Use of A Hydroalcoholic Extract of Moringa oleifera Leaves for the Green Synthesis of Bismuth Nanoparticles and Evaluation of Their Anti-Microbial and Antioxidant Activities

The employment of plant extracts in the synthesis of metal nanoparticles is a very attractive approach in the field of green synthesis. To benefit from the potential synergy between the biological activities of the Moringa oleifera and metallic bismuth, our study aimed to achieve a green synthesis of phytochemical encapsulated bismuth nanoparticles using a hydroalcoholic extract of M. oleifera leaves. The total phenolic content in the M. oleifera leaves extract used was 23.0 ± 0.3 mg gallic acid equivalent/g of dried M. oleifera leaves powder. The physical properties of the synthesized bismuth nanoparticles were characterized using UV-Vis spectrophotometer, FT-IR spectrometer, TEM, SEM, and XRD. The size of the synthesized bismuth nanoparticles is in the range of 40.4–57.8 nm with amorphous morphology. Using DPPH and phosphomolybdate assays, our findings revealed that the M. oleifera leaves extract and the synthesized bismuth nanoparticles possess antioxidant properties. Using resazurin microtiter assay, we also demonstrate that the M. oleifera leaves extract and the synthesized bismuth nanoparticles exert potent anti-bacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (estimated MIC values for the extract: 500, 250, 250, and 250 µg/mL; estimated MIC values for the bismuth nanoparticles: 500, 500, 500, and 250 µg/mL, respectively). Similarly, the M. oleifera leaves extract and the synthesized bismuth nanoparticles display relatively stronger anti-fungal activity against Aspergillus niger, Aspergillus flavus, Candida albicans, and Candida glabrata (estimated MIC values for the extract: 62.5, 62.5, 125, and 250 µg/mL; estimated MIC values for the bismuth nanoparticles: 250, 250, 62.5, and 62.5 µg/mL, respectively). Thus, green synthesis of bismuth nanoparticles using M. oleifera leaves extract was successful, showing a positive antioxidant, anti-bacterial, and anti-fungal activity. Therefore, the synthesized bismuth nanoparticles can potentially be employed in the alleviation of symptoms associated with oxidative stress and in the topic treatment of Candida infections.

agent [39]. Under a low X-ray dose, a radiosensitizer comprised of bismuth nanoparticles attached to antibodies was used to specifically and selectively target and kill multidrug resistant bacteria [39].
Herein, we attempted to achieve enhanced biological activities of bismuth nanoparticles via their synthesis using M. oleifera leaves' phytochemical constituents. To this end, we pursued green synthesis of bismuth nanoparticles using a hydroalcoholic extract of M. oleifera leaves, followed by their physical characterization using different techniques. We also assessed the antioxidant activity of M. oleifera leaves extract and of synthesized phytochemical encapsulated bismuth nanoparticles, as well as their anti-bacterial and anti-fungal activities against different species of bacteria and fungi. We anticipate that our findings will be significant in the employment of green-synthesized bismuth nanoparticles in several medical applications.

Phytochemical Analysis of the M. oleifera Leaves Extract
The qualitative evaluation of various chemical constituents in the M. oleifera leaves extract was performed using the test methods mentioned in Table 1. The protein content in the M. oleifera leaves extract was estimated to be 0.1% of the dry leaves powder. The total phenolic content in M. oleifera leaves extract was 23% of the dry leaves powder. An earlier study demonstrated that alanine, tyrosine, lysine, and threonine are among the major amino acids present in the M. oleifera leaves extract [40]. The concoction of M. oleifera leaves extract contains isomers of ceffeoylquinic acid, isomers of feruloylquinic acid, tannins, gallic acid, and several flavonoids like quercetin, kaempferol ( Figure S1) and their glycoside derivatives [41]. Using High-performance liquid chromatography (HPLC) analysis, isoquercetin, astragalin, and crypto-chlorogenic acid were identified in the ethanolic extract of M. oleifera leaves [42]. The hydroalcoholic extract of M. oleifera leaves (water/ethanol 50:50), contains numerous unexplored macromolecules. Collectively, such findings suggest that the M. oleifera leaves extract could serve as a nutritional supplement and a possible stabilizing agent for the synthesized bismuth nanoparticles. From the total phenolic content estimation of the M. oleifera leaves extract before and after the reaction (Table 2), Scheme 1 is proposed. The total phenolic content of 60 mg gallic acid equivalent from 10 g of dried M. oleifera leaves powder was used in the synthesis of 170 mg of bismuth nanoparticles from 0.04 M of bismuth (III) ion solution. The reducing and binding chemical entities present in the concoction aid in the formation and stabilization of the synthesized bismuth nanoparticles through encapsulation. A color change from light brown to dark brown indicates the successful formation of bismuth nanoparticles.

