# Bioactivities of the Green Synthesized Silver Nanoparticles Reduced UsingAllium cepa L Aqueous Extracts Induced Apoptosis in Colorectal Cancer Cell Lines

Authors: Ahmed A. H. Abdellatif; Amer Mahmood; Mansour Alsharidah; Hamdoon A. Mohammed; Salman Khalaf Alenize; Abdellatif Bouazzaoui; Osamah Al Rugaie; Abdullah M. Alnuqaydan; Rehan Ahmad; Mansoor-Ali Vaali-Mohammad; Musaad Alfayez; Thamer Bin Traiki; Khalid A. Al-Regaiey; Asmaa T. Ali; Yasser A. H. Hassan; Maha-Hamadien Abdulla
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Allium cepa L (A. cepa) extract is frequently used as an adjuvant food in cancer treatment. We hypothesized that it contains a source of anticancer activity. There is a need to synthesize the silver nanoparticles (AgNPs) using an environment-friendly green synthesis reduction method using an aqueous extract of A. cepa. The AgNPs-CEPA were prepared by reduction method using the aqueous extract of A. cepa. The formed AgNPs-CEPA were characterized for their sizes and charge distribution. The AgNP-CEPA was investigated for its antioxidant and anticancer properties. Cell viability was evaluated by MTT assay. Gene expression was evaluated by real-time polymerase chain reaction (RT-PCR), and apoptosis measurement was carried out by flow cytometry in AgNP-CEPA-treated cells. The results showed a uniform size for AgNPs-CEPA of 155±2.1 nm with a zeta potential of −37.3±−2.92 mv. The produced AgNPs-CEPA are biocompatible with anticancer action and a moderate level of antioxidant reactivity. AgNPs-CEPA showed better reducing activity for A. cepa extract compared to the AgNPs-CEPA. AgNP-CEPA treatment of human colorectal cancer cell lines (HT-29 and SW620) inhibited cell proliferation and altered Bcl2 family gene expression. Moreover, exposure of cell lines to AgNPs-CEPA resulted in the significant induction of apoptosis compared to A. cepa and AgNO3. These findings indicate that AgNP-CEPA induces apoptosis by inhibiting Bcl2 family gene expression, suggesting that this formula is a promising anticancer agent for treating colorectal cancer.


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## Body

## 1. Introduction

Metallic agents are the preferred chemotherapeutic therapy agents for certain malignancies. Finding new and effective chemotherapeutic drugs has led to the development of conventional and alternative therapies derived from plants. The use of nanoparticles in delivery, particularly superparamagnetic iron oxide nanoparticles (SPION), and silver nanoparticles (AgNPs), is increasing [1, 2]. The NPs have significant roles in transport, on-site transfer, dispersion, and safe internalizations of the medication to the organs and cells [3]. Natural conjugates, metal incorporations, and tagged nanoentities are also considered efficient anticancer treatments [4–6]. Several types of NPs are reported to be anticancer agents, such as gold nanoparticles, SPION, mesoporous nanosilica, and nanosilver [7, 8]. The biocompatible and biodegradable silver nanoparticles (AgNPs) are unique because of their chemical stability and pharmacological actions of anticancer, antiviral, antibacterial, and antifungal [9–11]. The AgNPs-based drugs have been tested against cancer cell lines [12], including human lung cancer cell lines A549 [13], KB cell lines [14], HT-29, HCT-116 and Caco-2 cell lines [15], HeLa and U937 cells [16], Hep-2 [17], prostate carcinoma cell lines (DU145), human ovarian carcinoma PC-3, SKOV3, human lymphocyte cells [18], neuroblastoma cells [19], human cervical cancer cells, prostate cancer, colon cancer COLO205 cell lines, and B16F10 mouse melanomas [20].To avoid the cytotoxicity of chemically synthesized AgNPs to healthy cells, a green synthesis methodology can be used through utilizing the plants’ extracts as the bioreduction catalyst [21, 22]. However, several chemical processes capable of producing nanosilver are available, including chemical reduction, electrochemical method, and microorganism-based reduction of AgNO3. The green synthesis through reduction by natural compounds is obtained from a safe aqueous extract containing a plethora of compounds from A. cepa. It seemed feasible, environmentally benign, robust, cost-effective, and safer than other traditional chemical processes or physical methods [23]. The green synthesis methods were environmentally safe and reproducible [24, 25]. Its components influence yield, particle size, and growth and have different improving activities [26]. The green synthesized AgNPs with anticancer activity as albumin-coated nanoentities are also available as berberine carrier anticancer agents [27]. AgNPs synthesized from plant extracts had shell capping, contributing to the formulation’s low toxicity and improved cytotoxicity to tumor cells [28].Allium cepa L (A. cepa), commonly known as an onion or bulb onion, is edible. It is a highly consumed culinary herb worldwide due to its food-specific flavor and antibacterial properties. Additionally, the plant is well-known for its multiple beneficial biological qualities related to complex sulfur compounds, thiosulfates, phenolics, and flavonoids [29]. These constituents are obtained by water and solvent extractions and steam distillation [30]. The phenolics and flavonoids, including gallic acid, ferulic acid, kaempferol, quercetin, and flavonoid glycosides, quercetin-3-gulocside, quercetin-4-gulocside, quercetin 3,4′-diglucoside, isorhamnetin 4′-glucoside, and isorhamnetin-3,4′-diglucoside, were found concentrated in the outer layers of the bulb [31]. Specifically, the aqueous extract of onion has been reported for the presence of quercetin or its glycosylated derivatives as the main flavonoid constituents (85-98% of the total flavonols in the extract), in which the quercetin aglycone (20-30%), quercetin-4′-monoglucoside and quercetin-3-monoglucodise (30-70%), and quercetin-3,4′-diglucoside (5-20%) were measured [32]. The total phenolic constituents in the aqueous extract of the plant were also measured at 47 mg/g of the extract [33]. In addition, the phenolics, gallic acid, chlorogenic acid, ferulic acid, quercetin, and kaempferol, were detected in the plant extract by the HPLC [34]. The onion bulb’s antioxidant activity was determined using the autoxidation of carotene/linoleic acid coupled reaction method and the DPPH assay, confirming the bulb’s positive benefits as a powerful antioxidant [35]. The potential of the A. cepa has also been applied in medicine as an anticancer agent [36–38]. AgNPs suppressed MCF-7 and HeLa cell growth and inflammation. Moreover, the treatment with AgNPs can reduce allergic disorders. AgNPs could potentially reduce inflammation, allergy diseases, and bacterial infection by increasing phagocytosis [39].The present study describes a rapid, facile, and robust green synthesis of AgNPs starting from silver nitrate. It concentrates aqueous extracts obtained fromA. cepa as a powerful anticancer AgNPs. The AgNPs-CEPA were prepared by reduction method using the aqueous extract of A. cepa. The AgNPs-CEPA were characterized for their sizes and charge distribution. The AgNP-CEPA was investigated for its antioxidant property and tested against HT-29 cancer cell lines for its cytotoxicity and antioxidant potential.


