# An Assessment of Anticancer Activity (MCF-7) and Free Radicals Scavenging Potential ofFerula Asafoetida Synthesized AgNPs through In Vitro Analyses

Authors: Nguyen Chi Thanh; Arunachalam Chinnathambi; Tahani Awad Alahmadi; Deepika Joshi; G. K. Jhanani; Adisu Haile
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Nanomaterials are increasingly being used in pharmaceutical industry to treat a variety of disorders and diseases. Hence, the aim of this research was to evaluate their antioxidant and anticancer activity of presynthesized and characterized plant-based AgNPs against ferric, DPPH, H2O2, and ABTS free radicals and MCF-7 cell line. Interestingly, the antioxidants assay results revealed that the increased concentration (100 μg mL-1) of AgNPs has remarkable antioxidant ability on ferric (78.20%), DPPH (89.61%), H2O2 (82.34%), and ABTS (67.95%) free radicals. These scavenging activities were almost close to the free radicals scavenging ability of vitamin C (ascorbic acid). Similarly, test AgNPs showed considerable cytotoxicity (83.15%) against MCF-7 cell line, and it was partially comparable with the cytotoxicity activity (91.35%) of positive control (Doxorubicin). These findings indicate that plant-mediated AgNPs have significant antioxidant and anticancer activity, and thus may be considered for biomedical applications pending the results of some in vivo experiments.


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## Body

## 1. Introduction

The recent advancement in biology’s understanding of free radicals and production of reactive oxygen species (ROS) is causing a healthcare revolution which assures a new era of diagnosis and treatment [1–3]. A free radical is any single-molecule species that can exist independently and has an extra electron in an atomic nucleus [4]. Because of the existence of an extra electron, most radicals generally have the following attributes. Numerous radicals are extremely reactive and volatile [5, 6]. They could either donate an electron to or receive an electron from these other atoms, consequently expected to behave as oxidizing agents or reducing agents [7, 8]. The human system is continuously fighting to avoid ageing [9]. Reactive oxygen species cause cell damage and disrupt homeostasis by damaging essential macromolecules. Free radicals can harm any type of molecule in the body, and the primary targets are triglycerides, nucleic acids, and polypeptides [10, 11]. Free radicals are produced by the human body’s natural vital metabolic pathways or by external factors such as X-rays, ozone, cigarette smoking, air pollutants, and hazardous chemicals [12]. Reactive oxygen species development occurs in cells on a continuous basis as a result of the nonenzymatic and enzymatic responses [1]. Enzymatic reactions engaged in the electron transport chain, phagocytosis, and prostaglandin production, as well as the cytochrome P-450 system, are all sources of free radicals [13]. Nonenzymatic reactions of O2 with organic molecules, and those begun by ionising interactions, can also produce free radicals [8]. Free radical reactions are supposed to induce progressive negative changes throughout the body as human’s age [14, 15]. These “normal” ageing changes occur in almost everyone. However, patterns influenced by genetic factors and ecological distinctions that mitigate reactive oxygen species damage are superimposed on the common approach [16, 17]. These manifest as illnesses at specific ages, which are established by genes and environmental influences [18]. Melanoma and coronary artery disease are two prominent “free radical” illnesses. Cancer emergence and progression are linked to chromosomal abnormalities and oncogene activation [19, 20]. Intrinsic free radical interactions, such as those induced by radiation exposure, may contribute in tumour formation [13]. Reactive oxygen and nitrogen species, as well as their biological metabolic products, are also essential in carcinogenesis [4]. ROS cause DNA damage because their reaction with genetic material contains strand break base alteration and DNA protein cross-links [10, 21]. Numerous works have suggested that reactive oxygen species play a role in oncogenesis [22], mutagenesis, and transition; it is obvious that their existence in the biological system could result to mutagenesis, transition, and inevitably cancer [23–25]. In 2020, there are 2.3 million cases of breast cancer as well as 685 000 deaths globally [26]. Since the end of 2020, there had been 7.8 million women who were suffered by breast malignancy in the last 5 five years, commonest cancer in the world [27]. Breast malignancy concerns women at any time of life upon adolescence in each and every country on the planet [28], with higher rates in later in life [29]. As one of the most prevalent form of cancer in Indian women, female in their early 30s to 50s are at a high risk of developing breast cancer, and the risk goes up until it reaches a peak by the moment those who are 50-64 years old [30]. One out of every twenty-eight Indian women will experience breast cancer in lifetime. It is superior in city person than in rural [1]. As a result, finding novel pharmaceutical agents with antioxidant potential to replace chemotherapy drugs used to treat cancer is urgently needed. As a result, researchers have recently focused on nanoparticles synthesized from biological sources that have significant antioxidant and anticancer activity [11]. To cope with this statement, the plant (Ferula asafoetida)-mediated silver nanoparticle (AgNPs) was used in this study. Since, genus of this plant has been recognized as possess significant quantity of pharmaceutically valuable phytochemicals as well as which can synthesize the AgNPs, and synthesized nanoparticles may possess remarkable biomedical application [31]. Hence, this research was performed to evaluate the antioxidant and anticancer activity of previously synthesized and characterized plant-mediated AgNPs.


