Pollen Bee Aqueous Extract-Based Synthesis of Silver Nanoparticles and Evaluation of Their Anti-Cancer and Anti-Bacterial Activities

: Bee pollens are rich source of essential amino acids and are often considered as complete food for human beings. Herein, we exploited the potential reducing abilities of Bee pollens extract for the eco-friendly preparation of silver nanoparticles (AgNPs-G). The resulting NPs were characterized using a combination of microscopic and spectroscopic techniques. The analyses conﬁrm the formation of spherical Ag NPs. AgNPs-G obtained from the aqueous extract of bee pollens was used to study their antibacterial properties against Gram-positive and Gram-negative microbes using the Minimum Inhibitory Concentration 50 (MIC 50 ) method. The antibacterial properties of AgNPs-G were compared to the properties of chemically synthesized Ag NPs (AgNPs-C) using sodium borohydride as a reducing agent. The green synthesized nanoparticles (AgNPs-G) exhibited a better antibacterial activity against most of the studied strains when compared to the chemically synthesized Ag NPs (AgNPs-C). In addition, the anti-cancer activity of Ag NPs was also studied against human liver and breast carcinoma cell lines by applying MTT-assay. The Ag NPs demonstrated considerable anticancer activity against the studied cell lines and exhibited high IC 50 values in both MCF-7 and HepG2 cell lines.


Introduction
Recently, the trend of exploiting the antioxidant properties of extracts of various natural products for the preparation of nanomaterials has gained considerable popularity [1]. Among the different nanoparticles, silver has gained the significant consideration of scientists and technologists because of Figure 1. Graphical representation of green synthesized silver nanoparticles using pollen bee extract (AgNPs-G), chemical synthesized silver nanoparticles using sodium borohydride (AgNPs-C) and evaluation of their antibacterial and anticancer activity.

Preparation of Bee Pollen Extract
Commercial bee pollen (First Elite 100% natural bee pollen) was procured from the Wadi-Al-Nahil local market, Riyadh, KSA. The extraction of bee pollen grain involved treating 1250 g of pollen grains with analytical grade 95% ethanol for 48 h at room temperature. The stirring with ethanol was continued with cycles of decantation and addition until exhaustion. The filtrate aliquots were mixed and filtered together to remove particulates and the resulting solution was subjected to evaporation on a rotary evaporator at 45 °C under reduced pressure to obtain a dark yellow gummy extract (yield-383 g, 30.6% w/w). About 80 g of the gummy mixture was dissolved in a mixture of 1 L distilled water and 30% methanol. This hydro-methanolic solution was subsequently subjected to fractionation using petroleum ether (3 × 0.5 L), dichloromethane (3 × 0.5 L), ethyl acetate (4 × 0.5 L), and n-butanol saturated with water (4 × 0.5 L). All the solvents used were distilled prior to use. Solvent-free residue was generated by concentrating each fraction under pressure, including pollen grain petroleum ether (PGp 25 g), dichloromethane (PGc 15.0 g,), ethyl acetate (PGe 10.0 g), and n-butanol (PGb 10.0 g,). The aqueous layer was separately concentrated to PGa (19.0 g) for further experiments involving generation of silver nanoparticles (Ag NPs).

Preparations of Silver Nanoparticles Using Bee Pollen Extract (AgNPs-G)
The aqueous extract of bee pollen grain (PGa) was used as reductant. An amount of 1 mL of aqueous bee pollen extract was poured into silver nitrate solution (84.935 mg) in a 100-mL flask. The mixture was allowed to stir for ~2 h at 90 °C. The change in color to dark brown was noted which was indicative of generation of Ag NPs. Confirmation was done using UV absorption spectroscopy between 380 and 450 nm. Chemically synthesized Ag NPs (AgNPs-C) were prepared using a Figure 1. Graphical representation of green synthesized silver nanoparticles using pollen bee extract (AgNPs-G), chemical synthesized silver nanoparticles using sodium borohydride (AgNPs-C) and evaluation of their antibacterial and anticancer activity.