Size and Morphology of the Synthesized Bismuth Nanoparticles
The high-resolution transmission electron microscopy (HRTEM) image of synthesized bismuth nanoparticles was captured using a JEOL-TEM 2100 plus electron microscope ( Figure 1). EDS (energy dispersive X-ray spectroscopy) studies indicate only the presence of elements in the sample ( Figure 2).
The synthesized encapsulated bismuth nanoparticles were washed only with ethanol and water, to avoid the use of harsh chemicals and thermal processes. Figure 1a shows the phytochemicals in the extract enclosing the nanoparticles as dark spots in the cloud of organic matrix. The right lower part of Figure 1a was enlarged to 20 nm in Figure 1b and further to 5 nm in Figure 1c. The selected area electron diffraction (SAED) of this portion was given in Figure 1d. The EDS, taken at the sample shown in Figure 1c, is given in Figure 2a

Size and Morphology of the Synthesized Bismuth Nanoparticles
The high-resolution transmission electron microscopy (HRTEM) image of synthesized bismuth nanoparticles was captured using a JEOL-TEM 2100 plus electron microscope ( Figure 1). EDS (energy dispersive X-ray spectroscopy) studies indicate only the presence of elements in the sample ( Figure 2).

Size and Morphology of the Synthesized Bismuth Nanoparticles
The high-resolution transmission electron microscopy (HRTEM) image of synthesized bismuth nanoparticles was captured using a JEOL-TEM 2100 plus electron microscope ( Figure 1). EDS (energy dispersive X-ray spectroscopy) studies indicate only the presence of elements in the sample ( Figure 2).
The synthesized encapsulated bismuth nanoparticles were washed only with ethanol and water, to avoid the use of harsh chemicals and thermal processes. Figure 1a shows the phytochemicals in the extract enclosing the nanoparticles as dark spots in the cloud of organic matrix. The right lower part of Figure 1a was enlarged to 20 nm in Figure 1b and further to 5 nm in Figure 1c. The selected area electron diffraction (SAED) of this portion was given in Figure 1d. The EDS, taken at the sample shown in Figure 1c, is given in Figure 2a    The synthesized bismuth nanoparticles were obtained in ethanol to give a colloidal solution. The electron diffraction pattern in Figure 1d explains the amorphous in nature of the product as nanoparticles agglomerate upon storage. EDS analysis ( Figure 2) confirmed the presence of elemental bismuth nanoparticles. The absence of other elemental peaks reflects the purity of the sample. As shown in Figure  3a, the SEM images suggest an approximate size of the synthesized bismuth particles in the range of 40 and 60 nm. It is envisaged to perform a TEM analysis to obtain the proper size of nanoparticles.
(a)   The synthesized bismuth nanoparticles were obtained in ethanol to give a colloidal solution. The electron diffraction pattern in Figure 1d explains the amorphous in nature of the product as nanoparticles agglomerate upon storage. EDS analysis ( Figure 2) confirmed the presence of elemental bismuth nanoparticles. The absence of other elemental peaks reflects the purity of the sample. As shown in Figure  3a, the SEM images suggest an approximate size of the synthesized bismuth particles in the range of 40 and 60 nm. It is envisaged to perform a TEM analysis to obtain the proper size of nanoparticles.
(a) The synthesized encapsulated bismuth nanoparticles were washed only with ethanol and water, to avoid the use of harsh chemicals and thermal processes. Figure 1a shows the phytochemicals in the extract enclosing the nanoparticles as dark spots in the cloud of organic matrix. The right lower part of Figure 1a was enlarged to 20 nm in Figure 1b and further to 5 nm in Figure 1c. The selected area electron diffraction (SAED) of this portion was given in Figure 1d. The EDS, taken at the sample shown in Figure 1c, is given in Figure 2a  The synthesized bismuth nanoparticles were obtained in ethanol to give a colloidal solution. The electron diffraction pattern in Figure 1d explains the amorphous in nature of the product as nanoparticles agglomerate upon storage. EDS analysis ( Figure 2) confirmed the presence of elemental bismuth nanoparticles. The absence of other elemental peaks reflects the purity of the sample. As shown in Figure 3a, the SEM images suggest an approximate size of the synthesized bismuth particles in the range of 40 and 60 nm. It is envisaged to perform a TEM analysis to obtain the proper size of nanoparticles.
However, SEM analysis suggests that phytochemical encapsulated bismuth nanoparticles tend to agglomerate upon storage for one week, as seen in Figure 3b,c.
X-ray diffraction (XRD) analysis confirms the amorphous nature of the synthesized bismuth nanoparticles ( Figure 4).
This substantiates the diffraction pattern observed in Figure 1d, suggesting that the synthesized bismuth nanoparticles are amorphous in morphology due to the presence of phytochemicals enclosing these nanoparticles.   However, SEM analysis suggests that phytochemical encapsulated bismuth nanoparticles tend to agglomerate upon storage for one week, as seen in Figure 3b,c.
X-ray diffraction (XRD) analysis confirms the amorphous nature of the synthesized bismuth nanoparticles ( Figure 4). This substantiates the diffraction pattern observed in Figure 1d, suggesting that the synthesized bismuth nanoparticles are amorphous in morphology due to the presence of phytochemicals enclosing these nanoparticles.