## 2. Materials and Methods

### 2.1. Brief Profile of Presynthesized and Characterized AgNPs

The AgNPs used in this study were presynthesized byFerula asafoetida extract and characterized as follows [8]. The shape and size of these AgNPs were discovered to be spherical and 41 nm, respectively, and it has been reported to have exceptional larvicidal and pupicidal activity against common mosquito vectors.


### 2.2. Preparation of Presynthesized AgNPs

To prevent aggregation, the suspension was stirred continuously for 30 min in a sonicator after diluting 1 mg of AgNPs in 1 mL distilled water. The stock AgNP solution was very well sonicated and dissolved in distilled water to achieve concentration levels of 25, 50, 75, and 100 g mL-1. Further research was conducted upon that diluted solution.


### 2.3. Antioxidant Potential Analyses

#### 2.3.1. The Fe3+ Reducing Potential

TheK3FeCN6 reducing competence of various concentrations (20-100 μg mL-1) of AgNPs were studied by method of Mahendran and Ranjitha [32] with slight modifications. In brief, about 1.5 mL of each dosage of AgNPs was amalgamated with 3.0 mL of 1% K3Fe(CN)6 (prepared in 200 mM phosphate buffer with pH 6.5). The reaction blend of each concentration was kept for 15 min at 55°C and cooled them immediately; furthermore, 2.5 mL of 10% TCA was blended and spun at 3000 g for 15 min. Then read at 700 nm by UV-vis. spec., and the IC50 was calculated as follows.
(1)Ferricreducing/scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


#### 2.3.2. The DPPH Assay

The DPPH scavenging potential of various above-mentioned concentrations of test AgNPs was determined through standard DPPH assay protocol [33]. Concisely, 1.2 mL of each dose of plant-mediated AgNPs was mixed with 10 mL of newly prepared DPPH (0.1 mM). The reaction mixture was kept for 30 min under darkness and then the color alteration was observed as purple into pale yellow. The final absorbance was recorded at 517 nm. The IC50 values of AgNPs and positive control were calculated using following equation.
(2)DPPHscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


#### 2.3.3. The H2O2 Assay

The H2O2 scavenging proficiency of various concentrations of AgNPs through typical assay protocol [34]. In brief, 1.2 mL of each dose of AgNPs was mixed with 0.8 mL of 40 mM of H2O2 dissolved in P buffer. Subsequently, the mix was kept for 10 min, later the reading was recorded at 230 nm. The IC50 and scavenging % was calculated as follows.
(3)H2O2scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


#### 2.3.4. The ABTS Assay

Concisely, 20μL of each concentration of test AgNPs were mixed with approximately 980 μL of ABTS solution. Under darkness, the reaction mixture (test and positive control) was left at room condition for 10 min. The OD of the incubated reaction blend was recorded at 734 nm. The following equation was applied to calculate the IC50 value AgNPs to scavenge the ABTS free radicals [35].
(4)ABTSscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


### 2.4. Anticancer Activity (MTT Assay)

The anticancer activity potential of plant-mediated AgNPs was evaluated through standard MTT test on MCF-7 malignancy cell line [36]. In brief, about 150 μL of MCF-7 (1×106) cells were added on 96-well plate containing predefined DMEM medium. These plates were left for 2 days at 37°C using CO2 (5%) incubator. Sterile PBS solution was used to remove the unattached cells from the plate and attached cells were treated with 100 μL of each quantity of AgNPs (performed in triplicates) and doxorubicin used as reference. Test and positive control treated plates were incubated for a day. About 20 μL of MTT reagent was blended after 24 h of incubation to each wells and incubated for 4 h and observed the formation of purple color precipitate. The medium and reagents present in the wells were aspirated after the 24 h of incubation and rinsed with 200 μL of 1X PBS buffer. The formazan crystals obtained from this study were blend in 100 μL of DMSO and, their absorbances were recorded at 570 nm using a microplate reader. The cytotoxicity percentage and IC50 values of AgNPs and positive control were determined through standard equation.


