 2.6.3. Fourier Transformed Infrared Spectroscopy (FTIR)

An infrared spectroscope (Spectrum One, Perkin Elmer, Waltham, MA, USA) with KBr was operated to explore the functional groups bound to the prepared nanoparticles within spectral range of 500 to 3500 cm-1.


## 2.6.4. Scanning Electron Microscopy (SEM)

The surface morphology of the produced NPs was examined with the help of SEM (Jeol JSM-6510LV). The ionic conductivity of the NPs was improved by sputtering them with platinum after they had been fastened to support.


## 2.7. Antioxidant Potential ofCarduus Edelbergii

The antioxidant activity ofCarduus edelbergii was assessed using the following assays.


### 2.7.1. DPPH Free Radical Scavenging Assay

In order to determine the antioxidant capacity of NPs, the commonly used antioxidant assay known as the DPPH (2,2-diphenyl-1-picrylhydrazyl) test was carried out in accordance with the recommended [26] protocol. Unique working solutions of ascorbic acid and NPs were made (100, 150, 300, 500, and 1000 μg/mL). The DPPH solution (3 mg in 100 mL) was produced, kept at 25 °C in a container completely covered with aluminum foil for 30 minutes, and then its absorbance at 517 nm was measured and calibrated to <1. The absorbance was spectrophotometrically determined after the sample/ascorbic acid solutions were poured to the DPPH solution. The capability of ascorbic acid/AgNPs to scavenge free radicals was calculated by using the formula below:
(4)Inhibition%=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


### 2.7.2. ABTS Free Radical Scavenging Assay

The ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) activity was carried out according to Re et al.’s [27] protocols. The stock solution was prepared by combination of 2.4 mM K2S2O8 and 7 mM ABTS solutions. The resultant mixture was diluted with methanol to attain the desired absorbance 0.900 (±0.02). The prepared solution was placed over night at 37 °C in total darkness and then measured the absorbance at 745 nm. After combining the ascorbic/NPs solution (300 μL; 100, 150, 300, 500, and 1000 μg/mL) with the ABTS working solution and incubation for 6 minutes, the absorbance was measured. Ascorbic acid was used as a reference in the control. The following equation was used to compute the % scavenging of ABTS free radicals:
(5)Percentscavenging=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


## 2.7.1. DPPH Free Radical Scavenging Assay

In order to determine the antioxidant capacity of NPs, the commonly used antioxidant assay known as the DPPH (2,2-diphenyl-1-picrylhydrazyl) test was carried out in accordance with the recommended [26] protocol. Unique working solutions of ascorbic acid and NPs were made (100, 150, 300, 500, and 1000 μg/mL). The DPPH solution (3 mg in 100 mL) was produced, kept at 25 °C in a container completely covered with aluminum foil for 30 minutes, and then its absorbance at 517 nm was measured and calibrated to <1. The absorbance was spectrophotometrically determined after the sample/ascorbic acid solutions were poured to the DPPH solution. The capability of ascorbic acid/AgNPs to scavenge free radicals was calculated by using the formula below:
(4)Inhibition%=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


## 2.7.2. ABTS Free Radical Scavenging Assay

The ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) activity was carried out according to Re et al.’s [27] protocols. The stock solution was prepared by combination of 2.4 mM K2S2O8 and 7 mM ABTS solutions. The resultant mixture was diluted with methanol to attain the desired absorbance 0.900 (±0.02). The prepared solution was placed over night at 37 °C in total darkness and then measured the absorbance at 745 nm. After combining the ascorbic/NPs solution (300 μL; 100, 150, 300, 500, and 1000 μg/mL) with the ABTS working solution and incubation for 6 minutes, the absorbance was measured. Ascorbic acid was used as a reference in the control. The following equation was used to compute the % scavenging of ABTS free radicals:
(5)Percentscavenging=Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol×100.


## 2.8. Antibacterial Activity

Five distinct strains of bacteria, includingEscherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Staphylococcus epidermis, and Acetobacter spp., were evaluated against the produced AgNPs and plant extract. The agar-well diffusion test was used to conduct an in vitro investigation of their antibacterial potential. Using a cotton swab, bacteria were inoculated onto agar plates in a quick manner. To the appropriate wells, 20 μL of each sample, i.e., standard antibiotic amoxicillin (10 mg/mL), plant extract (10 mg/mL), and Carduus edelbergii–mediated AgNPs (10 mg/mL), and DMSO were introduced. Prior to usage, all equipments to be utilized in the experiment were adequately sterilized. Plates were stored in incubator for 24 hours at room temperature. The zones of inhibition were measured in millimeters after a 24-hour incubation period.


## 2.9. Antifungal Activity

The slant approach was used for antifungal activity. A conical flask’s was sealed with a cotton stopper after pouring 50 mL of dH2O to 3.5 g SDA to prepare SDA media. By autoclaving the medium, test tubes, and wire loop for 15 minutes at 121 °C and 15 psi, all of the instruments were thoroughly sterilized. The slants within the laminar flow hood were established by pouring 60 mL of SDA medium into test tubes. To assess the zones of inhibition, 60 μL from each prepared sample, i.e., Carduus edelbergii–mediated AgNPs, plant extract, standard antibiotic (Terbinafine; Saffron Pharmaceuticals Pvt. Ltd.), and DMSO, was put into the appropriate wells.