Fourier Transform Infrared Spectroscopy (FT-IR) of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
The FT-IR spectra of the M. oleifera leaves extract and the synthesized bismuth nanoparticles are shown in Figures 5 and 6, respectively. This vibration spectroscopy data can be used to identify the biomolecules implicated in the synthesis of bismuth nanoparticles. The bands around 3400 cm −1 and 1630 cm −1 are broad in the M. oleifera leaves extract ( Figure 5), corresponding to the vibration mode of hydroxyl group, mostly found in polyphenolic molecules such as tannins, flavonoids, and glycoside derivatives. The FT-IR spectrum of the synthesized bismuth nanoparticles ( Figure 6) depicts a sharp band at 3431 cm −1 , corresponding to the N-H vibration mode. The fingerprint region at 1269 cm −1 explains the binding of the organic matrix to the synthesized bismuth nanoparticles. This comparison reflects the participation of the hydroxyl group in the synthesis of bismuth nanoparticles. Moreover, the binding characteristics of amino groups in the M. oleifera leaves extract are observed in Figures 5 and 6. This could be the reason for agglomeration of the synthesized bismuth nanoparticles to give an amorphous morphology.  The UV-Vis absorption spectrum of the M. oleifera leaves extract is shown in Figure 7, λmax at 390 nm. The UV-Vis spectrum of the synthesized bismuth nanoparticles reconstituted in dimethyl sulfoxide (DMSO) solvent is shown in Figure 8, λmax at 270 nm. The peak relative to the phytochemicals is slightly  The UV-Vis absorption spectrum of the M. oleifera leaves extract is shown in Figure 7, λmax at 390 nm. The UV-Vis spectrum of the synthesized bismuth nanoparticles reconstituted in dimethyl sulfoxide (DMSO) solvent is shown in Figure 8, λmax at 270 nm. The peak relative to the phytochemicals is slightly decreased, due to changes in poly-hydroxyl compounds responsible for bio-reduction and the The UV-Vis absorption spectrum of the M. oleifera leaves extract is shown in Figure 7, λ max at 390 nm. The UV-Vis spectrum of the synthesized bismuth nanoparticles reconstituted in dimethyl sulfoxide (DMSO) solvent is shown in Figure 8, λ max at 270 nm. The peak relative to the phytochemicals is slightly decreased, due to changes in poly-hydroxyl compounds responsible for bio-reduction and the encapsulation of the product, and the peak maximum is shifted to the right. The phytochemical complexed with bismuth are enriched in the Figure 8, and the absorbance at 220 and could be terpenes or polyphenolic species.