Preparation of Bee Pollen Extract
Commercial bee pollen (First Elite 100% natural bee pollen) was procured from the Wadi-Al-Nahil local market, Riyadh, KSA. The extraction of bee pollen grain involved treating 1250 g of pollen grains with analytical grade 95% ethanol for 48 h at room temperature. The stirring with ethanol was continued with cycles of decantation and addition until exhaustion. The filtrate aliquots were mixed and filtered together to remove particulates and the resulting solution was subjected to evaporation on a rotary evaporator at 45 • C under reduced pressure to obtain a dark yellow gummy extract (yield-383 g, 30.6% w/w). About 80 g of the gummy mixture was dissolved in a mixture of 1 L distilled water and 30% methanol. This hydro-methanolic solution was subsequently subjected to fractionation using petroleum ether (3 × 0.5 L), dichloromethane (3 × 0.5 L), ethyl acetate (4 × 0.5 L), and n-butanol saturated with water (4 × 0.5 L). All the solvents used were distilled prior to use. Solvent-free residue was generated by concentrating each fraction under pressure, including pollen grain petroleum ether (PGp 25 g), dichloromethane (PGc 15.0 g), ethyl acetate (PGe 10.0 g), and n-butanol (PGb 10.0 g). The aqueous layer was separately concentrated to PGa (19.0 g) for further experiments involving generation of silver nanoparticles (Ag NPs).

Preparations of Silver Nanoparticles Using Bee Pollen Extract (AgNPs-G)
The aqueous extract of bee pollen grain (PGa) was used as reductant. An amount of 1 mL of aqueous bee pollen extract was poured into silver nitrate solution (84.935 mg) in a 100-mL flask. The mixture was allowed to stir for~2 h at 90 • C. The change in color to dark brown was noted which was indicative of generation of Ag NPs. Confirmation was done using UV absorption spectroscopy between 380 and 450 nm. Chemically synthesized Ag NPs (AgNPs-C) were prepared using a reported method [27]. Briefly, NaBH 4 was added slowly to the aqueous AgNO 3 solution under gentle stirring at room temperature. After some time, the color of the solution changed and a black precipitate was obtained, which was isolated by centrifuge. The AgNPs-C was identified by UV spectroscopy ( Figure S1) and an XRD analysis ( Figure S2) is provided in the Supplementary Information.

Assessing Roles of the Prepared Bee Pollen Extract Ag NPs
A Perkin Elmer 1000 Fourier-transform infrared spectroscopy (FTIR) instrument was used to study both reducing and stabilizing properties of the prepared bee pollen extract. The sample for analysis was subjected to repeat washing prior to FTIR analysis to remove the unbound chemical constituents of the pollen extract. Peaks at specific positions were studied to determine the success of binding of the phytomolecules from the bee pollen aqueous extract to the prepared silver nanoparticles.

Assessing Size and Morphology of the Ag NPs
The size and morphology of the Ag NPs was analyzed by using transmission electron microscopy (TEM). A TEM analysis was carried out by using a JEM 1101 transmission electron microscope (JEOL, Tokyo, Japan).

UV-Vis Analysis
Optical measurements were carried out by using a UV-visible spectrophotometer (Perkin Elmer lambda 35, Waltham, MA, USA).

Antibacterial Activity Screening of the Nanoparticles (NPs)
The screening of the antibacterial potency of Ag NPs was performed using the classical agar disk diffusion and minimum inhibitory concentration (MIC 50 ) methods. Four American Type Culture Collection (ATCC) bacterial strains, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis were procured from the Department of Microbiology, College of Pharmacy, King Saud University. All the strains were sub-cultured in freshly prepared nutrient broth and post observation of growth was standardized as per 0.5 McFarland turbidity standards. The inoculum was further plated on nutrient agar plates using the spread plate technique and a 6-mm sterile borer was used to bore 4 wells in the ready plate. To each of the wells, 100 µL of 100 µg/mL each of Ag NPs and ampicillin was added and the fourth well remained as the negative control. The plates were incubated at 37 • C and the zone of inhibition was noted for each of the added agents.
To determine the MIC 50 , the microdilution technique was used, which involves the use of an agar dilution process, as described in a previous report, with slight modifications [28]. Samples of Ag NPs and ampicillin were diluted using sterile nutrient broth from 100 µg/mL to 3.72 µg/mL in a 96-well plate. Each of the dilutions was inoculated with 10 µL of the bacterial culture containing approximately 1.0 × 10 6 CFU/mL. Inoculation for each strain was done in triplicates followed by incubation at 37 • C for a period of 16 h. Growth control and negative control were also set up in parallel and incubated without the addition of the inoculum. The level of growth in each of the test groups was compared to the growth control and the MIC was expressed as the highest dilution that exhibited growth inhibition, judged by the lack of turbidity in the sample.