## 2. Materials and Methods

### 2.1. Materials

Silver nitrate (AgNO3) was purchased from VBBN Company (Hong Kong, China). Sulfuric acid was purchased from Severn Biotech Ltd, Kidderminster, United Kingdom. Sodium phosphate and Whatman filter paper Grade 1, 11 μm (1×3 cm), were purchased from Mumbai, Maharashtra, India. Ammonium molybdate was purchased from Sarkhej, Ahmedabad. Gujarat, India. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO (Dimethylsulfoxide) were purchased from Sigma-Aldrich (St. Louis, USA). Human colorectal cancer cell lines (HT-29 and SW620 cells) were purchased from ATCC (Manassas, Virginia, UK). Roswell Park Memorial Institute medium (RPMI-1640) (GIBCO, by Thermo Fisher Scientific, NY, USA) supplemented with 10% FBS and 1% penicillin and streptomycin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Invitrogen Ltd 3 (Fountain Drive, Inchinnan Business Park, Paisley, PA4 9RF, UK). All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. All glassware was washed using Millipore water. All chemicals were of analytical grades.


### 2.2. Plant Materials and Preparation of the Aqueous Extract ofA. cepa

The onion (Allium cepa L, A. cepa) was purchased from the local market in Buraydah, Al Qassim, Saudi Arabia, and identified by the local botanists at the Department of Plant Production and Protection, College of Agriculture, Qassim University. A. cepa bulbs were sliced into pieces of ~4 mm thickness. The A. cepa pieces were dried in an oven at 50°C temperatures for three days using the convective air-drying method. The aqueous A. cepa extract was prepared by continuous stirring of 1 gm dried A. cepa with 100 ml of distilled water at 25±1°C for 4 h. The aqueous extract was purified using a Whatman filter paper Grade 1, 11 μm (1×3 cm) [40].


### 2.3. Preparation of the AgNPs-CEPA

The AgNP-CEPA was prepared according to the previously reported method with some modification for better results [41]. Briefly, 1 mM stock solution of silver nitrate (AgNO3) was prepared, stirred with the scheduled aqueous extraction of A. cepa using a multiple-stirrer digital magnetic stirrer (VELP Scientifica Srl, Italy). The stirring at 600 rpm has been optimized at room temperature at 25±1°C. The synthesis of AgNPs (AgNPs-CEPA) was confirmed by observing a color change from colorless to yellowish. Then, the synthesized nanoparticles were collected and stored in a dark place away from light.


### 2.4. Characterization of AgNPs-CEPA

#### 2.4.1. Size and Charge

The size and charge of the produced AgNPs-CEPA were measured using a Malvern Zetasizer Nano, Malvern Instruments GmbH (Herrenberg, Germany). Data were presented as the average of the three different measurements of the same AgNPs. The surface charge of AgNPs-CEPA was determined by zeta potential measurements with the same equipment. AgNP-CEPA was put through a laser beam of 623 nm, and the angle was adjusted to 90° at 25°C [42, 43]. The results presented are the average measurements of the runs with standard deviation.


#### 2.4.2. Ultraviolet-Visible Spectroscopy

Ultraviolet-visible (UV-vis) spectrophotometry (Jasco, UV-630, Japan) was used to analyze theA. cepa extract and AgNPs-CEPA. AgNPs were put through a laser lamp and the angle was 90° at wavelengths ranging from 200 to 800 nm. The absorption range of reaction solutions was reported as a function of reaction time. We used distal water as a reference or blank, and then, we put the samples which contained particles in the instrument [44, 45].


#### 2.4.3. Fourier-Transform Infrared Spectroscopy

The study was aimed at proving the formation of AgNPs-CEPA. FTIR measurements were used to analyze the compatibility of biomolecules associated with AgNP formation. It was measured with a Bruker Tensor 27 FTIR spectrophotometer (Varian Company model: 640-IR, Australia) [42]. The FTIR spectra were recorded, and the absorption peaks were observed at 400-4000 cm−1.


#### 2.4.4. SEM Analysis

Scanning electron microscopy (FESEM, supra 55-Carl Zeiss, Germany) was used to study the morphology and size of the synthesized nanoparticles AgNPs-CEPA [44].


### 2.5. Antioxidant Activity Screening

As a comparable assay, threein vitro methods were conducted to evaluate the antioxidant activity of the A. cepa extract and formulated AgNPs of the extract (AgNPs-CEPA). The procedures were performed in triplicate for all experiments, and the antioxidant activity of CEPA and AgNPs-CEPA was calculated using standard calibration curves of Trolox for each method.


#### 2.5.1. Total Antioxidant Capacity (TAC)

Total antioxidant capacity (TAC) of the CEPA and AgNPs-CEPA was conducted according to Aroua et al. [46]. 200 μl of all samples (final concentration 200 μg) was added to the freshly prepared acidic molybdate reagent (2 ml). The mixture was then vigorously shaken and heated on the water bath for 90 min at 85°C and cooled to room temperature (25°C). The absorbance of the blue color that arises was measured at 695 nm, and the total antioxidant activity of the samples was calculated using the Trolox standard calibration curve.


#### 2.5.2. DPPH Scavenging Activity (DPPH-SA)

DPPH scavenging activity (DPPH-SA) was conducted according to Mohammed et al. [47] with minor modification. The DPPH in a concentration of 300 μM (1 ml) was added to 1 ml of A. cepa and AgNPs-CEPA (final concentration 200 μg). The mixture was vortexed and stood in a dark place for 30 min at room temperature to develop the violet color which measures spectrophotometrically at 517 nm. The DPPH-SA activity of the prepared nanoparticles AgNPs-CEPA and A. cepa aqueous extract was measured in equivalents to the Trolox using its calibration curve.


#### 2.5.3. DPPH Scavenging Activity (DPPH-SA)

In the ferric reducing antioxidant power (FRAP) method, the FRAB reagent was prepared according to the process of Benzie and Strain [48]. Accurately, 2 ml of the FRAB reagent was added to 0.1 ml of the samples (final concentration 200 μg). The mixture was incubated for 30 min at room temperature (25°C), and the absorbance was recorded at 593 nm. The FRAP of the prepared nanoparticles AgNPs-CEPA and A. cepa were measured in equivalents to the Trolox using its calibration curve.