Antioxidant Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
Using DPPH assay against ascorbic acid (standard), the antioxidant activity percentage (AA%) of the M. oleifera leaves extract and the synthesized bismuth nanoparticles was assessed. As shown in Table 3, the M. oleifera leaves extract exerted considerable antioxidant potential, while the synthesized bismuth nanoparticles displayed relatively weaker antioxidant activity. These results were supported with total antioxidant capacity measured by phosphomolybdate assay (Table 4).

Antioxidant Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
Using DPPH assay against ascorbic acid (standard), the antioxidant activity percentage (AA%) of the M. oleifera leaves extract and the synthesized bismuth nanoparticles was assessed. As shown in Table 3, the M. oleifera leaves extract exerted considerable antioxidant potential, while the synthesized bismuth nanoparticles displayed relatively weaker antioxidant activity. These results were supported with total antioxidant capacity measured by phosphomolybdate assay (Table 4).

Antioxidant Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
Using DPPH assay against ascorbic acid (standard), the antioxidant activity percentage (AA%) of the M. oleifera leaves extract and the synthesized bismuth nanoparticles was assessed. As shown in Table 3, the M. oleifera leaves extract exerted considerable antioxidant potential, while the synthesized bismuth nanoparticles displayed relatively weaker antioxidant activity. These results were supported with total antioxidant capacity measured by phosphomolybdate assay (Table 4).

Anti-Bacterial Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
The potential anti-bacterial activity of synthesized bismuth nanoparticles was evaluated against four bacterial species (E. coli, K. pneumoniae, S. aureus, and E. faecalis). The classification of these species of bacteria and their localization in the human body is given in Table 5. The estimated minimum inhibitory concentration (MIC) values measured in presence of the M. oleifera leaves extract and the bismuth nanoparticles are given in Table 6. The growth of the indicated species of bacteria in the presence of different concentrations (7.8-1000 µg/mL) of M. oleifera leaves extract and the bismuth nanoparticles is shown in Table 7. The resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized bismuth nanoparticles are shown in Figure 9. In presence of the M. oleifera leaves extract, the estimated MIC values against E. coli, K. pneumoniae, S. aureus, and E. faecalis were found to be in the range of 250-500 µg/mL ( Table 6). The estimated MIC values (250-500 µg/mL) in presence of the synthesized bismuth nanoparticles were similar to that of M. oleifera leaves extract (Table 6): the bismuth nanoparticles have comparable anti-bacterial activities (Table 6 and Figure 9), albeit a portion of the phytochemicals is complexed to the nanoparticles, as seen by the reduction in antioxidant power (Table 2). Hence, in the product bismuth nanoparticles, a reduction in TAC and AA% was observed with loss of some functional group during transformation of the phytochemicals, responsible for bio-reduction and the encapsulation of nanoparticles. This loss may account also to the difference in inhibition found, on K. pneumoniae and S. aureus, and on Aspergillus species.  Table 7. Anti-bacterial activity data (growth (+) and no growth (-)).