## 2.10. Cytotoxic Assay

Methanol extract and AgNPs were tested for their cytotoxic effects utilizing brine shrimp lethality bioassays. In a dark chamber, 1 mg shrimp eggs were cultured in 4% seawater (w/v). A central porous wall allowed shrimp to enter the lit chamber after hatching. Methanol was used to generate working solutions of all samples with varying concentrations (100-1000μg/mL). A total of three trials were conducted for each of the mentioned concentration. For the removal of methanol, the test tubes were held at 25 °C for 24 hours. 10 shrimp were put in each test tube and incubated for 24 hours at 25 °C. After that, the shrimps in each test tube were counted, and Abbot’s equation was applied to compute the percentage of death. Then, the experimental and control groups were compared.
(6)%Mortality=Experimental−control/control×100.


## 2.11. Statistical Analysis

GraphPad Prism software was used for statistical analysis.


## 3. Results and Discussion

### 3.1. Phytochemical Screening

Phytochemical assessment gives extensive familiarity concerning the chemical compounds which have effectiveness and are responsible for therapeutic characteristics. It is the preliminary step for a chemist to know about phytochemicals [28]. Plants contain bioactive phytoconstituents that are beneficial to both humans and animals for various essential physiological functions include flavonoids, alkaloids, phenolic compounds, tannins, and oils [29]. The methanol extract of aerial parts of Carduus edelbergii demonstrated the existence of alkaloids, carbohydrates, tannins, flavonoids, phenols, proteins, amino acids, saponins, and phytosterols, whereas diterpenes were found to be absent in the crude extract (Table 1). Patra et al. [30], Srivastava and Leelavathi [31], and Kumar et al. [32] detected similar types of phytoconstituents in Hygrophila spinosa, Catunaregam spinosa, and Crocus sativus respectively and suggested that initial phytochemical screening is crucial to the purification of bioactive plant constituents. They almost have no adverse effects and play a vital role in human health maintenance.Table 1
Phytochemical screening of an extract of aerial parts ofCarduus edelbergii.
PhytochemicalsTest nameChemicalResultsAlkaloids(a) Mayer’s testPotassium mercuric iodidePositiveCarbohydrates(a) Molisch’s testAlcoholicα-naphtholPositivePhenolsFerric chloride testFerric chloridePositiveTanninsGelatin testSodium chloride containing solution of 1% gelatinPositiveFlavonoid(a) Alkaline reagent testSodium hydroxidePositive(b) Lead acetate testLead acetatePositiveLegal’s testSodium nitropruside in pyridine and sodium hydroxideSaponinsFroth testDistilled waterPositiveFoam testWaterPositiveProteins and amino acids(a) Xanthoproteic testConc. Nitric acidPositive(b) Ninhydrin testNinhydrin reagentPositivePhytosterolsSalkowski’s testChloroformPositiveLibermann Burchard’s testChloroformPositiveDiterpenesCopper acetate testWater copper acetateNegative


### 3.2. Elemental Analysis

The elemental analysis ofCarduus edelbergii was shown in Table 2. It showed that among macro-elements, the potassium was found in the highest amount (K: 7.66.5±0.17), followed by sodium (Na: 589±1.7) and magnesium (Mg: 285.2±0.12). These minerals help in regulation of heart and nervous system functions, as well as blood pressure, glycogen, and glucose metabolism. When combined with salt, potassium aids in maintaining a healthy water-acid balance in blood and tissues [33].Table 2
Elemental analysis of an extract of aerial parts ofCarduus edelbergii.
S.no.Elements%1Potassium766.5±0.172Sodium589±1.73Magnesium285.2±0.124Lead2.3±0.115Manganese27.5±0.176Cobalt17.5±0.177Nickel14.1±0.578Zinc9.5±0.17Mean±SEM.Among micro-elements, manganese (Mn:27.5±0.17) was maximum, followed by cobalt (Co: 17.5±0.17), nickel (Ni: 14±0.57), zinc (Zn: 9.5±0.17), and lead (Pb: 2.3±0.11). It is no more secret that antioxidant enzymes need manganese as a key building block. Nutritional deficiencies such as an inadequate intake of zinc-rich foods or chronic illness may all contribute to zinc insufficiency. Pb is a neurotoxin that impairs mental and intellectual development and leads to behavioral problems [34, 35]. Mineral needs vary with animal species, as they need considerable quantities of calcium for bone construction and maintenance and appropriate nerve and muscle functions [36–38].