### 2.6. Cell Culture

Human colorectal cancer cell lines (HT-29 and SW620 cells) were cultured in Roswell Park Memorial Institute medium (RPMI-1640) (GIBCO, by Thermo Fisher Scientific, NY, USA) supplemented with 10% FBS and 1% penicillin and streptomycin. All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2.


### 2.7. Cell Viability Assay

Cell viability was quantified using the MTT assay as previously described method [35]. Cells (5×103 per well) were plated in a 96-well plate (Corning, NY, USA). 24 h after incubation, the cells were set as the control and incubated with an aqueous solution of A. cepa extract, AgNO3, and AgNPs-CEPA with varying concentrations (5–100 μg/ml) for 24 h. AgNPs-CEPA were incubated in the form of an aqueous solution as originally prepared. The A. cepa dried extract was suspended in the Millipore water before incubation with cells. 10 μl of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mM) solution was mixed with the cells and incubated for 2-3 h at 37°C in a 5% CO2 atmosphere. 100 μl of DMSO was put into each well, and the crystals were dissolved with careful pipetting. A Synergy 2 multimode microplate reader was used (Biotech, VA, USA). Each experiment was performed three times.


### 2.8. Flow Cytometry for Apoptosis

Apoptosis and necrosis were determined in the same manner as previously described [35]. HT-29 and SW620 cells were seeded in a 6-well plate at a density of 1×105/well for 24 h. The next day, cells were treated with different concentrations of aqueous solutions of A. cepa extract, AgNO3, and AgNPs-CEPA for 24 h. The cells were extracted and washed twice with ice-cold PBS after being harvested. Apoptosis detection was conducted using an Annexin V/Dead cell apoptosis kit (Cat# V13242, Thermo Fisher Scientific Inc., Waltham Massachusetts) to detect cell death. The pellet was resuspended in 1X binding buffer and treated for 15 minutes in the dark at room temperature with Annexin V-FITC (5 l) and 1 l propidium iodide. The data analysis was collected using Cell Quest Pro Ver 6.0 BD FACS CALIBUR (BD Biosciences, San Jose, CA USA).The primer sequences are as follows: Bax, F: CCC TTT TGC TTC AGG GTT TC and R: TCT TCT TCC AGA TGG TGA GTG; Bcl-2, F: ACG AGT GGG ATG GGG GAG ATG TG and R: GCG GTA GCG GCG GGA GAA GTC; Bcl-xL, F: CTG AAT CGG AGA TGG AGA CC and R: TGG GAT GTC AGG TCA CTG AA; and Mcl-1, F: AGA AAG CTG CAT CGA ACC AT and R: CC AGC TCC TAC TCC AGC AAC.


### 2.9. Physical Stability

A three-month storage period at25.0±0.5°C and 4.0±0.5°C was used to evaluate the physical stability of the prepared AgNPs-CEPA. The physical parameters such as color, shape, particle size, and zeta potential were measured before and after storage to determine their stability [49].


### 2.10. Statistical Analysis

All experiments were performed in triplicate, and statistical means and standard errors were calculated. The statistical significance of differences between values of the treated and untreated (control) groups was evaluated by two-way ANOVA. The differences withp<0.05 were considered significant. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.


## 2.1. Materials

Silver nitrate (AgNO3) was purchased from VBBN Company (Hong Kong, China). Sulfuric acid was purchased from Severn Biotech Ltd, Kidderminster, United Kingdom. Sodium phosphate and Whatman filter paper Grade 1, 11 μm (1×3 cm), were purchased from Mumbai, Maharashtra, India. Ammonium molybdate was purchased from Sarkhej, Ahmedabad. Gujarat, India. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO (Dimethylsulfoxide) were purchased from Sigma-Aldrich (St. Louis, USA). Human colorectal cancer cell lines (HT-29 and SW620 cells) were purchased from ATCC (Manassas, Virginia, UK). Roswell Park Memorial Institute medium (RPMI-1640) (GIBCO, by Thermo Fisher Scientific, NY, USA) supplemented with 10% FBS and 1% penicillin and streptomycin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Invitrogen Ltd 3 (Fountain Drive, Inchinnan Business Park, Paisley, PA4 9RF, UK). All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. All glassware was washed using Millipore water. All chemicals were of analytical grades.


## 2.2. Plant Materials and Preparation of the Aqueous Extract ofA. cepa

The onion (Allium cepa L, A. cepa) was purchased from the local market in Buraydah, Al Qassim, Saudi Arabia, and identified by the local botanists at the Department of Plant Production and Protection, College of Agriculture, Qassim University. A. cepa bulbs were sliced into pieces of ~4 mm thickness. The A. cepa pieces were dried in an oven at 50°C temperatures for three days using the convective air-drying method. The aqueous A. cepa extract was prepared by continuous stirring of 1 gm dried A. cepa with 100 ml of distilled water at 25±1°C for 4 h. The aqueous extract was purified using a Whatman filter paper Grade 1, 11 μm (1×3 cm) [40].


## 2.3. Preparation of the AgNPs-CEPA

The AgNP-CEPA was prepared according to the previously reported method with some modification for better results [41]. Briefly, 1 mM stock solution of silver nitrate (AgNO3) was prepared, stirred with the scheduled aqueous extraction of A. cepa using a multiple-stirrer digital magnetic stirrer (VELP Scientifica Srl, Italy). The stirring at 600 rpm has been optimized at room temperature at 25±1°C. The synthesis of AgNPs (AgNPs-CEPA) was confirmed by observing a color change from colorless to yellowish. Then, the synthesized nanoparticles were collected and stored in a dark place away from light.


## 2.4. Characterization of AgNPs-CEPA

### 2.4.1. Size and Charge

The size and charge of the produced AgNPs-CEPA were measured using a Malvern Zetasizer Nano, Malvern Instruments GmbH (Herrenberg, Germany). Data were presented as the average of the three different measurements of the same AgNPs. The surface charge of AgNPs-CEPA was determined by zeta potential measurements with the same equipment. AgNP-CEPA was put through a laser beam of 623 nm, and the angle was adjusted to 90° at 25°C [42, 43]. The results presented are the average measurements of the runs with standard deviation.