## 2.1. Brief Profile of Presynthesized and Characterized AgNPs

The AgNPs used in this study were presynthesized byFerula asafoetida extract and characterized as follows [8]. The shape and size of these AgNPs were discovered to be spherical and 41 nm, respectively, and it has been reported to have exceptional larvicidal and pupicidal activity against common mosquito vectors.


## 2.2. Preparation of Presynthesized AgNPs

To prevent aggregation, the suspension was stirred continuously for 30 min in a sonicator after diluting 1 mg of AgNPs in 1 mL distilled water. The stock AgNP solution was very well sonicated and dissolved in distilled water to achieve concentration levels of 25, 50, 75, and 100 g mL-1. Further research was conducted upon that diluted solution.


## 2.3. Antioxidant Potential Analyses

### 2.3.1. The Fe3+ Reducing Potential

TheK3FeCN6 reducing competence of various concentrations (20-100 μg mL-1) of AgNPs were studied by method of Mahendran and Ranjitha [32] with slight modifications. In brief, about 1.5 mL of each dosage of AgNPs was amalgamated with 3.0 mL of 1% K3Fe(CN)6 (prepared in 200 mM phosphate buffer with pH 6.5). The reaction blend of each concentration was kept for 15 min at 55°C and cooled them immediately; furthermore, 2.5 mL of 10% TCA was blended and spun at 3000 g for 15 min. Then read at 700 nm by UV-vis. spec., and the IC50 was calculated as follows.
(1)Ferricreducing/scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


### 2.3.2. The DPPH Assay

The DPPH scavenging potential of various above-mentioned concentrations of test AgNPs was determined through standard DPPH assay protocol [33]. Concisely, 1.2 mL of each dose of plant-mediated AgNPs was mixed with 10 mL of newly prepared DPPH (0.1 mM). The reaction mixture was kept for 30 min under darkness and then the color alteration was observed as purple into pale yellow. The final absorbance was recorded at 517 nm. The IC50 values of AgNPs and positive control were calculated using following equation.
(2)DPPHscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


### 2.3.3. The H2O2 Assay

The H2O2 scavenging proficiency of various concentrations of AgNPs through typical assay protocol [34]. In brief, 1.2 mL of each dose of AgNPs was mixed with 0.8 mL of 40 mM of H2O2 dissolved in P buffer. Subsequently, the mix was kept for 10 min, later the reading was recorded at 230 nm. The IC50 and scavenging % was calculated as follows.
(3)H2O2scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


### 2.3.4. The ABTS Assay

Concisely, 20μL of each concentration of test AgNPs were mixed with approximately 980 μL of ABTS solution. Under darkness, the reaction mixture (test and positive control) was left at room condition for 10 min. The OD of the incubated reaction blend was recorded at 734 nm. The following equation was applied to calculate the IC50 value AgNPs to scavenge the ABTS free radicals [35].
(4)ABTSscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


## 2.3.1. The Fe3+ Reducing Potential

TheK3FeCN6 reducing competence of various concentrations (20-100 μg mL-1) of AgNPs were studied by method of Mahendran and Ranjitha [32] with slight modifications. In brief, about 1.5 mL of each dosage of AgNPs was amalgamated with 3.0 mL of 1% K3Fe(CN)6 (prepared in 200 mM phosphate buffer with pH 6.5). The reaction blend of each concentration was kept for 15 min at 55°C and cooled them immediately; furthermore, 2.5 mL of 10% TCA was blended and spun at 3000 g for 15 min. Then read at 700 nm by UV-vis. spec., and the IC50 was calculated as follows.
(1)Ferricreducing/scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