Cytotoxic Activity Ag NPs
The cell lines and cell cultures utilized to study the anticancer activity of the as-prepared Ag NPs included a human liver carcinoma cell line (HepG2) and a breast cancer cell line (MCF-7). Both were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 7% fetal bovine serum (FBS) and 1% each of streptomycin and penicillin. The culturing was done at 37 • C in 5% CO 2 supply incubator. Monolayer sub-culturing was done at routine intervals and for each round of experiment, the exponentially growing cells were utilized.
Anticancer activity was assessed by the MTT (3-[4,5-dimethylthiazole-2-y]-2,5-diphenyltetrazolium bromide) colorimetric assay [29]. The activity of the Ag NPs was evaluated on both cell lines and the IC 50 was evaluated by the cytotoxicity test. Each cell suspension of about 5 × 10 4 cells were seeded in a 96-well flat plate containing DMEM with 5% FBS and different concentrations of the NPs: 31 µg/mL, 62 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL. This mixture was incubated at 37 • C for a period of 24 h in a 5% CO 2 incubator. Post incubation, the cells were subjected to a wash serum free medium and a 100 mL of 5 mg/mL of MTT solution was added followed by incubation for 4-5 h. Post MTT incubation, the cells were again washed with phosphate buffer saline, and 100 µL of dimethyl sulfoxide (DMSO) was added to solubilize the unbound formazan. The plates were read at 570 nm absorbance in a plate reader (Biotech, USA) and the entire experiment was run in triplicates. The IC 50 was determined by the color intensity of the generated formazan dye proportionally to the number of viable cells in the well.

UV-Vis Analysis
Ag NPs were prepared under eco-friendly conditions by the reduction of AgNO 3 in water using the bee pollen aqueous extract as a reducing agent. This environmentally friendly preparation was carried out without using any harmful reducing or stabilizing agents, since the bee pollen extract not only reduced but also functionalized the surface of the resultant Ag NPs. On adding the pollen extract to AgNO 3 solution, the color of the reaction mixture slowly turned to dark brown which points towards the effective synthesis of Ag NPs. Initially, the formation of Ag NPs was confirmed by a UV analysis ( Figure 2). Ag NPs commonly show absorption in the visible range of the spectrum from 380 to 450 nm, depending on the quality of NPs (size and shape). In this case, a broad absorption peak appeared at~440 nm, as shown in Figure 2, which confirms the formation of Ag NPs. Monolayer sub-culturing was done at routine intervals and for each round of experiment, the exponentially growing cells were utilized. Anticancer activity was assessed by the MTT (3-[4,5-dimethylthiazole-2-y]-2,5-diphenyltetrazolium bromide) colorimetric assay [29]. The activity of the Ag NPs was evaluated on both cell lines and the IC50 was evaluated by the cytotoxicity test. Each cell suspension of about 5 × 10 4 cells were seeded in a 96-well flat plate containing DMEM with 5% FBS and different concentrations of the NPs: 31 µg/mL, 62 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL. This mixture was incubated at 37 °C for a period of 24 h in a 5% CO2 incubator. Post incubation, the cells were subjected to a wash serum free medium and a 100 mL of 5 mg/mL of MTT solution was added followed by incubation for 4-5 h. Post MTT incubation, the cells were again washed with phosphate buffer saline, and 100 µL of dimethyl sulfoxide (DMSO) was added to solubilize the unbound formazan. The plates were read at 570 nm absorbance in a plate reader (Biotech, USA) and the entire experiment was run in triplicates. The IC50 was determined by the color intensity of the generated formazan dye proportionally to the number of viable cells in the well.