### 2.4.2. Ultraviolet-Visible Spectroscopy

Ultraviolet-visible (UV-vis) spectrophotometry (Jasco, UV-630, Japan) was used to analyze theA. cepa extract and AgNPs-CEPA. AgNPs were put through a laser lamp and the angle was 90° at wavelengths ranging from 200 to 800 nm. The absorption range of reaction solutions was reported as a function of reaction time. We used distal water as a reference or blank, and then, we put the samples which contained particles in the instrument [44, 45].


### 2.4.3. Fourier-Transform Infrared Spectroscopy

The study was aimed at proving the formation of AgNPs-CEPA. FTIR measurements were used to analyze the compatibility of biomolecules associated with AgNP formation. It was measured with a Bruker Tensor 27 FTIR spectrophotometer (Varian Company model: 640-IR, Australia) [42]. The FTIR spectra were recorded, and the absorption peaks were observed at 400-4000 cm−1.


### 2.4.4. SEM Analysis

Scanning electron microscopy (FESEM, supra 55-Carl Zeiss, Germany) was used to study the morphology and size of the synthesized nanoparticles AgNPs-CEPA [44].


## 2.4.1. Size and Charge

The size and charge of the produced AgNPs-CEPA were measured using a Malvern Zetasizer Nano, Malvern Instruments GmbH (Herrenberg, Germany). Data were presented as the average of the three different measurements of the same AgNPs. The surface charge of AgNPs-CEPA was determined by zeta potential measurements with the same equipment. AgNP-CEPA was put through a laser beam of 623 nm, and the angle was adjusted to 90° at 25°C [42, 43]. The results presented are the average measurements of the runs with standard deviation.


## 2.4.2. Ultraviolet-Visible Spectroscopy

Ultraviolet-visible (UV-vis) spectrophotometry (Jasco, UV-630, Japan) was used to analyze theA. cepa extract and AgNPs-CEPA. AgNPs were put through a laser lamp and the angle was 90° at wavelengths ranging from 200 to 800 nm. The absorption range of reaction solutions was reported as a function of reaction time. We used distal water as a reference or blank, and then, we put the samples which contained particles in the instrument [44, 45].


## 2.4.3. Fourier-Transform Infrared Spectroscopy

The study was aimed at proving the formation of AgNPs-CEPA. FTIR measurements were used to analyze the compatibility of biomolecules associated with AgNP formation. It was measured with a Bruker Tensor 27 FTIR spectrophotometer (Varian Company model: 640-IR, Australia) [42]. The FTIR spectra were recorded, and the absorption peaks were observed at 400-4000 cm−1.


## 2.4.4. SEM Analysis

Scanning electron microscopy (FESEM, supra 55-Carl Zeiss, Germany) was used to study the morphology and size of the synthesized nanoparticles AgNPs-CEPA [44].


## 2.5. Antioxidant Activity Screening

As a comparable assay, threein vitro methods were conducted to evaluate the antioxidant activity of the A. cepa extract and formulated AgNPs of the extract (AgNPs-CEPA). The procedures were performed in triplicate for all experiments, and the antioxidant activity of CEPA and AgNPs-CEPA was calculated using standard calibration curves of Trolox for each method.


### 2.5.1. Total Antioxidant Capacity (TAC)

Total antioxidant capacity (TAC) of the CEPA and AgNPs-CEPA was conducted according to Aroua et al. [46]. 200 μl of all samples (final concentration 200 μg) was added to the freshly prepared acidic molybdate reagent (2 ml). The mixture was then vigorously shaken and heated on the water bath for 90 min at 85°C and cooled to room temperature (25°C). The absorbance of the blue color that arises was measured at 695 nm, and the total antioxidant activity of the samples was calculated using the Trolox standard calibration curve.


### 2.5.2. DPPH Scavenging Activity (DPPH-SA)

DPPH scavenging activity (DPPH-SA) was conducted according to Mohammed et al. [47] with minor modification. The DPPH in a concentration of 300 μM (1 ml) was added to 1 ml of A. cepa and AgNPs-CEPA (final concentration 200 μg). The mixture was vortexed and stood in a dark place for 30 min at room temperature to develop the violet color which measures spectrophotometrically at 517 nm. The DPPH-SA activity of the prepared nanoparticles AgNPs-CEPA and A. cepa aqueous extract was measured in equivalents to the Trolox using its calibration curve.


### 2.5.3. DPPH Scavenging Activity (DPPH-SA)

In the ferric reducing antioxidant power (FRAP) method, the FRAB reagent was prepared according to the process of Benzie and Strain [48]. Accurately, 2 ml of the FRAB reagent was added to 0.1 ml of the samples (final concentration 200 μg). The mixture was incubated for 30 min at room temperature (25°C), and the absorbance was recorded at 593 nm. The FRAP of the prepared nanoparticles AgNPs-CEPA and A. cepa were measured in equivalents to the Trolox using its calibration curve.


## 2.5.1. Total Antioxidant Capacity (TAC)

Total antioxidant capacity (TAC) of the CEPA and AgNPs-CEPA was conducted according to Aroua et al. [46]. 200 μl of all samples (final concentration 200 μg) was added to the freshly prepared acidic molybdate reagent (2 ml). The mixture was then vigorously shaken and heated on the water bath for 90 min at 85°C and cooled to room temperature (25°C). The absorbance of the blue color that arises was measured at 695 nm, and the total antioxidant activity of the samples was calculated using the Trolox standard calibration curve.


## 2.5.2. DPPH Scavenging Activity (DPPH-SA)

DPPH scavenging activity (DPPH-SA) was conducted according to Mohammed et al. [47] with minor modification. The DPPH in a concentration of 300 μM (1 ml) was added to 1 ml of A. cepa and AgNPs-CEPA (final concentration 200 μg). The mixture was vortexed and stood in a dark place for 30 min at room temperature to develop the violet color which measures spectrophotometrically at 517 nm. The DPPH-SA activity of the prepared nanoparticles AgNPs-CEPA and A. cepa aqueous extract was measured in equivalents to the Trolox using its calibration curve.


## 2.5.3. DPPH Scavenging Activity (DPPH-SA)

In the ferric reducing antioxidant power (FRAP) method, the FRAB reagent was prepared according to the process of Benzie and Strain [48]. Accurately, 2 ml of the FRAB reagent was added to 0.1 ml of the samples (final concentration 200 μg). The mixture was incubated for 30 min at room temperature (25°C), and the absorbance was recorded at 593 nm. The FRAP of the prepared nanoparticles AgNPs-CEPA and A. cepa were measured in equivalents to the Trolox using its calibration curve.