### 3.3. Proximate Analysis

The proximate analysis ofCarduus edelbergii aerial parts (leaves, stem and flowers) was presented in Table 3. The results showed that the crude fibers content of flower is highest (70.5%) than stem (67.0%) and leaves (70.0%). Uza and Dastagir [39] reported the highest fiber contents in Astragalus scorpiurus and reported that maximum fiber contents reduces the risk of microorganisms attack and thus protects herbal drugs from deterioration. Fiber intake assists to maintain good health and lessens cholesterol level [40]. Crude fat is more in flower (3.0%) than stem and leaves. Moreover, in the present study, fat contents were lowest when compared to other nutrients. The low crude fat content indicates that Carduus edelbergii is a poor source of lipids. According to Egwuche et al. [41, 42], most medicinal plants are poor sources of lipids. This may be useful for individuals on weight-reducing diets, or those suffering from hyperlipidemia, obesity, and cardiovascular complications.Table 3
Proximate analysis of extracts of aerial parts ofCarduus edelbergii.
Plant partsDry matter (%)Moisture (%)Crude protein (%)Crude fat (%)Crude fiber (%)Total ash (%)Total carbohydrates (%)Gross energy (k cal/100 g)Flowers98.5 ± 0.12.4 ± 0.18.7 ± 0.023.0 ± 0.170.5 ± 0.15.6 ± 2.03.2 ± 2.395.566Stem97.6 ± 0.62.3 ± 0.18.6 ± 0.022.4 ± 0.167.0 ± 1.57.3 ± 1.27.3 ± 1.291.873Leaves97.7 ± 0.12.2 ± 0.18.5 ± 0.011.5 ± 0.170.0 ± 1.09.6 ± 1.45.0 ± 3.385.445Mean ± Standard deviation mean.Total carbohydrates contents are highest in stem (7.3%) compared to the rest of plant parts. Carbohydrates serve in diet as a rich source of energy as well as a raw material to different industries for the developments of various products. All the plant parts have higher gross energy, but flowers possess the highest energy (95.566 kcal/100 g) followed by stem (91.873 kcal/100 g) and leaves (85.445 k cal/100 g). These are important nutrients for curing the health of living organisms from different ailments and help in maintaining health in proper condition from adverse effects of environment [41, 42].


### 3.4. Biosynthesis of AgNPs

In nanotechnology, the longer shelf life of NPs is attributed to secondary metabolites that have proven to act as reducing and capping agents. In the current study, addition ofCarduus edelbergii extract to precursor salt (AgNO3) solution resulted in change in the color of the aqueous mixture. The change in color is a primary indication of initiation of AgNPs synthesis and is evident in Figure 1(a), while the schematic illustration of the synthesis of AgNPs is presented in Figure 1(b). Once the process of AgNPs formation was completed, the AgNPs were isolated and stored in Eppendorf tubes for characterization of AgNPs morphology, size, elemental composition, and the nature of capping agents involved in the biosynthesis of AgNPs.Figure 1
(a) Preparation of colloidal solution of AgNPs. (b) Schematic illustrations of AgNPs.
(a)(b)Other studies agree with our current results which reported that the change in the color is a sign of the synthesis of AgNPs which take place owing to surface plasmon resonance of the metal nanoparticles [43, 44].


### 3.5. UV-Vis Spectrophotometric Analysis

UV-vis spectrophotometry was used to determine the existence of metallic NPs in a sample and to provide its absorption spectra. Figure2 shows the spectral analysis of the synthesized AgNPs. It revealed the characteristic peaks (λmax) at 415 nm for AgNPs which confirms the presence of the respective AgNPs in the sample. These results are similar to the previous reports of Mock et al., [45]. Rivallan et al. [46] reported comparable absorption spectrum between 410 and 450 nm.Figure 2
UV-vis spectrophotometric analysis of theCarduus edelbergii extract mediated AgNPs.


### 3.6. X-Ray Diffraction Analysis

Upon the confirmation of the presence of AgNPs using UV-vis spectrophotometry, the AgNPs were subjected to X-ray diffraction analysis to examine the crystalline nature of the prepared AgNPs. Figure3 indicates the XRD pattern of AgNPs. AgNPs peaks were acquired at 27.79°, 32.26°, 38.19°, 46.26°, 54.78°, 57.47°, 64.37°, and 76.65° which corresponds to 210, 122, 111, 231, 142, 241, 220, and 311 planes of face centered cubic (fcc) crystal structure. The results are well in line with the data reported by Hashmi et al. [47] for biosynthesized AgNPs using Justicia peploides and Withania coagulans. Some additional peaks were also found in the X-ray diffractogram which may be owing to phytochemicals. The XRD results of AgNPs are in agreement with Khandel et al. [48].Figure 3
X-ray diffractogram of theCarduus edelbergii extract mediated AgNPs.