UV-Vis Analysis
Ag NPs were prepared under eco-friendly conditions by the reduction of AgNO3 in water using the bee pollen aqueous extract as a reducing agent. This environmentally friendly preparation was carried out without using any harmful reducing or stabilizing agents, since the bee pollen extract not only reduced but also functionalized the surface of the resultant Ag NPs. On adding the pollen extract to AgNO3 solution, the color of the reaction mixture slowly turned to dark brown which points towards the effective synthesis of Ag NPs. Initially, the formation of Ag NPs was confirmed by a UV analysis ( Figure 2). Ag NPs commonly show absorption in the visible range of the spectrum from 380 to 450 nm, depending on the quality of NPs (size and shape). In this case, a broad absorption peak appeared at ~440 nm, as shown in Figure 2, which confirms the formation of Ag NPs.

XRD Analysis
The formation and crystallinity of the as-prepared Ag NPs was also confirmed by using the powder diffraction technique. The XRD spectrum in Figure 3 indicates the presence of a

XRD Analysis
The formation and crystallinity of the as-prepared Ag NPs was also confirmed by using the powder diffraction technique. The XRD spectrum in Figure 3 indicates the presence of a face-centered cubic (fcc) structure for the Ag NPs. Particularly, the presence of distinct peaks at various positions,  (111) is the most intense peak, which may correspond to the direction of the growth of nanocrystals. In addition to the characteristic's peaks of Ag NPs, the XRD pattern also contains various additional peaks. These peaks can be attributed to the inorganic residual moieties that may be present in the bee pollen grain extract. The residual compounds may have been bound to the surface of the NPs and thus, contributed to the additional reflections in the XRD pattern of Ag-NPs synthesized by bee pollen grains extract.  (111) is the most intense peak, which may correspond to the direction of the growth of nanocrystals. In addition to the characteristic's peaks of Ag NPs, the XRD pattern also contains various additional peaks. These peaks can be attributed to the inorganic residual moieties that may be present in the bee pollen grain extract. The residual compounds may have been bound to the surface of the NPs and thus, contributed to the additional reflections in the XRD pattern of Ag-NPs synthesized by bee pollen grains extract.

FT-IR Analysis
Furthermore, FT-IR was also used to investigate the dual role of bee pollen grains as a reductant and stabilizer. For this purpose, the FT-IR sample was washed repeatedly prior to the measurement to remove the unbound chemical constituents of pollen extract. Figure 4 demonstrates the FT-IR spectrum of bee pollen grains-mediated synthesized Ag NPs. A careful analysis of the spectrum indicates the presence of hydroxyl rich phytomolecules such as flavonoids and polyphenols, which are known to demonstrate reducing and stabilizing abilities. These phytomolecules may have reduced the silver precursor to produce Ag NPs. The FT-IR spectrum of green synthesized Ag NPs exhibits various peaks at different positions, such as the peaks at 3430 cm −1 and 2955 cm −1 , which are related to the presence of hydrogen-bonded OH groups and C-H groups, respectively. Other peaks representing the C−H, C−C, and C−O stretching of the aromatics are present at 1743, 1620 and 1407 cm −1 , respectively. Moreover, peaks indicating the occurrence of C−O stretching of carboxylic acids, alcohols and ether and ester groups are located at 1260 and 1074 cm −1 . Furthermore, the peaks around 1743 cm −1 can be attributed to the C=O group of aldehydes, ketone, or COOH groups. The presence of these peaks in the IR spectrum of Ag NPs confirms the successful binding of the phytomolecules of bee pollen extracts to the surface of nanoparticles as stabilizing ligands.