## 2.6. Cell Culture

Human colorectal cancer cell lines (HT-29 and SW620 cells) were cultured in Roswell Park Memorial Institute medium (RPMI-1640) (GIBCO, by Thermo Fisher Scientific, NY, USA) supplemented with 10% FBS and 1% penicillin and streptomycin. All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2.


## 2.7. Cell Viability Assay

Cell viability was quantified using the MTT assay as previously described method [35]. Cells (5×103 per well) were plated in a 96-well plate (Corning, NY, USA). 24 h after incubation, the cells were set as the control and incubated with an aqueous solution of A. cepa extract, AgNO3, and AgNPs-CEPA with varying concentrations (5–100 μg/ml) for 24 h. AgNPs-CEPA were incubated in the form of an aqueous solution as originally prepared. The A. cepa dried extract was suspended in the Millipore water before incubation with cells. 10 μl of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mM) solution was mixed with the cells and incubated for 2-3 h at 37°C in a 5% CO2 atmosphere. 100 μl of DMSO was put into each well, and the crystals were dissolved with careful pipetting. A Synergy 2 multimode microplate reader was used (Biotech, VA, USA). Each experiment was performed three times.


## 2.8. Flow Cytometry for Apoptosis

Apoptosis and necrosis were determined in the same manner as previously described [35]. HT-29 and SW620 cells were seeded in a 6-well plate at a density of 1×105/well for 24 h. The next day, cells were treated with different concentrations of aqueous solutions of A. cepa extract, AgNO3, and AgNPs-CEPA for 24 h. The cells were extracted and washed twice with ice-cold PBS after being harvested. Apoptosis detection was conducted using an Annexin V/Dead cell apoptosis kit (Cat# V13242, Thermo Fisher Scientific Inc., Waltham Massachusetts) to detect cell death. The pellet was resuspended in 1X binding buffer and treated for 15 minutes in the dark at room temperature with Annexin V-FITC (5 l) and 1 l propidium iodide. The data analysis was collected using Cell Quest Pro Ver 6.0 BD FACS CALIBUR (BD Biosciences, San Jose, CA USA).The primer sequences are as follows: Bax, F: CCC TTT TGC TTC AGG GTT TC and R: TCT TCT TCC AGA TGG TGA GTG; Bcl-2, F: ACG AGT GGG ATG GGG GAG ATG TG and R: GCG GTA GCG GCG GGA GAA GTC; Bcl-xL, F: CTG AAT CGG AGA TGG AGA CC and R: TGG GAT GTC AGG TCA CTG AA; and Mcl-1, F: AGA AAG CTG CAT CGA ACC AT and R: CC AGC TCC TAC TCC AGC AAC.


## 2.9. Physical Stability

A three-month storage period at25.0±0.5°C and 4.0±0.5°C was used to evaluate the physical stability of the prepared AgNPs-CEPA. The physical parameters such as color, shape, particle size, and zeta potential were measured before and after storage to determine their stability [49].


## 2.10. Statistical Analysis

All experiments were performed in triplicate, and statistical means and standard errors were calculated. The statistical significance of differences between values of the treated and untreated (control) groups was evaluated by two-way ANOVA. The differences withp<0.05 were considered significant. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.


## 3. Results

The prepared AgNPs utilizing the aqueous extracts of theA. cepa showed a color change from dark green to light green, which indicated the formed nanosilver configuration due to Surface Plasmon Resonance (SPR) excitations of the AgNPs as reflected in the UV-vis spectrum of the product. The color change of the aqueous extract to the synthesized AgNPs confirmed the successful synthesis of AgNPs. For A. cepa, the aqueous extract color is white and it has been changed to olive yellowish color, which indicates AgNP-CEPA formation (Figure 1). The differential light scattering (DLS) recorded uniform AgNPs with symmetrical peaks of size 155±2.1 nm (Figure 2(a)). The zeta potentials of AgNPs-CEPA were also measured in phosphate buffer at pH 7. The AgNPs-CEPA had negative surface charge values of −37.3±2.92 mv (Figure 2(b)). All PDIs (polydispersity indices) were below 0.145, representing a stable colloidal system. The SEM showed AgNPs-CEPA with cubic shapes with an average diameter of 150–250 nm (Figure 2(c)).Figure 1
The schematic diagram for the preparation of AgNPs-CEPA.Figure 2
Nanoparticle characterization of the reduced AgNPs usingA. cepa: (a) size distribution by mean diameter as determined using DLS, (b) apparent zeta potential as determined by DLS, and (c) SEM examination analysis.
(a)(b)(c)


### 3.1. Ultraviolet-Visible Spectroscopy

The results showed an absorption spectrum ofA. cepa at 390 nm (Figure 3). Moreover, the UV–vis absorption spectrum of the produced AgNPs-CEPA showed an absorbance peak at 398 nm due to the excitation of SPR in the formed AgNPs. One of the most essential features in the optical absorbance spectra of metal nanoparticles is SPR, which is due to collective electron oscillation around the surface mode of the particles [41]. A single SPR band in the absorption spectra of the produced AgNPs indicates its spherical shape [50].Figure 3
UV–vis absorption spectrum of the produced AgNPs-CEPA and aqueous extract ofA. cepa.


### 3.2. Fourier-Transform Infrared Spectroscopy

As shown in Figure4, for AgNPs-CEPA, A. cepa, and AgNO3, the FTIR showed shared similar peaks with their AgNP-CEPA peaks between 3000 and 3600cm−1which indicates O-H stretching and C-H is bending at 1385-1380cm−1cm−1, C-N is stretching at 1000-1400cm−1, and C=O is stretching at 1750-1735cm−1.Figure 4
FT-IR spectrum of the biosynthesized silver nanoparticles AgNPs-CEPA.


### 3.3. Antioxidant Activity

The data mentioned in Table1 described the antioxidant activity of the A. cepa extract and formulated AgNPs-CEPA. The results showed insignificant variation between the A. cepa extract and its prepared nanoparticles in the TAA and DPPH-SA. However, FRAP showed better reducing activity for the A. cepa extract compared to the prepared AgNP-CEPA nanoparticles.Table 1
The antioxidant activities forA. cepa extract and synthesized silver nanoparticles AgNPs-CEPA using different antioxidant agents.
NameTAADPPH-SAFRAPA. cepa55.49±0.912.23±0.3614.78±0.20AgNPs-CEPA58.85±4.391.91±0.2013.37±0.17All the measurements were conducted in triplicate; mean and standard deviations were calculated. TAA: total antioxidant activity in mg Trolox equivalents per gram of the dried plant powder; FRAP: ferric reducing antioxidant power in mg Trolox equivalent per gram of the dried plant powder; DPPH-SA: 2,2-diphenyl-1-picrylhydrazyl-scavenging activity in mg Trolox equivalent per gram of the dried plant powder.