Anti-Fungal Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
The potential anti-fungal activity of synthesized bismuth nanoparticles was evaluated against four fungal species (A. niger, A. flavus, C. albicans, and C. glabrata). Ketoconazole (10 µg/500 µL) was used as a positive control, while water-ethanol (1:1) solution and DMSO solvent served as negative controls for the M. oleifera leaves extract and the bismuth nanoparticles, respectively. The Potato Dextrose Broth was also used as a negative control. The classification of these species of fungi and their localization in the human body is given in Table 8. The minimum inhibitory concentration (MIC) values measured in presence of the M. oleifera leaves extract and the bismuth nanoparticles are given in Table 6b. The growth of the indicated species of fungi in presence of different concentrations (7.8-1000 µg/mL) of M. oleifera leaves extract and the bismuth nanoparticles is shown in Table 9. The resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized bismuth nanoparticles are shown in Figure 10. In presence of the M. oleifera leaves extract, the estimated MIC values against A. niger, A. flavus, C. albicans, and C. glabrata were found to be 62.5, 62.5, 125, and 250 µg/mL, respectively (Table 6b). In presence of the synthesized bismuth nanoparticles, the estimated MIC values against A. niger, A. flavus, C. albicans, and C. glabrata were found to be 250, 250, 62.5, and 62.5 µg/mL, respectively (Table 6b). These findings indicate that the synthesized bismuth nanoparticles displayed more effective anti-fungal activity against C. albicans and C. glabrata compared to the M. oleifera leaves extract (Tables 6b and 9, and Figure 10). Candida species are normally present on human skin, and their infections are common in skin and other tissues due to dysbiosis and suppression of immune responses. Future in vivo analysis is required to shed light on the possible employment of the encapsulated phytochemical synthesized bismuth nanoparticles in the treatment of candidiasis, but we anticipate that they could serve as a promising therapeutic candidate to treat candidiasis after considering their pharmacokinetics.

Anti-Fungal Activity of the M. oleifera Leaves Extract and the Synthesized Bismuth Nanoparticles
The potential anti-fungal activity of synthesized bismuth nanoparticles was evaluated against four fungal species (A. niger, A. flavus, C. albicans, and C. glabrata). Ketoconazole (10 µg/500 µL) was used as a positive control, while water-ethanol (1:1) solution and DMSO solvent served as negative controls for the M. oleifera leaves extract and the bismuth nanoparticles, respectively. The Potato Dextrose Broth was also used as a negative control. The classification of these species of fungi and their localization in the human body is given in Table 8. The minimum inhibitory concentration (MIC) values measured in presence of the M. oleifera leaves extract and the bismuth nanoparticles are given in Table 6. The growth of the indicated species of fungi in presence of different concentrations (7.8-1000 µg/mL) of M. oleifera leaves extract and the bismuth nanoparticles is shown in Table 9. The resazurin microtiter assay plates for the M. oleifera leaves extract and the synthesized bismuth nanoparticles are shown in Figure 10. In presence of the M. oleifera leaves extract, the estimated MIC values against A. niger, A. flavus, C. albicans, and C. glabrata were found to be 62.5, 62.5, 125, and 250 µg/mL, respectively (Table 6). In presence of the synthesized bismuth nanoparticles, the estimated MIC values against A. niger, A. flavus, C. albicans, and C. glabrata were found to be 250, 250, 62.5, and 62.5 µg/mL, respectively (Table 6). These findings indicate that the synthesized bismuth nanoparticles displayed more effective anti-fungal activity against C. albicans and C. glabrata compared to the M. oleifera leaves extract (Tables 6 and 9, and Figure 10). Candida species are normally present on human skin, and their infections are common in skin and other tissues due to dysbiosis and suppression of immune responses. Future in vivo analysis is required to shed light on the possible employment of the encapsulated phytochemical synthesized bismuth nanoparticles in the treatment of candidiasis, but we anticipate that they could serve as a promising therapeutic candidate to treat candidiasis after considering their pharmacokinetics.  Table 9. Anti-fungal activity data (growth (+) and no growth (-)). No.

Preparation of the M. oleifera Leaves Extract
The leaves of M. oleifera were collected from the wild shrub regions in Rajapalayam, Tamil Nadu, India. The plant was identified by the facility at Asthagiri Herbal Research Foundation, Chennai, India. The identified specimen vouchers (AHRF/HERBARIUM/021) were deposited at Asthagiri Herbal Research Foundation, Chennai, India. The country's common breed was used in the study. The genetically-modified varieties of M. oleifera (PKM-1 and PKM-2) were not used in our study. Fresh M. oleifera leaves were collected and shade-dried. The dried leaves were smoothly crushed. The M. oleifera leaves powder was stored at room temperature. A total of 10 g of M. oleifera leaves powder was taken and soaked in 100 mL water-ethanol (1:1) solution. The mixture was macerated for 1 h and the M. oleifera leaves extract was filtered through Whatman filter paper 1. The M. oleifera leaves extract was used immediately for the preparation of bismuth nanoparticles and other experimental analyses.