FT-IR Analysis
Furthermore, FT-IR was also used to investigate the dual role of bee pollen grains as a reductant and stabilizer. For this purpose, the FT-IR sample was washed repeatedly prior to the measurement to remove the unbound chemical constituents of pollen extract. Figure 4 demonstrates the FT-IR spectrum of bee pollen grains-mediated synthesized Ag NPs. A careful analysis of the spectrum indicates the presence of hydroxyl rich phytomolecules such as flavonoids and polyphenols, which are known to demonstrate reducing and stabilizing abilities. These phytomolecules may have reduced the silver precursor to produce Ag NPs. The FT-IR spectrum of green synthesized Ag NPs exhibits various peaks at different positions, such as the peaks at 3430 cm −1 and 2955 cm −1 , which are related to the presence of hydrogen-bonded OH groups and C-H groups, respectively. Other peaks representing the C−H, C−C, and C−O stretching of the aromatics are present at 1743, 1620 and 1407 cm −1 , respectively. Moreover, peaks indicating the occurrence of C−O stretching of carboxylic acids, alcohols and ether and ester groups are located at 1260 and 1074 cm −1 . Furthermore, the peaks around 1743 cm −1 can be attributed to the C=O group of aldehydes, ketone, or COOH groups. The presence of these peaks in the IR spectrum of Ag NPs confirms the successful binding of the phytomolecules of bee pollen extracts to the surface of nanoparticles as stabilizing ligands.

TEM Analysis
The size and morphology of the bee pollen grain extract-synthesized Ag NPs were determined using a TEM analysis. Figure 5 demonstrates the formation of spherical shape Ag NPs, with an average size of ~10-30 nm. Mostly, the NPs are well dispersed, whereas some of them are slightly agglomerated. The few large particles seen in Figure 5b (~44 nm) could be due to the agglomeration of smaller particles; however, the majority of the particles in the TEM images are less than 30 nm in size.

Antibacterial Activity of the Ag NPs
The antibacterial properties of Ag NPs were measured using the disk diffusion method involving both Gram positive and negative organisms such as B. subtilis, S. aureus, P. aeruginosa and E. coli. The activity of AgNPs-G was compared with the commercially available ampicillin and chemically synthesized Ag NPs (AgNPs-C). For the Gram-positive organisms, AgNPs-G exhibited a comparable zone of inhibition corresponding to known antibiotic ampicillin at 18 mm and 17 mm for B. subtilis and S. aureus. Indeed, in the case of B. subtilis, the AgNPs-G demonstrated an even higher effect as compared to ampicillin at 14 mm. Moreover, in the case of Gram-negative organisms, the effect of AgNPs-G was highest against P. aeruginosa at 18 mm, while E.coli exhibited high inhibition with ampicillin at 22 mm. However, the chemically synthesized Ag NPs (AgNPs-C) exhibited much

TEM Analysis
The size and morphology of the bee pollen grain extract-synthesized Ag NPs were determined using a TEM analysis. Figure 5 demonstrates the formation of spherical shape Ag NPs, with an average size of~10-30 nm. Mostly, the NPs are well dispersed, whereas some of them are slightly agglomerated. The few large particles seen in Figure 5b (~44 nm) could be due to the agglomeration of smaller particles; however, the majority of the particles in the TEM images are less than 30 nm in size.

TEM Analysis
The size and morphology of the bee pollen grain extract-synthesized Ag NPs were determined using a TEM analysis. Figure 5 demonstrates the formation of spherical shape Ag NPs, with an average size of ~10-30 nm. Mostly, the NPs are well dispersed, whereas some of them are slightly agglomerated. The few large particles seen in Figure 5b (~44 nm) could be due to the agglomeration of smaller particles; however, the majority of the particles in the TEM images are less than 30 nm in size. Figure 5. (a,b) TEM images of the green synthesized AgNPs-G using the aqueous extract of bee pollen grains, (c) particle size distribution of AgNPs-G.