### 3.4. Cell Cytotoxicity

Human colorectal cancer cell line HT-29 cells were treated with different concentrations of the aqueous solutions ofA. cepa extract, AgNO3, and AgNPs-CEPA for 48 h. AgNP-CEPA was found to inhibit % viability of HT-29 in a dose-dependent manner starting at 20 μM (Figure 5(a)). We used another colorectal cancer cell line of metastatic origin, SW620 cells, to confirm this finding. Exposing SW620 cells with A. cepa extract, AgNO3, and AgNPs-CEPA resulted in significant dose inhibition of percent viability starting at 10 μM (Figure 5(b)). AgNO3 alone was found to have some inhibitory effect at higher concentrations (100 μM) in both cell lines. These findings thus demonstrate that AgNP-CEPA has anticancer potential in human colorectal cancer cell lines [51].Figure 5
Cell viability assay of two different cancer cell lines. (a) Anticancer activity ofA. cepa extract, AgNO3, and AgNPs-CEPA in HT-29 cells and (b) anticancer activity of A. cepa extract, AgNO3, and AgNPs-CEPA in SW620 cells. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)


### 3.5. Quantitative PCR

HT-29 and SW620 cells were treated with different concentrations ofA. cepa extract, AgNO3, and AgNPs-CEPA for 48 h. We looked at the apoptosis and antiapoptosis marker genes by quantitative RT-PCR. We clearly showed that AgNO3 and AgNPs-CEPA downregulate antiapoptotic genes BCL2 and BCL-Xl and increase apoptotic gene BAX significantly. All genes were normalized to the expression of GAPDH that was used as the housekeeping gene (Figure 6).Figure 6
Quantitative real-time PCR gene expression analysis of proapoptosis and antiapoptotic genes.∗p≤0.05 denotes statistically significant differences.We also investigated the effect ofA. cepa extract, AgNO3, and AgNPs-CEPA on inducing apoptosis by flow cytometry in two human colorectal cancer cell lines. Treatment of HT-29 with different concentrations of AgNPs-CEPA was associated with increased cell death. As shown in Figures 7(a) and 7(b), AgNPs-CEPA induced 29.5 and 88.6% apoptosis at 10 and 20 μg/ml concentrations, respectively, compared to control cells (1.3%). A. cepa extract was found to induce 3.8% and 10.7%. Treatment with AgNO3 resulted in 1% and 3.12% apoptosis induction. Furthermore, the effect of AgNP-CEPA-mediated cell death was studied in metastatic colorectal cancer cell lines SW620. AgNP-CEPA treatment of SW620 cells induced 68.6% and 74% total cell death compared to 1.07% in the control cells at 10 and 20 μg/ml concentrations (Figures 8(a) and 8(b)). Recent strategies for anticancer development target-specific biomarkers are required for cancer cell survival, thereby inducting cell apoptosis and thus affecting cancer cells selectively with minimum effect on normal cells. Among these targets is the antiapoptotic protein Bcl2 family. AgNP-CEPA treatment of HT-29 and SW620 cells resulted in a significant increase in apoptosis/cell death compared to A. cepa and AgNPs-CEPA. These findings thus indicate that AgNP-CEPA effectively inhibits cell proliferation and at the same time induces apoptosis by altering Bcl2 family gene expression.Figure 7
Apoptosis assay by flow cytometry of HT29 cancer cell lines with or without AgNO3, A. cepa, and AgNPs-CEPA. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)Figure 8
Apoptosis assay by flow cytometry of HT29 cancer cell lines with or without AgNO3, A. cepa, and AgNPs-CEPA. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)


### 3.6. Physical Stability

The physical stability of all prepared AgNPs-CEPA was investigated for three months at25±1°C and 4.0±1°C, respectively. The results revealed no difference in color or morphology between the two conditions studied. Furthermore, the prepared AgNPs-CEPA showed a nonsignificant (p≥0.05; ANOVA/Tukey) change in particle sizes, PDI, and zeta potentials, which agreed with the results obtained by Fernando et al. [52].


## 3.1. Ultraviolet-Visible Spectroscopy

The results showed an absorption spectrum ofA. cepa at 390 nm (Figure 3). Moreover, the UV–vis absorption spectrum of the produced AgNPs-CEPA showed an absorbance peak at 398 nm due to the excitation of SPR in the formed AgNPs. One of the most essential features in the optical absorbance spectra of metal nanoparticles is SPR, which is due to collective electron oscillation around the surface mode of the particles [41]. A single SPR band in the absorption spectra of the produced AgNPs indicates its spherical shape [50].Figure 3
UV–vis absorption spectrum of the produced AgNPs-CEPA and aqueous extract ofA. cepa.


## 3.2. Fourier-Transform Infrared Spectroscopy

As shown in Figure4, for AgNPs-CEPA, A. cepa, and AgNO3, the FTIR showed shared similar peaks with their AgNP-CEPA peaks between 3000 and 3600cm−1which indicates O-H stretching and C-H is bending at 1385-1380cm−1cm−1, C-N is stretching at 1000-1400cm−1, and C=O is stretching at 1750-1735cm−1.Figure 4
FT-IR spectrum of the biosynthesized silver nanoparticles AgNPs-CEPA.


## 3.3. Antioxidant Activity

The data mentioned in Table1 described the antioxidant activity of the A. cepa extract and formulated AgNPs-CEPA. The results showed insignificant variation between the A. cepa extract and its prepared nanoparticles in the TAA and DPPH-SA. However, FRAP showed better reducing activity for the A. cepa extract compared to the prepared AgNP-CEPA nanoparticles.Table 1
The antioxidant activities forA. cepa extract and synthesized silver nanoparticles AgNPs-CEPA using different antioxidant agents.
NameTAADPPH-SAFRAPA. cepa55.49±0.912.23±0.3614.78±0.20AgNPs-CEPA58.85±4.391.91±0.2013.37±0.17All the measurements were conducted in triplicate; mean and standard deviations were calculated. TAA: total antioxidant activity in mg Trolox equivalents per gram of the dried plant powder; FRAP: ferric reducing antioxidant power in mg Trolox equivalent per gram of the dried plant powder; DPPH-SA: 2,2-diphenyl-1-picrylhydrazyl-scavenging activity in mg Trolox equivalent per gram of the dried plant powder.