Preparation of the M. oleifera Leaves Extract
The leaves of M. oleifera were collected from the wild shrub regions in Rajapalayam, Tamil Nadu, India. The plant was identified by the facility at Asthagiri Herbal Research Foundation, Chennai, India. The identified specimen vouchers (AHRF/HERBARIUM/021) were deposited at Asthagiri Herbal Research Foundation, Chennai, India. The country's common breed was used in the study. The genetically-modified varieties of M. oleifera (PKM-1 and PKM-2) were not used in our study. Fresh M. oleifera leaves were collected and shade-dried. The dried leaves were smoothly crushed. The M. oleifera leaves powder was stored at room temperature. A total of 10 g of M. oleifera leaves powder was taken and soaked in 100 mL water-ethanol (1:1) solution. The mixture was macerated for 1 h and the M. oleifera leaves extract was filtered through Whatman filter paper 1. The M. oleifera leaves extract was used immediately for the preparation of bismuth nanoparticles and other experimental analyses.

Synthesis of the Bismuth Nanoparticles
Bismuth nitrate pentahydrate (1 g) was dissolved in 20 mL de-mineralized water and added to 80 mL of M. oleifera leaves extract. The reaction mixture was stirred for 3 h at 60 • C. The synthesized bismuth nanoparticles were collected by using centrifugation and repeatedly washed with de-mineralized water. The encapsulated phytochemical synthesized bismuth nanoparticles were dried at 105 • C for 1 h, and subsequently reconstituted in DMSO solvent.

Characterization of the Size and Morphology of the Synthesized Bismuth Nanoparticles
The completion of synthesis was characterized using a UV-3600 Plus UV-Vis double beam spectrophotometer (Shimadzu, Kyoto, Japan). The formation of bismuth nanoparticles and the role of biomolecules in this synthesis were confirmed using an ALPHA-E FTIR spectrometer (Bruker, Billerica, MA, USA). The crystalline nature of the synthesized bismuth nanoparticles was ascertained by XRD using a XRD-6000 diffractometer (Shimadzu, Kyoto, Japan). The size of the synthesized bismuth nanoparticles was measured by SEM using a FEI Quanta 200 scanning electron microscope, with the field emission gun (FEG) feature for better resolution (ThermoFisher Scientific, Waltham, MA, USA). The morphology assessment of the synthesized nanoparticles was performed by transmission electron microscopy (TEM) using a JEOL-TEM-2100 plus transmission electron microscopy (JEOL, Tokyo, Japan). The sample was dispersed in ethanol, coated on the grid, and dried for TEM analysis along with EDS analysis. In previous published work, several authors described improvement of silver nanoparticles, by means of a nitroxide coating to make silver NPs [43], through immobilization of TEMPO moieties on the surface of various materials and synthesis of novel hybrid nanostructures [44], and through synthesis of gold nanoparticles densely coated with nitroxide spins [45]. Although this is a preliminary work, in the next step we will perform thermogravimetric and XPS analyses to give a proper characterization of the synthesized materials and to assess the content of bismuth in the synthesized materials. We will deepen study of this area by applying this method in the future research. We are confident that the antifungal activity toward Candida species may provide a temporary therapeutic application.

Phytochemical Analysis of the M. oleifera Leaves Extract
The qualitative analysis of biomolecules present in the M. oleifera leaves extract was carried out for the presence of alkaloids, tannins, flavonoids, steroids, saponins, polyphenols, glycosides, carbohydrates, proteins, and amino acids. The total phenolic content in the M. oleifera leaves extract was estimated as gallic acid equivalent by Folin-Ciocalteu polyphenol assay [46]. The protein content in the M. oleifera leaves extract was estimated by using Lowry's method [47].