Antibacterial Activity of the Ag NPs
The antibacterial properties of Ag NPs were measured using the disk diffusion method involving both Gram positive and negative organisms such as B. subtilis, S. aureus, P. aeruginosa and E. coli. The activity of AgNPs-G was compared with the commercially available ampicillin and chemically synthesized Ag NPs (AgNPs-C). For the Gram-positive organisms, AgNPs-G exhibited a comparable zone of inhibition corresponding to known antibiotic ampicillin at 18 mm and 17 mm for B. subtilis and S. aureus. Indeed, in the case of B. subtilis, the AgNPs-G demonstrated an even higher effect as compared to ampicillin at 14 mm. Moreover, in the case of Gram-negative organisms, the effect of AgNPs-G was highest against P. aeruginosa at 18 mm, while E.coli exhibited high inhibition with ampicillin at 22 mm. However, the chemically synthesized Ag NPs (AgNPs-C) exhibited much Figure 5. (a,b) TEM images of the green synthesized AgNPs-G using the aqueous extract of bee pollen grains, (c) particle size distribution of AgNPs-G.

Antibacterial Activity of the Ag NPs
The antibacterial properties of Ag NPs were measured using the disk diffusion method involving both Gram positive and negative organisms such as B. subtilis, S. aureus, P. aeruginosa and E. coli. The activity of AgNPs-G was compared with the commercially available ampicillin and chemically synthesized Ag NPs (AgNPs-C). For the Gram-positive organisms, AgNPs-G exhibited a comparable zone of inhibition corresponding to known antibiotic ampicillin at 18 mm and 17 mm for B. subtilis and S. aureus. Indeed, in the case of B. subtilis, the AgNPs-G demonstrated an even higher effect as compared to ampicillin at 14 mm. Moreover, in the case of Gram-negative organisms, the effect of AgNPs-G was highest against P. aeruginosa at 18 mm, while E.coli exhibited high inhibition with ampicillin at 22 mm. However, the chemically synthesized Ag NPs (AgNPs-C) exhibited much lower antibacterial activities when compare to both AgNPs-G and ampicillin. The results are summarized in Table 1. The MIC 50 study was also done by the microdilution technique involving agar dilution. In the case of B. subtilis and S. aureus (Gram-positive bacteria), AgNP-G exhibited the lowest MIC 50 at 3.15 µg/mL, while for the P. aeruginosa (Gram-negative), the MIC 50 was detected to be 6.75 µg/mL. In all the cases, AgNP-G was found to have the lowest MIC 50 in comparison to ampicillin. The graphical presentation of the MIC 50 outcome is highlighted in Figure 6. lower antibacterial activities when compare to both AgNPs-G and ampicillin. The results are summarized in Table 1. The MIC50 study was also done by the microdilution technique involving agar dilution. In the case of B. subtilis and S. aureus (Gram-positive bacteria), AgNP-G exhibited the lowest MIC50 at 3.15 µg/mL, while for the P. aeruginosa (Gram-negative), the MIC50 was detected to be 6.75 µg/mL. In all the cases, AgNP-G was found to have the lowest MIC50 in comparison to ampicillin. The graphical presentation of the MIC50 outcome is highlighted in Figure 6.

Anticancer Cytotoxic Activity of AgNPs-G by MTT Assay
The anticancer activity assessment of AgNPs-G was done by utilizing a human liver cancer and breast cancer cell line, HepG2 and MCF-7, by the MTT assay. The outcome of the assay for AgNPs-G is shown in Figure 7. In the case of AgNPs-G, a less than 25% viability was noted for both MCF-7 and HepG2 at a concentration of 500 µg/mL which is better in comparison to that of the AgNPs-C (AP) wherein a viability of over 25% was seen at the same concentration.
The IC50 analysis for AgNPs-G and AgNPs-C was also done on both the cell lines as part of the anticancer activity assessment. The results are shown in Table 2. The AgNPs-G are exhibited a low IC50 for both cell lines HepG2 and MCF-7 at 47.6 +/− 1.5 µg/mL and 96 +/− 3.5 µg/mL in comparison to Ag NPs.