## 3.4. Cell Cytotoxicity

Human colorectal cancer cell line HT-29 cells were treated with different concentrations of the aqueous solutions ofA. cepa extract, AgNO3, and AgNPs-CEPA for 48 h. AgNP-CEPA was found to inhibit % viability of HT-29 in a dose-dependent manner starting at 20 μM (Figure 5(a)). We used another colorectal cancer cell line of metastatic origin, SW620 cells, to confirm this finding. Exposing SW620 cells with A. cepa extract, AgNO3, and AgNPs-CEPA resulted in significant dose inhibition of percent viability starting at 10 μM (Figure 5(b)). AgNO3 alone was found to have some inhibitory effect at higher concentrations (100 μM) in both cell lines. These findings thus demonstrate that AgNP-CEPA has anticancer potential in human colorectal cancer cell lines [51].Figure 5
Cell viability assay of two different cancer cell lines. (a) Anticancer activity ofA. cepa extract, AgNO3, and AgNPs-CEPA in HT-29 cells and (b) anticancer activity of A. cepa extract, AgNO3, and AgNPs-CEPA in SW620 cells. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)


## 3.5. Quantitative PCR

HT-29 and SW620 cells were treated with different concentrations ofA. cepa extract, AgNO3, and AgNPs-CEPA for 48 h. We looked at the apoptosis and antiapoptosis marker genes by quantitative RT-PCR. We clearly showed that AgNO3 and AgNPs-CEPA downregulate antiapoptotic genes BCL2 and BCL-Xl and increase apoptotic gene BAX significantly. All genes were normalized to the expression of GAPDH that was used as the housekeeping gene (Figure 6).Figure 6
Quantitative real-time PCR gene expression analysis of proapoptosis and antiapoptotic genes.∗p≤0.05 denotes statistically significant differences.We also investigated the effect ofA. cepa extract, AgNO3, and AgNPs-CEPA on inducing apoptosis by flow cytometry in two human colorectal cancer cell lines. Treatment of HT-29 with different concentrations of AgNPs-CEPA was associated with increased cell death. As shown in Figures 7(a) and 7(b), AgNPs-CEPA induced 29.5 and 88.6% apoptosis at 10 and 20 μg/ml concentrations, respectively, compared to control cells (1.3%). A. cepa extract was found to induce 3.8% and 10.7%. Treatment with AgNO3 resulted in 1% and 3.12% apoptosis induction. Furthermore, the effect of AgNP-CEPA-mediated cell death was studied in metastatic colorectal cancer cell lines SW620. AgNP-CEPA treatment of SW620 cells induced 68.6% and 74% total cell death compared to 1.07% in the control cells at 10 and 20 μg/ml concentrations (Figures 8(a) and 8(b)). Recent strategies for anticancer development target-specific biomarkers are required for cancer cell survival, thereby inducting cell apoptosis and thus affecting cancer cells selectively with minimum effect on normal cells. Among these targets is the antiapoptotic protein Bcl2 family. AgNP-CEPA treatment of HT-29 and SW620 cells resulted in a significant increase in apoptosis/cell death compared to A. cepa and AgNPs-CEPA. These findings thus indicate that AgNP-CEPA effectively inhibits cell proliferation and at the same time induces apoptosis by altering Bcl2 family gene expression.Figure 7
Apoptosis assay by flow cytometry of HT29 cancer cell lines with or without AgNO3, A. cepa, and AgNPs-CEPA. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)Figure 8
Apoptosis assay by flow cytometry of HT29 cancer cell lines with or without AgNO3, A. cepa, and AgNPs-CEPA. ∗p≤0.05 and ∗∗p≤0.01 denote statistically significant differences.
(a)(b)


## 3.6. Physical Stability

The physical stability of all prepared AgNPs-CEPA was investigated for three months at25±1°C and 4.0±1°C, respectively. The results revealed no difference in color or morphology between the two conditions studied. Furthermore, the prepared AgNPs-CEPA showed a nonsignificant (p≥0.05; ANOVA/Tukey) change in particle sizes, PDI, and zeta potentials, which agreed with the results obtained by Fernando et al. [52].