### 3.7. FR-IR Spectral Analysis

The functional groups of bioactive compounds present inCarduus edelbergii extract and thier reducing and stabilizing capabilities, were evaluated by FTIR analysis. The FTIR spectra of plant extract and AgNPs were documented (Figures 4(a) and 4(b)). An O-H stretching and bending bond was found to be a major FTIR peak in the plant crude extract at 3248 cm-1 which indicates that phenols are present. Two distinct modes of C-H stretching, symmetric and asymmetric, are associated with the peak at 2934 cm-1, which is attributed to –CH2- stretching (methylene). The C=C stretch at 1600 cm-1 indicates the conjugation of two aromatic rings of phenols. Aromatic rings are shown by the 923 cm-1 peak, which is out of plan C-H bending and vibration. Flavorings, such as flavonoids, may be found in plant extracts that absorb in this infrared range. The IR spectra of the produced AgNPs show that plant biomolecules such as alkaloids, phenolic compounds, amino acids, carbohydrates, and notably tannins interacted with AgNPs, most likely via their oxygen capabilities, which act as reducing and capping agents. These findings are in line with those of previous reports [49, 50].Figure 4
(a) FTIR of theCarduus edelbergii extract. (b) FTIR of the Carduus edelbergii extract mediated AgNPs.
(a)(b)


### 3.8. SEM Analysis

Figure5 represents the SEM analysis of the AgNPs which shows that they were spherical shaped, and properly distributed with no aggregation.Figure 5
SEM of theCarduus edelbergii extract mediated AgNPs.The biocompatible wrapping molecules coupled to the AgNPs established hydrogen bonds and electrostatic interactions, which are visible in the SEM pictures of the assembled AgNPs [51]. Congruent results were found in earlier studies in which the FE-SEM micrograph reveals crystallized spherical AgNPs [52].


### 3.9. Antioxidant Activities

The antioxidant potency ofCarduus edelbergii extract and its mediated AgNPs was measured through DPPH and ABTS assays. The results of these assays are given as follows.


#### 3.9.1. ABTS Free Radical Scavenging Activity

Both extract and AgNPs revealed considerable scavenging efficacy against ABTS free radicals (Table4). The mentioned activity was found to be dose dependent. Moteriya and Chanda,[53, 54] documented 23–95% ABTS cation radical scavenging activity and 8.8 Mg−1 FRAP activity of AgNPs synthesized using C. pulcherrima flower extract.Table 4
ABTS free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on ABTS radicalsAgNPsExtractAscorbic acid10053.11±0.00243.29±0.01487.24±0.04615064.30±0.00549.93±0.01990.98±0.02730070.31±0.04351.62±0.05191.63±0.03150077.79±0.02352.93±0.01993.36±0.022100082.27±0.00953.14±0.03293.44±0.016Mean±SEM.


#### 3.9.2. DPPH Free Radical Scavenging Activity

Antioxidant characteristics of extract and its mediated AgNPs were also evaluated against DPPH free radicals (Table5). Both AgNPs and DPPH displayed dose-dependent free radical scavenging potential, similar to ABTS activity. DPPH free radical scavenging ability of the extract and NPs may be due to the presence of phenolic and polyphenolic compounds. Similar findings were reported by other researchers due to the presence of bioactive constituents like tannins, alkaloids, and flavonoids in Piper longum [53, 54], Iresine herbstii [55], Chenopodium murale [56], Morinda pubescens [57], Nelumbo nucifera [58], and Areca catechu [59] silver nanoparticles.Table 5
DPPH free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on DPPH radicalsAgNPsExtractAscorbic acid10053.11 ± 0.2546.21 ± 0.5187.24 ± 2.615064.30 ± 0.5149.74 ± 0.6190.98 ± 1.730071.32 ± 0.2150.83 ± 0.4491.63 ± 3.050077.79 ± 0.2351.49 ± 0.2493.36 ± 2.2100082.27 ± 0.9153.14 ± 0.5493.44 ± 2.0Data is represented asmean±standarddeviation.


### 3.10. Antibacterial Activity

Table6 indicates the antibacterial efficacy of the prepared AgNPs against selected strains of bacteria. Carduus edelbergii mediated AgNPs revealed significant antibacterial properties against the selected strains, i.e., Klebsiella aerogenes, Staphylococcus epidermis, Escherichia coli, Staphylococcus aureus, and Acetobacter spp. AgNPs proved to be most potent against K. aerogenes and S. aureus with larger zone of inhibition. AgNPs also showed excellent antibacterial potency against S. epidermis, E. coli and Acetobacter spp. The zones of inhibitions were recorded for S. epidermis, S. aureus, K. aerogenes, Acetobacter spp., and E. coli and compared the results to that of the standard (amoxicillin). The results indicated that both AgNPs and extract are efficient antibacterial agents and the small size of Ag nanoparticles make them capable to infiltrate easily through the bacteria’s outer cell wall, come into the cell, devastating the respiratory sequence, and finally bacterial cell death. Congruent results were reported in previous studies [16]. Ovais et al. [4] also reported similar results while working on the inhibition zone and antibacterial activity of Ag nanoparticles.Table 6
Antibacterial potential ofCarduus edelbergii extract and its mediated AgNPs.
Bacterial speciesAmoxicillin (1μg/mL)AgNPsExtractKlebsiella aerogenes15.4±0.0212.2±0.059.9±0.04Staphylococcus epidermis16.2±0.0411.1±0.098.7±0.20Staphylococcus aureus14.7±0.0712.3±0.048.8±0.71Acetobacter spp.15.1±0.0211.8±0.098.2±0.12Escherichia coli15.7±0.0711.6±0.059.3±0.21Mean±SEM.