Anticancer Cytotoxic Activity of AgNPs-G by MTT Assay
The anticancer activity assessment of AgNPs-G was done by utilizing a human liver cancer and breast cancer cell line, HepG2 and MCF-7, by the MTT assay. The outcome of the assay for AgNPs-G is shown in Figure 7. In the case of AgNPs-G, a less than 25% viability was noted for both MCF-7 and HepG2 at a concentration of 500 µg/mL which is better in comparison to that of the AgNPs-C (AP) wherein a viability of over 25% was seen at the same concentration.
lower antibacterial activities when compare to both AgNPs-G and ampicillin. The results are summarized in Table 1. The MIC50 study was also done by the microdilution technique involving agar dilution. In the case of B. subtilis and S. aureus (Gram-positive bacteria), AgNP-G exhibited the lowest MIC50 at 3.15 µg/mL, while for the P. aeruginosa (Gram-negative), the MIC50 was detected to be 6.75 µg/mL. In all the cases, AgNP-G was found to have the lowest MIC50 in comparison to ampicillin. The graphical presentation of the MIC50 outcome is highlighted in Figure 6.

Anticancer Cytotoxic Activity of AgNPs-G by MTT Assay
The anticancer activity assessment of AgNPs-G was done by utilizing a human liver cancer and breast cancer cell line, HepG2 and MCF-7, by the MTT assay. The outcome of the assay for AgNPs-G is shown in Figure 7. In the case of AgNPs-G, a less than 25% viability was noted for both MCF-7 and HepG2 at a concentration of 500 µg/mL which is better in comparison to that of the AgNPs-C (AP) wherein a viability of over 25% was seen at the same concentration.
The IC50 analysis for AgNPs-G and AgNPs-C was also done on both the cell lines as part of the anticancer activity assessment. The results are shown in Table 2. The AgNPs-G are exhibited a low IC50 for both cell lines HepG2 and MCF-7 at 47.6 +/− 1.5 µg/mL and 96 +/− 3.5 µg/mL in comparison to Ag NPs. The IC 50 analysis for AgNPs-G and AgNPs-C was also done on both the cell lines as part of the anticancer activity assessment. The results are shown in Table 2. The AgNPs-G are exhibited a low IC50 for both cell lines HepG2 and MCF-7 at 47.6 +/− 1.5 µg/mL and 96 +/− 3.5 µg/mL in comparison to Ag NPs.