DPPH Assay (Antioxidant Activity Percentage-AA%)
The antioxidant activity percentage (AA%) (scavenging activity) of the M. oleifera leaves extract and the synthesized bismuth nanoparticles was assessed by using DPPH free radical scavenging assay. A total of 1 mg of ascorbic acid (standard) was dissolved in 1 mL of methanol. Different aliquots (serial dilution) of the ascorbic acid solution (0.1-0.5 mL), corresponding to 100-500 µg, were used for calibration. To each tube containing ascorbic acid solution, 1 mL of 0.1 mM DPPH radical solution in ethanol was added, and the final volume was adjusted to 4 mL using ethanol. The stock solutions for the M. oleifera leaves extract and the synthesized bismuth nanoparticles were prepared by dissolving 1 mg of each sample in 1 mL of an appropriate solvent (methanol for the M. oleifera leaves extract; DMSO for the synthesized bismuth nanoparticles). Different aliquots from the stock solutions (0.1-0.5 mL), corresponding to 100-500 µg, were added to all tubes except the blank tube (control). The volume in each tube was adjusted to 3 mL using ethanol. A total of 1 mL of 0.1 mM DPPH radical solution in ethanol was added to each tube,. The blank tube (control) was prepared by mixing 3 mL of ethanol and 1 mL of DPPH radical solution in ethanol. All tubes were incubated for 30 min at room temperature, and absorbance at 517 nm was recorded. AA% was determined using the following formula: AA% = {(absorbance of blank) − (absorbance of sample)/(absorbance of blank)} × 100 (1)

Phosphomolybdenum Assay (Total Antioxidant Capacity-TAC)
The total antioxidant capacity (TAC) of the M. oleifera leaves extract and the synthesized bismuth nanoparticles was assessed by phosphomolybdenum assay as ascorbic acid equivalent. A total of 1 mg of ascorbic acid (standard) was dissolved in 1 mL of methanol. Different aliquots (serial dilution) of the ascorbic acid solution (0.1-0.5 mL), corresponding to 100-500 µg, were prepared. The volume in each tube was adjusted to 4 mL using distilled water. The stock solutions for the M. oleifera leaves extract and the synthesized bismuth nanoparticles were prepared by dissolving 1 mg of each sample in 1 mL of an appropriate solvent (methanol for the M. oleifera leaves extract; DMSO for the synthesized bismuth nanoparticles). Different aliquots from the stock solutions (0.1-0.5 mL), corresponding to 100-500 µg, were added to all tubes except the blank tube (control). The volume in each tube was adjusted to 3 mL using distilled water. To each tube, 1 mL of phosphomolybdenum reagent (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) was added. The volume in each tube was adjusted to 4 mL using distilled water. After incubation for 90 min at 95 • C, absorbance at 695 nm was recorded. The calibration curve for the ascorbic acid solution (standard) was plotted for the absorbance at 695 nm against known amounts of ascorbic acid with the phosphomolybdate reagent in order to express the TAC values as ppm equivalent of ascorbic acid. Using the ascorbic acid calibration curve, the TAC values for the M. oleifera leaves extract and the synthesized bismuth nanoparticles were calculated and expressed as a ppm equivalent of ascorbic acid.

Anti-Bacterial Activity using Resazurin Microtiter Assay
The bacterial cultures of E. coli, K. pneumoniae, S. aureus, and E. faecalis were obtained from the Royal Bio Research Centre, Chennai, Tamil Nadu, India.The most rapid and inexpensive way to screen several microorganism isolates at the same time, with better correlation in comparison to other techniques, is the resazurin microtiter assay [48][49][50]. The resazurin solution was prepared by dissolving a 270 mg tablet of resazurin in 40 mL of sterile distilled water. The test was carried out in 96-well plates under aseptic conditions. A volume of 100 µL of sample containing 10 mg/mL was transferred into the first well of the plate. Afterwards, 50 µL of nutrient broth was added to all other wells, and the tested sample was serially diluted. Subsequently, 10 µL of resazurin solution and 10 µL of bacterial suspension were added to each well. To prevent dehydration, the plates were wrapped with cling film and incubated at 37 • C for 18-24 h. The color change was visually observed. A purple to pink (or colorless) color change was considered as being positive, indicating cell growth (i.e., (+) means growth and (-) means no growth). MIC was recorded at the lowest concentration whereby a color change occurred. Streptomycin (10 µg/500 µL) served as a positive control, while water-ethanol (1:1) solution and DMSO solvent were used as negative controls for the M. oleifera leaves extract and the bismuth nanoparticles, respectively. The nutrient broth was also used as a negative control.