### 3.11. Antifungal Activity

Three strains, i.e.,Aspergillus niger, Aspergillus flavus and Fusarium solani were used in the present study. The antifungal activity was determined by measuring the % growth inhibition of the selected strains of fungi (Figure 6). AgNPs expressed effective antifungal activities against Aspergillus flavus and Aspergillus niger. Plant extract also indicated efficient antifungal activity against Aspergillus flavus and Aspergillus niger. The antifungal activities of AgNPs and extract were compared to terbinafine. These results are similar to the previous reports [60, 61].Figure 6
Antifungal potential ofCarduus edelbergii extract and its mediated AgNPs.


### 3.12. Cytotoxic Assay

Cytotoxicity results are presented in Table7. The data revealed that the cytotoxic activities of AgNPs increase with increasing concentrations. Plant extracts also indicated similar behavior of dose dependent activity. Plant extract showed 96.47±2.4 LD50 value as compared to AgNPs which showed 40.44±2.9μg/mL mean that AgNPs are more potent. Similar effects were reported by Meretoudi et al. [62]. Other studies have been reported similar findings [63–65].Table 7
Cytotoxic potential ofCarduus edelbergii extract and its mediated AgNPs.
Concentration (μg/mL)ExtractAgNPs15070.4±4.5100±4.310050.2±3.780.3±5.15030.6±1.650.5±2.11020.8±0.730.7±1.5LD5096.47±2.140.44±2.0LD70152.34±4.3.77.84±3.2LD90211.54±7.5126.51±5.3Mean±SEM.


## 3.1. Phytochemical Screening

Phytochemical assessment gives extensive familiarity concerning the chemical compounds which have effectiveness and are responsible for therapeutic characteristics. It is the preliminary step for a chemist to know about phytochemicals [28]. Plants contain bioactive phytoconstituents that are beneficial to both humans and animals for various essential physiological functions include flavonoids, alkaloids, phenolic compounds, tannins, and oils [29]. The methanol extract of aerial parts of Carduus edelbergii demonstrated the existence of alkaloids, carbohydrates, tannins, flavonoids, phenols, proteins, amino acids, saponins, and phytosterols, whereas diterpenes were found to be absent in the crude extract (Table 1). Patra et al. [30], Srivastava and Leelavathi [31], and Kumar et al. [32] detected similar types of phytoconstituents in Hygrophila spinosa, Catunaregam spinosa, and Crocus sativus respectively and suggested that initial phytochemical screening is crucial to the purification of bioactive plant constituents. They almost have no adverse effects and play a vital role in human health maintenance.Table 1
Phytochemical screening of an extract of aerial parts ofCarduus edelbergii.
PhytochemicalsTest nameChemicalResultsAlkaloids(a) Mayer’s testPotassium mercuric iodidePositiveCarbohydrates(a) Molisch’s testAlcoholicα-naphtholPositivePhenolsFerric chloride testFerric chloridePositiveTanninsGelatin testSodium chloride containing solution of 1% gelatinPositiveFlavonoid(a) Alkaline reagent testSodium hydroxidePositive(b) Lead acetate testLead acetatePositiveLegal’s testSodium nitropruside in pyridine and sodium hydroxideSaponinsFroth testDistilled waterPositiveFoam testWaterPositiveProteins and amino acids(a) Xanthoproteic testConc. Nitric acidPositive(b) Ninhydrin testNinhydrin reagentPositivePhytosterolsSalkowski’s testChloroformPositiveLibermann Burchard’s testChloroformPositiveDiterpenesCopper acetate testWater copper acetateNegative


## 3.2. Elemental Analysis

The elemental analysis ofCarduus edelbergii was shown in Table 2. It showed that among macro-elements, the potassium was found in the highest amount (K: 7.66.5±0.17), followed by sodium (Na: 589±1.7) and magnesium (Mg: 285.2±0.12). These minerals help in regulation of heart and nervous system functions, as well as blood pressure, glycogen, and glucose metabolism. When combined with salt, potassium aids in maintaining a healthy water-acid balance in blood and tissues [33].Table 2
Elemental analysis of an extract of aerial parts ofCarduus edelbergii.
S.no.Elements%1Potassium766.5±0.172Sodium589±1.73Magnesium285.2±0.124Lead2.3±0.115Manganese27.5±0.176Cobalt17.5±0.177Nickel14.1±0.578Zinc9.5±0.17Mean±SEM.Among micro-elements, manganese (Mn:27.5±0.17) was maximum, followed by cobalt (Co: 17.5±0.17), nickel (Ni: 14±0.57), zinc (Zn: 9.5±0.17), and lead (Pb: 2.3±0.11). It is no more secret that antioxidant enzymes need manganese as a key building block. Nutritional deficiencies such as an inadequate intake of zinc-rich foods or chronic illness may all contribute to zinc insufficiency. Pb is a neurotoxin that impairs mental and intellectual development and leads to behavioral problems [34, 35]. Mineral needs vary with animal species, as they need considerable quantities of calcium for bone construction and maintenance and appropriate nerve and muscle functions [36–38].