## 4. Discussion

The study was aimed at using the aqueous extract of theA. cepa for reducing AgNO3 to AgNPs-CEPA and investigate the antioxidant and anticancer activities. The synthesis of AgNPs-CEPA was confirmed as the color changed from colorless to yellowish color. These results agreed with those reported previously by Safaepour et al. [53], who reported the same color change from colorless to dark brown, indicating the formation of AgNPs. Percent of phenolics, flavonoids, quercetin aglycone, and calcium in the peel of A. cepa can play a role in forming AgNPs. A. cepa has been reported to possess dietary fibers and antioxidant activities. The brown skin of an onion peel extract contains different phytochemical constituents such as dietary fiber, phenolics, and flavonoids. In the development and stabilization of AgNPs, this phytochemical plays a significant role [31]. Thus, A. cepa (onion) is encouraged as a good and protective diet against cancer. In this research, A. cepa extract was used to reduce Ag+ ions in the aqueous solution of silver nitrate (equation (1)). In addition, a color transition signaling is due to the successful formation of AgNPs. The shift in the state of matter from the molecular level to the nanoscale includes a change in color due to the stimulation of SPR vibrations, which occurs due to the transition.
(1)Ag++e−=Ag0In the current preparation, the particle sizes were observed to increase, reflecting various involved factors’ roles. They were validated by the color changes in the preparation recorded with the UV-vis absorption spectra of the colored prepared solutions. Fuku et al. [54] outlined that the colors of all plasmonic NPs can be scientifically altered, owing to changes in the SPR absorption wavelength by varying the size and morphology of the particles. The AgNPs-CEPA had a size of 155±2.1 nm and a negative surface charge −37.3±2.92 mv with a color change from colorless to yellowish brown. The high negative zeta potential indicated that the particles are of the highest electrically stabilized nature to resist aggregation. Similar results were reported by Moraes et al. [55], as the size, PDI, and zeta potential of nanoparticles are parameters that indicate the stability of NPs. The surface charges upon NPs play significant roles in the stabilization of the NPs, whereas the degree of zeta potential is an investigative parameter of the colloidal stability of the system [56]. The PDI was 0.145, supposedly indicating the (near) homogeneity of the particles’ sizes in the sample of the nanocolloid. It was reported that PDIs lower than 0.2 are ideal because the size distribution of NPs falls within a small range of sizes [57]. A significant technique used to validate the formation of metal nanoparticles in an aqueous solution is UV-visible spectroscopy. UV-vis absorption spectrum showed an absorbance peak at 390 and 398 nm for A. cepa and AgNPs-CEPA, respectively. The appearance in the absorption spectra of generated AgNPs of a single SPR band indicates their spherical form [41]. The FTIR absorption spectroscopy confirmed the presence of A. cepa covered silver colloid of the present synthesis. The FTIR bands showed the typical peaks for A. cepa for the surface related to Ag0. In an earlier study, Sunkar and Nachiyar [58] also confirmed IR absorption peaks in the ranges of 3510 cm-1 and 1636 cm-1 for the AgNP-citrate preparation by trisodium citrate reduction method.The band at 3424 cm-1 correlates to O-H extending H-bonded alcohols and phenols. Peaks of 2921 cm-1 correlate to carboxylic acids with O-H stretch. N-H bend primary amines correspond to the assignment at 1625 cm-1. C-N stretching of the aromatic amine group corresponds to the highest at 1387 cm-1, and the bands detected at 1061 and 971 cm-1 relate to C-N stretching alcohols, carboxylic acids, ethers, and esters [41]. Therefore, peaks were presented for proteins and metabolites such as terpenoids with functional aldehydes, ketones, alcohols, and carboxylic acids accompanying the synthesized AgNPs [59]. Moreover, the amino acid residue carbonyl group has a greater capacity to bind metal, suggesting that proteins can keep molecules in groups and stabilize AgNPs in the aqueous medium [60].The results obtained for the antioxidant activity of the aqueousA. cepa extract and the formulated AgNP-CEPA particles demonstrated potential and similar reducing power for the molybdenum (VI) to molybdenum (V) in the TAA with 55.49±0.91 and 58.85±4.39 mg Trolox equivalents per gram of the dried plant powder, respectively. The scavenging activity of the extract and AgNP-CEPA particles also showed nonsignificant variations in the DPPH-SA at 2.23±0.36 and 1.91±0.20 mg Trolox equivalents per gram of the dried plant powder, respectively. However, results obtained for FRAP assay showed a little higher activity for the onion extract compared to AgNP-CEPA particles at 14.78±0.20 and 13.37±0.17 mg Trolox equivalents per gram of the dried plant powder, respectively. The overall antioxidant results demonstrated in Table 1 revealed that AgNP-CEPA nanoparticles retained the antioxidant power of the plant extract.However, the AgNP-CEPA was more toxic compared toA. cepa, and this may be due to the high toxicity of AgNO3. Because antioxidant and anticancer are two different events, anticancer activity indeed depends on antioxidant activity. Still, other pathways influence the viability, like apoptosis which different stress factors can induce. Therefore, it is possible that the antioxidant activity does not correlate with the anticancer activity which is already supported in previous publications [61]. The prospects of AgNPs synthesized from the A. cepa are an anticancer agent owing to its potent antioxidant potential due to high contents of phenolics and flavonoids [62]. They also have strong reducing possibilities due to various sulfur compounds in the aqueous extract, together with the elemental silver at a nanosized scale. Moreover, the shell capping of the chemical components of the A. cepa made the combination a potent candidate for anticancer evaluation [23]. Gomaa reported anticancer activity of AgNPs synthesized by A. cepa extract against various cancer cell lines from the breast, hepatocellular, and colon [41]. We are writing here for the first time the anticancer property of AgNPs synthesized with A. cepa extract against colorectal cancer cell (CRC) lines from different stages, i.e., adenocarcinoma CRC cell line (HT-29) and metastatic CRC cell line (SW620). We also increased the concentration of AgNPs from 0.1 mM by Gomaa to 1 mM with safe effects on cells.Cancer cell survival is regulated by genes that regulate cell proliferation and apoptosis. Bcl2 family proteins are divided into antiapoptotic proteins, Bcl2 and BclxL, and proapoptotic proteins, Bax, Bak, and Bid, which play a vital role in the apoptosis regulation [63]. Cancer cells’ fate is determined by the balance of proapoptotic and antiapoptotic proteins. Bcl2 and BclxL overexpression inhibits apoptosis and enhances cancer cell growth, making them intriguing targets for cancer therapy development. This study found that AgNPs-CEPA inhibited the Bcl2 and BclxL gene expression in a dose-dependent manner in colorectal cancer cells as a novel result for future targeting of cell Bcl2 and BclxL overexpression. As a result, the suppression of Bcl2 and BclxL increases the vulnerability of cancer cells to apoptosis.Further, treatment with AgNPs-CEPA increases proapoptotic gene expression like Bax. AgNPs-CEPA could be effective as an anticancer agent by targeting the Bcl2 family protein. Blocking cell proliferation is primarily mediated by apoptosis that plays an important role in fighting cancer [64]. As a result, apoptosis is a common target for cancer therapy techniques. In this study, we found that AgNPs-CEPA could trigger a significant increase in apoptosis in HT-29 and SW620 cells compared to control and A. cepa extract alone. Induction of apoptosis by anticancer therapy is the most known way to kill cancer cells. AgNP-CEPA was significantly effective in inducing apoptosis by targeting Bcl2 family proteins which holds promise as anticancer therapeutics. Moreover, the higher death of cells treated with AgNPs-CEPA is expected due to the synergistic effect produced due to the reduction of A. cepa to Ag0 [65–67]. The size used in cell biology is considered a larger size. According to research, using the smallest feasible nanoparticles may not be the greatest idea. The larger particles demonstrated higher resistance against external influences than smaller NPs. The particle size substantially impacted the in vitro toxicity, as samples with a larger particle size preserved more of their activity against mammalian cells [68]. Moreover, nanoparticle size and shape are known to affect biological activity [69, 70]. AgNPs, according to the findings of this study, could be a highly effective cancer therapy for a variety of cancers, as well as an alternate treatment for inflammation prevention by increasing autophagy [71].


## 5. Conclusion

The green synthesized AgNPs with their unique physical properties provide an alternative for the chemically produced AgNPs. The synthesized AgNPs using an aqueous extract ofA. cepa were successfully synthesized with uniform size and good stability. The nanoparticles also showed antibreast cancer activity and could further be developed as a template for other anticancer actions. AgNP-CEPA inhibits cell viability and reduced antiapoptotic genes. AgNP-CEPA has anticancer properties that could be used to treat colorectal adenocarcinoma and mCRC. These results are very promising in the field of nanoparticles for anticancer activity. We encourage using onion and AgNPs-CEPA as a dietary food for humans. Still, additional investigations to confirm these properties are needed in vivo study.



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*Source: 1746817-2022-02-04.xml*