## 2.3.2. The DPPH Assay

The DPPH scavenging potential of various above-mentioned concentrations of test AgNPs was determined through standard DPPH assay protocol [33]. Concisely, 1.2 mL of each dose of plant-mediated AgNPs was mixed with 10 mL of newly prepared DPPH (0.1 mM). The reaction mixture was kept for 30 min under darkness and then the color alteration was observed as purple into pale yellow. The final absorbance was recorded at 517 nm. The IC50 values of AgNPs and positive control were calculated using following equation.
(2)DPPHscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


## 2.3.3. The H2O2 Assay

The H2O2 scavenging proficiency of various concentrations of AgNPs through typical assay protocol [34]. In brief, 1.2 mL of each dose of AgNPs was mixed with 0.8 mL of 40 mM of H2O2 dissolved in P buffer. Subsequently, the mix was kept for 10 min, later the reading was recorded at 230 nm. The IC50 and scavenging % was calculated as follows.
(3)H2O2scavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100


## 2.3.4. The ABTS Assay

Concisely, 20μL of each concentration of test AgNPs were mixed with approximately 980 μL of ABTS solution. Under darkness, the reaction mixture (test and positive control) was left at room condition for 10 min. The OD of the incubated reaction blend was recorded at 734 nm. The following equation was applied to calculate the IC50 value AgNPs to scavenge the ABTS free radicals [35].
(4)ABTSscavenging%=Absorbanceofcontrol‐AbsorbanceoftestAbsorbanceofcontrolX100.


## 2.4. Anticancer Activity (MTT Assay)

The anticancer activity potential of plant-mediated AgNPs was evaluated through standard MTT test on MCF-7 malignancy cell line [36]. In brief, about 150 μL of MCF-7 (1×106) cells were added on 96-well plate containing predefined DMEM medium. These plates were left for 2 days at 37°C using CO2 (5%) incubator. Sterile PBS solution was used to remove the unattached cells from the plate and attached cells were treated with 100 μL of each quantity of AgNPs (performed in triplicates) and doxorubicin used as reference. Test and positive control treated plates were incubated for a day. About 20 μL of MTT reagent was blended after 24 h of incubation to each wells and incubated for 4 h and observed the formation of purple color precipitate. The medium and reagents present in the wells were aspirated after the 24 h of incubation and rinsed with 200 μL of 1X PBS buffer. The formazan crystals obtained from this study were blend in 100 μL of DMSO and, their absorbances were recorded at 570 nm using a microplate reader. The cytotoxicity percentage and IC50 values of AgNPs and positive control were determined through standard equation.


## 3. Results and Discussion

### 3.1. Ferric Reducing Efficiency

The plant-mediated AgNPs showed moderate ferric reducing potential, and it was dose dependent, since the ferric reducing percentage was gradually increased from 21.08% to 78.20% at the concentration of 20 to 100μg mL-1 , respectively. This value was statistically significant at P<0.05 (Figure 1). These reducing values were moderately comparable to the ferric reducing activity of standard, since it was found as 81.3% at 100 μg mL-1 concentration. The value of IC50 of AgNPs that reducing the ferric compound was found as 44.97 μg mL-1 as well as it had been average while compared to ferric reducing activity of control (26.33 μg mL-1) (Figure 1). These results suggest that the plant-mediated AgNPs moderately reduced the colorless Fe3+-2,4,6-tripyridyl-s-triazine compound to the deep blue colored Fe2+-2,4,6-tripyridyl-s-triazine compound in the acidic condition [37]. The increased absorbance value related to the intensive blue formation in the reduction reaction, so the increased color density strongly suggests that the reduction of Fe3+ to Fe2+ [38]. However, the ferric reducing potential of the test compounds was not completely determine the antioxidant potential of them, since other compounds which possess redox potential also reduce the Fe3+ to Fe2+, and it might increase the FRAP value [39]. However, not even all antioxidants reduce Fe3+ at a rate fast enough to be measured. This ferric reduction can also induced by the bioactive phytochemicals [40] such as saponins, and other steroidal molecules present over the surface of AgNPs [41].Figure 1
Ferric reducing activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.