## 3.3. Proximate Analysis

The proximate analysis ofCarduus edelbergii aerial parts (leaves, stem and flowers) was presented in Table 3. The results showed that the crude fibers content of flower is highest (70.5%) than stem (67.0%) and leaves (70.0%). Uza and Dastagir [39] reported the highest fiber contents in Astragalus scorpiurus and reported that maximum fiber contents reduces the risk of microorganisms attack and thus protects herbal drugs from deterioration. Fiber intake assists to maintain good health and lessens cholesterol level [40]. Crude fat is more in flower (3.0%) than stem and leaves. Moreover, in the present study, fat contents were lowest when compared to other nutrients. The low crude fat content indicates that Carduus edelbergii is a poor source of lipids. According to Egwuche et al. [41, 42], most medicinal plants are poor sources of lipids. This may be useful for individuals on weight-reducing diets, or those suffering from hyperlipidemia, obesity, and cardiovascular complications.Table 3
Proximate analysis of extracts of aerial parts ofCarduus edelbergii.
Plant partsDry matter (%)Moisture (%)Crude protein (%)Crude fat (%)Crude fiber (%)Total ash (%)Total carbohydrates (%)Gross energy (k cal/100 g)Flowers98.5 ± 0.12.4 ± 0.18.7 ± 0.023.0 ± 0.170.5 ± 0.15.6 ± 2.03.2 ± 2.395.566Stem97.6 ± 0.62.3 ± 0.18.6 ± 0.022.4 ± 0.167.0 ± 1.57.3 ± 1.27.3 ± 1.291.873Leaves97.7 ± 0.12.2 ± 0.18.5 ± 0.011.5 ± 0.170.0 ± 1.09.6 ± 1.45.0 ± 3.385.445Mean ± Standard deviation mean.Total carbohydrates contents are highest in stem (7.3%) compared to the rest of plant parts. Carbohydrates serve in diet as a rich source of energy as well as a raw material to different industries for the developments of various products. All the plant parts have higher gross energy, but flowers possess the highest energy (95.566 kcal/100 g) followed by stem (91.873 kcal/100 g) and leaves (85.445 k cal/100 g). These are important nutrients for curing the health of living organisms from different ailments and help in maintaining health in proper condition from adverse effects of environment [41, 42].


## 3.4. Biosynthesis of AgNPs

In nanotechnology, the longer shelf life of NPs is attributed to secondary metabolites that have proven to act as reducing and capping agents. In the current study, addition ofCarduus edelbergii extract to precursor salt (AgNO3) solution resulted in change in the color of the aqueous mixture. The change in color is a primary indication of initiation of AgNPs synthesis and is evident in Figure 1(a), while the schematic illustration of the synthesis of AgNPs is presented in Figure 1(b). Once the process of AgNPs formation was completed, the AgNPs were isolated and stored in Eppendorf tubes for characterization of AgNPs morphology, size, elemental composition, and the nature of capping agents involved in the biosynthesis of AgNPs.Figure 1
(a) Preparation of colloidal solution of AgNPs. (b) Schematic illustrations of AgNPs.
(a)(b)Other studies agree with our current results which reported that the change in the color is a sign of the synthesis of AgNPs which take place owing to surface plasmon resonance of the metal nanoparticles [43, 44].


## 3.5. UV-Vis Spectrophotometric Analysis

UV-vis spectrophotometry was used to determine the existence of metallic NPs in a sample and to provide its absorption spectra. Figure2 shows the spectral analysis of the synthesized AgNPs. It revealed the characteristic peaks (λmax) at 415 nm for AgNPs which confirms the presence of the respective AgNPs in the sample. These results are similar to the previous reports of Mock et al., [45]. Rivallan et al. [46] reported comparable absorption spectrum between 410 and 450 nm.Figure 2
UV-vis spectrophotometric analysis of theCarduus edelbergii extract mediated AgNPs.


## 3.6. X-Ray Diffraction Analysis

Upon the confirmation of the presence of AgNPs using UV-vis spectrophotometry, the AgNPs were subjected to X-ray diffraction analysis to examine the crystalline nature of the prepared AgNPs. Figure3 indicates the XRD pattern of AgNPs. AgNPs peaks were acquired at 27.79°, 32.26°, 38.19°, 46.26°, 54.78°, 57.47°, 64.37°, and 76.65° which corresponds to 210, 122, 111, 231, 142, 241, 220, and 311 planes of face centered cubic (fcc) crystal structure. The results are well in line with the data reported by Hashmi et al. [47] for biosynthesized AgNPs using Justicia peploides and Withania coagulans. Some additional peaks were also found in the X-ray diffractogram which may be owing to phytochemicals. The XRD results of AgNPs are in agreement with Khandel et al. [48].Figure 3
X-ray diffractogram of theCarduus edelbergii extract mediated AgNPs.