Anti-Fungal Activity Using Resazurin Microtiter Assay
The fungal cultures of A. niger, A. flavus, C. albicans, and C. glabrata were obtained from the Royal Bio Research Centre, Chennai, Tamil Nadu, India.The resazurin microtiter assay was performed as described above (Section 3.6). Ketoconazole (10 µg/500 µL) was used as a positive control, while water-ethanol (1:1) solution and DMSO solvent served as negative controls for the M. oleifera leaves extract and the bismuth nanoparticles, respectively. The Potato Dextrose Broth was also used as a negative control.

Conclusions
Our experimental approach led to the successful green synthesis of bismuth nanoparticles using a hydroalcoholic extract of M. oleifera leaves. UV-Vis absorption and FT-IR spectrometry confirmed the formation of bismuth nanoparticles and the role of different biomolecules present in the M. oleifera leaves extract in the synthesis of bismuth nanoparticles. The presence of elemental bismuth was affirmed by EDS analysis. XRD analysis confirmed the crystalline nature of the synthesized bismuth nanoparticles. SEM analysis revealed that the size of the synthesized bismuth nanoparticles is in the range of 40.4-57.8 nm. As revealed by TEM with EDS, the synthesized bismuth nanoparticles were confirmed to display an amorphous morphology. DPPH and phosphomolybdenum assays revealed that both the M. oleifera leaves extract and the synthesized bismuth nanoparticles exert considerable antioxidant activity. However, the antioxidant potential of the synthesized bismuth nanoparticles is weaker than that of the M. oleifera leaves extract, most likely due to a loss of some phenolic constituents. Our study further demonstrated that the M. oleifera leaves extract and the synthesized bismuth nanoparticles play a potent anti-bacterial role against E. coli, K. pneumoniae, S. aureus, and E. faecalis (MIC values for the extract: 500, 250, 250, and 250 µg/mL; MIC values for the bismuth nanoparticles: 500, 500, 500, and 250 µg/mL, respectively), with a more profound inhibitory effect of the bismuth nanoparticles, compared to the M. oleifera leavesextract, against E. faecalis. Moreover, our findings also revealed that the M. oleifera leaves extract and the synthesized bismuth nanoparticles display relatively stronger anti-fungal activity against A. niger, A. flavus, C. albicans, and C. glabrata (MIC values for the extract: 62.5, 62.5, 125, and 250 µg/mL; MIC values for the bismuth nanoparticles: 250, 250, 62.5, and 62.5 µg/mL, respectively), with a promising and exploitable inhibitory activity against Candida species. This indicates that the anti-fungal effects of the M. oleifera leaves extract against A. niger and A. flavus is stronger than that of the synthesized bismuth nanoparticles, while the latter exerts more potent anti-fungal effects on C. albicans and C. glabrata. Obviously, our green synthesis approach to obtain bismuth nanoparticles using a hydroalcoholic extract of M. oleifera leaves did not jeopardize the bismuth nanoparticles' anti-bacterial and anti-fungal activities. Collectively, our study implies that the synthesized bismuth nanoparticles can be potentially used in the amelioration of oxidative stress and various microbial infections. In particular, the synthesized bismuth nanoparticles seem to be powerful anti-fungal agents against cases of candidiasis including, but not limited to oral thrush, urinary yeast infection, genital infection, intra-abdominal candidiasis, acute hematogenous candidiasis, fungemia, invasive candidiasis, endocarditis, and endophthalmitis. One advantage of green synthesis of nanoparticles is that it yields non-toxic products and it reduces the production of wasteful material. Another advantage of green synthesis of nanoparticles is that the encapsulating material being polyphenols has a therapeutic value compared to the incinerated nanoparticles that are synthesized by using conventional thermal-chemical methods.