### 3.2. DPPH Scavenging Assay

The increased concentration of AgNPs demonstrated remarkable DPPH free radicals scavenging activity, and it showed about 89.61% of scavenging at 100μg mL-1 concentration, and this value was statistically significant at P<0.05 (Figure 2). Furthermore, this was moderately comparable with the DPPH radicals scavenging potential of ascorbic acid (98.45%) at increased concentration. Interestingly, the IC50 of AgNPs, which suppressing the DPPH radicals was calculated as 34.03 μg mL-1 as well as it was comparable with scavenging activity potential of standard (28.49 μg mL-1).Figure 2
DPPH radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.The obtained results were significantly greater than the previous report of Sudha et al. [42], they reported that the AgNPs scavenging about 67% of DPPH at 500 μg mL-1 concentration. Previous report also stated that the increased concentration of AgNPs showed maximum scavenging potential [32]. The potential DPPH scavenging ability of AgNPs previously disclosed has confirmed the notion of our current study [37]. The phytochemicals which coated over the surface of AgNPs can reduce the complex DPPH radicals into hydrazine (reduced) form that leads to color conversion from purple to pale yellow [38].


### 3.3. H2O2 Scavenging Assay

The result obtained from this AgNPs H2O2 scavenging assay stated that the H2O2 scavenging competence of test AgNPs was directly related to the concentration used them. Since, the maximum (82.34%) H2O2 radicals scavenging were found at increased concentration of AgNPs. Thus, this was a dose dependent reaction and obtained H2O2 scavenging percentage was statistically significant at P<0.03 (Figure 3).Figure 3
H2O2 radicals scavenging ability of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates. ∗: indicates the statistically significant at P<0.03.Furthermore, this result suggests that the AgNPs effectively scavenging (82.34%) the H2O2 than the positive standard (81.68). The IC50 value of AgNPs and standard ascorbic acid, which scavenging H2O2 were calculated as 38.31 and 33.82 μg mL-1. Sundararajan et al. [43] reported that Cardiospermum halicacabum mediated AgNPs showed 73.6% of H2O2 scavenging activity at increased concentration (100 μg mL-1). The spherical shaped AgNPs possess excellent antioxidant activity than other shapes, since they possess large surface area thus they can easily absorb/accept the electrons from hydroxyl radicals [44]. The phenolic compounds which coated on the large surface area of spherical-shaped AgNPs might possess significant H2O2 radicals scavenging activity [45]. Furthermore, the plant-mediated AgNPs possess remarkable H2O2 radicals scavenging activity and probably it could be site specific (-OH) and dose dependent [46].


### 3.4. ABTS Scavenging Assay

The ABTS radical scavenging assay possesses more benefits over the DPPH radical assay, since it can be applied in both aqueous and organic formulations [38]. The ABTS assay outcome revealed that, tested AgNPs demonstrated considerable ABTS scavenging potential up to 67.95% at increased concentration of 100 μg mL-1 (Figure 4). This scavenging activity was remarkably compared with ascorbic acid (71.42%) at a dosage of 100 μg mL-1. Furthermore, the IC50 of AgNPs scavenging ABTS free radicals had been calculated as 38.32 μg mL-1. It had been partially match with IC50 value (24.321 μg mL-1) of ascorbic acid. Datkhile et al. [47] reported that the AgNPs mediated from microbial sources showed 84.59% of ABTS radical scavenging activity [47]. Another report stated that the plant-mediated AgNPs showed about 61% of ABTS activity at a dosage of 50 μg mL-1 [38].Figure 4
ABTS radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.The possible mechanism involved in this AgNP-based ABTS radicals scavenging activity is by transferring electron from dark blue colored 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (cation) (ABTS•+) into colorless ABTS [48].