Discussion
Pollen produced by the anther of the plant is collected by bees mixed with their salivary secretion and nectar and stored in bee colonies wherein it serves as a principle food source, also termed "bee-collected pollen" [30]. Beekeeping is a major growing business in Saudi Arabia and the knowledge on floral resources helps beekeepers maintain colony strength to harvest high-quality as well as high-yield bee products including honey [31]. Providing a map of nectar and pollen sources to beekeepers in their respective areas plays a crucial role in devising a plan for colony management and identifying alternate back-up sources to move colonies during certain periods as per need and emergency [32]. Honeybee pollen, which is a result of the agglutination of floral pollens and salivary secretion of the bees, has proven to be an excellent food supplement in humans, depending on the geography of origin, as it highly influences the chemical composition. An increasing number of studies have focused on identifying the pharmacological benefits of each pollen variety in the respective geographical location to identify the utility of bee products for humans beyond food supplementation. Phenols and flavonoids have been widely studied for their antimicrobial and antioxidant activities. Bee pollen consumption has been linked to many benefits, like tackling anemia, depression, impaired immune function, memory loss, ageing, and impotency [33].
Our study focused on identifying the antibacterial and anticancer activity of Ag NPs prepared by using a commercial pollen variety from Riyadh, Saudi Arabia. Bee pollen extract was prepared using analytical-grade solvents and a series of filtration and concentration steps. Furthermore, the aqueous part of the filtrate was utilized for the preparation of AgNPs-G, the characteristics and success of which were assessed using different analytical techniques, including UV absorption spectroscopy, XRD pattern, FTIR and TEM. The as-prepared NPs were then tested for antibacterial activity using the traditional disk diffusion method and MIC 50 , while anticancer activity was determined using human cancer cell lines and IC 50 .
An antibacterial activity assessment of AgNPs-G was performed in comparison to chemically synthesized Ag NPs (AgNP-C) and ampicillin using ATCC strains of Gram positive organisms like B.subtilis and S.aureus as well as Gram negative organisms like P.aeruginosa and E.coli. Many studies have assessed the effect of methanolic and ethanolic bee pollen extract on different organisms, and one such study which assessed the antibacterial activity against S.aureus detected the highest zone of inhibition in the case of 70% methanol extract of 15% pollen [34]. Another study which evaluated the biological activity of Helianthus annuus L. bee pollen against Gram positive and negative organisms detected a good zone of inhibition against P.aeruginosa using both dried and frozen extracts [35]. In this study, the Gram negative and positive organisms exhibited comparable resistance towards the AgNPs-G except for E.coli, which exhibited maximum inhibition against the positive-control ampicillin. The as-prepared nanoparticles exhibited a better inhibition activity in comparison to the positive control for B.subtilis and P.aeruginosa. A study on antibacterial and antioxidant activity of bee products on different Gram positive and negative organisms detected S.aureus and E.coli to be more resistant to the tested bee products [36]. In our case, the MIC 50 study for each of the AgNPs-G, AgNPs-C and positive control also detected the least for AgNP-G against the ATCC test organisms studied.
The results indicate that the as-prepared AgNPs-G obtained from commercial pollen is a promising candidate for use in food packaging as well as the pharmaceutical industry.
The anticancer activity of bee pollens is attributed to different peptides which induce in vitro apoptosis in many transformed human cancer cell lines, including those from the lung, liver, prostate, and bladder. [37]. Previous studies have detected the steroid fraction of chloroform extract from Brassica campestris bee pollen to exhibit strong cytotoxic activity on human prostate cancer cells by the stimulation of TNF-alpha secretion and apoptosis induction [38]. A study on the anticancer activity of Ag NPs of stingless bee propolis exhibited significant activity against A549 human lung cancer cells and the IC50 value was identified to be 38 µg/mL. Nanoparticles associated with drug delivery systems have been found to be promising in cancer because of their ability to cross the biological barrier and achieve therapeutic concentration in tumors even in low dosage of drug administration and Ag NPs have gained widespread attention in this space [39]. Ag NPs have shown great potential in reducing angiogenesis and oxidative stress. A study that assessed the potential of Ag NPs prepared by using aqueous extract of rapeseed flower pollens against different cancer cell lines detected cytotoxic activity towards MDA-MB-231 and MCF-7 cells [40]. Another study which documented the effect of Ag NPS obtained by using honeybee extract detected 60% inhibition against colon cancer by down regulating the expression of Bcl2 and the surviving gene [41].
In our study AgNPs-G were generated and their anticancer activity was assessed using human a liver carcinoma cell line (HepG2) and a breast cancer cell line (MCF-7). The IC 50 studies done using AgNPs-G, AgNPs-C. AgNPs-G showed them to be active at low concentrations of 47.6 +/− 1.5 µg/mL against HepG2 and 96 +/− 3.5 µg/mL against MCF-7 in comparison to Ag NPs which showed a high concentration at 310 +/− 5.5 µg/mL and 214 +/− 5 µg/mL. A viability study by MTT assay detected a greater reduction by AgNPs-G as compared to AgNPs-C. The prepared nanoparticles were found to be effective against the studied cancer cell lines albeit at IC50 higher than the positive control, indicating their potential use against human cancer.

Conclusions
Bee pollens are considered as high-energy food, which is obtained from the plant flower pollens collected by the bees. It is an apitherapeutic product containing~22.7% proteins, which includes essential amino acids, lipids inclusive of essential fatty acids and phenolic compounds. In this study, eco-friendly synthesis of Ag NPs was reported by an aqueous extract of bee pollen, which contains a variety of potential reducing agents, including polyphenols and flavonoids. Spherically shaped, highly crystalline Ag NPs (AgNPs-G) were prepared in the absence of any hazardous chemical or capping agents. The biosynthesized Ag NPs demonstrated excellent antioxidant properties, which were confirmed by testing the antibacterial and anticancer properties of nanoparticles against various bacterial strains and cell lines. Indeed, in many cases, the AgNPs-G exhibited superior antioxidant properties when compared to the chemically synthesized AgNPs-C and commercially available ampicillin.