## 3.7. FR-IR Spectral Analysis

The functional groups of bioactive compounds present inCarduus edelbergii extract and thier reducing and stabilizing capabilities, were evaluated by FTIR analysis. The FTIR spectra of plant extract and AgNPs were documented (Figures 4(a) and 4(b)). An O-H stretching and bending bond was found to be a major FTIR peak in the plant crude extract at 3248 cm-1 which indicates that phenols are present. Two distinct modes of C-H stretching, symmetric and asymmetric, are associated with the peak at 2934 cm-1, which is attributed to –CH2- stretching (methylene). The C=C stretch at 1600 cm-1 indicates the conjugation of two aromatic rings of phenols. Aromatic rings are shown by the 923 cm-1 peak, which is out of plan C-H bending and vibration. Flavorings, such as flavonoids, may be found in plant extracts that absorb in this infrared range. The IR spectra of the produced AgNPs show that plant biomolecules such as alkaloids, phenolic compounds, amino acids, carbohydrates, and notably tannins interacted with AgNPs, most likely via their oxygen capabilities, which act as reducing and capping agents. These findings are in line with those of previous reports [49, 50].Figure 4
(a) FTIR of theCarduus edelbergii extract. (b) FTIR of the Carduus edelbergii extract mediated AgNPs.
(a)(b)


## 3.8. SEM Analysis

Figure5 represents the SEM analysis of the AgNPs which shows that they were spherical shaped, and properly distributed with no aggregation.Figure 5
SEM of theCarduus edelbergii extract mediated AgNPs.The biocompatible wrapping molecules coupled to the AgNPs established hydrogen bonds and electrostatic interactions, which are visible in the SEM pictures of the assembled AgNPs [51]. Congruent results were found in earlier studies in which the FE-SEM micrograph reveals crystallized spherical AgNPs [52].


## 3.9. Antioxidant Activities

The antioxidant potency ofCarduus edelbergii extract and its mediated AgNPs was measured through DPPH and ABTS assays. The results of these assays are given as follows.


### 3.9.1. ABTS Free Radical Scavenging Activity

Both extract and AgNPs revealed considerable scavenging efficacy against ABTS free radicals (Table4). The mentioned activity was found to be dose dependent. Moteriya and Chanda,[53, 54] documented 23–95% ABTS cation radical scavenging activity and 8.8 Mg−1 FRAP activity of AgNPs synthesized using C. pulcherrima flower extract.Table 4
ABTS free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on ABTS radicalsAgNPsExtractAscorbic acid10053.11±0.00243.29±0.01487.24±0.04615064.30±0.00549.93±0.01990.98±0.02730070.31±0.04351.62±0.05191.63±0.03150077.79±0.02352.93±0.01993.36±0.022100082.27±0.00953.14±0.03293.44±0.016Mean±SEM.


### 3.9.2. DPPH Free Radical Scavenging Activity

Antioxidant characteristics of extract and its mediated AgNPs were also evaluated against DPPH free radicals (Table5). Both AgNPs and DPPH displayed dose-dependent free radical scavenging potential, similar to ABTS activity. DPPH free radical scavenging ability of the extract and NPs may be due to the presence of phenolic and polyphenolic compounds. Similar findings were reported by other researchers due to the presence of bioactive constituents like tannins, alkaloids, and flavonoids in Piper longum [53, 54], Iresine herbstii [55], Chenopodium murale [56], Morinda pubescens [57], Nelumbo nucifera [58], and Areca catechu [59] silver nanoparticles.Table 5
DPPH free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on DPPH radicalsAgNPsExtractAscorbic acid10053.11 ± 0.2546.21 ± 0.5187.24 ± 2.615064.30 ± 0.5149.74 ± 0.6190.98 ± 1.730071.32 ± 0.2150.83 ± 0.4491.63 ± 3.050077.79 ± 0.2351.49 ± 0.2493.36 ± 2.2100082.27 ± 0.9153.14 ± 0.5493.44 ± 2.0Data is represented asmean±standarddeviation.


## 3.9.1. ABTS Free Radical Scavenging Activity

Both extract and AgNPs revealed considerable scavenging efficacy against ABTS free radicals (Table4). The mentioned activity was found to be dose dependent. Moteriya and Chanda,[53, 54] documented 23–95% ABTS cation radical scavenging activity and 8.8 Mg−1 FRAP activity of AgNPs synthesized using C. pulcherrima flower extract.Table 4
ABTS free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on ABTS radicalsAgNPsExtractAscorbic acid10053.11±0.00243.29±0.01487.24±0.04615064.30±0.00549.93±0.01990.98±0.02730070.31±0.04351.62±0.05191.63±0.03150077.79±0.02352.93±0.01993.36±0.022100082.27±0.00953.14±0.03293.44±0.016Mean±SEM.