### 3.5. Anticancer Activity of AgNPs

The results obtained from the MTT assay on MCF-7 cells revealed that the plant-mediated test AgNPs exhibited significant cytotoxicity on this cancerous cell by inhibiting the cell proliferation. Furthermore, the dose dependent cell proliferation inhibition by AgNPs was observed on MCF-7. Since at increased concentration (100μg mL-1) of AgNPs showed around as 83.15% of cytotoxicity, and it was partially comparable with the cytotoxicity activity (91.35%) of Doxorubicin. The obtained cytotoxicity percentage of test AgNPs were found as statically significant at P<0.05 (Figure 5).Figure 5
H2O2 radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates. ∗∗: indicates the statistically significant at P<0.05.TheIC50 value of AgNP-based cytotoxicity on MCF-7 cell line was determined as 38.556 μg mL-1. However, it showed maximum cytotoxicity, and it was rather close up to the positive control; the IC50 of test showed significantly lower than the Doxorubicin. The cytotoxicity activity was found as dose dependent [49]. These findings are partially correlated with the results of Gomathi et al. [50], and they reported that Tamarindus indica mediated AgNPs showed remarkable cytotoxicity against MCF-7 cell line, and IC50 value was reported as 20 μg mL-1 [50]. AgNPs are prospective anticancer therapy since they influence the cell cycle, diminish cancer cell propagation, cause oxidative stress, as well as cause apoptosis [51]. Bethu et al. [52] reported that the AgNPs derived from biological sources cause cytotoxicity in SKOV3 cells by inducing oxidative stress by stimulating the accumulation of ROS, lipid peroxidation, and reducing the glutathione quantity [52].


## 3.1. Ferric Reducing Efficiency

The plant-mediated AgNPs showed moderate ferric reducing potential, and it was dose dependent, since the ferric reducing percentage was gradually increased from 21.08% to 78.20% at the concentration of 20 to 100μg mL-1 , respectively. This value was statistically significant at P<0.05 (Figure 1). These reducing values were moderately comparable to the ferric reducing activity of standard, since it was found as 81.3% at 100 μg mL-1 concentration. The value of IC50 of AgNPs that reducing the ferric compound was found as 44.97 μg mL-1 as well as it had been average while compared to ferric reducing activity of control (26.33 μg mL-1) (Figure 1). These results suggest that the plant-mediated AgNPs moderately reduced the colorless Fe3+-2,4,6-tripyridyl-s-triazine compound to the deep blue colored Fe2+-2,4,6-tripyridyl-s-triazine compound in the acidic condition [37]. The increased absorbance value related to the intensive blue formation in the reduction reaction, so the increased color density strongly suggests that the reduction of Fe3+ to Fe2+ [38]. However, the ferric reducing potential of the test compounds was not completely determine the antioxidant potential of them, since other compounds which possess redox potential also reduce the Fe3+ to Fe2+, and it might increase the FRAP value [39]. However, not even all antioxidants reduce Fe3+ at a rate fast enough to be measured. This ferric reduction can also induced by the bioactive phytochemicals [40] such as saponins, and other steroidal molecules present over the surface of AgNPs [41].Figure 1
Ferric reducing activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.


## 3.2. DPPH Scavenging Assay

The increased concentration of AgNPs demonstrated remarkable DPPH free radicals scavenging activity, and it showed about 89.61% of scavenging at 100μg mL-1 concentration, and this value was statistically significant at P<0.05 (Figure 2). Furthermore, this was moderately comparable with the DPPH radicals scavenging potential of ascorbic acid (98.45%) at increased concentration. Interestingly, the IC50 of AgNPs, which suppressing the DPPH radicals was calculated as 34.03 μg mL-1 as well as it was comparable with scavenging activity potential of standard (28.49 μg mL-1).Figure 2
DPPH radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.The obtained results were significantly greater than the previous report of Sudha et al. [42], they reported that the AgNPs scavenging about 67% of DPPH at 500 μg mL-1 concentration. Previous report also stated that the increased concentration of AgNPs showed maximum scavenging potential [32]. The potential DPPH scavenging ability of AgNPs previously disclosed has confirmed the notion of our current study [37]. The phytochemicals which coated over the surface of AgNPs can reduce the complex DPPH radicals into hydrazine (reduced) form that leads to color conversion from purple to pale yellow [38].


## 3.3. H2O2 Scavenging Assay

The result obtained from this AgNPs H2O2 scavenging assay stated that the H2O2 scavenging competence of test AgNPs was directly related to the concentration used them. Since, the maximum (82.34%) H2O2 radicals scavenging were found at increased concentration of AgNPs. Thus, this was a dose dependent reaction and obtained H2O2 scavenging percentage was statistically significant at P<0.03 (Figure 3).Figure 3
H2O2 radicals scavenging ability of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates. ∗: indicates the statistically significant at P<0.03.Furthermore, this result suggests that the AgNPs effectively scavenging (82.34%) the H2O2 than the positive standard (81.68). The IC50 value of AgNPs and standard ascorbic acid, which scavenging H2O2 were calculated as 38.31 and 33.82 μg mL-1. Sundararajan et al. [43] reported that Cardiospermum halicacabum mediated AgNPs showed 73.6% of H2O2 scavenging activity at increased concentration (100 μg mL-1). The spherical shaped AgNPs possess excellent antioxidant activity than other shapes, since they possess large surface area thus they can easily absorb/accept the electrons from hydroxyl radicals [44]. The phenolic compounds which coated on the large surface area of spherical-shaped AgNPs might possess significant H2O2 radicals scavenging activity [45]. Furthermore, the plant-mediated AgNPs possess remarkable H2O2 radicals scavenging activity and probably it could be site specific (-OH) and dose dependent [46].


## 3.4. ABTS Scavenging Assay

The ABTS radical scavenging assay possesses more benefits over the DPPH radical assay, since it can be applied in both aqueous and organic formulations [38]. The ABTS assay outcome revealed that, tested AgNPs demonstrated considerable ABTS scavenging potential up to 67.95% at increased concentration of 100 μg mL-1 (Figure 4). This scavenging activity was remarkably compared with ascorbic acid (71.42%) at a dosage of 100 μg mL-1. Furthermore, the IC50 of AgNPs scavenging ABTS free radicals had been calculated as 38.32 μg mL-1. It had been partially match with IC50 value (24.321 μg mL-1) of ascorbic acid. Datkhile et al. [47] reported that the AgNPs mediated from microbial sources showed 84.59% of ABTS radical scavenging activity [47]. Another report stated that the plant-mediated AgNPs showed about 61% of ABTS activity at a dosage of 50 μg mL-1 [38].Figure 4
ABTS radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates.∗∗: indicates the statistically significant at P<0.05.The possible mechanism involved in this AgNP-based ABTS radicals scavenging activity is by transferring electron from dark blue colored 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (cation) (ABTS•+) into colorless ABTS [48].


## 3.5. Anticancer Activity of AgNPs

The results obtained from the MTT assay on MCF-7 cells revealed that the plant-mediated test AgNPs exhibited significant cytotoxicity on this cancerous cell by inhibiting the cell proliferation. Furthermore, the dose dependent cell proliferation inhibition by AgNPs was observed on MCF-7. Since at increased concentration (100μg mL-1) of AgNPs showed around as 83.15% of cytotoxicity, and it was partially comparable with the cytotoxicity activity (91.35%) of Doxorubicin. The obtained cytotoxicity percentage of test AgNPs were found as statically significant at P<0.05 (Figure 5).Figure 5
H2O2 radicals scavenging activity of various concentrations of AgNPs. The graph was plotted with mean and standard error (±SE) of triplicates. ∗∗: indicates the statistically significant at P<0.05.TheIC50 value of AgNP-based cytotoxicity on MCF-7 cell line was determined as 38.556 μg mL-1. However, it showed maximum cytotoxicity, and it was rather close up to the positive control; the IC50 of test showed significantly lower than the Doxorubicin. The cytotoxicity activity was found as dose dependent [49]. These findings are partially correlated with the results of Gomathi et al. [50], and they reported that Tamarindus indica mediated AgNPs showed remarkable cytotoxicity against MCF-7 cell line, and IC50 value was reported as 20 μg mL-1 [50]. AgNPs are prospective anticancer therapy since they influence the cell cycle, diminish cancer cell propagation, cause oxidative stress, as well as cause apoptosis [51]. Bethu et al. [52] reported that the AgNPs derived from biological sources cause cytotoxicity in SKOV3 cells by inducing oxidative stress by stimulating the accumulation of ROS, lipid peroxidation, and reducing the glutathione quantity [52].


## 4. Conclusion

The results obtained from this study conclude that the presynthesized and characterized plant-based AgNPs possess considerable antioxidant activity against DPPH, H2O2, and ABTS free radicals. Furthermore, this AgNPs showed dose dependent antioxidant activity, since at 100 μg mL-1 concentration, it showed remarkable free radicals scavenging activity. This AgNPs also possess considerable cytotoxic activity against MCF-7 cell line at increased dosage of 100 μg mL-1 as well as comparable to cytotoxicity activity of Doxorubicin. Hence, the acquired results suggest that the plant fabricated AgNPs may be considered for further in vivo study to understand their possible biomedical potential to use for the welfare of humans.



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*Source: 6095005-2022-10-10.xml*