## 3.9.2. DPPH Free Radical Scavenging Activity

Antioxidant characteristics of extract and its mediated AgNPs were also evaluated against DPPH free radicals (Table5). Both AgNPs and DPPH displayed dose-dependent free radical scavenging potential, similar to ABTS activity. DPPH free radical scavenging ability of the extract and NPs may be due to the presence of phenolic and polyphenolic compounds. Similar findings were reported by other researchers due to the presence of bioactive constituents like tannins, alkaloids, and flavonoids in Piper longum [53, 54], Iresine herbstii [55], Chenopodium murale [56], Morinda pubescens [57], Nelumbo nucifera [58], and Areca catechu [59] silver nanoparticles.Table 5
DPPH free radical scavenging potency ofCarduus edelbergii extract and its mediated AgNPs.
Concentrations (μg/mL)Scavenging ability on DPPH radicalsAgNPsExtractAscorbic acid10053.11 ± 0.2546.21 ± 0.5187.24 ± 2.615064.30 ± 0.5149.74 ± 0.6190.98 ± 1.730071.32 ± 0.2150.83 ± 0.4491.63 ± 3.050077.79 ± 0.2351.49 ± 0.2493.36 ± 2.2100082.27 ± 0.9153.14 ± 0.5493.44 ± 2.0Data is represented asmean±standarddeviation.


## 3.10. Antibacterial Activity

Table6 indicates the antibacterial efficacy of the prepared AgNPs against selected strains of bacteria. Carduus edelbergii mediated AgNPs revealed significant antibacterial properties against the selected strains, i.e., Klebsiella aerogenes, Staphylococcus epidermis, Escherichia coli, Staphylococcus aureus, and Acetobacter spp. AgNPs proved to be most potent against K. aerogenes and S. aureus with larger zone of inhibition. AgNPs also showed excellent antibacterial potency against S. epidermis, E. coli and Acetobacter spp. The zones of inhibitions were recorded for S. epidermis, S. aureus, K. aerogenes, Acetobacter spp., and E. coli and compared the results to that of the standard (amoxicillin). The results indicated that both AgNPs and extract are efficient antibacterial agents and the small size of Ag nanoparticles make them capable to infiltrate easily through the bacteria’s outer cell wall, come into the cell, devastating the respiratory sequence, and finally bacterial cell death. Congruent results were reported in previous studies [16]. Ovais et al. [4] also reported similar results while working on the inhibition zone and antibacterial activity of Ag nanoparticles.Table 6
Antibacterial potential ofCarduus edelbergii extract and its mediated AgNPs.
Bacterial speciesAmoxicillin (1μg/mL)AgNPsExtractKlebsiella aerogenes15.4±0.0212.2±0.059.9±0.04Staphylococcus epidermis16.2±0.0411.1±0.098.7±0.20Staphylococcus aureus14.7±0.0712.3±0.048.8±0.71Acetobacter spp.15.1±0.0211.8±0.098.2±0.12Escherichia coli15.7±0.0711.6±0.059.3±0.21Mean±SEM.


## 3.11. Antifungal Activity

Three strains, i.e.,Aspergillus niger, Aspergillus flavus and Fusarium solani were used in the present study. The antifungal activity was determined by measuring the % growth inhibition of the selected strains of fungi (Figure 6). AgNPs expressed effective antifungal activities against Aspergillus flavus and Aspergillus niger. Plant extract also indicated efficient antifungal activity against Aspergillus flavus and Aspergillus niger. The antifungal activities of AgNPs and extract were compared to terbinafine. These results are similar to the previous reports [60, 61].Figure 6
Antifungal potential ofCarduus edelbergii extract and its mediated AgNPs.


## 3.12. Cytotoxic Assay

Cytotoxicity results are presented in Table7. The data revealed that the cytotoxic activities of AgNPs increase with increasing concentrations. Plant extracts also indicated similar behavior of dose dependent activity. Plant extract showed 96.47±2.4 LD50 value as compared to AgNPs which showed 40.44±2.9μg/mL mean that AgNPs are more potent. Similar effects were reported by Meretoudi et al. [62]. Other studies have been reported similar findings [63–65].Table 7
Cytotoxic potential ofCarduus edelbergii extract and its mediated AgNPs.
Concentration (μg/mL)ExtractAgNPs15070.4±4.5100±4.310050.2±3.780.3±5.15030.6±1.650.5±2.11020.8±0.730.7±1.5LD5096.47±2.140.44±2.0LD70152.34±4.3.77.84±3.2LD90211.54±7.5126.51±5.3Mean±SEM.


## 4. Conclusions

Silver has been used for its biological potential for centuries. With the advent of nanotechnology, new frontiers were introduced in scientific research. Silver in its nano form has much greater biological potency as compared to its bulk similitude. Moreover, the introduction of green nanotechnology further enhanced the biological potential of AgNPs. In this regard, the current study focused on the green synthesis of AgNPs usingCarduus edelbergii extract which has not yet been explored to its full potential in terms of phytochemistry. However, the phytochemical screening, elemental and proximate analysis revealed that the plant is rich in a plethora of important phytochemicals. The green synthesized silver nanoparticles using Carduus edelbergii extract has potent antioxidant, cytotoxic, antibacterial, and antifungal activity which indicates its potential use against microbes and scavenging of free radicals.



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*Source: 8540149-2022-09-13.xml*